WO1994017826A1 - Vaccinal polypeptides - Google Patents

Abstract

This invention provides vaccine compositions capable of conferring multi-strain immunity against influenza A and influenza B. This invention also provides methods of increasing expression and improving homogeneity of H3HA2 protein, fragments thereof, and fusion proteins containing same, as well as novel nucleotide sequences encoding these proteins and fragments.

Description

VACCINAL POLYPEPTIDES

Field of the Invention

The present invention relates generally to polypeptides useful in vaccine compositions and more specifically to vaccine compositions useful in providing immunity against influenza A and influenza B in an animal. The present invention also relates generally to a method of enhancing expression of polypeptides and, more specifically, to a method of enhancing influenza protein expression and homogeneity in E. coli.

Background of the Invention

Influenza virus infection causes acute respiratory disease in man, horses, swine and fowl, sometimes of pandemic proportions. Influenza viruses are orthomyxoviruses and, as such, have envelope virions of 80 to 120 nanometers in diameter, with two different glycoprotein spikes. Three types, A, B and C, infect humans. Type A viruses have been responsible for the majority of human epidemics in modern history, although there are also sporadic outbreaks of Type B infections. Known swine, equine, and avian viruses have mostly been Type A, although Type C viruses have also been isolated from swine.

The Type A viruses are divided into subtypes based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) surface

glycoproteins. Within Type A, subtypes H1 ("swine flu"), H2 ("asian flu"), and H3 ("Hong Kong flu") are predominant in human infections. In swine, the predominant influenza A subtypes are H1 and H3; in horses, H3 and H7; and in avians, H5 and H7. Presently only one Type B virus has been identified, with no subtypes.

Genetic "drift" or "shift", i.e., rapid and unpredictable change in the antigen, occurs at approximately yearly intervals, and affects antigenic determinants in the HA and NA proteins. Therefore, it has not been possible to prepare a

"universal" influenza virus vaccine using conventional killed or attenuated viruses, that is, a vaccine which is non-strain specific. Recently, attempts have been made to prepare such universal, or semi-universal, vaccines from reassortant viruses prepared by crossing different strains. More recently, such attempts have involved recombinant DNA techniques focusing primarily on the HA protein.

There remains a need in the art for vaccine formulations and compositions capable of inducing protective responses in animals against influenza viruses. The expression of recombinant proteins in bacterial systems, particularly E. coli, is highly desirable because it can be used to produce large amounts of the desired proteins relatively inexpensively. However, high level expression of several eukaryotic proteins in E. coli has not been achieved for reasons including, among others, unfavorable codon usage and toxicity of the gene product [U. Brinkmann et al., Gene, 85:109-114 (1989)]. Methods of overcoming these impediments to high-level expression in bacteria have been described, but are not universally applicable.

For example, Brinkmann et al., cited above, described low-level expression of certain genes, such as human tissue-type plasminogen activator or gp41 of human immunodeficiency virus, which the authors attributed to the presence of the rare triplets AGA and AGG which encode arginine (Arg) in unexpectedly high amounts in the gene (3.2%). However, other eukaryotic genes, such as the NS1 gene of influenza virus, contain greater than 3% of such triplets yet express at high levels in E. coli [Young et al, Proc. Natl. Acad. Sci., 80:6105-6109 (1983)].

Another group, Spanjaard et al., Nucl. Acids Res., 18(17):5031-5036 (1990) describe a translation shift in about 50% of ribosomes after tandem (double) AGA and AGG codons in cloned tRNA genes, but observed no frame shifts following single AGG or AGA codons. The authors attribute this frame shift to tRNA depletion. There also remains a need in the art for improved methods of producing vaccinal polypeptides capable of inducing protective responses in animals against influenza viruses. Summary of the Invention

The present invention provides compositions containing, and methods for use of a protein which is capable of inducing protection in animals and avians against challenge with more than one strain of influenza Type A and influenza Type B.

Thus, one aspect of the invention provides a DNA sequence encoding a modified purified recombinant protein. The DNA sequence of the invention encodes a modified protein sequence derived from the HA2 subunit of a selected hemagglutinin (HA) protein. In one embodiment, the sequence is derived from an H3N2 subtype influenza virus. These H3N2 fusion proteins are capable of inducing T cell responses in the absence of neutralizing antibodies. In another embodiment, a DNA sequence of this invention encodes a modified protein sequence derived from the HA2 subunit from a Type B influenza virus. Still further embodiments include DNA sequences obtained as described for the two above viruses, where the sequences are derived from other Type A influenza strains infecting animals as well as humans. Such viruses include, without limitation, Type A subtypes of H1, H2, H3, H4, H5, H6 and H7.

In another aspect, the invention provides a DNA sequence encoding a recombinant fusion protein, in which the desired Type A subtype HA2 subunit sequence or a portion thereof, is fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein. One embodiment includes the H3N2 subtype HA2 subunit sequence described above fused in frame to another protein or fragment capable of enhancing expression thereof. Another embodiment of such a fusion protein comprises a Type B HA2 sequence, described above, or a portion thereof, fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein. Additionally, other Type A subtype HA2 sequences can be similarly used. It is desirable that this fusion partner protein be an influenza protein sequence or fragment thereof.

In still another aspect, a protein encoded by a DNA sequence of the invention is provided. The protein may be a protein sequence derived from the HA2 subunit of an HA protein from a selected Type A subtype virus. Desirably the subtype virus is an H3N2. In another embodiment, the protein may be derived from the HA subunit of a Type B influenza virus. Other embodiments include H5 or H7 subtypes. Additionally, preferred embodiments include fusion proteins comprising a protein sequence derived from the HA2 subunit of an HA protein from a Type A virus, e.g., an H3N2 subtype, or from a Type B virus fused in frame to a selected influenza sequence. The proteins of this invention are particularly useful in inducing protection in mammals, especially humans, against challenge by Type B or an H3N2 subtype of influenza A. The proteins employing other Type A subtypes, e.g., H5 and H7, are useful in inducing protection in animals against influenza viruses.

In another aspect, the invention provides a method of recombinantly producing the fusion proteins of the invention, and a method of purifying the same.

In a further aspect, the invention provides a vaccine composition containing a purified protein of the invention, as described above. Such a vaccine composition may include a fusion protein of the invention. In other embodiments of the invention, the vaccine compositions contain an H3HA2 protein of the invention and other influenza antigens; a Type B HA2 protein of the invention and other influenza antigens; or both an H3HA2 protein, a BHA2 protein and other influenza antigens. In a preferred embodiment for human use, a combination vaccine of the invention will contain an H3HA2 and a BHA2 protein of the invention in combination with influenza antigens derived from the other Type A influenza virus subtypes, H1 and H2. An embodiment for use in animals may contain an H5HA2 or H7HA2 protein, among others.

A further aspect of this invention is a method for inducing in an animal protection against influenza Type A, influenza Type B, influenza Type C, or combinations thereof, which comprises internally administering to the animal an effective immunogenic amount of a vaccine composition of the present invention.

Still a further aspect of this invention is a method for inducing in an animal protection against multiple strains of influenza Types A and B which comprises internally administering to the animal an effective immunogenic amount of a vaccine composition of the present invention.

In another aspect, the present invention provides a method of enhancing in E. coli the expression of influenza vaccinal proteins characterized by a naturally-occurring amino acid pattern comprising Arg-Arg-Xaa-Xaa-Arg [SΕQ ID NO:8]. In this pattern, Arg is arginine, Xaa is any amino acid, and at least one of the arginines in the naturally-occurring sequence is encoded by the rare nucleic acid triplets AGG or AGA.

In one embodiment, the method of the invention involves mutating one or more of these AGG or AGA codons to a preferred arginine codon and expressing the mutated sequence in E. coli. Surprisingly, it has been found that this modification, which does not result in a change in the encoded amino acid sequence, can increase the expression and homogeneity of an influenza protein in E. coli significantly.

In another embodiment, the method of this invention involves increasing the expression of the above-identified proteins by inserting into the host cell tRNA molecules capable of translating the native rare arginine codons. Thus, the E. coli host cells are modified such that they are capable of efficiently translating the rare, native arginine codons.

In another aspect, the present invention provides novel nucleic acid sequences of influenza proteins which contain the nucleotide sequence CGn-CGn-

Xaa-Xaa-CGn, where n represents a nucleotide selected from the group consisting of T, C, A or G [SΕQ ID NO:9], in place of the native nucleotide sequence AGr- AGr-Xaa-Xaa-AGr, where r represents the nucleotides A or G [SΕQ ID NO: 10]. When expressed in E. coli, these sequences result in increased expression of the encoded protein as compared to the native sequence.

In still another aspect, the invention provides the novel modified nucleic acid sequences described above fused in the same reading frame to another DNA sequence encoding a polypeptide or protein, i.e., a fusion partner, which may further enhance the expression of, or immunogenicity of, the encoded influenza protein. It is desirable that the fusion partner be an influenza protein sequence or fragment thereof.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings

Fig. 1 illustrates the nucleic acid sequences of the HA2 portions of (a) A/Udorn [SEQ ID NO: 1], (b) A/Victoria [SEQ ID NO: 3], (c) A/PR/8/34 [SEQ ID NO: 5], and (d) a consensus sequence [SEQ ID NO: 7]. Dashes indicate the same nucleotide as the consensus sequence. Different nucleotides from that of the consensus sequence are reported in lower case letters. Dots indicate no

corresponding nucleotide when compared to the consensus sequence.

Fig. 2 illustrates the nucleic acid and amino acid sequences of

H3C13, NS1_{(1 -81)}H3HA2_{(1 -221 )} fusion protein [SEQ ID NO: 9 & 10], with the mutant nucleic acid sequences of H3C13mut5855 [SEQ ID NO: 58] illustrated above the sequence of the unmodified H3HA2 portion.

Fig. 3 illustrates the nucleic acid and amino acid sequences of the NS1 _(1-81)H3HA2_(77-221) fusion protein [SEQ ID NO: 11 & 12].

Fig. 4 illustrates the nucleic acid and amino acid sequences of the Type B fusion protein, NS1_{(1 -42)}HA2_(41-223). [SEQ ID NO: 13 & 14].

Fig. 5 illustrates the pOTS208NS1BLmut2 vector nucleic acid sequences [SEQ ID NO: 54] encoding the amino acid sequences [SEQ ID NO: 55] of the mutant NS_(1-81)BLHA2_{( 1-223)}(m et-leu) fusion protein, with the nucleic acid sequences of the coding region NS_(1-81)BLHA2_{(1 -223)} [SEQ ID NO: 56] and native amino acid sequences [SEQ ID NO: 57], which include a Met in amino acid position 98, illustrated above the modified BLHA2 sequences.

Fig. 6 illustrates the nucleic acid [SEQ ID NO: 17] and amino acid [SEQ ID NO:18] sequences of the H1N1 fusion protein, NS1_{( 1-81 )}HA2_(65-222), also known as flu D.

Fig. 7 illustrates the naturally-occurring nucleic acid sequence [SEQ ID NO:1] and corresponding amino acid sequence [SEQ ID NO:2] of the HA2 portion of the H3N2 virus, A/Udorn.

Fig. 8 illustrates the naturally-occurring nucleic acid sequence [SEQ

ID NO:3] and corresponding amino acid sequence [SEQ ID NO:60] of the HA2 portion of the H3N2 virus, A/Victoria. Detailed Description of the Invention

The present invention provides novel proteins, DNA sequences, pharmaceutical vaccine compositions, and methods of use thereof for conferring protection in vaccinated mammals against one strain, or desirably multiple strains, of influenza viruses. The proteins and vaccine compositions of the present invention demonstrate the ability to stimulate or produce a protective immune response which is capable of recognizing an influenza virus or influenza virus- infected cells and protecting the vaccinated mammal against disease caused thereby. This protective response is desirably a T cell response, produced in the substantial absence of vaccine-induced neutralizing antibody.

While the proteins and DNA sequences specifically described herein are directed to the H3HA2 and BHA2 sequences originating from viral strains to which humans are susceptible, it is expected that similar sequences and molecules can be prepared for veterinary applications. For example, selected HA2 sequences obtained from Type A viral strains, e.g., H5HA2, H7HA2 and other strains of interest may be obtained following the teachings described herein for the exemplified H3HA2 and BHA2 sequences. One of skill in the art should understand that this invention is not limited to the exemplified protein and DNA sequences, even though the following disclosure is limited to the two latter sequences for simplicity. Such additional viral HA2 subunits are expected to share the biological characteristics of the exemplified sequences.

Thus, this invention provides a protein or fragment thereof characterized by an amino acid sequence derived from the HA2 subunit of an HA protein, e.g., from a H3N2 subtype virus. As used herein, a "fragment" of the HA2 subunit is an amino acid sequence derived from the HA2 subunit which is characterized by having an immunogenic determinant of the HA2 subunit. Such a fragment is desirably at least about 8 amino acids in length.

The H3 proteins of the invention are capable of inducing T helper cells, particularly cytotoxic T lymphocytes, in the absence of neutralizing antibodies. Among H3N2 subtype strains of influenza A include A/Udorn and A/Victoria viruses. Other H3N2 virus strains of influenza A may also produce HA proteins for use in vaccine compositions according to this invention. Fig. 1 compares the nucleic acid sequences of the HA2 portions of the A/Udorn [SEQ ID NO: 1] and A/Victoria [SEQ ID NO: 3] strains with the nucleic acid sequence of an H1N1 subtype virus, A/PR/8/34 [SEQ ID NO: 5]. A consensus sequence [SEQ ID NO: 7] was computer generated, and may likewise be useful in producing proteins according to this invention. This consensus sequence [SEQ ID NO: 7] can be constructed by a commercially available computerized sequence analysis program, such as Genetics Computers Group [University of Wisconsin].

Proteins according to this invention may include unfused HA2 subunits of the influenza A viruses, particularly H3N2 subtype. For example, in one embodiment, a protein of the invention contains amino acids 1-221 of a selected H3HA2 subunit. In another embodiment, a protein of the invention contains amino acids 77-221 of the H3HA2 subunit. Other fragments of this HA2 amino acid sequence characterized by the ability to stimulate similar immunological activity in an immunized animal are also encompassed by this invention.

Proteins of this invention also include fusion proteins comprising a protein sequence derived from the HA2 subunit of an HA protein from a Type A virus, e.g., an H3N2 subtype virus, fused in frame to another protein or protein fragment capable of enhancing expression of the fusion protein. It is desirable that this fusion "partner" protein be an influenza protein sequence or fragment thereof derived from the same or another strain of influenza virus as the HA protein or protein fragment. Preferably, this fusion partner protein is all or a portion of the influenza virus NS1 protein or an HA2 subunit protein.

In the embodiments exemplified herein, the NS1 portion of the fusion protein is derived from an HlNl subtype virus, A/PR/8/34. For example, in one embodiment, the NS1 portion may comprise amino acid residues 1 to 42 of H1NS1. In another embodiment the NS1 portion may comprise amino acid residues 1 to 81 of the selected virus. The HA2 fragment may alternatively be fused to a portion of the NS1 peptide derived from a selected Type A virus, e.g., an H3 subtype virus (H3HA2), or a Type B (BHA2) virus.

However, other non-influenza fusion proteins may also produce desirable fusion proteins with the H3N2, or other Type A, or Type B protein or portion thereof. Thus, in still another alternative embodiment, as discussed below, the HA2 fragment may be fused to any peptide capable of enhancing its expression in the host cell selected. One of skill in the art may readily select a fusion "partner" protein or fragment taking into account the desired host cell and utilizing the teachings herein. The fusion proteins of the present invention are not limited by the selection of the "partner" protein or fragment to which the HA2 fragment is fused.

In yet another embodiment, the present invention provides a modified protein containing a portion of the HA2 subunit of a Type B influenza virus. Currently, the preferred human virus strain is B/Lee/40. However, the vaccinal proteins of this invention are not limited to this Type B strain, and other strains infecting other species, or other as yet unidentified Type B virus strains, may be used to produce the HA2 protein. These Type B HA2 proteins may be fused to a fusion "partner" protein or protein fragment, as described above for the H3HA2 proteins of this invention, or remain unfused.

In the construction of a fusion protein according to this invention, a linker sequence may optionally be inserted between the two fused sequences, i.e., between the NS1 portion and the HA2 portion. This optional linker may provide space between the two linked sequences. Alternatively, this linker sequence may encode, if desired, a polypeptide which is selectively cleavable or digestible by conventional chemical or enzymatic methods. For example, the selected cleavage site may be an enzymatic cleavage site, including sites for cleavage by a proteolytic enzyme, such as enterokinase, factor Xa, trypsin, collagenase, and thrombin.

Alternatively, the cleavage site in the linker may be a site capable of being cleaved upon exposure to a selected chemical, e.g., cyanogen bromide or hydroxylamine. The cleavage site, if inserted into a linker useful in the fusion sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose.

A presently preferred example of an H3 fusion protein of this invention is NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO: 10], which comprises the first 81 amino acids of NS1 fused to amino acids 1 to 221 of the H3HA2 subunit (amino acids 1-221). (Fig. 2) Another exemplary fusion protein, NS1_{( 1-81)}H3HA2_{(77-221 )} [SEQ ID NO: 12], comprises the first 81 amino acids of NS1 fused to amino acids 77 to 221 of the truncated H3HA2 subunit. (Fig. 3)

A present preferred example of a Type B fusion protein of this invention is NS1_{( 1 -42)}BHA2_(41-223) [SEQ ID NO: 14], which comprises the first 42 amino acids of NS1 fused to amino acids 41 to 223 of the truncated BHA2 subunit. (Fig. 4) Another fusion protein of this invention is NS1_{( 1 -81)}BHA2_{(1 - 223)} [SEQ ID NO: 57], which contains the first 81 amino acids of NS1 fused to amino acids 1 to 223 of the BHA2 subunit. (Fig. 5) Another preferred fusion protein of the invention is NS1_{( 1-81 )}BHA2_{( 1 -223)}(met-leu) SEQ ID NO: 55, which contains the same amino acid sequence as NS1_(1-81)BHA2_(1-223), with the exception that the internal methionine residue at position 98 of the fusion protein has been changed to a leucine. (Fig. 5)

These proteins, fusion proteins, and similar proteins encoded by the below-described DNA sequences are referred to collectively herein as H3HA2 proteins. The NS1_{( 1-81 )}H3HA2_{(1 -221)} protein [SEQ ID NO: 10] of the invention has a three-dimensional structure which is substantially similar to that of the NS1_{( 1-81 )}HA2_{(1 -222)} protein [SEQ ID NO: 16] derived from the H1N1 subtype virus (C13). However, the amino acid sequence of the NS1_{( 1 - 81 )}H3HA2_{( 1 -221 )} protein [SEQ ID NO: 10] has only approximately 50%

homology with the amino acid sequence of C13 protein [SEQ ID NO: 16].

Additionally, as illustrated in Fig. 1, the nucleic acid sequence of the H3HA2_1-221 protein derived from A/Udorn (nucleotides 25-560 from that virus) [SEQ ID NO: 1] has only approximately 60% homology with the nucleic acid sequence of the HlHA2_1-222 protein derived from strain A/PR/8/34 (nucleotides 1872-2407 from A/PR/8/34) [SEQ ID NO: 5]. However, the nucleic acid sequence of H3HA2_1-221 from A/Udorn (nucleotides 1-499 of A/Udorn) [SEQ ID NO: 1] has approximately 99% homology with the nucleic acid sequence of H3HA2_{1 -221} from

A/Victoria/H3/75 (nucleotides 1226-1725 of A/Victoria) [SEQ ID NO: 3] [Fiers et al, Cell, 19:683-696 (1980)].

Analogs of the HA2 peptides from a Type A virus, e.g., an H3, or Type B viruses, included within the definition of this invention, include truncated polypeptides (including fragments) and HA2 polypeptides, e.g. mutants that retain the epitopes and thus the biological activity of HA2. It is anticipated that, because the NS1 portion of the fusion peptide provides a means of expressing the protein at high levels and does not appear to play as significant a role in the immunological responses to the HA2 fusion proteins as does the HA2 portion, any number of analogs of this fusion partner can be made.

Typically, the analogs of the HA2 peptides and/or the fusion partner differ by only 1 to about 4 codon changes. Other examples of analogs include polypeptides with minor amino acid variations from the natural amino acid sequence of HA2; in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on its activity, especially if the replacement does not involve an amino acid at an epitope of the HA2 polypeptide. The construction of such analogs, given the description herein and conventional methods of protein modification known to one of skill in the art, are believed to be encompassed by this invention.

Currently, it is theorized that the HA2 portion of the fusion peptide

(e.g., H3HA2_1-221, H3HA2_77-221 and BHA2_41-223) confers the majority of the necessary epitopes for antibody binding or T cell (particularly CTL) targeting.

Once these epitope sequences are precisely identified, portions of the HA2 sequence which are not part of these epitopes may be altered without significantly affecting the bioactivity of the fusion protein.

The present invention also encompasses DNA sequences of this invention encoding the above-described proteins and fusion proteins, the sequences characterized by having an immunogenic determinant of a modified HA2 subunit of an HA protein, derived from a Type A virus, e.g., an H3 subtype, or Type B virus. Other DNA sequences of this invention encode such HA2 subunits, optionally fused to a DNA sequence encoding a protein or peptide which is capable of enhancing expression of the protein in a selected host cell. For example, the consensus sequence illustrated in Fig. 1(d) may provide a source of HA2 DNA. The currently preferred embodiment provides a DNA sequence encoding a Type A virus, e.g., an H3 or Type B HA2 protein or fragment thereof fused in frame to a DNA sequence encoding a portion of the nonstructural influenza protein 1 (NS1).

Coding sequences for the HA2, NS1, and other viral proteins of influenza virus can be prepared synthetically or can be derived from viral RNA or from available cDNA-containing plasmids by known techniques. For example, in addition to the above-cited references, a DNA coding sequence for HA from the

A/Japan/305/57 strain was cloned, sequenced and reported by Gething et al, Nature, 287:301-306 (1980). An HA coding sequence for strain A/NT/60/68 was cloned as reported by Sleigh et al, and by Both et al, in Developments in Cell Biology, Elsevier Science Publishing Co., pages 69-79 and 81-89, respectively, (1980). An HA coding sequence for strain A/WSN/33 was cloned as reported by Davis et al,

Gene, 10:205-218 (1980); and by Hiti et al, Virology, 111: 113-124 (1981). An HA coding sequence for fowl plague virus was cloned as reported by Porter et al and by Emtage et al, both in Developments in Cell Biology, cited above, at pages 39-49 and 157-168. Also, influenza viruses, including other strains, subtypes, and types are available from clinical specimens and from public depositories, such as the

American Type Culture Collection (ATCC), Rockville, Maryland, U.S.A. Allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) of DNA sequences encoding the H3HA2 or BHA2 protein sequences are also included in the present invention, as well as analogs or derivatives thereof. Similarly, DNA sequences which code for H3 or other Type A or Type B HA2 proteins of the invention but which differ in codon sequence due to the degeneracies of the genetic code or variations in the DNA sequence encoding H3HA2, other Type A or BHA2 proteins which are caused by point mutations or by induced modifications to enhance the activity, half-life or production of the peptide encoded thereby are also encompassed in the invention. Suitably, this invention provides certain silent mutations to the coding sequences for NS1_{(1 -81)}H3HA2_(1-221), which have been found to increase expression yields. See Fig. 2. Further, the NS1_{( 1-81 )}BHA2_{(1 - 223)}(met-leu)-encoding sequence, BC13mut2, in addition to modifying the codon encoding amino acid position 98 of the fusion protein (position 17 of the HA2 portion), contains a number of silent modifications designed to increase protein expression. See Fig. 5.

Also covered by this invention are DNA sequences which hybridize under stringent conditions with the DNA sequences encoding the HA2 subunit proteins, e.g., H3HA2 or BHA2 proteins, of this invention. DNA sequences which hybridize under non-stringent conditions with the disclosed sequences, but which encode proteins or fragments retaining the biological activities of the H3HA2 or BHA2 proteins, are also included in this invention. Typical conditions for stringent or non-stringent hybridization are known to those of skill in the art. [See, e.g., Sambrook et al, Molecular Cloning. A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, NY (1989)].

The fusion proteins of the invention may be prepared by conventional genetic engineering and recombinant techniques known to those of skill in the art. Similarly, the proteins may be purified from expression in host cell or vector systems by conventional means.

Preferably, however, the recombinantly-produced fusion proteins of the invention are purified as described herein. Generally, method of purification involves (step 1) the isolation of the proteins, (step 2) enzymatic digestion and extraction, (step 3) urea extraction, (step 4) solubilization, reduction, and DEAE chromatography, (step 5) reverse phase chromatography, (step 6) precipitation, and (step 7) desalting and preparation of the final product. More specifically, the host cells containing the fusion proteins are disrupted, either chemically or by mechanical means. Preferably the cells are lysed by osmotic shock. Following centrifugation, the resulting pellet (P1) is subjected to nuclease digestion extraction and centrifuged to yield pellet 2 (P2). A second extraction step is then performed using urea (pH 6) and the mixture centrifuged to yield pellet 3 (P3). P3 is then solubilized and reduced. Preferably, solubilization is performed using urea at pH 12.5 and reduction is via DTT DEAE chromatography followed by SDS elution. The resulting DEAE product is further reduced, preferably using DTT, and subjected to reverse phase chromatography. The reverse phase product is then precipitated by adjusting to pH 6 and centrifuged. The precipitated product is resolubilized, preferably with urea at pH 12.5, and subjected to G25

chromatography. The resulting G25 product is then filtered (e.g. with a 0.2 micron filter) to yield the final product. Further details of this method are provided in Example 17 below.

Systems for cloning and expression of the vaccinal polypeptide of this invention in various microorganisms and cells, including, for example, E. coli, Bacillus, Streptomyces, Saccharomyces, mammalian and insect cells, are known and available from private and public laboratories and depositories and from commercial vendors. The preferred host is E. coli because it can be used to produce large amounts of desired proteins safely and cheaply. To circumvent the requirement of ampicillin for plasmid selection in production fermentations, a desirable method of production employs an alternative expression system in which the β-lactamase coding sequence is wholly or partially replaced by a coding sequence for an alternative selectable marker such as, for example, kanamycin or chloramphenicol.

Thus, the polypeptide employed in the presently preferred

embodiment is preferably expressed in E. coli. A suitable strain, LW14, has the following genotype: galE::Tnl0λCI857 bio- uvrB-; phenotypically, strain LW 14 requires biotin for growth, is sensitive to UV light and DNA damaging agents, and cannot use galactose as a carbon source. Construction of this strain is described in the examples below.

To aid in expression of the H3 or other Type A subunit or Type B HA2 peptides or fusion protein described above, these protein sequences or fragments thereof may also be fused to a polypeptide capable of enhancing expression of these fragments in the selected host system. Ordinarily, such a peptide would contain a leader sequence fragment that provides for secretion of the Type A subunit fragment, e.g., the H3HA2 fragment, or Type B HA2 fragment in the host cell. The leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. There may be processing sites encoded between the leader sequence and the Type A subtype or Type B HA2 fragment that can be cleaved either in vivo or in vitro. Alternatively, a promoter sequence may be linked directly with the DNA molecule encoding the HA2 fragment. Such polypeptides, promoter and leader sequences are known to those of skill in the art and may be readily selected for expression in the selected host.

Construction of expression systems, including expression vectors and transformed host cells are thus within the art. See, generally, methods described in standard texts, such as Sambrook et al, Molecular Cloning A Laboratory Manual, 2d edit., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). The present invention is therefore not limited to any particular expression system or vector, nor to any particular purification process from cell lysates or cell medium.

The proteins and fusion proteins of this invention may be employed in vaccine compositions. Pharmaceutical vaccine compositions of this invention, therefore, contain an effective immunogenic amount of a selected HA2 protein, e.g., H3HA2 or BHA2 protein, of the invention in admixture with a suitable adjuvant in a nontoxic and sterile pharmaceutically acceptable carrier.

Suitable carriers for vaccine use are well known to those of skill in the art. However, exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, squalene, and water. Additionally, the carrier or diluent may include a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax.

Optionally, suitable chemical stabilizers may be used to improve the stability of the pharmaceutical preparation. Suitable chemical stabilizers are well known to those of skill in the art and include, for example, citric acid and other agents to adjust pH, chelating or sequestering agents, and antioxidants.

While any aluminum adjuvant may be used in the vaccine

compositions of this invention, two desirable adjuvants are available commercially, i.e., REHSORPTAR™ adjuvant [Armour Pharmaceuticals, Kankakee, IL] and REHYDRAGEL™ adjuvant [Reheis Chemical Co., Berkeley Heights, NJ]. These products are aluminum hydroxide gels which contain approximately 2% w/v AI₂O₃, which is equivalent to approximately 10.6 mg/ml Al .

Vaccine compositions of this invention may employ an immunogenic amount of a purified recombinant protein as described above. A preferred embodiment of the vaccine of the invention is composed of an aqueous suspension or solution containing the recombinant HA2 protein molecule, e.g., H3HA2 or BHA2, together with an adjuvant, preferably an aluminum, most preferably aluminum hydroxide, buffered at physiological pH, in a form ready for injection. A preferred protein for use in these vaccine compositions includes a protein

comprising amino acid residues 1 to 81 from NS1 fused to C-terminal amino acid residues 1-221 from the hemagglutinin subunit 2 (HA2) from influenza A, subtype H3N2. Another preferred vaccine composition of this invention employs a purified recombinant protein made up of amino acid residues 1 to 81 from NS1 fused to amino acid residues 77-221 of the HA2 from influenza A, subtype H3N2. Still another preferred vaccine composition of this invention employs a purified recombinant protein made up of amino acid residues 1 to 42 fused to amino acid residues 41-223 of the HA2 from influenza B.

Vaccine compositions of the invention may also employ an immunogenic amount of a recombinant protein of the invention in combination with other influenza antigens. Suitable influenza antigens for combination in a vaccine composition with the proteins of this invention may be derived from Type A, H1 subtype viruses and may include the recombinant fusion proteins described in detail in copending U. S. Patent Application Ser. No. 07/387,200, filed July 28, 1989 and its corresponding European Patent Application No. 366, 238, published May 2, 1990; and in co-pending U. S. Patent Application Ser. No. 07/387,558, filed July 28, 1989 and its corresponding European Patent Application No. 366,239, published May 2, 1990. The C13 protein (NS1_(1-81)HA2_(1-222)) [SEQ ID NO: 15 & 16], D protein (NS1_(1-81)HA2_(65-222)) [SEQ ID NO: 17 & 18] and other fusion proteins derived from the HlNl influenza virus subtype and the recombinant expression and purification thereof are disclosed in detail in these applications, and in the parent applications identified in this application, all of which are incorporated by reference herein.

More specifically, suitable H1 subtype immunogenic proteins include

C13 (NS1_{( 1-81 )}-D-L-S-R-HA2_{( 1-222)}) [SEQ ID NO: 15 & 16], D (NS1_{(1 -81 )}-Q- I-P-HA2_(65-222)) [SEQ ID NO: 17 & 18], C13 short (NS1_(1-42)-M-D-L-S-R- HA2_(1-222)) [SEQ ID NO: 19 & 20], D short (NS1_{( 1 -42)}-M-D-H-M-L-T-S-T-R-S- HA2_(66-222)) [SEQ ID NO: 21 & 22], A (NS1_(1-81)-Q-I-P-HA2_(69-222)) [SEQ ID NO: 23 & 24], C (NS1_(1-81)-Q-I-P-HA2_(81-222)) [SEQ ID NO: 25 & 26], ΔD (NS1_{(1 -81 )}HA2_(150-222)) [SEQ ID NO: 27], Δ13 (NS1_{( 1-81 )}-D-L-S-R-HA2_{(1 - 70)}-S-C-L-T-A-Y-H-R) [SEQ ID NO: 28], M (NS1_{( 1-81 )}-Q-I-P-HA2_{(65- 196)}-G- G-S-Y-S-M-E-H-F-R-W-G-K-P-V) [SEQ ID NO: 29], ΔM (NS1_(1-81)-Q-I-P- HA2_{(65- 196)}-G-G-S-Y-S-M-L-V-N) [SEQ ID NO: 30], ΔM+ (NS1_{(1 -81)}-Q-I-P- HA2_(65-200)-L-V-L-L) [SEQ ID NO: 31 & 32]. These HlNl fusion proteins are described in published European Patent Application 366,238 and in copending U.S. Patent Application Ser. No. 07/751,896. Other suitable HI proteins consist of unfused polypeptides, such as H1HA266-222 [SEQ ID NO: 33 & 34] which is disclosed in co-pending U. S. Patent Application Ser. No. 07/751,898, incorporated herein by reference. Thus, one desirable combination vaccine to provide protection against Type A influenza contains NS1_(1-81)H3HA2_(1-221) protein [SEQ ID NO: 9 & 10] of the invention, one or more proteins derived from subtype H1N1 as described above, and an aluminum adjuvant.

Preferably, a combination vaccine of the invention will contain an immunogenic amount of the H3 fusion protein of the invention in combination with immunogenic amounts of influenza antigens derived from the other Type A influenza virus subtypes, including among others, H1, H2, H3, H4, H5, H6; and H7, as well as a Type B fusion protein of the invention.

A currently preferred combination vaccine of the invention contains the H3 subtype fusion protein NS1_{( 1 -81 )}H3HA2_{( 1 -221 )} [SEQ ID NO: 10], the B subtype fusion protein NS1_{(1 -81 )}BHA2_{( 1 -223)}(met-leu) [SEQ ID NO: 55], and the H1 subtype fusion protein NS1_{( 1 -81 )}HA2(65-222) [SEQ ID NO: 18]. Studies have shown that such a combination vaccine is protective against challenge with H1, H3 and Type B influenza viruses in mice.

Other preferred combination vaccines would include the NS1_{(1 - 81 )}H3HA2_{(77-221 )} protein [SEQ ID NO: 12] or the NS1_{(1 -81 )}BHA2_(1-223) [SEQ ID NO: 57] in combination with one or more additional influenza antigens derived from the type or subtype influenza viruses described above. These combination vaccines will protect against influenza infections caused by both Type A and Type B influenza viruses. Still other combination vaccine compositions will employ other proteins described herein.

The compositions of the present invention are advantageously made up in a dose unit form adapted for the desired mode of administration. Each unit will contain, at a minimum, a predetermined quantity of the selected HA2 subunit protein, e.g., H3HA2 protein and/or BHA2 protein, and adjuvant calculated to produce the desired therapeutic effect in optional association with a pharmaceutical diluent, carrier or vehicle.

Dosage protocol can be optimized in accordance with standard vaccination practices. Typically, the vaccine will be administered intramuscularly, although other routes of administration may be used, such as intradermal. It is expected that an effective immunogenic amount of a protein, fusion protein or combination of proteins of this invention for average adult humans is in the range of 1 to 1000 micrograms. Another desirable immunogenic amount ranges between 50 to 500 micrograms. Most preferably, the proteins of the invention are in admixture with the same amount or more adjuvant to form a vaccine composition.

While the proteins described herein have been particularly developed for use in humans (e.g., the H3HA2 and BHA2 sequences), it is expected that due to species cross-reactivity, these vaccines will be useful in other animals, particularly swine. Additionally, similar molecules can be prepared for equine and avian veterinary applications utilizing the HA2 proteins from other strains to which animals are susceptible. Combination vaccines for use in swine would preferably include protections against both H1 and H3 viruses. Combination vaccines for use in equine would preferably include protection against H3 and H7 viruses.

Combination vaccines for use in avian species would preferably confer protection against H5 and H7 viruses. Appropriate dosages can be determined by one skilled in veterinary medicine.

It will be understood, however, that the specific effective

immunogenic amount for any particular patient will depend upon a variety of factors including the age, general health, sex, and diet of the vaccinee; the species of the vaccinee; the time of administration; the route of administration; interactions with any other drugs being administered; and the degree of protection being sought.

The vaccine can be administered initially in late summer or early fall and can be readministered two to six weeks later, if desirable, or periodically as immunity wanes, for example, every two to five years. Of course, as stated above, the administration can be repeated at suitable intervals if necessary or desirable.

The present invention provides methods for producing enhanced expression and improved homogeneity of influenza viral proteins and polypeptides in E. coli. Also provided are novel modified nucleotide sequences which encode these influenza proteins and are useful in the methods of production.

Preferably, the influenza proteins or polypeptides produced according to the invention include the complete HA2 protein of the hemagglutinin antigen (HA) of a selected H3N2 influenza virus, a complete HA protein of an H3HA2 virus, fragments thereof, and fusion proteins containing the complete H3HA2 protein or desired fragments thereof fused in the same reading frame with a selected fusion partner polypeptide or protein. These proteins are characterized by having the native amino acid sequence pattern described above.

By the term "fragment" is meant a subunit of HA, or a span of contiguous amino acids from the complete protein capable of stimulating an antigenic or protective immunogenic response in an animal. A fragment may contain at least about 8 amino acids from the selected influenza protein, and can contain up to the number of amino acids which make up the entire protein. When the term 'fragment' is used herein to modify a nucleotide sequence, it refers to nucleotide sequences which encode the above-defined amino acid fragments.

Native (or naturally-occurring) nucleotide sequences which encode certain influenza proteins are characterized by a nucleotide sequence pattern encoding the fragment Arg-Arg-Xaa-Xaa-Arg [SEQ ID NO:61]. Arg represents arginine and Xaa represents any amino acid in this formula. Hereafter, this five amino acid sequence is referred to as Formula I.

Formula I sequences are typically encoded by native nucleotide sequences of the formula of codons AGr-AGr-Xaa-Xaa-AGr, where r represents the nucleotides A or G and Xaa represent any codon [SEQ ID NO:63]. Hereafter, this five codon nucleotide sequence is referred to as Formula II. Specifically, the native nucleic acid sequence encoding a subtype H3N2 influenza virus protein, fusion protein, or a fragment or subunit thereof, specifically the HA2 portions of H3N2 virus strains, is characterized by a Formula II sequence.

Among H3N2 subtype strains of influenza A characterized by this nucleotide fragment Formula II include the A/Udom and A/Victoria viruses. Figs. 7 and 8 provide the native nucleic acid sequences of the HA2 portions of the A/Udorn [SEQ ID NO: 1] and A/Victoria [SEQ ID NO: 3] strains. Other H3N2 virus strains of influenza A may also provide native nucleotide sequences containing Formula II, which sequences are susceptible to the modifications described herein.

Additional examples of native nucleotide sequences encoding proteins whose expression may be enhanced according to this invention are those native sequences which encode certain fragments of influenza proteins including the fragment spanning amino acids 1 to about amino acids 221 of H3HA2 [Fig. 7 SEQ ID NO:2 and Fig. 8 SEQ ID NO:3]; the fragment spanning from about amino acid 77 to about amino acid 221 [Fig. 7 SEQ ID NO:69 and Fig. 8 SEQ ID NO:70], or other desirable fragments. Other desirable fragments of this H3HA2 amino acid sequence include those characterized by the ability to stimulate immunological activity in an immunized animal similar to that stimulated by use of the entire 221 amino acid sequence of H3HA2.

Nucleotide sequences encoding fusion proteins which contain fragments of the native nucleotide sequences encoding these influenza proteins or subunits, e.g., the fusion protein NS1_{( 1-81 )}H3HA2_{(1 -221)} [SEQ ID NO:10], can also be characterized by the Formula II nucleotide sequence. Thus these fusion proteins are also desirable for enhanced expression according to the method of this invention. The inventors have discovered that when native nucleotide sequences of influenza proteins, which sequences comprise Formula II, are expressed in E. coli, a frame shift of one nucleotide after the third triplet in Formula II in the native sequence occurs, resulting in the increased translation of truncated proteins. It has been surprisingly found that by application of a method of the present invention, the expression and homogeneity of the influenza protein is increased significantly.

The methods of this invention involve enhancing the expression of proteins characterized by the amino acid pattern of Formula I, which proteins have a native nucleotide sequence of Formula II. According to one embodiment of the method of this invention, a native nucleotide sequence encoding a selected influenza protein or fragment, which sequence comprises Formula II, is modified by mutating one or more of the rare AGG or AGA arginine codons of Formula II to a preferred Arg codon. A preferred arginine codon for use in replacing a native AGA or AGG codon according to this invention is defined herein by the codons CGT, CGG, CGA and CGC. Of these codons, CGT and CGC are currently the most preferred. The modified influenza protein-encoding nucleotide sequence is then expressed in an E. coli expression system, resulting in enhanced expression in comparison to that obtained by expression of the native nucleotide sequence encoding the same protein in the same expression system.

The enhanced protein expression occurs even though the mutation does not result in a change in the encoded amino acid sequence of the protein. By the terms 'enhanced expression' or 'enhanced protein expression' is meant an expression level of at least 40% higher than the expression level of the protein encoded by the native, non-mutated nucleotide sequence comprising Formula II, when expressed in E. coli.

While not wishing to be bound by theory, the inventors believe that the enhanced expression levels are obtained because the silent mutation of the AGA or AGG to a preferred arginine codon in Formula II eliminates the frame shift mutation found in the unmutated nucleotides encoding these proteins, thus substantially reducing the production of truncated messages (proteins). It is believed that the resulting influenza proteins are more homogeneous when expressed in an E. coli expression system according to this invention.

In a second embodiment of the method of the invention, the expression of the proteins containing arginines encoded by the rare codons AGG and AGA (i.e. proteins encoded by amino acid and nucleotide sequences characterized by Formulae I and II) can be increased by inserting into the host in which expression is desired one or more genes for tRNA molecules which are capable of properly translating the AGG and AGG arginine codons. Preferably the host cells are E. coli.

This method can be accomplished as follows. A gene for a tRNA molecule described above can be selected from among known gene sequences. The genes and tRNA molecules which can translate the rare Arg codons identified above are known and readily available to one of skill in the art. See, e.g., [P. Saxena and J. Walker, J. Bacteriol., 174(6): 1956-1964 (Mar. 1992)].

According to conventional techniques, these genes may be placed on a plasmid which will increase the copy number of these genes and therefore the tRNA molecules encoded by these genes. Alternatively, these sequences can be genetically engineered and placed on the host cell chromosome behind an appropriate promoter element in such a manner that the effective concentration of these tRNA molecules is increased inside the cell. Conventional texts describe the techniques useful in this method [See, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual. 2d edition, Cold Spring Harbor, New York (1989)].

The insertion of the tRNA genes into the host cell expressing the protein increases the concentration of these tRNA molecules inside the host cells which are naturally deficient for these tRNA molecules. This allows the host cells to translate these rare arginine codons in an efficient manner, eliminating the production of the truncated or lower molecular weight species of the fusion protein observed in the unmodified host cell. Thus, this method may be used to increase expression of a protein in host cells lacking sufficient amounts of the appropriate tRNA to permit efficient expression of the protein. Use of this method obviates the need to modify the sequences encoding the selected protein, and thus provides an alternative method to the first embodiment described above.

As another aspect of this invention novel modified nucleotide sequences are provided, which in E. coli expression systems, can be employed to produce the encoded influenza proteins, subunits, fragments and fusion proteins described above according to the first embodiment of the method of this invention. The proteins encoded by these nucleotides are produced at levels of expression enhanced over that of the native sequences, by about forty percent or more. The novel nucleotide sequences of the invention are characterized by comprising the nucleotide sequence CGn-CGn-Xaa-Xaa-CGn, where n represents a nucleotide selected from the group consisting of T, C, A or G [SΕQ ID NO:62], in place of the Formula II fragment in the native nucleotide sequence encoding the selected influenza protein or fragment. The nucleotide fragment identified by the formula above is referred to herein for simplicity as Formula III. For example, a modified DNA sequence of the invention comprises the Formula El nucleotide sequence and may encode the amino acid sequences identified specifically above, e.g., Fig. 7 [SEQ ID NO:2], Fig. 8 [SEQ ID NO:3]; Fig. 7 [SEQ ID NO:69] and Fig. 8 [SEQ ED NO:70], or other fragments.

In one example of the present invention, the nucleic acid sequence encoding the HA2 subunit protein which contains the native sequence of Formula II has been provided with three silent mutations, which have changed each of the three native arginine-encoding AGG codons each to a preferred arginine codon CGT. These codons encode amino acid numbers 123, 124 and 127 of the H3HA2 subunit protein of the A/Udorn strain identified in Fig. 7. The same codons (and amino acid numbers) are altered in the A/Victoria strain identified in Fig. 8 to provide another example of a modified nucleotide sequence according to this invention.

Thus, with reference to each of Figs. 7 and 8, the native nucleotide sequences encoding the HA2 subunit proteins of the aforementioned viruses [SEQ ID NO:1 and 60], are modified according to this invention at nucleotides 367, 370, and 379. At each of these nucleotide sites, the native A (adenine) is changed to a C (cytosine) and the native nucleotides at sites 369, 372 and 381 in each sequence are changed from a G (guanine) to a T (thymine), resulting in preferred Arg codons.

Other nucleotide sequences encoding the influenza vaccinal polypeptides described herein, or other such influenza proteins or subunits characterized by Formula II may be mutated into novel nucleotide sequences of this invention, i.e., by mutating Formula II into Formula III within those sequences using the first embodiment of the methods of this invention. The silent mutations described herein may be inserted at analogous regions in each nucleotide sequence.

The novel modified H3HA2 nucleotide sequences, whether alone or in association with a nucleotide sequence encoding a fusion partner of a fusion protein of the invention are useful in E. coli expression systems. The novel nucleotide sequences of the invention will also encode analogs of the H3HA2 peptides, such as truncated polypeptides (including fragments) and H3HA2 polypeptides, e.g. mutants that retain the epitopes and thus the biological activity of

H3HA2. Where the nucleotide sequence encodes a fusion protein, it is anticipated that, because the non-HA2 fusion partner, e.g., NS1 as described below, the fusion peptide provides a means of expressing the protein at high levels and does not appear to play as significant a role in the immunological responses to the HA2 fusion proteins as does the HA2 portion, any number of analogs of this fusion partner can be made. Typically, the analogs of the nucleotide sequences encoding the HA2 peptides and/or the fusion partner may differ by only 1 to about 4 codon changes, in addition to the nucleotide mutations to the above-identified fragment. Other sequences of this invention include modified nucleotide sequences which encode polypeptides with minor amino acid variations from the natural amino acid sequence of HA2. For example, conservative amino acid replacements may be introduced by altering, deleting or replacing codons of the native sequence, in addition to altering those codons in Formula II according to one embodiment of this method.

Conservative replacements are those that take place within a family of amino acids that are related in their side chains and are well known in the art. For example, it is reasonable to expect that an isolated replacement of a selected amino acid with a conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on the activity of the protein, especially if the replacement does not involve an amino acid at an epitope of the HA2 polypeptide.

The construction of modified nucleotide sequences and proteins or fusion proteins, given the description herein and conventional methods of protein modification known to one of skill in the art, are believed to be encompassed by this invention.

The novel modified nucleotide sequences of this invention are further characterized by encoding an immunogenic determinant of a modified HA2 subunit of an HA protein, derived from an H3N2 subtype. The encoded protein may contain all or a portion of the H3N2 HA2 sequence, including the Formula I amino acid sequence. The currently preferred embodiment provides a novel DNA sequence encoding an H3HA2 protein or fragment thereof fused in frame to a DNA sequence encoding a portion of the nonstructural influenza protein 1 (NS1). One modified fusion protein-encoding nucleotide sequence is obtained by making mutations according to this invention in the nucleotide sequence encoding the fusion protein NS1_{( 1-81 )}H3HA2_{(1 -221)} [SEQ ID NO:10]. Upon mutation, the nucleotide sequence [SEQ ID NO:58] for this fusion protein [SEQ ID NO: 10] is referred to herein as pOTS208NS1H3mut5585.

The modified coding sequences for the HA2 proteins, as well as the coding sequences for NS1 and other viral proteins of influenza virus can be prepared synthetically or can be derived from viral RNA or from available cDNA-containing plasmids by known techniques. For example, see references known to the art which disclose the nucleotide coding sequences for HA from the A/Japan/305/57 strain [Gething et al, Nature, 287:301-306 (1980)]; strain A/NT/60/68 [Sleigh et al., and Both et al., in Developments in Cell Biology, Elsevier Science Publishing Co., pages 69-79 and 81-89, respectively, (1980)]; strain A/WSN/33 [Davis et al, Gene, 10:205-218 (1980); Hiti et al., Virology, 111:113-124 (1981)]; and fowl plague virus [Porter et al. and by Emtage et al., both in Developments in Cell Biology, cited above, at pages 39-49 and 157-168]. Also, influenza viruses, including other strains, subtypes and types, are available from clinical specimens and from public depositories, such as the American Type Culture Collection (ATCC), Rockville, Maryland, U.S.A.

Novel modified nucleotide sequences of this invention may also include allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) of DNA sequences encoding the H3HA2 protein sequences, and the Formula III fragment [SEQ ID NO:62]. Similarly, DNA sequences having the Formula III fragment, which sequences encode other H3N2 HA2 proteins of the invention include sequences which differ in codon sequence outside of Formula II due to degeneracies of the genetic code or variations in the DNA sequence encoding H3HA2 proteins. Such codon differences may be caused by point mutations or by induced modifications to enhance the activity, half- life or production of the peptide encoded thereby. Also covered by this invention are DNA sequences characterized by the above modification of Formula II into Formula III, which hybridize under stringent conditions with the DNA sequences encoding the HA2 subunit proteins, e.g., H3HA2 proteins, of this invention. DNA sequences which hybridize under non-stringent conditions with the disclosed sequences, but which encode proteins or fragments retaining the biological activities of the H3HA2 proteins, are also included in this invention. Typical conditions for stringent or non-stringent hybridization are known to those of skill in the art [See, e.g., Sambrook et al, cited above].

The actual techniques for producing the mutations described herein are now conventional to the art of genetic engineering, and are readily known and available to one of skill in the art. See, e.g., Sambrook et al, cited above. Such conventional techniques include, for example, site directed mutagenesis, which is available in commercial kits from, e.g. Clonetech and Promega Corporation. Other suitable techniques include, e.g., total gene synthesis and removing the fragment and replacing it with a synthetically generated, mutated fragment. It is anticipated that similar modifications to any H3HA2 sequence having an analogous codon pattern will result in the enhanced expression in E. coli, exemplified by the modified H3HA2 sequence. The mutations described herein are preferentially developed for increased expression of the influenza protein or fusion protein in E. coli, which is the preferred host because it can be used to produce the desired proteins safely and cheaply. To circumvent the requirement of ampicillin for plasmid selection in production fermentations, a preferred method of production which uses the modified nucleotide sequences of this invention employs an alternative expression system in which the β-lactamase coding sequence is wholly or partially replaced by a coding sequence for an alternative selectable marker, such as, kanamycin or

chloramphenicol.

To aid in expression of the H3HA2 peptides or fusion proteins, these protein sequences or fragments thereof may also be fused to a polypeptide capable of further enhancing expression of these fragments in the selected host system.

Ordinarily, such a peptide would contain a leader sequence fragment that provides for secretion of the H3HA2 subunit fragment, in the host cell. The leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. There may be processing sites encoded between the leader sequence and the H3HA2 fragment that can be cleaved either in vivo or in vitro. Alternatively, a promoter sequence may be linked directly with the DNA molecule encoding the H3HA2 fragment. Such polypeptides, promoter and leader sequences are known to those of skill in the art and may be readily selected for expression in the selected host.

Construction of bacterial expression systems, preferably E. coli expression systems, including expression vectors and transformed host cells are also within the skill of the art. See, generally, methods described in standard texts, such as Sambrook et al, cited above. The present invention is therefore not limited to any particular vector, nor to any particular purification process from cell lysates or cell medium.

Influenza proteins encoded by the modified nucleotide sequence may be expressed in enhanced manner according to the first embodiment of the method of this invention, or the influenza proteins may be expressed in an enhanced manner by translation from their native sequences by the second embodiment of the method.

Additionally, the methods of this invention may be used to enhance the expression of a fusion protein which comprises a protein sequence encoded by the modified nucleotide sequence containing Formula III in place of Formula II in the native nucleotide sequence encoding an HA2 subunit of an HA protein from an H3N2 subtype virus, fused in frame to another protein or protein fragment (a "fusion partner") capable of enhancing expression of the fusion protein. One of skill in the art may readily select a fusion partner protein or fragment taking into account the desired host cell, i.e., E. coli, and utilizing the teachings herein. For the purposes of this invention, the H3HA2 fragment or sequence encoded by a modified nucleotide sequence as described above or the native sequence used in the second embodiment of this method may be fused to any peptide capable of further enhancing its expression in the host cell selected or of increasing its immunogenicity. The method of the present invention does not limit the nature of the "partner" protein or fragment to which the H3HA2 fragment is fused to provide the enhanced expression of the resulting fusion protein.

For example, the influenza protein or fragment bearing the amino acid sequence of Formula I may be fused to a number of conventionally known and used "partner" proteins [See, general texts on expression such as Current Protocols in Molecular Biology, Vol. 2, suppl. 10, publ. John Wiley and Sons, New York, NY, pp. 16.4.1-16.8.1 (1990); Smith et al, Gene, 67:31-40 (1988); U. S. Patent No. 4,801,536, among others]. However, it may be desirable that this fusion "partner" protein be an influenza protein sequence or fragment thereof derived from the same or another strain of influenza virus as the HA protein or protein fragment.

Preferably, this fusion partner protein is all or a portion of the influenza virus NS1 gene or an HA2 subunit.

In such a fusion protein, a linker sequence may be inserted optionally between the two sequences, i.e., between the sequence encoding the fusion partner and the HA2 protein encoded by the modified nucleotide sequence of this invention or the native sequence for expression according to the second embodiment of the method. This optional linker may provide space between the two protein sequences; and may encode a polypeptide or contain a cleavage site, which is selectively cleavable or digestible by conventional chemical or enzymatic methods. An example of a fusion protein whose expression can be enhanced by a method of this invention is NS1_{( 1-81 )}H3HA2_{(1 -221)} illustrated in Fig. 2 [SEQ ID NO: 10], which comprises the first 81 amino acids of NS1 (derived from an HlNl subtype virus, A/PR/8/34) fused to the sequences spanning amino acid 1 to 221 of the H3HA2 subunit (amino acids 1-221) via an optional four amino acid linker sequence.

Another exemplary fusion protein, NS1_{( 1-81 )}H3HA2_{(77-221 )} SEQ ID NO:72, comprises the first 81 amino acids of NS1 fused to the sequences spanning amino acid 77 to 221 of the truncated H3HA2 subunit. In other embodiments, the NS1 portion may comprise the sequence spanning amino acid residues 1 to amino acids 42 of H1N1. The HA2 fragment may alternatively be fused to a portion of the NS1 peptide derived from a selected Type A virus, e.g., an H3 subtype virus (H3N2). These proteins, their native nucleotide sequences, and their uses, are described in co-pending U.S. application 07/837,773, filed February 18, 1992, which is incorporated by reference.

As described below in the examples, the host cells used to express these fusion proteins may be modified by the second embodiment of the method of this invention to contain tRNA molecules capable of translating the rare arginine codons of Formula II. See, e.g., Example 25. Alternatively, the nucleic acid sequence encoding these and other suitable H3HA2 proteins or H3HA2-containing proteins, i.e. those comprising a native Formula II sequence [SEQ ED NO:9], may be modified by the first embodiment of the method of this invention to replace

Formula II with the Formula HI sequence to increase the expression of the encoded protein in E. coli according to the method of this invention.

The proteins and fusion proteins whose expression is enhanced by the methods of this invention may be employed in vaccine compositions. Several of the specific influenza proteins or fusion proteins described herein, which are produced according to the methods of this invention, have demonstrated the ability to stimulate or produce a protective immune response capable of recognizing an influenza virus or influenza virus-infected cells and protecting the vaccinated mammal against disease caused thereby. This protective response is desirably a T cell response, produced in the substantial absence of vaccine-induced neutralizing antibody. Such H3HA2 proteins and fusion proteins are capable of inducing T helper cells, particularly cytotoxic T lymphocytes, in the absence of neutralizing antibodies.

Pharmaceutical vaccine compositions can contain an effective immunogenic amount of a selected H3HA2 protein produced according to this invention or encoded by a modified nucleotide sequence of this invention in admixture with a suitable adjuvant in a non toxic and sterile pharmaceutically acceptable carrier. Suitable carriers for vaccine use, as well as other vaccine formulation additives and adjuvants, are well known to those of skill in the art. See, e.g., European Patent Application No. 366, 238, published May 2, 1990; and

European Patent Application No. 366,239, published May 2, 1990. Such

compositions may be effectively administered to human and animal patients to induce the appropriate immune response. The details of dosage and treatment using such compositions are also described in the above-cited published patent

applications.

The following examples illustrate methods for preparing H3HA2 and BHA2 fusion proteins of the invention and demonstrate the subtype specific protection against heterologous virus induced upon vaccination with the H3HA2 proteins. The following examples also illustrate methods for preparing the modified DNA sequences of the invention. All of these examples are illustrative only and do not limit the scope of the invention.

EXAMPLE 1 - PLASMID pMS3H3HA

Plasmid pFV88 contains the entire 221 amino acid length HA from A/Udorn, an H3 subtype virus [C. J. Lai et al, Proc. Natl. Acad. Sci. USA, 77:210- 214 (1980)], which HA nucleic acid sequence is illustrated in Fig. 1 [SEQ ID NO: 1]. This plasmid was cut with Pst I. The resulting 1900 bp fragment, which contains the entire HA (HA1 and HA2) fragment and some GC tailing, was then inserted into pUC18 [Bethesda Research Laboratories]. The resulting plasmid is termed pMS3 or pMS3H3HA. EXAMPLE 2 - pMG1

Plasmid pAPR801 is a pBR322-derived cloning vector which carries the NS1 coding region (A/PR/8/34). It is described by Young et al, in The Origin of Pandemic Influenza Viruses, ed. by W. G. Laver, Elsevier Science Publishing Co. (1983).

Plasmid pAS 1 is a pBR322-derived expression vector which contains the P_L promoter, an N utilization site (to relieve transcriptional polarity effects in the presence of N protein), and the ell ribosome binding site including the ell translation initiation codon followed immediately by a BamHI site. It is described by Rosenberg et al, in Methods Enzymol., 101:123-138 (1983).

Plasmid pAS 1ΔEH was prepared by deleting a non-essential EcoRI-

Hindlll region of pBR322 origin from pASl. A 1236 base pair BamHI fragment of pAPR801, containing the NS1 coding region in 861 base pairs of viral origin and 375 base pairs of pBR322 origin, was inserted into the BamHI site of pAS 1ΔEH. The resulting plasmid, pAS1ΔEH/801, expresses authentic NS1 (230 amino acids). The plasmid has an Ncol site between the codons for amino acids 81 and 82 and an

Nrul site 3' to the NS sequences. The BamHI site between amino acids 1 and 2 is retained.

Plasmid pMG27N, a pAS1 derivative [MoI. Cell. Biol., 5:1015-1024 (1985)], was cut with BamHI and Sad and ligated to a BamHI/NcoI fragment encoding the first 81 amino acids of NS1 from pAS 1ΔEH801 and a synthetic DNA Ncol/Sacl fragment of the following sequence: SEQ ID NO: 35:

5'-CATGGATCATATGTTAACAGATATCAAGGCCTGACTGACTGAGAGCT-

3'

SEQ ID NO: 36:

3'- CTAGTATACAATTGTCTATAGTTCCGGACTGACTGACTC -5'.

The resulting plasmid, pMG1, allows the insertion of DNA fragments after the first 81 amino acids of NS1 in any of the three reading frames within the synthetic linker fragment followed by termination codons in all three reading frames.

EXAMPLE 3 - pMG1H3HA

Plasmid pMG1, described above in Example 2, was digested with Ncol and Xbal, releasing a 54 bp fragment, which was discarded. Plasmid pMS3H3HA, described in Example 1 above, was digested with Hhal and Xbal, and a 701 bp fragment containing the coding sequence for the HA2 subunit of influenza strain A/Udorn (H3N2) was isolated, as illustrated in Fig. 1 [SEQ ID NO: 1].

Synthetic oligonucleotides were annealed to generate an Ncol 5' overhang sequence (at the 5' end) and a Hhal 3' overhang sequence (at the 3' end). The sequence of these oligonucleotides is as follows:

SEQ ID NO: 37: 5'-CATGGGCGCCCATATGGGCATATTCGGCG-3'

SEQ ID NO: 38: 3'- CCGCGGGTATACCCGTATAAGCC-5'.

The annealing reaction was performed as follows. The annealing mixture was made up of 2.5μL each of 5' oligo (1.3 μg/μL), the 3' oligo (1.2 μg/μL), and added water (15 μL) to a final volume of 20 μL. The reaction tubes were then placed in 4 mL culture tubes containing water which had been heated to 65°C for 10 minutes and allowed to cool down slowly. The tubes were then put on ice and used immediately for ligation.

This three part ligation generates pMGlH3HA2_(1-221) [SEQ ID NO: 9] which codes for the first 81 amino acids of NS1 fused to four amino acids donated from the linker and amino acids 1-221 of the HA2 subunit. This sequence is illustrated in Fig. 2 [SEQ ID NO: 9 & 10]. This molecule is also designated NS1_{( 1-81 )}H3HA2_{(1 -221)} [SEQ ID NO: 9 & 10] or H3C13.

EXAMPLE 4 - NS1_{( 1-81 )}H3HA2_{(77-22 1 )} TSEO ID NO: 11 & 121

pMS3H3HA, described in Example 1 above, was digested with

EcoRI and end-filled (Klenow). Subsequently, the vector was digested with Xbal. A 487 bp fragment, which contains the coding sequence for amino acids 77-221 of the HA2 subunit, was isolated and ligated to the Hpal and Xbal sites of pMGl. The resulting vector codes for a fusion polypeptide containing amino acids 1-81 of NS1 fused to amino acids 77-221 of the HA2 subunit. This molecule has been termed NS1_{( 1-81 )}H3HA2_{(77-221 )} and is illustrated in Fig. 3 [SEQ ID NO: 11 & 12].

EXAMPLE 5 - pMG₄₂BLHA2

To derive a vector similar to pMGl (described in Example 2), which contains the coding region for the first 42 amino acids of NS1 rather than the first 81 amino acids of NS1, pMGl was digested with BamHI and Ncol and ligated to the BamHI/NcoI fragment encoding amino acids 2 to 42 of NS1 from pNS1₄₂TGF α. pNS1₄2TGFα is derived when pAS1ΔEH801 is cut with Ncol and SalI and ligated to a synthetic DNA encoding human TGFα as an Ncol/Sall fragment.

pNS1₄₂TGFCI encodes a protein comprised of the first 42 amino acids of NS1 and the mature TGFα sequence. The NS1 portion of pNS142TGFα contains an amino acid change from Cys to Ser at amino acid 13.

The resulting plasmid, termed PMG42A, was then modified to contain an alternative synthetic linker after the NS1₄₂ sequence with a different set of restriction enzyme sites within which to insert foreign DNA fragments into the three reading frames after the NS1₄₂. This linker has the following sequence: SEQ ID NO: 39:

5'-

CATGGATCATATGTTAACAAGTACTCGATATCAATGAGTGACTGAAGCT- 3'

SEQ ID NO: 40:

3'- CTAGTATACAATTGTTCATGAGCTATAGTTACTCACTGACT -5'.

The resulting plasmid is called pMG₄₂B. This vector is needed to contain the neomycin phosphotransferase- 1 (NPT-1) gene which confers kanamycin resistance.

As described in Shatzman and Rosenberg, Met. Enzymol., 152:661- 673 (1987), pOTS207 is a pAS derived cloning vector which carries the kanamycin resistance gene from Tn903 [Berg et al, Microbiology, ed. D. Schlessinger, pp. 13- 15, American Society for Microbiology (Washington, DC 1978); Nomura et al, The Single-Stranded DNA Phages, ed. D. Denhardt et al, pp.467-472, Cold Spring Harbor Laboratory (New York 1978); Castellazzi et al, Molecul. Gen. Genet., 117:211-218 (1982)]. It was constructed by digesting plasmid pUC8 [Yanisch- Perron et al, Gene, 33:103-119 (1985)], with BamHI and ligated to a BcII fragment containing the kanamycin gene from Tn903. The resulting plasmid, pUC8-Kan, was digested with EcoRI and Pstl, and the fragment containing the kanamycin gene was inserted between the EcoRI and Pstl sites of pOTSV [Shatzman and Rosenberg, cited above]. The resulting plasmid is pOTS207.

The pOTS207 was digested with EcoRI and Pstl, and the 1467 bp fragment containing the kanamycin resistance gene was isolated. Synthetic oligonucleotides:

SEQ ID NO: 41: 5' AATTCGTACCTA 3'

SEQ ID NO: 42: 3' GCATGGATCTAG 5'

were made to link the NPT-1 gene to pMG42B vector. pMG₄₂B was digested with

Bglll and Pstl. The EcoRI/Pstl NPT-1 gene fragment and the synthetic oligo linker were ligated to the digested pMG₄₂B. The resulting plasmid, pMG₄₂Kn allows fusions, in three different reading frames, to the NS_{1 -42} gene, while allowing antibiotic selection with kanamycin.

Plasmid pBHA is a pBR322-derived vector, containing the complete nucleotide sequence of the HA gene of a Type B influenza virus (B/Lee/40). It is described by Krystal et al, Proc. Natl. Acad. Sci. USA, 79:4900-4804 (1982).

pBHA was digested with Rsal and a 813 bp fragment containing the HA subunit was isolated. This fragment was ligated into plasmid pMG₄₂-Kn (described above) that had been digested with Seal. During the cloning, a nucleotide base (T) was deleted from the Seal recognition site shifting the gene out of the reading frame. The vector was digested with Ncol, and filled-in using Klenow, putting the gene back into the reading frame.

The resulting construct, pMG₄₂BLHA2 [SEQ ID NO: 14], expresses a fusion polypeptide containing amino acids 1-42 of NS1 and 41-233 of the HA2 subunit. This construct contains the Cys to Ser change at amino acid 13 of the NS1 portion of the fusion peptide.

In preliminary studies with this construct, vaccinated laboratory mice demonstrated protection from challenge with Type B influenza in the absence of neutralizing antibody for the virus. EXAMPLE 6 - PREPARING SEED VIRUS AND RAISING ANTISERA

The seed virus, A/Udorn, was prepared according to the procedures described in P. Palese and J. Schulman, Virol., 57:227-237 (1974). Briefly, this technique is as follows.

Influenza virus strain A/Udorn was inoculated in 10-day old embryonated hen's eggs into the allantoic cavity. The eggs were incubated for

24-48 hours at 35°C then chilled at 4°C overnight. A portion of the eggshell over the airsac was removed and the allantoic fluid was aseptically removed using a 10-ml syringe. The fluid was centrifuged at low speed (3,000 x g) to remove particulates. This clarified supernatant was centrifuged at high speed using an SW28 Beckman rotor at 27,000 rpm (4°C for 90 minutes), resulting in the virus pellet. The virus was resuspended in 10 mM Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA and repelleted as before. The virus was layered on 30-60% sucrose gradient in 1 mM EDTA (NTE) and spun for 3-5 hours at 25,000 rpm. The band in the middle of the tube was withdrawn, diluted in NTE and centrifuged at 27,000 rpm for 90 minutes. The pellet was suspended in phosphate-buffered saline (PBS). These viral particles were used as immunogens for preparation of antisera.

Antisera was prepared as follows. 100-200 micrograms of purified virus in complete Freund's adjuvant w ected into the subscapula of a New Zealand White rabbit. A second injection in incomplete Freund's adjuvant was done 4 weeks later, and the animals were bled and antisera collected 7-10 days later. EXAMPLE 7 - EXPRESSION OF H3HA2 FUSION PROTEINS

A. NS1_{( 1-81 )}H3HA2_{(1 -221)} [SEQ ID NO: 9 & 101

The plasmid pMGlH3HA2_{(1 -221)} [SEQ ID NO: 9] was transfected into E. coli strain AR58 [SmithKline Beecham Pharmaceuticals]. Cultures were grown at 32°C to mid-log phase at which time cultures were shifted to 39.5°C for 2 hours. The E. coli cell pellets containing the recombinant polypeptide were then stored at -70°C until used.

Production of the NS1_{( 1-81 )}H3HA2_{(1 -221)} protein [SEQ ID NO: 10] was confirmed by Western blot analysis [Towbin et al, Proc. Natl. Acad. Sci. U.S.A., 76:4350 (1979)] using antisera prepared against A/Udorn virus, as described in Example 5. A major immunoreactive species was found at a molecular weight of 35,050 daltons.

B. NS1_{( 1-81 )}H3HA2_{(77-221 )} [SEQ ID NO: 11 & 121

The plasmid encoding the NS1_{( 1-81 )}H3HA2_{(77-221 )} peptide [SEQ ID NO: 12] was expressed as described in part A above. Production of this peptide was confirmed by Western blot analysis, as described above. A major

immunoreactive species was found at a molecular weight of 26,697 daltons.

EXAMPLE 8 - PARTIAL PURIFICATION OF H3HA2 FUSION PROTEINS

E. coli cell pellets containing the recombinant polypeptides, prepared as described in Example 6, were stored at -70°C until used. E. coli cells were thawed and resuspended in lysis buffer A (50 mM Tris-HCl, 5% glycerol, 2 mM EDTA and 0.1 mM DTT, pH 8.0) at 10 mL/gram. The stirred suspension was then treated with lysozyme (0.2 mg/mL) for 45 minutes at room temperature and sonicated 2x for 2-3 minutes each time by a Sonicator. The resultant suspension was treated with 0.1% DOC for 60 minutes at 4°C, then centrifuged at 25,000 x g. The pellet was resuspended by sonication in 50 mM glycine pH 10.0, 5% glycerol, 2 mM EDTA and then the suspension was treated with 1% Triton X-100 [J.T. Baker Chemicals Co.] at 4°C for 60 minutes and centrifuged as above.

The resulting pellet was solubilized in 50 mM Tris, 8 M urea, pH 8.0 and centrifuged to remove any insoluble material. This solubilized material is dialyzed against 10 mM Tris, 1 mM EDTA, pH 8.0 followed,

again, by centrifugation of insoluble material. The solubilized material is designated as "crude" material and is used in in vitro and in vivo mouse assays. At this point, the material is approximately 40 - 50% pure.

The "crude" material was electrophoresed through an SDS-PAGE and the appropriate H3HA2 protein bands were visualized by KC1 staining according to D. Hager et al, Anal. Biochem, 109:76-86 (1980). The band was cut-out and eluted electrophoretically by the "S&S Elutrap Electro-Separation System" [Schleicher & Schuell]. The electro-eluting buffer was the Tris-glycine. A concentrated and eluted sample was obtained and exhaustively dialyzed against 0.01 M NH₄HCO₃ and 0.02% SDS [M. Hunkapiller et al, Method. Enzvmol., 91:227-236 (1983)]. This sample was frozen quickly by dry ice and lyophilized to complete dryness.

The lyophilized material was brought back into solution using 50 mM Tris pH 8.0 and used for in vitro and in vivo mouse assays.

Following this gel elution step, the protein is usually greater than 75% pure.

EXAMPLE 9 - CONSTRUCTION OF POTS208 VECTORS

pOTSV is described in Devara et al, Cell, 36: 43-49 (1984).

Briefly, this vector is a pAS1 derivative with t-oop inserted at the Nrul site and a synthetic oligonucleotide encoding Sad, Xhol and Xbal restriction sites inserted at the Sail site (which is destroyed).

A. pOTS208

pOTS208 was prepared by digesting pOTSV with EcoRI and Seal, followed by fill in reaction using Klenow. Tn5 Plasmid DNA [described in R. Jorgensen et al., Mol. Gen. Genet., 177:65-72 (1979)] was digested with HindIII and Smal, followed by a fill in reaction using Klenow yielding a 1323 bp fragment encoding for neomycin phosphotransferase-2 gene (NPT-2). This fragment is described in detail in Rothstein et al., Cell, 19:795-805 (1980) and Jorgensen, cited above. This fragment and the above digested vector were ligated together to create pOTS208, which is kanamycin resistant.

B. pOTS208H3C13

pMGlH3HA2( 1-221) (Example 3) was digested with BamHI and Xbal, releasing two fragments: an 806 bp BamHI fragment and a 160 bp

BamHI/Xbal fragment. These fragments together code for NS1_(1-81)H3HA2_{( 1- 221)}. A three part ligation between the two fragments and BamHI/Xbal digested ρOTS208 (part A) yield pOTS208H3C13 which utilizes NPT-2 for kanamycin resistance.

C. pOTS208NS181Nco

pOTS208H3C13 (part B) was digested with Bglll. pSelect

[Promega] was cut with BamHI and ligated with the Bglll fragment, resulting in pSelectNPTII102. Transformation into E. coli JM101 [ATCC E. coli 33876] was followed by selection on kanamycin and tetracycline plates. KanR was conferred by the NPT2 region from pOTS208H3Cl 3. Some lambda sequence was also on the Bglll fragment. Oligo 4852, SEQ ID NO: 49 GCATCGCCATGAGTCACGACG, was used to mutate the Ncol site to CCATGA in pSelectNPTII102, resulting in pSelectNPTII102-8. This vector was cut with BstEII and BssHII. pOTS208H3C13 was cut with BstEII, BssHII and Sphl, and fragment exchange generated

pOTS208NS 181H3HA2-26. This clone has the Ncol site of NPT2 mutated.

pOTS208NS181NS181H3HA2-26 was cut with Ncol and Sail, filled in and ligated with Linker 1041 [New England Biolabs] to insert a Kpnl site and regenerate the Ncol site. This step also deletes the H3C13 region. The unique Xbal site of the parent pOTS208 vector is downstream of the deletion. The resulting vector is pOTS208NS181Nco.

EXAMPLE 10 - MODIFICATION OF GENE ENCODING H3HA2 FUSION PROTEIN

In order to increase yield of the H3HA2 protein, silent mutations to certain rare arginine codons were made to the coding sequence of the H3HA2 protein. These nucleotide changes resulted in no change in the protein sequence.

A mutant H3C13 protein was prepared by mutating the nucleotide sequences of the fusion protein prepared according to Example 3 above. Site directed mutagenesis using the Altered Sites System [Promega Corporation] according to the manufacturer's directions was used to change nucleotide numbers, 622, 625, and 634 (A to C) and 624, 627, and 636 (G to T) of nucleotide sequences [SEQ ID NO:9] encoding the NS1_(1-81)H3HA2_{(1 -221)} fusion protein of Fig. 2 [SEQ ID NO: 10], thereby changing the codons at these regions from AGG to CGT, both encoding Arg. These changes correspond to nucleotide numbers 367, 370, and 379 (A to C) and 369, 372, and 381 (G to T) of the HA2 fragment of Fig. 2 [SEQ ID NO: 58].

Fig. 2 illustrates the modified nucleotide sequences of the fusion protein [SEQ ID NO: 10] by contrast with the nucleotide sequence [SEQ ID NO: 9] of the "unmodified" fusion gene (nucleotide changes above sequences of unmodified gene). Mutagenesis on this sequence was carried out according to the method provided with the pSelect kit from Promega.

A. NS1_(1-81)H3HA2_{(1 -221)} [SEO ID NO: 10]

Briefly, cloning for the mutagenesis was performed as follows. The pSelect plasmid [Promega] and pMG1H3HA2 (Example 3) were each digested with HindIII. These two plasmids were ligated together and selected on tetracycline plates. The resulting vector is pSe1H3HA2. Mutagenesis was performed according to Promega's kit. The following oligonucleotide was used: SEQ ID NO: 43:

5'-AAACTGTTTG AAAAAACACG TCGTCAACTG CGTGAAAATG

CTGACGACAT GGGC -3'.

Clones were verified by restriction endonuclease HincII. The resulting plasmid, pSe1H3HA2mut5585 was digested with Ncol and Xbal, and a 748 bp fragment coding for the H3HA2mut5585 polypeptide was isolated.

pOTS208NS181Nco (Example 9C) was digested with NcoI and

Xbal. The ligation of linear pOTS208NS181Nco and the 748 bp fragment resulted in pOTS208NS1H3mut5585 [SEQ ID NO:7]. This vector codes for the

polypeptide, NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO:10].

B. Expression of mutated gene encoding H3C13 protein

The plasmid of A was transfected into E. coli strain AR58

[SmithKline Beecham]. Cultures are grown at 32°C to mid-log phase at which time cultures are shifted to 39.5°C for two hours. The E. coli cell pellets containing the recombinant polypeptide are then stored at -70°C until used. Production of the NS1_(1-81)H3HA2_{(1 -221)} protein [SEQ ID NO:10] is confirmed by Western blot analysis [Towbin et al., Proc. Natl. Acad. Sci. U.S.A., 76 :4350 ( 1979)] using antisera prepared against A/Udorn virus, as described in Example 4. A major immunoreactive species is expected at a molecular weight of approximately 35,00 daltons.

The expression levels obtained are about 50-100% higher than those obtained by the expression of the unmodified coding sequences in the same expression system. C. Construction of Alternative H3 Mutant

pSe1H3HA2mut5585 (part A) was subjected to site-directed mutagenesis, as described above. Oligo SEQ ID NO: 44

TGTGACAATGCTTGCATCGGTTCAATCCGTAATGGTACTTATGACCA TGATG, was used and clones were verified by restriction endonuclease Rsal. The resulting plasmid, pSe1H3HA2mut2 was digested with Ncol and Xbal, and an approximately 748 bp fragment encoding for the H3HA2mut2 polypeptide was isolated. pOTS208NS181Nco was digested with Ncol and Xbal. The ligation of linear pOTS208NS181Nco (Example 9C) and the 748bp fragment resulted in pOTS208NS1H3mut2. This vector codes for the NS1_(1-81)H3HA2_{(1 -221)} polypeptide [SEQ ID NO: 10].

Example 11 - PLASMID pD

Plasmid pAS1_EH/801 (described above in Example 2) was cut with Bglll, end-filled with DNA polymerase I (DNApolI; Klenow), and ligated closed, thus eliminating the Bglll site. The resulting plasmid pBgl- was digested with Ncol, end-filled with DNApolI (Klenow), and ligated to a Bglll linker. The resulting plasmid, pB4, contains a Bglπ site within the NS1 coding region. Plasmid pB4 was digested with Bgiπ and ligated to a synthetic DNA linker of the sequence:

SEQ ID NO: 45: 5'-GATCCCGGGTGACTGACTGA -3'

SEQ ID NO: 46: 3'- GGCCCACTGACTGACTCTAG-5'.

The resulting plasmid, pB4+, permits insertion of DNA fragments within the linker following the coding region for first 81 amino acids of NS1 followed by termination codons in all three reading frames. Plasmid pB4+ was digested with Xmal (cuts within linker), end-filled (Klenow), and ligated to a 520 base pair PvuII/Hindlll, end-filled fragment derived from the HA2 coding region. The resulting plasmid, pD, codes for a protein [SEQ ID NO: 18] comprised of the first 81 amino acids of NS1, three amino acids derived from the synthetic DNA linker (Gln-He-Pro), followed by amino acids 65-222 of the HA2.

Expression is obtained by transfecting pD into a desired E. coli strain, preferably LW14, using standard techniques. Purification may be by standard techniques or, preferably, as described in Example 18 below.

EXAMPLE 12 - H3 SUBTYPE HETEROLOGOUS PROTECTION ELICITED BY VACCINATION WITH NS1_(1-81)H3HA2_{(1 -221)} TSEO ID NO: 101

Mice (NIH/Swiss; 15 per group) were vaccinated subcutaneously with 50 or 10 μg NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO: 9 & 10] in aluminum hydroxide on days 0 and 21. The mice were boosted intraperitoneally on day 42 with the protein without adjuvant. On day 47, mice were challenged intranasally with 2 - 3 LD50 doses of either A/PR/8/34 (H1N1) or A/HK/68 (H3N2) virus, and survival was monitored through day 21. This represents a heterologous challenge (A/PR/8/34) and an H3 heterosubtypic challenge, since the NS1_(1-81)H3HA2_{(1 - 221 )} construct [SEQ ID NO: 9 & 10] was derived from A/Udorn/72 cDNA. The control group received adjuvant (CFA) only.

The results in Table 1 below show that survival in mice vaccinated with NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO: 10] and challenged with A/HK/68 (80-93%) was significantly higher than in control mice which were injected with adjuvant only (26% survival). In contrast, vaccination with NS1_(1-81)H3HA2_{(1 - 221 )} [SEQ ID NO: 10] did not confer protection against challenge with A/PR/8/34, an H1N1 strain (0-26% survival). Thus protection elicited by NS1_(1-81)H3HA2_{( 1 - 221 )} [SEQ ID NO: 10] is selective for antigenically diverse virus strains within the H3 subtype.

Likewise, vaccination with the D protein (NS1_{( 1 -81 )}HA2_(65-222) [SEQ ID NO: 18], derived from the HlNl subtype) elicits protection from heterosubtypic challenge with HlNl, but not the H3N2 subtype [S. Dillon et al, Nature, (1992); Mbawuike et al, Faseb. J., 5:A1362 (abs. 5749 and Table 1)]. These results in outbred mice also suggest that the response to the HI and H3 proteins will not be restricted to a- limited number of individuals with certain major

histocompatibility alleles, and therefore the vaccine will be effective in a majority of individuals.

Table 1

Percent Survival After Challenge:

Vaccination of mice with live homologous (A/HK/68) virus provided complete or partial protection, reflecting protection mediated by neutralizing antibody (homologous H3N2 challenge) and/or CTL (heterologous H1N1 challenge), respectively.

Duration of protective immunity was tested by immunizing mice subcutaneously with the recombinant influenza protein plus adjuvant on days 0 and 21. Some mice were also given an ip injection of the protein

(without adjuvant) on day 42. Mice were challenged with A/HK/68 (H3N2) on day 47, four weeks after the second injection. Control mice were immunized as described above for Table 1, where an ip injection was given at week 6 (5 days prior to challenge). The results in Table 2 show that CB6F1 mice (15 per group) were significantly protected when challenged with the A/HK/68 heterologous H3 virus strain 5-28 days after the last injection.

Table 2

EXAMPLE 13 - TYPE A CROSS-PROTECTION WITH D AND H3C13

PROTEIN

Mice (CB6F1) were divided randomly into six groups, with fifteen in each group. The mice were injected subcutaneously with proteins in Al⁺³ (100 μg) on days 0 and 21, and then were challenged with 2-3 LD₅₀ doses of virus on day 49. Survival was monitored through day 21. The results of this study are illustrated in Table 3 below. For convenience, NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO: 10] is referred to as H3C13 in the table below.

vs. control group

** p < 0.03 vs. control group

This data demonstrates that mice immunized with a mixture of the D protein and H3C13 protein in aluminum adjuvant were protected against challenge with either A/PR/8/34 (H1) or A/HK/68 (H3) virus. In contrast, mice immunized with the D protein were protected against HI but not H3 challenge. Likewise, mice immunized with the H3C13 protein were protected against the H3 but not the H1 challenge. Therefore, the combination of the D protein and the H3C13 proteins elicited protection against the currently circulating subtypes of influenza A virus. Thus, this combination represents a subtype cross-protective vaccine.

EXAMPLE 14 - CREATION OF pEA181KNRBS3 VECTOR

pMG1 (Example 2) and pMG42Kn (Example 5) were both digested with BamHI and Ncol. A 236 BamHI/NcoI fragment containing the coding sequence for amino acid sequence spanning residues 1 to 81 of the NS1 gene was isolated from pMGl. The digested pMG42Kn and the 236 bp fragment were ligated together and transformants were selected on LB and kanamycin agar plates. The resulting vector pMG181Kn(cII) maintains all regulatory elements of pMG42Kn except the NS1 (aa1-42) sequence is replaced with the NS1 (aa1-81) sequence.

pMG181Kn(cII) described above was digested with BstXI and BamHI. The folio wing linker en coding ribosome binding site (RB S3) is cloned in the digested vector, replacing the ell RBS. The linker sequence is:

5' TAAGGAGGATATAACATATG [SEQ ID NO: 47]

3' T GG AATTCCTCCTATATTGTATACCT AG 5' [SEQ ID NO: 48].

BstXI BamHI

The resulting vector is pMG181KnRBS3.

To generate pEA181KnRBS3, a 1.2 kb EcoRI/Bglll fragment from similarly digested pOTSV containing the lambda rexArexB region was cloned into mpl8 [Gibco/Bethesda Research Labs] and mutagenized to create silent mutations in the two Ndel sites in this region. The mutations were CATATG to CATGTG in both sites. One site is in the rexA and the other in the rexB. The mutagenized fragment was inserted into both EcoRI/Bglll digested pMG181Kn(cII) and similarly digested pMG181KnRBS3, resulting in the plasmids pEA181Kn(cII) and pEA181KnRBS3, respectively. pEA181KnRBS3 has the useful properties of the pMG vectors, plus the additional attribute of nalidixic acid induction.

An EcoRI/Pstl fragment containing the ampR gene of pBR322 was then inserted into EcoRI/Pstl digested pEA181Kn(cII) and pEA181KnRBS3 to create pEA181CIIamp and pEA181RBS3amp, respectively. These plasmids are rexB-i- and should be nalidixic acid inducible, in contrast to pMGl and its descendants, which are rexB- and cannot be induced with nalidixic acid. The mutant EcoRI/Bglll region was functionally examined by cloning it into a pMG1 vector carrying galK and demonstrating induction of galK with nalidixic acid.

EXAMPLE 15 - CREATION OF VECTOR FOR PRODUCTION OF NS1_(1-

_{81 )}BHA2_(1-223)

Plasmid pOTS208BLeeHA2 was created as follows. An EcoRI fragment encoding the B/Lee HA region from plasmid pBHA (Example 5) was cloned into pSelect to generate pSelectPBHAS2. Site-directed mutagenesis inserted an Ncol site at the start of HA2, resulting in an N-terminus: MET GLY PHE PHE, and a C terminus of SER ILE CYS LEU. The resulting construct is called pSelectPBHAS2-B1. This plasmid was cut with Ncol and Xbal (a site in the polylinker of pSelect downstream of the HA gene), and ligated into Ncol/Xbal digested pEA181KnRBS3, described above, to generate pEA181BLeeB1-1. A BamHI/EcoRI, filled in, fragment was cut out of pEA181BLeeB1-1 and ligated into pOTS208 (Example 9A), that had been digested with Xbal, filled in, and BamHI. The EcoRI and Xbal sites were regenerated by the ligation. This extra cloning step was necessary because there was no convenient cloning site to fuse the gene to NS_(1-81) in pOTS208. pEA181KnRBS3 (described above) and pOTS208 have unique BamHI and Xbal sites to facilitate fragment exchanges.

A BamHI/Xbal fragment of about 1011 bp encoding the NS_{( 1 - 81)}BLHA2_(1-223) sequence from plasmid pOTS208BLeeHA2 was isolated and ligated into vector pSelect- 1 [Promega], which was also digested with BamHI and Xbal. The resulting construct is called pSe1BC13. This vector contains the coding sequence for NS1_(1-81)BHA2_{( 1-223)}, also termed BC13 [SEQ ID NO: 57].

EXAMPLE 16 - CREATION OF VECTOR FOR PRODUCTION OF BC13mut2

Mutagenesis was carried out on the pSelBC13 using Promega's protocol and oligonucleotide 5492, SEQ ID NO: 50

GGAGGATGGGAAGGACTCATTGCAGGTTGG. This mutagenesis changed the ATG codon within the HA2 portion of the molecule to CTC (MET to LEU). The resulting plasmid is called pSelBC13mut5492. This plasmid was then digested with Ncol and Xbal, releasing a digestion fragment encoding for HA2, and ligated into pOTS208NS 18 INco (Example 9C) that had been digested with Ncol and Xbal.

The resulting construct, pOTS208NS1BLHA2mut5492 codes for the same polypeptide of pOTS208BLeeHA2, (i.e. BC13), except the internal translation start is eliminated at amino acid position 98 of the fusion protein. This protein is NS1_{( 1 - 81)}BHA2_{(1 -223)}(met-leu) [SEQ ID NO: 55].

A HindIII fragment of approximately 1 kb encoding NS1 (amino acid residues 7-81) and BLee HA2 (amino acid residues 1-223) and which contained the MET to LEU changes from plasmid pOTS208NS1BLHA2mut5492 was isolated. This fragment was ligated into the Hindlll site of vector pSelect- 1, resulting in pSelBC13mut5492. Mutagenesis was carried out using Promega's protocol and the following oligos 5920, 5921 and 5939, respectively:

SEQ ID NO: 51

CTCTGCTGTAGAAATCGGTAACGGTTGCTTTGAAACCAAAC SEQ ID NO: 52

GGTTTCTTGGAAGGTGGTTGGGAAGGTCTCATTGCAGGTTGGCACGG

SEQ ID NO: 53

GCTTTCCAACGAAGGTATCAATCAACAGTGAAGACGAGCATCTCTTGG.

This mutagenesis created the following silent codon changes in the

HA2 region:

The codons for GLY at positions 93, 94, 97, 187, 215, and 217 were each mutated from GGG to GGT; the codons for ILE at positions 188, 189, and 214 were each changed from ATA to ATC; the codon for ASP at position 193 was changed from GAT to GAC; and the codon for ASN at position 216 was changed from AAT to AAC.

The resulting plasmid was called pSelBC13mut2. This plasmid was then digested with Ncol and Xbal, releasing a fragment of about 775 bp encoding for HA2. This fragment was ligated into pOTS208NS181Nco (described above), that had been digested with Ncol and Xbal. The resulting construct,

pOTS208NS1BLmut2 (see Fig. 5 [SEQ ID NO: 54]), codes for the same polypeptide [SEQ ID NO: 55] as pOTS208NS1BLHA2mut5492, except for the silent codon changes. EXAMPLE 17 - EXPRESSION OF FUSION PROTEIN

pOTS208NS1BLmut2 [SEQ ID NO: 54] is transfected into a suitable host cell, preferably an E. coli strain and expressed essentially as described for the H3 proteins described above. Strain LW14 is a derivative of E. coli K-12 strain W3110 [ATCC E. coli 27325]. The transducing phage PI [E. coli ATCC 25404- B1] was grown on E. coli K-12 strain AR58, described above, the genotype of which is thr-galΕ::Tn10 λCI857 bio-uvrB- rpsL. Phenotypically, strain AR58 requires threonine, biotin for growth, is sensitive to UV light and DNA damaging agents, cannot use galactose as a carbon source, and is resistant to streptomycin. Strain W3110, a prototroph, is incubated with the phage and plated onto a medium containing tetracycline to select for the transduction of the Tn10 element. The P1 phage picks up the segment of DNA containing the Tn10 and brings with it the λ CI857 bio- uvrB-. Thus the strain LW14 has the following genotype: galE::Tn10λ CI857 bio- uvrB-. Phenotypically, strain LW14 requires biotin for growth, is sensitive to UV light and DNA damaging agents, and cannot use galactose as a carbon source. EXAMPLE 18 - PURIFICATION OF BC13mut2

E. coli whole cells transformed with the pOTS208NS1BLmut2 plasmid [SEQ ID NO: 54] as described in Example 16 above were recovered after fermentation by centrifugation or tangential flow filtration, washed to remove media, and stored at -70°C until use.

A. Step 1: Lvsis and centrifugation (Isolation)

E. coli cells, 500 gm wet cell weight (WCW), were thawed and suspended in 4-7 volumes (2L) of buffer containing 0.025 M Tris-HCl, 0.005 M EDTA, pH 8.0. Chicken egg lysozyme (Calbiochem; suspension at 100 mg/ mL) was added to a final concentration of 1 g/L and the preparation stirred with a Tekmar mixer at room temperature for 1 hour.

The lysate was centrifuged at 15,000 x g f or 1 hour at 4°C and the supernatant discarded. The pellet (PI) was resuspended in 5 mL per gram of original wet cell weight of buffer consisting of 0.025 M Tris-HCl, 0.002 M MgCl₂, pH 8.0 (about 2.5L).

The yield of this step was 90-100% by SDS-PAGE analysis, and 65- 100% by RP-HPLC for product.

B. Step 2: Nuclease digestion and extraction

The preparation was treated with benzonase to digest nucleic acids, then extracted with nonionic detergents to reduce the levels of E. coli contaminants in the pellet. Benzon nuclease, 0.2 mL per L of suspension, was added to the suspension, which was then stirred at room temperature for 1 hr. The sample was diluted with one volume of cold water containing 2% w/v Triton X-100 and 0.2% deoxycholate and stirred for 30 min at or below 15°C. Centrifugation was repeated as in step 1 and the supernatant discarded.

C. Step 3: Urea extinction

The pellet (P2) was extracted with 5 mL/gm WCW of cold 0.025 M NaH₂PO₄, 0.025 M Tris-HCl, pH 6.0, containing 4 M urea and 10 mM

dithiothreitol (DTT). The Tekmar was used at a very low speed to mix, and temperature held below 15°C. The sample was stirred at 4°C for 1 hr. then centrifuged as in step 1. The supernatant (S3) was discarded. The pellet (P3) must be stored in the freezer until use.

D. Step 4: Solubilization. reduction, and DEAE

chromatography

The P3 pellet was solubilized and applied to anion exchange chromatography. This step removes remaining nucleic acid and major host cell proteins. P3 was suspended to 5 mL per gm WCW in .01 M Tris base, 8M urea (pH not adjusted). DTT was added to 25 mM. The pH was then adjusted to 12.5 using 6N NaOH, stirring for 15 min at room temperature, immediately followed by a 5- fold dilution of the same with 10 mM boric acid containing 25 mM DTT. If needed, the sample may be diluted to keep conductivity below 2mS/cm. The pH was adjusted to 9.0 and the sample stirred for up to 2 hour at room temperature.

The pH 12.5 treatment was necessary to complete solubilization of the B/Lee protein. However since carbamylation may occur under these conditions, the time was controlled very carefully. In addition, the pH 9 adjusted sample was unstable and cannot be held.

The sample (no more than 12 mg total protein per mL of resin) was then loaded onto a 14 x 250 cm (0.75L) DEAE Toyopearl 650M column

equilibrated with buffer A. All steps were performed at room temperature at a linear velocity of 100 cm/hr. The column was washed sequentially with 2-3 column volumes of buffers B, C, and D, then eluted with buffer E. When protein began to elute from the column, flow was stopped for 15-20 minutes to improve the efficiency of elution of the B/Lee product; then the peak of product protein was collected. The column was cleaned with buffer D followed by 0.5 N NaOH.

The yield of this step was 85-90% by SDS-PAGE or Western blot analysis, and was estimated at 65-70% by RP-HPLC assay for product.

E. Step 5: Pretreatment and reverse phase chromatography

The buffer E eluate from step 4 was adjusted to no more than 1 g/L protein concentration and made 2% in SDS, 30 mM DTT, 0.1% M Tris, 5 mM EDTA, pH 9, then heated at either: 90°C for 60, 95°C for 30 min, or 100°C for 25 minutes, using a heat exchanger or water bath. This treatment was necessary to break up aggregates and prepare the sample for RP chromatography. The sample was cooled to room temperature and 2-propanol was added to 10% v/v.

The sample was injected on an Amberchrome reverse phase column equilibrated in 10% 2-propanol/0.2% trifluoroacetic acid (TFA)/water. The gradient shown in Table 1 was used to elute the column. Fractions containing product were analyzed by analytical RP-HPLC, pooled, and held at 4°C. The column was 25cm in height and was run at a linear velocity of 75-80 cm/hr at ambient temperature. An Amicon Vantage column, 9 cm in diameter, was used. The loading capacity of the column was 2 g/L.

The reverse phase column step has a yield of 30-80% (60-80% is typical). F. Step 6: Precipitation

The pH of the RP eluate was adjusted to 6.0 +/- 0.5 using 1 N NaOH. After 10-15 min of stirring at room temperature, the precipitate was collected by centrifugation at 16,000 x g for 30 min at 4°C. The precipitate was resuspended to approximately 6-8 mg/mL protein concentration in 25 mM Tris, 8 M urea. DTT was added to 25 mM, and the sample stirred for 30 min at room temperature. The pH was adjusted to 12.5 and stirring repeated for 15 min, immediately followed by pH adjustment to 9.0 using HCl.

Alternately, the precipitate was suspended in buffer containing 0.1 M Tris-HCl, 2% SDS, 0.01 M EDTA, pH 8.0-9.0. DTT was added to 25 mM, and stirred 15-30 min until the solution was clear and all of the precipitate had dissolved. The sample was immediately taken to the next step.

G. Step 7: Desalting and preparation of final product.

A 7 x 10 cm column was packed with Sephadex G25M (Pharmacia) at room temperature. It was equilibrated with 3-7 column volumes of 25 mM Tris- HCl, pH 9.,0, containing 5% w/v mannitol. Sample, at 6-10 mg/mL protein concentration, is injected on the column (20-25% of total column volume, i.e. 80- 100 mL per injection). The column was developed at 150 cm/hr linear velocity and the product desalted into the column buffer. The final product can be stored at 4°C.

The yield of steps 6 and 7 together was no less than 90%.

The product of the purification process was recovered at an overall yield of about 20-40%, and was over 95% pure by SDS-PAGE and RP-HPLC analysis. The final yield is about 3 g/500 g well cell weight.

A: 0.2% TFA . in water

B: 99.8% 2-propanol/0.2% TFA H. Purification of FluD, NS1_(1-81)HA2_{(65-222 )}

FluD (Example 10) may be purified in much the same manner as the B/Lee with the following parameter alterations. For DEAE chromatography, the FluD column was equilibrated in 8M urea, 50 mM Tris, 25 mM borate at pH 9.0. After the sample is loaded, sequential washes are performed with the following buffers: 4M urea in Tris-borate pH 9.0, 4 M urea and 0.4 M NaCl in Tris-borate pH 9.0, and Tris-borate pH 9.0. The product is eluted with a step elution of 2% SDS, 0.1 to 0.25 M NaCl, in Tris-borate pH 9.0. Prior to RPLC, the protein concentration is adjusted to 1 mg/mL or less, the product is heated at 95°C for 30 minutes, and cooled, and 2-propanol is added to 10% v/v. The column is then loaded. RPLC is then performed on Amberchrome resin, as described above for B/Lee. Up to 2-3 mg of protein are loaded per ml of resin. The final yield is about 4 g/500 g wet cell weight. EXAMPLE 19 - 3-PART INFLUENZA VACCINE

A recombinant vaccine was formulated to contain 1 μg each of the recombinant proteins NS1_(1-81)HA2_(65-222) (Example 11), NS1_(1-81)H3HA2_{(1 - 221 )}mut5255 (Example 10), and the BC13mut2 (described in Example 15 above) in Al⁺³ (100 μg) plus 3-o-deacylated monophosphoryl-lipid A (3D-MPL) (5 μg) [described in U.S. Patent No. 4,912,093; commercially available from Ribi

Immunochem Research, Inc., Hamilton, Montana]. Prior to inclusion in the recombinant vaccine, the influenza proteins were purified as described in Example 15 above to remove any contaminating bacterial proteins, DNA, and endotoxin.

Mice (female, CB6F1) were divided randomly into groups with 15 mice per group. The mice w ere injected subcutaneously on days 0 and 21 with the recombinant vaccine. A group of control mice were injected with the same dose of Al/MPL without antigen according to the same schedule. Mice were challenged with 3-5 LD₅₀ doses of virus on day 49 and survival was monitored through day 21 post-challenge. In the following table showing these results, N.D. = not done and under the antigens, H1 = NS1_(1-81)HA2_(65-222)) H3 = NS1_{( 1-81)}H3HA2_{( 1 - 221)}mut5855 and B = NS1_(1-81)BLHA2_{(1 -221)}mut2. Table 5

Type A and B Cross-Protection in Mice Immunized with a Combination of

Recombinant HA2 Antigens

* p≤ 0.001 vs. control group

** p≤ 0.01 vs. control group

1 p > 0.05 (not statistically different than control group)

The data in Table 5 above results from two experiments that demonstrate that mice vaccinated with the combination of H1, H3, and Type B HA2 antigens were protected against all three virus challenges (H1, H3 and Type B) (>73- 100% survival vs. 0-7% in controls). The H1 and H3 antigens in Al/MPL were subtype protective when administered individually as shown in Table 5. The Type B construct administered without the other antigens was only protective in one study (Exp. 1; 33% survival vs. 0% survival in controls but protected 80% of the mice in a second study, Exp. 2). Thus, preliminary data shows equivocal data on the stabihty of the Type B construct when formulated in Al/MPL in the absence of the other HA2 antigens. Studies are ongoing to confirm the stability of the construct in other formulations and in NIH/Swiss mice to confirm activity in an outbred system.

Although each antigen contains the NS1_(1-81) regions from

A/PR/8/34 (H1) virus, protections against H1 challenge was only achieved with the D protein which contains the H1HA2 region as well. Thus, the H3HA2 and Type B HA2 portions of each chimeric antigen are responsible for conferring subtype- specific protection. Thus, the combined HA2 constructs provide cross-protections for all currently circulating influenza Type A (H1 and H3 subtypes) and Type B viruses.

Survival of NIH/Swiss outbred mice immunized with the mutant NS_{( 1-81)}BHA2_{( 1 -223)}(met-leu) (not shown) showed activity at 100 micrograms (73% survival), but reduced activity at lower doses. This confirms earlier studies in outbred mice showing reduced potency relative to H1 or H3 constructs (which are active at≥ 1 microgram per dose). In contrast, in CB6F1 inbred mice, an inverse dose response or no dose response is seen with NS_{(1-81 )}BHA2_(1-223)(met-leu).

EXAMPLE 20 - PLASMID pMS3H3HA

Plasmid pFV88 contains the entire 221 amino acid length HA2 from A/Udorn, an H3 subtype virus [C. J. Lai et al, Proc. Natl. Acad. Sci. USA, 77:210- 214 (1980)], which HA2 nucleic acid sequence is illustrated in Fig. 7 [SEQ ID NO: 1]. This plasmid was cut with Pst I. The resulting 1900 bp fragment, which contains the entire HA (HA1 and HA2) fragment and some GC tailing, was then inserted into pUC18 [Bethesda Research Laboratories]. The resulting plasmid is termed pMS3 or pMS3H3HA. EXAMPLE 21 - pMG1

Plasmid pAPR801 is a pBR322-derived cloning vector which carries the NS1 coding region (A/PR/8/34). It is described by Young et al, in The Origin of Pandemic Influenza Viruses, ed. by W. G. Laver, Elsevier Science Publishing Co. (1983).

Plasmid pAS1 is a pBR322-derived expression vector which contains the PL promoter, an N utilization site (to relieve transcriptional polarity effects in the presence of N protein) and the ell ribosome binding site including the cll translation initiation codon followed immediately by a BamHI site. It is described by Rosenberg et al, in Methods Enzymol., 101:123-138 (1983).

Plasmid pAS1ΔEH was prepared by deleting a non-essential EcoRI -

Hindlll region of pBR322 origin from pAS1. A 1236 base pair BamHI fragment of pAPR801, containing the NS1 coding region in 861 base pairs of viral origin and 375 base pairs of pBR322 origin, was inserted into the BamHI site of pAS1ΔEH. The resulting plasmid, pAS1ΔEH/801 expresses authentic NS1 (230 amino acids). The plasmid has an Ncol site between the codons for amino acids 81 and 82 and an Nrul site 3' to the NS sequences. The BamHH site between amino acids 1 and 2 is retained. Plasmid pMG27N, a pAS 1 derivative [Mol. Cell. Biol., 5:1015-1024 (1985)], was cut with BamHI and SacI and ligated to a BamHI/NcoI fragment encoding the first 81 amino acids of NS1 from pASlΔEH801 and a synthetic DNA Ncol/Sacl fragment of the following sequence:

SEQ ID NO: 10:

5'-CATGGATCATATGTTAACAGATATCAAGGCCTGACTGACTGAGAGCT-

3'

SEQ ID NO: 58:

3'- CTAGTATACAATTGTCTATAGTTCCGGACTGACTGACTC -5'

The resulting plasmid, pMGl, allows the insertion of DNA fragments after the first 81 amino acids of NS1 in any of the three reading frames within the synthetic linker fragment followed by termination codons in all three reading frames. EXAMPLE 22 - pMG1H3HA

Plasmid pMG1, described above in Example 21, was digested with Ncol and Xbal, releasing a 54 bp fragment, which was discarded. pMS3H3HA, described in Example 1 above, was digested with Hhal and Xbal, and a 701 bp fragment containing the coding sequence for the HA2 subunit of influenza strain A/Udorn (H3N2) was isolated, as illustrated in Fig. 1 [SEQ ID NO: 1].

Synthetic oligonucleotides were annealed to generate an Ncol 5' overhang sequence (at the 5' end) and a Hhal 3' overhang sequence (at the 3' end). The sequence of these oligonucleotides is as follows:

SEQ ID NO: 66: 5'-CATGGGCGCCCATATGGGCATATTCGGCG-3'

SEQ ID NO: 67: 3'- CCGCGGGTATACCCGTATAAGCC -5'

The annealing reaction was performed as follows. The annealing mixture was made up of 2.5μL each of 5' oligo (1.3 μg/μL), the 3' oligo (1.2 μg/μL), and added water (15 μL) to a final volume of 20 μL. The reaction tubes were then placed in 4 mL culture tubes containing water which had been heated to 65°C for 10 minutes and allowed to cool down slowly. The tubes were then put on ice and used immediately for ligation.

This three part ligation generates pMGlH3HA2_(1-221) [SEQ ID NO: 9] which codes for the first 81 amino acids of NS1 fused to four amino acids donated from the linker and amino acids 1-221 of the HA2 subunit. This sequence is illustrated in Fig. 2 [SEQ ID NO: 9 & 10]. This molecule is also designated NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO: 9 & 10], EXAMPLE 23 - PREPARING SEED VIRUS AND RAISING ANTISERA

The seed virus, A/Udorn, was prepared according to the procedures described in P. Palese and J. Schulman, Virol., 57:227-237 (1974). Briefly, this technique is as follows.

Influenza virus strain A/Udorn was inoculated in 10-day old embryonated hen's eggs into the allantoic cavity. The eggs were incubated for 24-48 hours at 35°C then chilled at 4°C overnight. A portion of the eggshell over the airsac was removed and the allantoic fluid was aseptically removed using a 10-ml syringe. The fluid was centrifuged at low speed (3,000 x g) to remove particulates. This clarified supernatant was centrifuged at high speed using an SW28 Beckman rotor at 27,000 rpm (4°C for 90 minutes), resulting in the virus pellet. The virus was resuspended in 10 mM Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA and repelleted as before. The virus was layered on 30-60% sucrose gradient in 1 mM EDTA (NTE) and spun for 3-5 hours at 25,000 rpm. The band in the middle of the tube was withdrawn, diluted in NTE and centrifuged at 27,000 rpm for 90 minutes. The pellet was suspended in phosphate-buffered saline (PBS). These viral particles were used as immunogens for preparation of antisera.

Antisera was prepared as follows. 100-200 micrograms of purified virus in complete Freund's adjuvant was injected into the subscapula of a New Zealand White rabbit. A second injection in incomplete Freund's adjuvant was done 4 weeks later, and the animals were bled 7-10 days later.

EXAMPLE 24 - MODIFICATION AND EXPRESSION OF H3HA2 FUSION PROTEINS

The modified nucleotide sequences encoding the H3HA2 proteins were prepared by mutating the nucleotide sequences of the fusion proteins prepared according to Example 22 above. Site directed mutagenesis using the Altered Sites System [Promega Corporation] according to the manufacturer's directions was used to change nucleotide numbers, 622, 625 and 634 (A to C) and 624, 627, and 636 (G to T) of nucleotide sequences [SEQ ID NO:9] encoding the NS1_(1-81)H3HA2_{( 1- 221)} fusion protein of Fig. 3 [SEQ ID NO:10], thereby changing the codons at these regions from AGG to CGT, both encoding Arg. These changes correspond to nucleotide numbers 367, 370 and 379 (A to C) and 369, 372 and 381 (G to T) of the HA2 fragment of Fig. 7 [SEQ ED NO:1].

Fig. 2 illustrates the modified nucleotide sequences of the fusion proteins [SEQ ID NO: 58] by contrast with the nucleotide sequence [SEQ ID NO:9] of the "unmodified" fusion proteins (nucleotide changes below and amino acid changes in above sequences of unmodified fusion protein). Mutagenesis on this sequence was carried out according to the method provided with the pSelect kit from Promega.

A. NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO: 10]

Briefly, cloning for the mutagenesis was performed as follows. The pSelect plasmid [Promega] and pMGlH3HA2 (Example 22) were each digested with Hindlll. These two plasmids were ligated together and selected on tetracycline plates. The resulting vector is pSelH3HA2. Mutagenesis was performed according to Promega's kit. The following oligonucleotide was used: SEQ ID NO:68:

5'-AAACTGTTTG AAAAAACACG TCGTCAACTG CGTGAAAATG

CTGACGACAT GGGC -3'.

Clones were verified by restriction endonuclease HincII. The resulting plasmid, pSe1H3HA2mut5585 was digested with Ncol and XbaI, and a 748 bp fragment coding for the H3HA2mut5585 polypeptide was isolated.

pOTS 208NS181 (Eco-740) was digested with Ncol and Xbal. The ligation of linear pOTS208NS181Nco and the 748 bp fragment resulted in pOTS208NS1H3mut5585 [SEQ ID NO:58]. This vector codes for the polypeptide, NS1_(1-81)H3HA2_{(1 -221)} [SEQ ID NO: 10]. B. Expression of mutated NS1_(1-81)H3HA2 proteins The plasmid of A was transfected into E. coli strain AR58 [SmithKline Beecham].

Cultures are grown at 32°C to mid-log phase at which time cultures are shifted to 39.5°C for two hours. The E. coli cell pellets containing the recombinant polypeptide are then stored at -70°C until used. Production of the NS1_{(1- 81 )}H3HA2_{(1 -221 )} protein [SEQ ID NO: 10] is confirmed by Western blot analysis [Towbin et al, Proc. Natl. Acad. Sci. U.S.A., 76:4350 (1979)] using antisera prepared against A/Udorn virus, as described in Example 23. A major

immunoreactive species is expected at a molecular weight of approximately 35,00 daltons.

The expression levels obtained are about 50-100% higher than those obtained by the expression of the unmodified coding sequences in the same expression system. EXAMPLE 25 - tRNA INSERTION INTO HOST CELLS EXPRESSING H3 PROTEIN

E. coli host cells containing H3N2 fusion protein obtained as described in Example 22 above were transformed using conventional techniques. See, e.g. Sambrook et al, cited above.

Briefly, a culture of E. coli strain MM294cI⁺ containing the plasmid pDC952 was grown overnight in Luria broth with chloramphenicol. The plasmid pDC952 carries the argU gene which encodes the tRNA that recognizes the

AGA/AGG codons [P. Saxena and J. Walker, J. Bacteriol., 174(6): 1956- 1964 (Mar. 1992)]. From this culture the plasmid pDC952 was prepared. A second culture of E. coli, strain AR13 [SmithKline Beecham] carrying the plasmid for the H3 flu antigen, was grown overnight in Luria broth with kanamycin. These cells were made competent for transformation as described below.

The H3/AR13 overnight culture was diluted 1:50 in LB and kanamycin (50 mL total) and incubated at 37°C until it reached an O.D.650 of 0.6. The culture was then transferred to a 50 mL conical tube and chilled at about 4°C. Following this, the tube was centrifuged in a TJ6 centrifuge (10 min; 2000-3000 rev/min), the pellet resuspended in 25 mL 100 mM CaCl₂, and placed on ice for about 30 minutes. The pellet was then centrifuged as described above and resuspended in about 2.5 mL 100 mM CaCl₂.

The competent cells were aliquoted (100 μl) into three separate sterile tubes. The first tube was the negative control and did not receive any DNA. The second tube was a positive control and 1 μl of plasmid pT₇II was added to the cells. To the third tube was added 3 μl of pDC952. These controls served to ensure that transformation occurred. Each tube of cells was mixed, placed on ice for 60 min., heat shocked at 37°C in a water bath for 2 minutes, and incubated in a 32°C water bath for 60 min. after adding 1 mL LB. The tubes were then microfuged for 1 minute and the supernatants poured off until only about 200 μL were left. The pellets were then resuspended in the remaining supernatant and plated as follows: (1) on LB and chloramphenicol, (2) on LB and ampicillin, and (3) on LB and chloramphenicol and kanamycin. The plates were then incubated at 32°C overnight.

Shake flasks were inoculated with the control strain, H3/AR13, and 4 transformants, pDC952/H3/AR13, and grown at 32°C to an optical density of 0.6 to 0.7 at which point the cultures were shifted to 39.5°C for 3 hours. Samples were taken at induction start (temperature shift to 39.5°C) and 3 hours post-induction. These samples were analyzed by high performance liquid chromatography (HPLC) and Western blotting. The results of these analyses indicated that expression of H3 had increased by as much as 80% and the presence of the argU gene had eliminated the lowest western positive band as compared with the wild-type constructs (H3/AR13). It is believed that these results were obtained by eliminating the frameshifting caused by tandem AGG rare arginine codons. Further, there did not appear to be any difference in product quality between the H3 mutant prepared according to Example 24, and the argU tRNA transformants made according to this Example.

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Shatzman, Allan

Scott, Miller

Dillon, Susan B.

Kane, James

(ii) TITLE OF INVENTION: Vaccinal Polypeptides

(iii) NUMBER OF SEQUENCES: 72

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SmithKline Beecham Corporation - Corporate

Patents

(B) STREET: U.S. Mailcode UW2220 - 709 Swedeland Road

(C) CITY: King of Prussia

(D) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19406-2799

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 149,150

(B) FILING DATE: 05-NOV-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 013,415

(B) FILING DATE: 01-FEB-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 108,914

(B) FILING DATE: 18-AUG-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 837,773

(B) FILING DATE: 18-FEB-1992

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 751,896

(B) FILING DATE: 30-AUG-1991 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 387,200

(B) FILING DATE: 28-JUL-1989

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 238,801

(B) FILING DATE: 02-NOV-1988

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 645,732

(B) FILING DATE: 30-AUG-1984

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Baumeister, Kirk

(B) REGISTRATION NUMBER: 33,833

(C) REFERENCE/DOCKET NUMBER: P50134 PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-270-5096

(B) TELEFAX: 215-270-5090

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 666 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..663

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GGC ATA TTC GGC GCA ATA GCA GGT TTC ATA GAA AAT GGT TGG GAG GGA 48 Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA AAT TCT GAG GGC ACA 96 Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA GCC ATC GAC CAA ATC 144 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

AAT GGG AAA CTG AAT AGG GTA ATC GAG AAG ACG AAC GAG AAA TTC CAT 192 Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His

50 55 60 CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG AGA ATT CAG GAC CTC 240 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu

65 70 75 80

GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT CTC TGG TCT TAC AAT GCG 288 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA ATT GAT CTG ACT GAC 336 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

TCG GAA ATG AAC AAA CTG TTT GAA AAA ACA AGG AGG CAA CTG AGG GAA 384 Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

AAT GCT GAG GAC ATG GGC AAT GGT TGC TTC AAA ATA TAC CAC AAA TGT 432 Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

GAC AAT GCT TGC ATA GGG TCA ATC AGA AAT GGG ACT TAT GAC CAT GAT 480 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

GTA TAC AGA GAC GAA GCA TTA AAC AAC CGG TTT CAG ATC AAA GGT GTT 528 Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

GAA CTG AAG TCA GGA TAC AAA GAC TGG ATC CTG TGG ATT TCC TTT GCC 576 Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

ATA TCA TGC TTT TTG CTT TGT GTT GTT TTG CTG GGG TTC ATC ATG TGG 624 Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205

GCC TGC CAG AAA GGC AAC ATT AGG TGC AAC ATT TGC ATT TGA 666

Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 221 amino acids

(B) TYPE: amino acid

( D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15 Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr 20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys 130 135 140

Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp 145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205

Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 666 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..663 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

GGC ATA TTC GGC GCA ATA GCA GGT TTC ATA GAA AAT GGT TGG GAG GGA 48

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA AAT TCC GAG GGC ACA 96

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA GCC ATC GAC CAA ATC 144 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

AAT GGG AAA CTG AAT AGG GTA ATC GAG AAG ACG AAC GAG AAA TTC CAT 192 Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His

50 55 60

CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG AGA ATT CAG GAC CTC 240 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu

65 70 75 80

GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT CTC TGG TCT TAC AAT GCG 288 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA ATT GAT CTG ACT GAC 336 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

TCG GAA ATG AAC AAA CTG TTT GAA AAA ACA AGG AGG CAA CTG AGG GAA 384 Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

AAT GCT GAG GAC ATG GGC AAT GGT TGC TTC AAA ATA TAC CAC AAA TGT 432 Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

GAC AAT GCT TGC ATA GGG TCA ATC AGA AAT GGG ACT TAT GAC CAT GAT 480 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

GTA TAC AGA GAC GAA GCA TTA AAC AAC CGG TTT CAG ATC AAA GGT GTT 528 Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

GAA CTG AAG TCA GGA TAC AAA GAC TGG ATC CTG TGG ATT TCC TTT GCC 576 Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

ATA TCA TGC TTT TTG CTT TGT GTT GTT TTG CTG GGG TTC ATC ATG TGG 624 Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205 GCC TGC CAA AAA GGC AAC ATT AGG TGC AAC ATT TGC ATT TGA 666

Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 221 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

1 5 10 15

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His

50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu

65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205 Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

(2 ) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 670 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..666

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

GGT CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA GGG GGA TGG ACT GGA 48 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly

1 5 10 15

ATG ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG AAT GAA CAG GGA TCA 96 Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser

20 25 30

GGC TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT GCC ATT AAC GGG ATT 144 Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile

35 40 45

ACA AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG AAC ATT CAA TTC ACA 192 Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr

50 55 60

GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA 240

Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu

65 70 75 80

AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA 288

Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala

85 90 95

GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC 336 Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp

100 105 110

TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT 384 Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn

115 120 125

AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT 432 Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys

130 135 140 GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC 480

Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro

145 150 155 160

AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG 528

Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val

165 170 175

AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT 576 Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr

180 185 190

GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC 624 Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe

195 200 205

TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC 666

Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

210 215 220

TGAG 670

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 222 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly

1 5 10 15

Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser

20 25 30

Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile

35 40 45

Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr

50 55 60

Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu

65 70 75 80

Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala

85 90 95 Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp

100 105 110

Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn

115 120 125

Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys

130 135 140

Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro

145 150 155 160

Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val

165 170 175

Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr

180 185 190

Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe

195 200 205

Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

210 215 220

(2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 670 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..670

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GGCATATTCG GCGCAATAGC AGGTTTCATA GAAAATGGTT GGGAGGGAAT GATAGACGGT 60

TGGTACGGTT TCAGGCATCA AAATTCNGAG GGCACAGGAC AAGCAGCAGA TCTTAAAAGC 120 ACTCAAGCAG CCATCGACCA AATCAATGGG AAACTGAATA GGGTAATCGA GAAGACGAAC 180

GAGAAATTCC ATCAAATCGA AAAGGAATTC TCAGAAGTAG AAGGGAGAAT TCAGGACCTC 240

GAGAAATACG TTGAAGACAC TAAAATAGAT CTCTGGTCTT ACAATGCGGA GCTTCTTGTC 300

GCTCTGGAGA ACCAACATAC AATTGATCTG ACTGACTCGG AAATGAACAA ACTGTTTGAA 360

AAAACAAGGA GGCAACTGAG GGAAAATGCT GAGGACATGG GCAATGGTTG CTTCAAAATA 420 TACCACAAAT GTGACAATGC TTGCATAGGG TCAATCAGAA ATGGGACTTA TGACCATGAT 480 GTATACAGAG ACGAAGCATT AAACAACCGG TTTCAGATCA AAGGTGTTGA ACTGAAGTCA 540

GGATACAAAG ACTGGATCCT GTGGATTTCC TTTGCCATAT CATGCTTTTT GCTTTGTGTT 600

GTTTTGCTGG GGTTCATCAN NNTGTGGGCC TGCCANAAAG GCAACATTAG GTGCAACATT 660

TGCATTTGAN 670 (2) INFORMATION FOR SEQ ID NO : 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 222 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His

50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Xaa Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe

180 185 190 Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met 195 200 205

Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 918 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..918

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

ATG GGC GCC CAT ATG GGC ATA TTC GGC GCA ATA GCA GGT TTC ATA GAA 288

Met Gly Ala His Met Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu

85 90 95

AAT GGT TGG GAG GGA ATG ATA GAC GGT TGG TAC GGT TTC AGG CAT CAA 336 Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln

100 105 110 AAT TCT GAG GGC ACA GGA CAA GCA GCA GAT CTT AAA AGC ACT CAA GCA 384 Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala

115 120 125

GCC ATC GAC CAA ATC AAT GGG AAA CTG AAT AGG GTA ATC GAG AAG ACG 432 Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr

130 135 140

AAC GAG AAA TTC CAT CAA ATC GAA AAG GAA TTC TCA GAA GTA GAA GGG 480 Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly

145 150 155 160

AGA ATT CAG GAC CTC GAG AAA TAC GTT GAA GAC ACT AAA ATA GAT CTC 528 Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu

165 170 175

TGG TCT TAC AAT GCG GAG CTT CTT GTC GCT CTG GAG AAC CAA CAT ACA 576 Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr

180 185 190

ATT GAT CTG ACT GAC TCG GAA ATG AAC AAA CTG TTT GAA AAA ACA AGG 624 Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg

195 200 205

AGG CAA CTG AGG GAA AAT GCT GAG GAC ATG GGC AAT GGT TGC TTC AAA 672 Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys

210 215 220

ATA TAC CAC AAA TGT GAC AAT GCT TGC ATA GGG TCA ATC AGA AAT GGG 720 Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly

225 230 235 240

ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA GCA TTA AAC AAC CGG TTT 768 Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe

245 250 255

CAG ATC AAA GGT GTT GAA CTG AAG TCA GGA TAC AAA GAC TGG ATC CTG 816 Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu

260 265 270

TGG ATT TCC TTT GCC ATA TCA TGC TTT TTG CTT TGT GTT GTT TTG CTG 864

Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu

275 280 285

GGG TTC ATC ATG TGG GCC TGC CAA AAA GGC AAC ATT AGG TGC AAC ATT 912 Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile

290 295 300

TGC ATT 918 Cys Ile

305 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gly Ala His Met Gly Ile Pne Gly Ala Ile Ala Gly Phe Ile Glu

85 90 95 Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln

100 105 110

Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala

115 120 125

Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr 130 135 140

Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly 145 150 155 160

Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu

165 170 175 Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr

180 185 190

Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg

195 200 205

Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys 210 215 220

Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly 225 230 235 240 Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe

245 250 255 Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu

260 265 270

Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu

275 280 285

Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile

290 295 300

Cys Ile

305

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 690 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..690

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80 ATG GAT CAT ATG TTA ATT CAG GAC CTC GAG AAA TAC GTT GAA GAC ACT 288 Met Asp His Met Leu Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr

85 90 95

AAA ATA GAT CTC TGG TCT TAC AAT GCG GAG CTT CTT GTC GCT CTG GAG 336 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu

100 105 110

AAC CAA CAT ACA ATT GAT CTG ACT GAC TCG GAA ATG AAC AAA CTG TTT 384 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe

115 120 125

GAA AAA ACA AGG AGG CAA CTG AGG GAA AAT GCT GAG GAC ATG GGC AAT 432 Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn

130 135 140

GGT TGC TTC AAA ATA TAC CAC AAA TGT GAC AAT GCT TGC ATA GGG TCA 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser

145 150 155 160

ATC AGA AAT GGG ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA GCA TTA 528 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu

165 170 175

AAC AAC CGG TTT CAG ATC AAA GGT GTT GAA CTG AAG TCA GGA TAC AAA 576 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys

180 185 190

GAC TGG ATC CTG TGG ATT TCC TTT GCC ATA TCA TGC TTT TTG CTT TGT 624 Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys

195 200 205

GTT GTT TTG CTG GGG TTC ATC ATG TGG GCC TGC CAA AAA GGC AAC ATT 672 Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile

210 215 220

AGG TGC AAC ATT TGC ATT 690

Arg Cys Asn Ile Cys Ile

225 230

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 230 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Asp His Met Leu Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr

85 90 95

Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu

100 105 110

Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe

115 120 125

Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 130 135 140

Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser 145 150 155 160 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu

165 170 175

Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys

180 185 190

Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys

195 200 205

Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile

210 215 220

Arg Cys Asn Ile Cys Ile

225 230 (2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 699 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 1..699

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TCC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Ser Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC ATG CAT GGA TCA TAT GTT 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met His Gly Ser Tyr Val

35 40 45

AAC AAG ACA CAA GAA GCT ATA AAC AAG ATA ACA AAA AAT CTC AAC TAT 192 Asn Lys Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Tyr

50 55 60

TTA AGT GAG CTA GAA GTA AAA AAC CTT CAA AGA CTA AGC GGA GCA ATG 240 Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met

65 70 75 80

AAT GAG CTT CAC GAC GAA ATA CTC GAG CTA GAC GAA AAA GTG GAT GAT 288 Asn Glu Leu His Asp Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp

85 90 95

CTA AGA GCT GAT ACA ATA AGC TCA CAA ATA GAG CTT GCA GTC TTG CTT 336

Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu

100 105 110

TCC AAC GAA GGG ATA ATA AAC AGT GAA GAT GAG CAT CTC TTG GCA CTT 384

Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu

115 120 125

GAA AGA AAA CTG AAG AAA ATG CTT GGC CCC TCT GCT GTA GAA ATA GGG 432

Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly

130 135 140

AAT GGG TGC TTT GAA ACC AAA CAC AAA TGC AAC CAG ACT TGC CTA GAC 480

Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp

145 150 155 160

AGG ATA GCT GCT GGC ACC TTT AAT GCA GGA GAT TTT TCT CTT CCC ACT 528

Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Asp Phe Ser Leu Pro Thr

165 170 175

TTT GAT TCA TTA AAC ATT ACT GCT GCA TCT TTA AAT GAT GAT GGC TTG 576 Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu

180 185 190 GAT AAT CAT ACT ATA CTG CTC TAC TAC TCA ACT GCT GCT TCT AGC TTG 624

Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu

195 200 205

GCT GTA ACA TTA ATG ATA GCT ATC TTC ATT GTC TAC ATG GTC TCC AGA 672

Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg

210 215 220

GAC AAT GTT TCT TGT TCC ATC TGT CTG 699 Asp Asn Val Ser Cys Ser Ile Cys Leu

225 230

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 233 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Ser Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met His Gly Ser Tyr Val

35 40 45

Asn Lys Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Tyr

50 55 60

Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met

65 70 75 80

Asn Glu Leu His Asp Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp

85 90 95

Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu

100 105 110

Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu

115 120 125

Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly

130 135 140

Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp

145 150 155 160 Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Asp Phe Ser Leu Pro Thr

165 170 175

Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu

180 185 190

Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu

195 200 205

Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg

210 215 220

Asp Asn Val Ser Cys Ser Ile Cys Leu

225 230

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 924 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..921

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

ATG GAT CTG TCC AGA GGT CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA 288 Met Asp Leu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu

85 90 95 GGG GGA TGG ACT GGA ATG ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG 336

Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln

100 105 110

AAT GAA CAG GGA TCA GGC TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT 384

Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn

115 120 125

GCC ATT AAC GGG ATT ACA AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG 432

Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met

130 135 140

AAC ATT CAA TTC ACA GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA 480

Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys

145 150 155 160

AGG ATG GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT 528

Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile

165 170 175

TGG ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT 576

Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr

180 185 190

CTG GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA 624

Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys

195 200 205

AGC CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG 672

Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu

210 215 220

TTC TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG 720

Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly

225 230 235 240

ACT TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA 768

Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu

245 250 255

AAG GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG 816

Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu

260 265 270

GCG ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG 864

Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu

275 280 285

GGG GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA 912

Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg

290 295 300 ATA TGC ATC TGA 924 Ile Cys Ile

305

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 307 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

Met Asp Leu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu

85 90 95

Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln

100 105 110

Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn

115 120 125

Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met

130 135 140

Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys

145 150 155 160

Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile

165 170 175

Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr

180 185 190 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys

195 200 205

Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu

210 215 220

Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly

225 230 235 240

Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu

245 250 255

Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu

260 265 270

Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu

275 280 285

Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg

290 295 300

Ile Cys Ile

305

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 729 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..726

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45 ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

ATG CAG ATC CCG GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG 288 Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg

85 90 95

ATG GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG 336 Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

100 105 110

ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG 384

Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

115 120 125

GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC 432

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser

130 135 140

CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC 480 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe

145 150 155 160

TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT 528 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr

165 170 175

TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG 576 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

180 185 190

GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG 624 Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala

195 200 205

ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG 672 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly

210 215 220

GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA 720 Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

225 230 235 240

TGC ATC TGA 729 Cys Ile (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 242 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg

85 90 95

Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

100 105 110

Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

115 120 125

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser 130 135 140

Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 145 150 155 160

Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr

165 170 175

Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

180 185 190

Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala

195 200 205

Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly

210 215 220 Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

225 230 235 240

Cys Ile

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 810 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..807

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC ATG GAT CTG TCC AGA GGT 144

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp Leu Ser Arg Gly

35 40 45

CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA GGG GGA TGG ACT GGA ATG 192

Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met

50 55 60

ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG AAT GAA CAG GGA TCA GGC 240 Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly

65 70 75 80

TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT GCC ATT AAC GGG ATT ACA 288

Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr

85 90 95

AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG AAC ATT CAA TTC ACA GCT 336

Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala

100 105 110

GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA AAT 384 Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn

115 120 125 AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA GAA 432

Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu

130 135 140

TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC TCA 480

Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser

145 150 155 160

AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT AAT 528 Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn

165 170 175

GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT GAC 576 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp

180 185 190

AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC AAA 624 Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys

195 200 205

TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG AAA 672

Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys

210 215 220

TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT GTC 720

Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val

225 230 235 240

GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC TGG 768 Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp

245 250 255

ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC TGA 810 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

260 265

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 269 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp Leu Ser Arg Gly 35 40 45

Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 50 55 60

Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 65 70 75 80 Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr

85 90 95

Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala

100 105 110

Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn

115 120 125

Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 130 135 140

Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser

145 150 155 160 Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn

165 170 175

Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp

180 185 190

Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys

195 200 205

Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys 210 215 220

Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val

225 230 235 240 Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp

245 250 255

Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

260 265

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 630 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 1..627

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC ATG GAT CAT ATG TTA ACA 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp His Met Leu Thr

35 40 45

AGT ACT CGA TCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG 192 Ser Thr Arg Ser Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met

50 55 60

GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA 240 Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr

65 70 75 80

TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT 288 Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp

85 90 95

TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA 336 Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln

100 105 110

TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC 384

Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr

115 120 125

CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT 432

His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr

130 135 140

GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA 480

Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val

145 150 155 160

GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC 528 Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile

165 170 175

TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA 576 Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala

180 185 190 ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC 624 Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys

195 200 205

ATC TGA 630 Ile

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 209 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Met Asp His Met Leu Thr

35 40 45

Ser Thr Arg Ser Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met

50 55 60

Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr

65 70 75 80

Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp

85 90 95

Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln

100 105 110

Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr

115 120 125

His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr

130 135 140

Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val

145 150 155 160

Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile

165 170 175 Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala

180 185 190

Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys

195 200 205

Ile (2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 717 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..714

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

ATG CAG ATC CCG GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA 288

Met Gln Ile Pro Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu

85 90 95

AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA 336 Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala

100 105 110 GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC 384

Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp

115 120 125

TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT 432

Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn

130 135 140

AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT 480 Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys

145 150 155 160

GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC 528 Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro

165 170 175

AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG 576 Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val

180 185 190

AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT 624

Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr

195 200 205

GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC 672

Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe

210 215 220

TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC 714

Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

225 230 235

TGA 717

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 238 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gln Ile Pro Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu

85 90 95

Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala

100 105 110

Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp

115 120 125

Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn 130 135 140

Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys 145 150 155 160

Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro

165 170 175

Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val

180 185 190

Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr

195 200 205

Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe

210 215 220

Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

225 230 235 (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 681 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..678 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

ATG CAG ATC CCG AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG 288 Met Gln Ile Pro Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

85 90 95

ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG 336 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

100 105 110

GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC 384 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser

115 120 125

CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC 432 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe

130 135 140

TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT 480 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr

145 150 155 160

TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG 528 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

165 170 175

GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG 576 Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala

180 185 190

ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG 624 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly

195 200 205 GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA 672 Ala Ile Se r Phe Trp Met Cys Se r Asn Gly Ser Leu Gln Cys Arg Ile

210 215 220

TGC ATC TGA 681 Cys Ile

225 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 226 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 26 :

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

Met Gln Ile Pro Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

85 90 95

Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

100 105 110

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser

115 120 125

Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe

130 135 140

Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr

145 150 155 160

Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

165 170 175 Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala 180 185 190 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly

195 200 205

Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

210 215 220

Cys Ile

225

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 158 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gln Ile Pro Val Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro

85 90 95

Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val

100 105 110

Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr

115 120 125

Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe 130 135 140

Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

145 150 155 (2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 163 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Asp Leu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu

85 90 95

Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln

100 105 110

Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn

115 120 125

Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met 130 135 140

Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Ser Cys Leu Thr Ala 145 150 155 160

Tyr His Arg

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 231 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg

85 90 95

Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

100 105 110

Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

115 120 125

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser 130 135 140

Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 145 150 155 160

Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr

165 170 175

Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

180 185 190

Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala

195 200 205

Ile Tyr Ser Thr Val Ala Ser Ser Gly Gly Ser Tyr Ser Met Glu His 210 215 220

Phe Arg Trp Gly Lys Pro Val

225 230 (2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 225 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gln Ile Pro Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg

85 90 95

Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

100 105 110

Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

115 120 125

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser

130 135 140

Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe

145 150 155 160

Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr

165 170 175

Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

180 185 190

Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala

195 200 205 Ile Tyr Ser Thr Val Ala Ser Ser Gly Gly Ser Tyr Ser Met Leu Val

210 215 220 Asn

225

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 912 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..912

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

ATG CAG ATC CCG GGT CTA TTT GGA GCC ATT GCC GGT TTT ATT GAA GGG 288

Met Gln Ile Pro Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly

85 90 95

GGA TGG ACT GGA ATG ATA GAT GGA TGG TAC GGT TAT CAT CAT CAG AAT 336

Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn

100 105 110

GAA CAG GGA TCA GGC TAT GCA GCG GAT CAA AAA AGC ACA CAA AAT GCC 384 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala

115 120 125 ATT AAC GGG ATT ACA AAC AAG GTG AAC TCT GTT ATC GAG AAA ATG AAC 432 lie Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn

130 135 140

ATT CAA TTC ACA GCT GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG 480 Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg

145 150 155 160

ATG GAA AAT TTA AAT AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG 528 Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

165 170 175

ACA TAT AAT GCA GAA TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG 576 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

180 185 190

GAT TTC CAT GAC TCA AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC 624 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser

195 200 205

CAA TTA AAG AAT AAT GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC 672 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe

210 215 220

TAC CAC AAG TGT GAC AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT 720 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr

225 230 235 240

TAT GAT TAT CCC AAA TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG 768 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

245 250 255

GTA GAT GGA GTG AAA TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG 816 Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala

260 265 270

ATC TAC TCA ACT GTC GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG 864 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly

275 280 285

GCA ATC AGT TTC TGG ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA 912 Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

290 295 300

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 304 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gln Ile Pro Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly

85 90 95

Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln Asn

100 105 110

Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala

115 120 125

Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 130 135 140

Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg 145 150 155 160

Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp

165 170 175

Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu

180 185 190

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser

195 200 205

Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe

210 215 220

Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr 225 230 235 240

Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys

245 250 255

Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala

260 265 270 Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly

275 280 285

Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

290 295 300

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 474 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..471

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

GTG GGT AAA GAA TTC AAC AAA TTA GAA AAA AGG ATG GAA AAT TTA AAT 48

Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn

1 5 10 15

AAA AAA GTT GAT GAT GGA TTT CTG GAC ATT TGG ACA TAT AAT GCA GAA 96

Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu

20 25 30

TTG TTA GTT CTA CTG GAA AAT GAA AGG ACT CTG GAT TTC CAT GAC TCA 144

Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser

35 40 45

AAT GTG AAG AAT CTG TAT GAG AAA GTA AAA AGC CAA TTA AAG AAT AAT 192

Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn

50 55 60

GCC AAA GAA ATC GGA AAT GGA TGT TTT GAG TTC TAC CAC AAG TGT GAC 240

Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp

65 70 75 80

AAT GAA TGC ATG GAA AGT GTA AGA AAT GGG ACT TAT GAT TAT CCC AAA 288

Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys

85 90 95

TAT TCA GAA GAG TCA AAG TTG AAC AGG GAA AAG GTA GAT GGA GTG AAA 336

Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys

100 105 110 TTG GAA TCA ATG GGG ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT GTC 384 Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val

115 120 125

GCC AGT TCA CTG GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC TGG 432 Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp

130 135 140

ATG TGT TCT AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC TGA 474 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

145 150 155

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 157 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn

1 5 10 15

Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu

20 25 30

Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser

35 40 45

Asn Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn

50 55 60

Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp

65 70 75 80

Asn Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys

85 90 95

Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys

100 105 110

Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val

115 120 125

Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp

130 135 140

Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile

145 150 155 (2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

CATGGATCAT ATGTTAACAG ATATCAAGGC CTGACTGACT GAGAGCT 47

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

CTCAGTCAGT CAGGCCTTGA TATCTGTTAA CATATGATC 39

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

CATGGGCGCC CATATGGGCA TATTCGGCG 29 (2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

CCGAATATGC CCATATGGGC GCC 23

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

CATGGATCAT ATGTTAACAA GTACTCGATA TCAATGAGTG ACTGAAGCT 49

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

TCAGTCACTC ATTGATATCG AGTACTTGTT AACATATGAT C 41 (2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

AATTCGTACC TA 12

(2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

GATCTAGGTA CG 12

(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

AAACTGTTTG AAAAAACACG TCGTCAACTG CGTGAAAATG CTGACGACAT GGGC 54

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

TGTGACAATG CTTGCATCGG TTCAATCCGT AATGGTACTT ATGACCATGA TG 52

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

GATCCCGGGT GACTGACTGA 20

(2) INFORMATION FOR SEQ ID NO : 46 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

GATCTCAGTC AGTCACCCGG 20

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

TAAGGAGGAT ATAACATATG 20 (2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

GATCCATATG TTATATCCTC CTTAAGGT 28

(2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 49 :

GCATCGCCAT GAGTCACGAC G 21

(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

GGAGGATGGG AAGGACTCAT TGCAGGTTGG 30 (2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

CTCTGCTGTA GAAATCGGTA ACGGTTGCTT TGAAACCAAA C 41

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

GGTTTCTTGG AAGGTGGTTG GGAAGGTCTC ATTGCAGGTT GGCACGG 47

(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

GCTTTCCAAC GAAGGTATCA ATCAACAGTG AAGACGAGCA TCTCTTGG 48

(2) INFORMATION FOR SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7616 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) ( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 1879..2790

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

AATTCTCATG TTTGACAGCT TATCATCGAT AAGCTTCAGT TGAAGATATT AAGAACAGCC 60 TCGCAGATGA CGAATCATTG GGATTCCCAT CTTTTTTGTT TGTTGAAGGC GACACCATTG 120

GTTTTGCCAG AACTGTTTTC GGGCCGACCA CATCCGATCT GACAGATTTT TTAATCGGGA 180

AAGGAATGTC ATTAAGCAGT GGAGAGCGCG TTCAGATAGA GCCACTGATG AGGGGAACCA 240

CCAAAGACGA TGTTATGCAT ATGCATTTCA TCGGCCGAAC AACGGTGAAG GTAGAAGCCA 300

AGCTACCTGT ATTTGGCGAT ATATTAAAGG TCTTAGGGGC AACAGATATT GAAGGGGAGC 360 TTTTTGACTC ATTGGATATA GTCATTAAGC CAAAATTTAA AAGGGATATA AAAAAGGTTG 420

CCAAGGATAT TATTTTTAAC CCGTCACCTC AATTTTCAGA CATTAGCCTG CGGGCAAAAG 480

ATGAGGCCGG AGATATTTTA ACAGAACATT ATCTATCAGA AAAAGGCCAT CTCTCAGCGC 540

CTCTGAACAA GGTCACCAAT GCTGAGATAG CTGAAGAGAT GGCATATTGC TACGCAAGAA 600

TGAAAAGTGA TATACTGGAA TGTTTTAAAA GGCAGGTGGG CAAAGTTAAG GATTAATTAT 660 CAGGAGTAAT TATGCGGAAC AGAATCATGC CTGGTGTTTA CATAGTAATA ATTCCTTACG 720

TTATCGTAAG CATTTGCTAT CTCCTTTTCC GCCACTACAT TCCTGGTGTT TCTTTTTCAG 780

CTCATAGAGA TGGTCTTGGG GCGACATTGT CATCATATGC AGGAACCATG ATTGCAATCC 840

TGATTGCTGC CTTGACGTTT CTAATCGGAA GCAGAACGCG CCGACTGGCC AAGATTAGAG 900

AGTATGGGTA TATGACATCG GTAGTTATTG TCTATGCCCT TAGTTTTGTT GAGCTTGGAG 960 CTTTGTTTTT CTGCGGGTTA TTGCTTCTTT CCAGCATAAG CGGCTACATG ATACCCACTA 1020

TCGCCATCGG CATTGCCTCT GCATCGTTCA TTCATATATG CATCCTTGTT TTCCAACTAT 1080

ATAATTTGAC CAGAGAACAA GAATAACCCG GCCTCAGCGC CGGGTTTTCT TTGCCTCACG 1140

ATCGCCCCCA AAACACATAA CCAATTGTAT TTATTGAAAA ATAAATAGAT ACAACTCACT 1200

AAACATAGCA ATTCAGATCT CTCACCTACC AAACAATGCC CCCCTGCAAA AAATAAATTC 1260 ATATAAAAAA CATACAGATA ACCATCTGCG GTGATAAATT ATCTCTGGCG GTGTTGACAT 1320

AAATACCACT GGCGGTGATA CTGAGCACAT CAGCAGGACG CACTGACCAC CATGAAGGTG 1380

ACGCTCTTAA AAATTAAGCC CTGAAGAAGG GCAGCATTCA AAGCAGAAGG CTTTGGGGTG 1440 TGTGATACGA AACGAAGCAT TGGCCGTAAG TGCGATTCCG GATTAGCTGC CAATGTGCCA 1500

ATCGCGGGGG GTTTTCGTTC AGGACTACAA CTGCCACACA CCACCAAAGC TAACTGACAG 1560

GAGAATCCAG ATGGATGCAC AAACACGCCG CCGCGAACGT CGCGCAGAGA AACAGGCTCA 1620

ATGGAAAGCA GCAAATCCCC TGTTGGTTGG GGTAAGCGCA AAACCAGTTC CGAAAGATTT 1680

TTTTAACTAT AAACGCTGAT GGAAGCGTTT ATGCGGAAGA GGTAAAGCCC TTCCCGAGTA 1740

ACAAAAAAAC AACAGCATAA ATAACCCCGC TCTTACACAT TCCAGCCCTG AAAAAGGGCA 1800

TCAAATTAAA CCACACCTAT GGTGTATGCA TTTATTTGCA TACATTCAAT CAATTGTTAT 1860 CTAAGGAAAT ACTTACAT ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA 1911

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val

1 5 10

GAT TGC TTT CTT TGG CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA 1959 Asp Cys Phe Leu Trp His Val Arg Lys Arg Val Ala Asp Gln Glu Leu

15 20 25

GGT GAT GCC CCA TTC CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA 2007 Gly Asp Ala Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu

30 35 40

AGA GGA AGG GGC AGC ACC CTC GGT CTG GAC ATC GAG ACA GCC ACA CGT 2055 Arg Gly Arg Gly Ser Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg

45 50 55

GCT GGA AAG CAG ATA GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG 2103 Ala Gly Lys Gln Ile Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu

60 65 70 75

GCA CTT AAA ATG ACC ATG GGT TTC TTC GGA GCT ATT GCT GGT TTC TTG 2151 Ala Leu Lys Met Thr Met Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu

80 85 90

GAA GGT GGT TGG GAA GGT CTC ATT GCA GGT TGG CAC GGA TAC ACA TCT 2199 Glu Gly Gly Trp Glu Gly Leu Ile Ala Gly Trp His Gly Tyr Thr Ser

95 100 105

CAT GGA GCA CAT GGA GTG GCA GTG GCA GCA GAC CTT AAG AGT ACA CAA 2247 His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln

110 115 120

GAA GCT ATA AAC AAG ATA ACA AAA AAT CTC AAC TAT TTA AGT GAG CTA 2295 Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Tyr Leu Ser Glu Leu

125 130 135

GAA GTA AAA AAC CTT CAA AGA CTA AGC GGA GCA ATG AAT GAG CTT CAC 2343 Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asn Glu Leu His

140 145 150 155 GAC GAA ATA CTC GAG CTA GAC GAA AAA GTG GAT GAT CTA AGA GCT GAT 2391 Asp Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp

160 165 170

ACA ATA AGC TCA CAA ATA GAG CTT GCA GTC TTG CTT TCC AAC GAA GGT 2439 Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly

175 180 185

ATC ATC AAC AGT GAA GAC GAG CAT CTC TTG GCA CTT GAA AGA AAA CTG 2487 Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu

190 195 200

AAG AAA ATG CTT GGC CCC TCT GCT GTA GAA ATC GGT AAC GGT TGC TTT 2535 Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn Gly Cys Phe

205 210 215

GAA ACC AAA CAC AAA TGC AAC CAG ACT TGC CTA GAC AGG ATA GCT GCT 2583

Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala Ala

220 225 230 235

GGC ACC TTT AAT GCA GGA GAT TTT TCT CTT CCC ACT TTT GAT TCA TTA 2631

Gly Thr Phe Asn Ala Gly Asp Phe Ser Leu Pro Thr Phe Asp Ser Leu

240 245 250

AAC ATT ACT GCT GCA TCT TTA AAT GAT GAT GGC TTG GAT AAT CAT ACT 2679 Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr

255 260 265

ATA CTG CTC TAC TAC TCA ACT GCT GCT TCT AGC TTG GCT GTA ACA TTA 2727 Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu

270 275 280

ATG ATA GCT ATC TTC ATT GTC TAC ATG GTC TCC AGA GAC AAT GTT TCT 2775 Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser

285 290 295

TGT TCC ATC TGT CTG TGAGGGAGAT TAAGCCCTGT GTTTTCCTTT ACTGTAGTGC 2830 Cys Ser Ile Cys Leu

300

TCATTTGCTT GTCACCATTA CAAAGAAACG TTATTGAAAA ATGCTCTTGT TACTACTGAA 2890

TTCTAGAATC GATAAGCTTC GACCGATGCC CTTGAGAGCC TTCAACCCAG TCAGCTCCTT 2950 CCGGTGGGCG CGGGGCATGA CTATCGTCGC CGCACTTATG ACTGTCTTCT TTATCATGCA 3010

ACTCGTAGGA CAGGTGCCGG CAGCGCTCTG GGTCATTTTC GGCGAGGACC GCTTTCGCTG 3070

GAGCGCGACG ATGATCGGCC TGTCGCTTGC GGTATTCGGA ATCTTGCACG CCCTCGCTCA 3130

AGCCTTCGTC ACTGGTCCCG CCACCAAACG TTTCGGCGAG AAGCAGGCCA TTATCGCCGG 3190

CATGGCGGCC GACGCGCTGG GCTACGTCTT GCTGGCGTTC GTCCAGTAAT GACCTCAGAA 3250 CTCCATCTGG ATTTGTTCAG AACGCTCGGT TGCCGCCGGG CGTTTTTTAT TGGTGAGAAT 3310 CGCAGCAACT TGTCGCGCCA ATCGAGCCAT GTCGTCGTCA ACGACCCCCC ATTCAAGAAC 3370

AGCAAGCAGC ATTGAGAACT TTGGAATCCA GTCCCTCTTC CACCTGCTGA GACGCGAGGC 3430

TGGATGGCCT TCCCCATTAT GATTCTTCTC GCTTCCGGCG GCATCGGGAT GCCCGCGTTG 3490

CAGGCCATGC TGTCCAGGCA GGTAGATGAC GACCATCAGG GACAGCTTCA AGGATCGCTC 3550 GCGGCTCTTA CCAGCCTAAC TTCGATCACT GGACCGCTGA TCGTCACGGC GATTTATGCC 3610

GCCTCGGCGA GCACATGGAA CGGGTTGGCA TGGATTGTAG GCGCCGCCCT ATACCTTGTC 3670

TGCCTCCCCG CGTTGCGTCG CGGTGCATGG AGCCGGGCCA CCTCGACCTG AATGGAAGCC 3730

GGCGGCACCT CGCTAACGGA TTCACCACTC CAAGAATTGG AGCCAATCAA TTCTTGCGGA 3790

GAACTGTGAA TGCGCAAACC AACCCTTGGC AGAACATATC CATCGCGTCC GCCATCTCCA 3850 GCAGCCGCAC GCGGCGCATC TCGGGCAGCG TTGGGTCCTG GCCACGGGTG CGCATGATCG 3910

TGCTCCTGTC GTTGAGGACC CGGCTAGGCT GGCGGGGTTG CCTTACTGGT TAGCAGAATG 3970

AATCACCGAT ACGCGAGCGA ACGTGAAGCG ACTGCTGCTG CAAAACGTCT GCGACCTGAG 4030

CAACAACATG AATGGTCTTC GGTTTCCGTG TTTCGTAAAG TCTGGAAACG CGGAAGTCAG 4090

CGCCCTGCAC CATTATGTTC CGGATCTGCA TCGCAGGATG CTGCTGGCTA CCCTGTGGAA 4150 CACCTACATC TGTATTAACG AAGCGCTGGC ATTGACCCTG AGTGATTTTT CTCTGGTCCC 4210

GCCGCATCCA TACCGCCAGT TGTTTACCCT CACAACGTTC CAGTAACCGG GCATGTTCAT 4270

CATCAGTAAC CCGTATCGTG AGCATCCTCT CTCGTTTCAT CGGTATCATT ACCCCCATGA 4330

ACAGAAATTC CCCCTTACAC GGAGGCATCA AGTGACCAAA CAGGAAAAAA CCGCCCTTAA 4390

CATGGCCCGC TTTATCAGAA GCCAGACATT AACGCTTCTG GAGAAACTCA ACGAGCTGGA 4450 CGCGGATGAA CAGGCAGACA TCTGTGAATC GCTTCACGAC CACGCTGATG AGCTTTACCG 4510

CAGCTGCCTC GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC AGCTCCCGGA 4570

GACGGTCACA GCTTGTCTGT AAGCGGATGC CGGGAGCAGA CAAGCCCGTC AGGGCGCGTC 4630

AGCGGGTGTT GGCGGGTGTC GGGGCGCAGC CATGACCCAG TCACGTAGCG ATAGCGGAGT 4690

GTATACTGGC TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG 4750 TGTGAAATAC CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCTC TTCCGCTTCC 4810

TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA 4870

AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA 4930 AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG 4990

CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG 5050

ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT 5110

CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT 5170

TCTCAATGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC 5230

TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT 5290

GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT 5350 AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC 5410

TACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA 5470

AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT 5530

TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT 5590

ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA 5650 TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA 5710

AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC 5770

TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT 5830

ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC 5890

TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT 5950 GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA 6010

AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTGCAGG CATCGTGGTG 6070

TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT 6130

ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC 6190

AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT 6250 ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTAGCTTCA CGCTGCCGCA 6310

AGCACTCAGG GCGCAAGGGC TGCTAAAGGA AGCGGAACAC GTAGAAAGCC AGTCCGCAGA 6370

AACGGTGCTG ACCCCGGATG AATGTCAGCT ACTGGGCTAT CTGGACAAGG GAAAACGCAA 6430

GCGCAAAGAG AAAGCAGGTA GCTTGCAGTG GGCTTACATG GCGATAGCTA GACTGGGCGG 6490

TTTTATGGAC AGCAAGCGAA CCGGAATTGC CAGCTGGGGC GCCCTCTGGT AAGGTTGGGA 6550 AGCCCTGCAA AGTAAACTGG ATGGCTTTCT TGCCGCCAAG GATCTGATGG CGCAGGGGAT 6610 CAAGATCTGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT GATTGAACAA GATGGATTGC 6670

ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA 6730

CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT 6790

TTGTCAAGAC CGACCTGTCC GGTGCCCTGA ATGAACTGCA GGACGAGGCA GCGCGGCTAT 6850 CGTGGCTGGC CACGACGGGC GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG 6910

GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG 6970

CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC 7030

CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT CGAGCGAGCA CGTACTCGGA 7090

TGGAAGCCGG TCTTGTCGAT CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG 7150 CCGAACTGTT CGCCAGGCTC AAGGCGCGCA TGCCCGACGG CGAGGATCTC GTCGTGACTC 7210

ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG 7270

ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA 7330

TTGCTGAAGA GCTTGGCGGC GAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG 7390

CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC 7450 TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCAACCTG CCATCACGAG ATTTCGATTC 7510

CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT TTCCGGGACG CCGGCTGGAT 7570

GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC CACCCC 7616

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 304 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80 Met Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu

85 90 95

Gly Leu Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly

100 105 110

Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys

115 120 125

Ile Thr Lys Asn Leu Asn Tyr Leu Ser Glu Leu Glu Val Lys Asn Leu 130 135 140

Gln Arg Leu Ser Gly Ala Met Asn Glu Leu His Asp Glu Ile Leu Glu

145 150 155 160 Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln

165 170 175 Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu

180 185 190

Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly

195 200 205

Pro Ser Ala Val Glu Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys 210 215 220

Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala

225 230 235 240 Gly Asp Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala

245 250 255

Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr

260 265 270

Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe

275 280 285

Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu 290 295 300 (2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 915 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..912

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACC CTC GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

ATG GGT TTC TTC GGA GCT ATT GCT GGT TTC TTG GAA GGA GGA TGG GAA 288

Met Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu

85 90 95

GGA ATG ATT GCA GGT TGG CAC GGA TAC ACA TCT CAT GGA GCA CAT GGA 336

Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly

100 105 110

GTG GCA GTG GCA GCA GAC CTT AAG AGT ACA CAA GAA GCT ATA AAC AAG 384

Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys

115 120 125

ATA ACA AAA AAT CTC AAC TAT TTA AGT GAG CTA GAA GTA AAA AAC CTT 432 Ile Thr Lys Asn Leu Asn Tyr Leu Ser Glu Leu Glu Val Lys Asn Leu

130 135 140 CAA AGA CTA AGC GGA GCA ATG AAT GAG CTT CAC GAC GAA ATA CTC GAG 480 Gln Arg Leu Ser Gly Ala Met Asn Glu Leu His Asp Glu Ile Leu Glu

145 150 155 160

CTA GAC GAA AAA GTG GAT GAT CTA AGA GCT GAT ACA ATA AGC TCA CAA 528 Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln

165 170 175

ATA GAG CTT GCA GTC TTG CTT TCC AAC GAA GGG ATA ATA AAC AGT GAA 576 Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu

180 185 190

GAT GAG CAT CTC TTG GCA CTT GAA AGA AAA CTG AAG AAA ATG CTT GGC 624 Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly

195 200 205

CCC TCT GCT GTA GAA ATA GGG AAT GGG TGC TTT GAA ACC AAA CAC AAA 672

Pro Ser Ala Val Glu Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys

210 215 220

TGC AAC CAG ACT TGC CTA GAC AGG ATA GCT GCT GGC ACC TTT AAT GCA 720

Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala

225 230 235 240

GGA GAT TTT TCT CTT CCC ACT TTT GAT TCA TTA AAC ATT ACT GCT GCA 768 Gly Asp Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala

245 250 255

TCT TTA AAT GAT GAT GGC TTG GAT AAT CAT ACT ATA CTG CTC TAC TAC 816 Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr

260 265 270

TCA ACT GCT GCT TCT AGC TTG GCT GTA ACA TTA ATG ATA GCT ATC TTC 864 Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe

275 280 285

ATT GTC TAC ATG GTC TCC AGA GAC AAT GTT TCT TGT TCC ATC TGT CTG 912 Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu

290 295 300

TGA 915

(2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 304 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu

85 90 95

Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His Gly Ala His Gly

100 105 110

Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu Ala Ile Asn Lys

115 120 125

Ile Thr Lys Asn Leu Asn Tyr Leu Ser Glu Leu Glu Val Lys Asn Leu 130 135 140

Gln Arg Leu Ser Gly Ala Met Asn Glu Leu His Asp Glu Ile Leu Glu 145 150 155 160

Leu Asp Glu Lys Val Asp Asp Leu Arg Ala Asp Thr Ile Ser Ser Gln

165 170 175 Ile Glu Leu Ala Val Leu Leu Ser Asn Glu Gly Ile Ile Asn Ser Glu

180 185 190

Asp Glu His Leu Leu Ala Leu Glu Arg Lys Leu Lys Lys Met Leu Gly

195 200 205

Pro Ser Ala Val Glu Ile Gly Asn Gly Cys Phe Glu Thr Lys His Lys 210 215 220

Cys Asn Gln Thr Cys Leu Asp Arg Ile Ala Ala Gly Thr Phe Asn Ala 225 230 235 240

Gly Asp Phe Ser Leu Pro Thr Phe Asp Ser Leu Asn Ile Thr Ala Ala

245 250 255

Ser Leu Asn Asp Asp Gly Leu Asp Asn His Thr Ile Leu Leu Tyr Tyr

260 265 270

Ser Thr Ala Ala Ser Ser Leu Ala Val Thr Leu Met Ile Ala Ile Phe

275 280 285 Ile Val Tyr Met Val Ser Arg Asp Asn Val Ser Cys Ser Ile Cys Leu

290 295 300

(2) INFORMATION FOR SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 918 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

ATGGATCCAA ACACTGTGTC AAGCTTTCAG GTAGATTGCT TTCTTTGGCA TGTCCGCAAA 60

CGAGTTGCAG ACCAAGAACT AGGTGATGCC CCATTCCTTG ATCGGCTTCG CCGAGATCAG 120

AAATCCCTAA GAGGAAGGGG CAGCACTCTT GGTCTGGACA TCGAGACAGC CACACGTGCT 180

GGAAAGCAGA TAGTGGAGCG GATTCTGAAA GAAGAATCCG ATGAGGCACT TAAAATGACC 240

ATGGGCGCCC ATATGGGCAT ATTCGGCGCA ATAGCAGGTT TCATAGAAAA TGGTTGGGAG 300 GGAATGATAG ACGGTTGGTA CGGTTTCAGG CATCAAAATT CTGAGGGCAC AGGACAAGCA 360

GCAGATCTTA AAAGCACTCA AGCAGCCATC GACCAAATCA ATGGGAAACT GAATAGGGTA 420

ATCGAGAAGA CGAACGAGAA ATTCCATCAA ATCGAAAAGG AATTCTCAGA AGTAGAAGGG 480

AGAATTCAGG ACCTCGAGAA ATACGTTGAA GACACTAAAA TAGATCTCTG GTCTTACAAT 540

GCGGAGCTTC TTGTCGCTCT GGAGAACCAA CATACAATTG ATCTGACTGA CTCGGAAATG 600 AACAAACTGT TTGAAAAAAC ACGTCGTCAA CTGCGTGAAA ATGCTGAGGA CATGGGCAAT 660

GGTTGCTTCA AAATATACCA CAAATGTGAC AATGCTTGCA TAGGGTCAAT CAGAAATGGG 720

ACTTATGACC ATGATGTATA CAGAGACGAA GCATTAAACA ACCGGTTTCA GATCAAAGGT 780

GTTGAACTGA AGTCAGGATA CAAAGACTGG ATCCTGTGGA TTTCCTTTGC CATATCATGC 840

TTTTTGCTTT GTGTTGTTTT GCTGGGGTTC ATCATGTGGG CCTGCCAAAA AGGCAACATT 900 AGGTGCAACA TTTGCATT 918 (2) INFORMATION FOR SEQ ID NO: 59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 221 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 65 70 75 80

Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys

130 135 140

Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160

Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205

Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220 (2) INFORMATION FOR SEQ ID NO: 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 221 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:

Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15

Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr

20 25 30

Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile

35 40 45

Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His 50 55 60

Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 65 70 75 80 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala

85 90 95

Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp

100 105 110

Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu

115 120 125

Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys 130 135 140

Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp

145 150 155 160 Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val

165 170 175

Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala

180 185 190

Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly Phe Ile Met Trp

195 200 205 Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile

210 215 220

(2) INFORMATION FOR SEQ ID NO: 61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

Arg Arg Xaa Xaa Arg

1 5

(2) INFORMATION FOR SEQ ID NO: 62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

CGNCGNNNNN NNCGN 15

(2) INFORMATION FOR SEQ ID NO: 63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:

AGRAGRNNNN NNAGR 15 (2) INFORMATION FOR SEQ ID NO: 64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

CATGGATCAT ATGTTAACAG ATATCAAGGC CTGACTGACT GAGAGCT 47

(2) INFORMATION FOR SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:

CTCAGTCAGT CAGGCCTTGA TATCTGTTAA CATATGATC 39

(2) INFORMATION FOR SEQ ID NO: 66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

CATGGGCGCC CATATGGGCA TATTCGGCG 29 (2) INFORMATION FOR SEQ ID NO: 67:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:

CCGAATATGC CCATATGGGC GCC 23

(2) INFORMATION FOR SEQ ID NO: 68:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

AAACTGTTTG AAAAAACACG TCGTCAACTG CGTGAAAATG CTGACGACAT GGGC 54

(2) INFORMATION FOR SEQ ID NO: 69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 145 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:

Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp 1 5 10 15

Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile

20 25 30

Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg

35 40 45 Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile 50 55 60

Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr

65 70 75 80

Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln

85 90 95 Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp

100 105 110

Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly

115 120 125

Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys 130 135 140

Ile

145

(2) INFORMATION FOR SEQ ID NO: 70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 145 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:

Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp 1 5 10 15 Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile

20 25 30

Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg

35 40 45 Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile 50 55 60

Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr 65 70 75 80

Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln

85 90 95 Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp 100 105 110

Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val Val Leu Leu Gly

115 120 125

Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys

130 135 140

Ile

145

(2) INFORMATION FOR SEQ ID NO: 71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 690 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..690

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71

ATG GAT CCA AAC ACT GTG TCA AGC TTT CAG GTA GAT TGC TTT CTT TGG 48 Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

CAT GTC CGC AAA CGA GTT GCA GAC CAA GAA CTA GGT GAT GCC CCA TTC 96 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

CTT GAT CGG CTT CGC CGA GAT CAG AAA TCC CTA AGA GGA AGG GGC AGC 144

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

ACT CTT GGT CTG GAC ATC GAG ACA GCC ACA CGT GCT GGA AAG CAG ATA 192

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

GTG GAG CGG ATT CTG AAA GAA GAA TCC GAT GAG GCA CTT AAA ATG ACC 240 Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80 ATG GAT CAT ATG TTA ATT CAG GAC CTC GAG AAA TAC GTT GAA GAC ACT 288

Met Asp His Met Leu Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr

85 90 95

AAA ATA GAT CTC TGG TCT TAC AAT GCG GAG CTT CTT GTC GCT CTG GAG 336

Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu

100 105 110 AAC CAA CAT ACA ATT GAT CTG ACT GAC TCG GAA ATG AAC AAA CTG TTT 384 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe

115 120 125 GAA AAA ACA AGG AGG CAA CTG AGG GAA AAT GCT GAG GAC ATG GGC AAT 432 Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn

130 135 140 GGT TGC TTC AAA ATA TAC CAC AAA TGT GAC AAT GCT TGC ATA GGG TCA 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser

145 150 155 160 ATC AGA AAT GGG ACT TAT GAC CAT GAT GTA TAC AGA GAC GAA GCA TTA 528 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu

165 170 175 AAC AAC CGG TTT CAG ATC AAA GGT GTT GAA CTG AAG TCA GGA TAC AAA 576 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys

180 185 190 GAC TGG ATC CTG TGG ATT TCC TTT GCC ATA TCA TGC TTT TTG CTT TGT 624 Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys

195 200 205 GTT GTT TTG CTG GGG TTC ATC ATG TGG GCC TGC CAA AAA GGC AAC ATT 672 Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile

210 215 220 AGG TGC AAC ATT TGC ATT 690

Arg Cys Asn Ile Cys Ile

225 230 (2 ) INFORMATION FOR SEQ ID NO : 72 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 230 amino acids

(B) TYPE : amino acid

(D ) TOPOLOGY : linear

( ii ) MOLECULE TYPE : protein

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 72 :

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80

Met Asp His Met Leu Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr

85 90 95 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu

100 105 110

Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe

115 120 125

Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 130 135 140

Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser 145 150 155 160 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu

165 170 175 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys

180 185 190

Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys

195 200 205

Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile 210 215 220 Arg Cys Asn Ile Cys Ile 225 230

Claims

WHAT IS CLAIMED IS:

1. A vaccine for stimulating protection in animals against infection by influenza virus which comprises an effective amount of an

immunogenic fragment of the HA2 subunit of an HA protein selected from the group consisting of a Type A subtype influenza virus and a Type B influenza virus.

2. The vaccine according to claim 1 wherein said Type A subunit is H3N2.

3. The vaccine according to claim 1 wherein the polypeptide is fused to a second polypeptide.

4. The vaccine according to claim 3 wherein the second polypeptide comprises the N terminal amino acids of influenza NS1 protein.

5. The vaccine according to claim 1 wherein the immunogenic fragment of the HA2 subunit is selected from the group consisting of a peptide comprising amino acids 1 to 221 of the H3HA2 subtype, a peptide comprising amino acids 77 to 221 of the H3HA2 subtype, a peptide comprising amino acids 1 to 223 of the BHA2 Type, and a peptide comprising amino acids 41 to 223 of the BHA2 type.

6. The vaccine according to claim 5 comprising NS1 _{( 1 - 81)}H3HA2_{(1 -221)} SEQ ID NO: 10.

7. The vaccine according to claim 5 comprising NS1 _{( 1 - 81 )}H3HA2_(77-221) SEQ ID NO: 12.

8. The vaccine according to claim 5 comprising NS1 _{( 1 -}

₄₂₎BLHA2_{(41 -223)} SEQ ID NO: 14.

9. The vaccine according to claim 5 comprising NS1 _{( 1 - 81)}BLHA2_{(1 -223)} SEQ ID NO: 57.

10. The vaccine according to claim 5 comprising NS1 _{( 1 - 81 )}H3HA2_{(1 -221)} SEQ ID NO:10 and NS1_(1-81)BLHA2_{(1 -223)}(met-leu) SEQ ID NO: 55.

11. A protein comprising an immunogenic fragment of the HA2 subunit of an HA protein selected from the group consisting of Type A subtype or Type B influenza virus.

12. The protein according to claim 11 wherein said Type A subtype is H3N2.

13. The protein according to claim 11 wherein the peptide containing the immunogenic fragment is fused to a second peptide or protein.

14. The protein according to claim 13 wherein the second peptide comprises the N terminal amino acids of a NS1 protein.

15. The protein according to claim 11 wherein the immunogenic fragment of the HA2 subunit is selected from the group consisting of a peptide comprising amino acids 1 to 221 of the H3HA2 subunit, a peptide comprising amino acids 77 to 221 of the H3HA2 subunit, a peptide comprising amino acids 1-223 of the BHA2 subunit, and a peptide comprising amino acids 41-223 of the BHA2 subunit.

16. A polypeptide NS1_(1-81)H3HA2_{(1 -221)} SEQ ID NO: 10.

17. A polypeptide NS1_(1-81)H3HA2_(77-221) SEQ ID NO: 12.

18. A polypeptide NS1_(1-81)BLHA2_{(41 -223)} SEQ ID NO: 14.

19. A polypeptide NS1_(1-81)BLHA2_{( 1 -223)} SEQ ID NO: 57.

20. A polypeptide NS1_(1-81)BLHA2_{( 1-223)}(met-leu) SEQ ID

NO: 55.

21. A DNA molecule comprising a coding sequence for an immunogenic fragment of the HA2 subunit of an HA protein selected from the group consisting of a Type A subtype or Type B influenza virus.

22. The DNA molecule according to claim 21 wherein said Type

A subunit is H3N2.

23. The DNA molecule according to claim 22 comprising a coding sequence for the polypeptide NS1_(1-81)H3HA2_{(1 -221)} SEQ ID NO: 10.

24. The DNA molecule according to claim 21 comprising a coding sequence for the polypeptide NS1_(1-42)BLHA2_(41-223) SEQ ED NO: 14.

25. The DNA molecule according to claim 21 comprising a coding sequence for the polypeptide NS1_(1-81)H3HA2_(77-221) SEQ ID NO: 12.

26. The DNA molecule according to claim 21 comprising a coding sequence for the polypeptide NS1_(1-81)BLHA2_{( 1-223)} SEQ ID NO: 57.

27. A vector pOTS208NS lBLmut2 SEQ ED NO: 54.

28. A microorganism transformed with a DNA molecule comprising a coding sequence for an immunogenic fragment of the HA2 subunit of an HA protein selected from the group consisting of a Type A subtype or Type B influenza virus.

29. The microorganism according to claim 28 wherein said Type A subunit is H3N2.

30. The microorganism according to claim 28 wherein said DNA molecule comprises a coding sequence for the polypeptide NS1_(1-81)H3HA2_{( 1 - 221 )} SEQ ED NO: 10.

31. The microorganism according to claim 28 wherein said DNA molecule comprises a coding sequence for the polypeptide NS1_(1-81)BLHA2_{( 1- 223)} SEQ ID NO: 57.

32. The microorganism according to claim 28 wherein said DNA molecule comprises a coding sequence for the polypeptide NS1_(1-81)BLHA2_{( 1 - 223})(met-leu) SEQ ED NO: 55.

33. A combination vaccine for stimulating protection in animals against infection by influenza virus which comprises a first polypeptide having an immunogenic fragment of the HA2 subunit of an influenza H3 subtype virus and a second polypeptide selected from the group consisting of a polypeptide having an immunogenic fragment of the HA2 subunit of a Type B influenza virus, and a polypeptide having an immunogenic fragment of the HA2 subunit of an H1 subtype influenza virus, and a polypeptide having an immunogenic fragment of the HA2 subunit of an H2 subtype influenza virus.

34. The combination vaccine according to claim 33 wherein the first polypeptide is selected from the group consisting of NS1_(1-81)H3HA2_{(1 -221)}

SEQ ID NO: 10 and NS1_(1-81)H3HA2_(77-221) SEQ ID NO: 12.

35. The combination vaccine according to claim 33 wherein the second polypeptide is a polypeptide having an immunogenic fragment of the HA2 subunit of an H1 subtype influenza virus.

36. The combination vaccine according to claim 33 wherein said second polypeptide is selected from the group consisting of C13 SEQ ID NO: 16, D SEQ ED NO: 18, C13 short SEQ ID NO: 20, D short SEQ ID NO: 22, A SEQ ID NO: 24, C SEQ ID NO: 26, ΔD SEQ ID NO: 27, Δ13 SEQ ID NO: 28, M SEQ ID NO: 29, ΔM SEQ ID NO: 30, ΔM+ SEQ ID NO: 32, and H1HA266-222 SEQ ID NO: 34.

37. The combination vaccine according to claim 33 wherein said second polypeptide is NS1_(1-42)BLHA2_(41-223) SEQ ID NO: 14.

38. The combination vaccine according to claim 33 wherein said second polypeptide is NS1_(1-81)BLHA2_{( 1 -223)} SEQ ID NO: 57.

39. A combination vaccine for stimulating protection in animals against infection by influenza virus which comprises a first polypeptide having an immunogenic fragment of the HA2 subunit of an influenza H3 subtype virus, a second polypeptide having an immunogenic fragment of the HA2 subunit of an influenza B Type virus, and a third polypeptide selected from the group consisting of a polypeptide having an immunogenic fragment of the HA2 subunit of an H1 subtype influenza virus and a polypeptide having an immunogenic fragment of the HA2 subunit of an H2 subtype influenza virus.

40. The combination vaccine according to claim 39 wherein the first polypeptides is NS1_(1-81)H3HA2_{(1 -221)} SEQ ID NO: 10, the second polypeptide is NS1_(1-81)BHA2_{(1 -223)}(met-leu) SEQ ID NO: 57, and the third polypeptide is NS1_(1-81)HA2_(65-222) SEQ ID NO: 18.