DE4303744A1 - Canine growth hormone - Google Patents

Abstract

The present invention relates to canine growth hormone (dGH), its precursor and the functional equivalents thereof, to pharmaceutical compositions containing the growth hormone and to the use thereof for treating mammals, to DNA molecules which code for the growth hormone, and to vectors containing these DNA sequences. The invention additionally relates to the cloning of canine growth hormone cDNA with the aid of the polymerase chain reaction (PCR) and to expression thereof in E. coli.

Description

The present invention relates to dog growth hormone (dGH), its precursor and the functional equivalents thereof, the the growth agent containing pharmaceutical agents, the Use for the treatment of mammals, DNA molecules, which code for the growth hormone, as well as these DNA sequences containing vectors. The invention also relates to Cloning of dog growth hormone cDNA using the Polymerase chain reaction (PCR) and its expression in E. coli.

Mammalian growth hormones are single-stranded polypeptides that usually comprise 190 amino acids and a molecular weight have in the range of 22,000 daltons. They are from the anterior pituitary gland is produced and excreted responsible for stimulating linear growth in living beings [Daughaday, W.H. Growth hormones and somatomedins, in Daughaday, W.H. (Ed.), Endocrine Control of Growth, Elsevier, New York, 1981, pages 1-24].

Growth hormones are members of a family of hormones which also include prolactins and placental lactogens. This Hormones have overlapping structural [Catt et al, Science 157 (1967) 321; Sherwood, L.M., Proc. Natl. Acad, Sci, USA 58 (1967) 2307-2314 .; Niall et al. Proc. Natl. Acad. Sci. USA 68 (1971) 866-869], functional and immunological properties [Niall et al., Rec. Prog. Hormone Res. 29 (1973) 387-416] and it is assumed that their genes are derived from one have developed common precursor genes [Seeburg, P.H., DNA 1 (1982) 239-249; Miller, W.L. and Eberhardt, N.L., Endocrine Rev. 4 (1983) 97-130].

The nucleotide sequences were used for Growth hormone genes of the following species are determined: Human [DeNoto, F.M. et al., Nucleic Acids Res. 9 (1981) 3719-3730; Seeburg, P.H., DNA 1 (1982) 239-249], Rat [Barta, A. et al., Proc. Natl. Acad. Sci. USA 78: 4867-4871 (1981); Page, G.S. et al., Nucl. Acids Res. 9 (1981) 2087-2104], Schwein [Seeburg,  P.H. et al., DNA 2 (1983) 33-45], bovine [Woychik, R.P. et al., Nucl. Acids Res. 10 (1982) 7192-7210], sheep [Warwick, J. et al., Biochim. Biophys. Acta 1008 (1989) 247-250], goat [Yamano, Y et al., FEBS Lett. 228 (1988) 301-304], mink [Shoji. K. et al., Nucl. Acids Res. 18 (1990) 6424] and horse [Stewart, F. and Tuffnell, P.P., J. Mol. Endocr. 6 (1991) 189-196]. Isolation of the dog growth hormone gene (dGH) and the determination of its nucleotide sequence was in the state the technology has not been described so far.

A large number of publications are known from the prior art which deal with the cloning of the coding DNA for various growth hormones. The expression of the DNA for the production of certain recombinant growth hormones is also known. For example, EP 82 302 804.9 discloses a method for cloning bovine growth hormone. This is suitable for stimulating growth and milk production in cattle. Here, poly (A) mRNA from the anterior pituitary gland of bovine is used as a template for the enzymatic synthesis of ds cDNA. The ds cDNA is inserted into pBR322 and cloned into E. coli χ ¹⁷⁷⁶ . Clones which contain the DNA complementary to the bovine growth hormone mRNA are identified by colony hybridization with partially purified growth hormone cDNA. The cloned cDNA contains most of the 5'-untranslated section, the complete coding section for the signal peptide and the growth hormone and the entire 3'-untranslated section.

EP-A-0 047 600 discloses a DNA transfer vector which is one for the cattle growth hormone or for pre- Contains bovine growth hormone coding nucleotide sequence.

EP-A-0 068 646 describes a method for expressing the Structural gene for cattle growth hormone by cultivating the Yeast strain S. cerevisiae LL 20 (pBGH 1) in an aqueous Nutrient medium.  

Figure description

Fig. 1 shows a schematic representation of the experiment according to the invention for the PCR cloning of dGH. RNA extracted from the pituitary gland of dogs is isolated, reverse transcribed and then selectively amplified using the polymerase chain reaction (PCR). The amplified dGH cDNA is cut by restriction enzyme digestion specifically at sites which have been introduced by adding appropriate recognition sequences to the 5 'ends of the primers ( FIG. 2). The product is cloned into M13mp18 and M13mp19 for sequencing and then subcloned into pMAL vectors for expression in bacteria.

Fig. 2 shows the conserved cDNA portions of growth hormones of various origins downstream of the start codon (IC) and upstream of the stop codon (TC) and the ajar invention thereto PCR primer design. The cDNA sequences of different growth hormones (mink, pig, cattle and sheep) downstream of the translation start codon (ATG) and upstream of the translation stop codon (TAG) are compared with one another. A pronounced evolutionary conservation is noted. A consensus sequence is established for each of the conserved sections and the primers for the PCR reaction are prepared on this basis. In addition, individual specific restriction sites are added to the respective 5 'ends of the primers.

Fig. 3 shows the nucleotide sequence of the cDNA dGH. The differences between the nucleotide sequences from dog, mink, pig, cattle and sheep are listed. The primer sequences are shown in small letters.

Fig. 4 shows the nucleotide sequence and the deduced amino acid sequence of dGH. The differences between the nucleotide sequences from dog, mink, pig, cattle and sheep are listed. The primer sequences are shown in small letters.

Figure 5 illustrates the construction of the pMALc-dGH expression vector. The dGH cDNA flanked by Eco RI and HindIII cleavages from a recombinant M13 clone is isolated and inserted into pMALc at the same sites, giving pMALc-dGH. After introduction and induction of expression in E. coli, the recombinant plasmid produces a fusion protein (maltose binding protein with dog growth hormone: MBP-dGH).

Fig. 6 shows the electrophoresis of the fusion protein MBP-dGH. Protein samples are analyzed using SDS polyacrylamide gel electrophoresis and stained with Coomassie blue (A) or by blotting and subsequent immunodetection (B) (Western blotting). The individual samples are: total protein from lysed TBI / pMALc-dGH cells, after growth overnight without (lanes A1 and B1) or with (lanes A2 and B2) 5-15 hour IPTG induction. Lanes A3 and B4, cleaned inclusion bodies, containing MBP-dGH. Positive expression control: Expression of HGH (22Kd) after IPTG induction by TB1 / pBHX (lanes A4 and B3). Lane A5: IPTG induction of TB1 / pMALc (without dGH cDNA). The molecular weight standards (M) used in each case and their relative size in kd are given in Figures A and B, respectively.

The present invention relates to dog growth hormone (dGH), with an amino acid sequence which is essentially shown in Table I and which extends from amino acid residue +1 to amino acid residue +190, and the precursor polypeptide thereof, which additionally has an amino-terminal amino acid sequence which essentially is shown in Table I and ranges from amino acid residue -26 to amino acid residue -1, as well as the functional equivalents thereof that have growth hormone activity. In the amino acid sequence ^according to the invention, Val ⁺¹⁸⁴ can ^optionally be replaced by Gly ⁺¹⁸⁴ and Ser ⁺¹⁸⁶ can be replaced by Ala ⁺¹⁸⁶ .

Table I

Nucleotide sequence and deduced amino acid sequence from dGH

Techniques for producing functional equivalents of dGH are known to the average person skilled in the art. Starting from the Amino acid sequence or the corresponding DNA sequence can be the Functional dGH equivalents suitable for the person skilled in the art by addition, Substitution and / or deletion of individual amino acid residues in manufacture special positions.

According to a further embodiment of the present invention DNA sequences are provided which are for those mentioned above Encode polypeptides. The DNA molecules according to the invention in particular essentially comprise the DNA sequence according to Table I in whole or in part. A DNA is preferably Sequence provided that corresponds to the nucleotide sequence of position Corresponds to +90 to position +659 in Table I and for a Polypeptide encoded with dGH activity, the partial sequence

can optionally be replaced by the partial sequence GGGAGGC.

The invention also relates to a transfer vector for the Expression of dGH in a eukaryotic or prokaryotic Host system. This vector comprises a DNA coding for dGH Sequence as defined above. Preferably the vector is a plasmid of the pMAL family, in which the DNA coding for dGH Sequence for controlled expression inserted in E. coli is. A plasmid with the name is particularly preferred pMALc-dGH, which is part of the American Type Culture Collection the deposit number ATCC 69176 is deposited.

The invention also relates to a method of manufacture the DNA encoding dGH. This procedure includes the following Steps:

• a) extraction of the total mRNA from the pituitary of a dog;
• b) Preparation of a single-stranded cDNA using the Enzyme reverse transcriptase;
• c) Hybridization of the cDNA with for the growth hormone specific oligo nucleotide primers and the selective Amplification of the dGH cDNA using the polymerase  Chain reaction (PCR); and
• d) Isolation of the amplified cDNA.

Preferably, this method is carried out using the carried out the following primer:

or

Another embodiment of the present invention relates to a method for producing a polypeptide Growth hormone activity. This procedure includes the following Steps:

• a) the transformation of eukaryotic or prokaryotic Host cells with a vector described above;
• b) induction of expression of the exogenous DNA sequence;
• c) the isolation of the expressed product, which preferably a polypeptide with the amino acid sequence of dGH or its Is precursor, preferably after prior cell lysing; and
• d) if necessary, the cleavage of non-dGH peptide Fragments and / or the signal peptide sequence from Expression product. If necessary, you can in the usual way the expression product was renatured become.

The invention also relates to pharmaceutical agents, which is an effective amount of dGH or a functional Equivalent to it, possibly in combination with one pharmaceutically acceptable carrier or diluent contain.  

The invention also relates to the use of Peptide hormones according to the invention for treatment of infertility, chronic renal insufficiency, Growth hormone deficiency, atrophy, burns, aging, Osteoporosis and obesity.

The use of the invention is particularly preferred Hormones in veterinary compositions for Promotion of animal growth, to increase the feed Conversion efficiency, to stimulate milk production and Reduction in meat fat. Preferably the Hormones according to the invention for the treatment of Dogs and pigs used.

According to a preferred embodiment, the total RNA is isolated from canine pituitary glands for cloning the dGH cDNA [Chomczynski, P. and Sacchi, N., Anal. Biochem. 162 (1987) 156-159]. The total RNA is used to synthesize corresponding cDNA strands with the aid of the reverse transcriptase. The specific amplification of the dGH cDNA takes place with the help of the PCR using special primers. These were prepared on the basis of the consensus sequence determined by sequence comparison of related mammalian growth hormone cDNAs. Artificial restriction sites are added to the 5 'ends of the primers to facilitate the subsequent cloning steps required for sequencing and expression. For better control of the insertion, two different (only unique) restriction sites are selected (see FIG. 2). According to a particularly preferred embodiment, a single Eco RI interface is introduced by adding the oligonucleotide 5'-CTAGGAATTCC-3 'to the 5'-end of the derived 5'-consensus sequence. A single Hind III site is introduced by adding the oligonucleotide 5'-GGAAGCTT-3 'to the 5' end of the 3 'consensus sequence. In the 3′-primer used in later experiments, the HindIII cleavage was introduced with the help of the oligonucleotide 5′-TGCATGCAAAGCTT-3 ′. The amplified DNA is digested with EcoRI and HindIII to form cohesive ends. The product obtained is inserted into EcoRI and HindIII digested vectors M13mp18 and M13mp19 and using the enzyme method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 546-54671]. The DNA sequence of both strands is determined using two independent cDNA clones.

The coding dGH cDNA sequence consists of 651 nucleotides and corresponds to a typical mammalian growth hormone cDNA (cf. FIG. 3). It consists of 217 codons, of which 190 codons correspond to the mature hormone, 26 codons correspond to the signal peptide and at least one codon is the stop codon (cf. FIG. 4). If only the naturally occurring codons (+1 to +190) are taken into account, it is found that the deduced amino acid sequence for dGH exhibits a sequence homology of 100%, 98.4%, 90.5% and 89.5% with the growth hormones Pig, mink, beef or sheep has ( Fig. 4 in comparison with Table I).

Although dog growth hormone differs from pig growth hormone (pGH) at the DNA level in 37 nucleotide positions (33 neutral nucleotide swaps in the third codon position and four swaps in the first codon position) only results one of these changes to a change in the Amino acid sequence. However, since the only difference between dGH and pGH affect position -6 of the signal sequence of dGH, there is no difference between mature dGH and mature pGH be determined. According to the invention, the complete identity of two growth hormones from animals different order (Carnivora and Artiodactyla) detected.

After determining the dGH cDNA sequence and checking the additionally introduced restriction sites for their presence, the cDNA is isolated from the recombinant M13 clones by cutting with EcoRI and HindIII. The DNA fragment obtained is then subcloned into pMAL vectors, a plasmid pMAL-dGH being obtained. The expression vectors of the pMAL series (pMALc and pMALp) containing the tac promoter produce a fusion protein from maltose binding protein and Lac Z. FIG. 5 shows the cloning of dGH cDNA in M13 and the subcloning in the expression plasmid pMALc.

The recombinant plasmid (pMAL-dGH) produced in this way is introduced into E. coli TB1. The resulting clone is cultivated and incubated for expression with the inducer IPTG (isopropylthiogalactoside). The total bacterial protein is analyzed using polyacrylamide gel electrophoresis and Western blotting. Only in induced clones is the appearance of a new strong protein band, the size of which corresponds to the fusion protein (MBP-dGH). The band is stained in the prepared Western blots using an anti-bovine growth hormone antiserum ( FIG. 6).

The present invention is illustrated by the following examples described in more detail.

materials

Restriction enzymes and enzymes for DNA modification were obtained from Bethesda Research Laboratories Inc. (BRL, Gaithersburg, MD) and from New England Biolabs, Inc. (NEB, Beverly, MA). ³⁵ S-dATP was obtained from Amersham International (Buckinghamshire, UK). The cloning and sequencing vectors (pMALc and M13) were obtained from NEB. The sequencing kit was obtained from United States Biochemical. Inc. (USB, Cleveland, OH).

example 1 mRNA isolation

Pituitaries from dogs become liquid after isolation Nitrogen saved until RNA extraction. The total RNA was made using the acidic guanidine thiocyanate phenol Chloroform method [Chomcynski and Sacchi, 1987] isolated.  

Immediately after removal from the liquid nitrogen, this becomes Tissue thawed and at 4 ° C with 1 ml of solution D (4 M Guanidinium thiocyanate, 25 mM sodium citrate, pH 7; 0.5 sarcosyl, 0.1 M 2-mercaptoethanol) in a glass Teflon homogenizer homogenized and then in a 4 ml polypropylene tube given. One after the other 0.1 ml of 2 M is added to the homogenate Sodium acetate, pH 4, 1 ml phenol (water saturated) and 0.2 ml Add chloroform-isoamyl alcohol mixture (49: 1) and mix carefully after adding each reagent Invert the vessel. The one obtained in this way Suspension is shaken vigorously for 10 seconds and cooled 15 Minutes in ice. Samples are at 4 ° C for 20 minutes at 10,000 g centrifuged. After centrifugation, RNA is in the aqueous phase before, while DNA and protein in the interphase and the phenol phase can be found. The aqueous phase is in transfer a new tube, mix with 1 ml isopropanol and stored at -20 ° C for at least 1 hour for RNA to fail. After centrifugation at 10,000 g for 20 minutes the resulting RNA pellet is transferred in 0.3 ml of solution D. in a 1.5 ml Eppendorf tube and precipitated with 1 volume Isopropanol within one hour at -20 ° C. To Centrifuge in an Eppendorf centrifuge (10 minutes at 4 ° C.) the RNA pellet is resuspended in 75% ethanol, sedimented, dried in vacuo (15 minutes) and dissolved in 50 µl 0.5% SDS (10 minutes at 65 ° C). From 450 mg of frozen tissue isolate 1 mg of extracted total RNA.

Example 2 Synthesis of single strand cDNA (ss cDNA)

Single-strand cDNA is synthesized from 8 µg of total RNA Use of reverse transcriptase and random primers (Hexamer).

The microcentrifuge tube containing the total RNA (1 µg / µl) from the dog's pituitary is heated to 65 ° C for 10 minutes and then cooled by incubation in ice. In a sterile 1.5 ml microcentrifuge tube treated with diethyl pyrocarbonate (DEPC) in ice, 50 μl of a reaction mixture are introduced by the following additions: 10 μl FSB 5X (250 mM Tris.HCl pH 8.3, 375 mM KCl, 15 mM MgCl ₂ and 50 mM dithiothreitol), 3 µl dNTP's 8 mM mix, 1 µl dithiothreitol 1 M, 1 µl random primer (0.2 µg / µl), 8 µl total RNA (1 µg / µl), 22 µl DEPC -treated distilled water and 5 µl Maloney Murine Leukemia Virus (M-MLV) reverse transciptase (200 µl). Mix the contents of the tube and incubate for 1 hour at 37 ° C. Then put the jar on ice. The reaction product is extracted with phenol-chloroform-isoamyl alcohol (25: 245: 1) by adding one volume of this organic solvent mixture, shaking (vortex shaker), centrifuging for three minutes in an Eppendorf centrifuge at 10,000 rpm and transferring the aqueous phase into a new microcentrifuge tube. Add 0.1 volume of 3.5 M sodium acetate, pH 5.2 and mix. Precipitation of the cDNA-RNA hybrids is achieved by adding 2.5 volumes of ethanol (100%), mixing and incubating at -20 ° C over a period of at least 30 minutes. The nucleic acids are isolated by centrifugation at 12,000 rpm for 10 minutes. After removing the supernatant, the precipitate is washed with 0.5 ml of 70% cold ethanol. After centrifugation at 10,000 rpm for 4 minutes, the supernatant is discarded and the precipitate is dried in a speed vacuum concentrator. Finally, the precipitate is resuspended in 20 µl of autoclaved, distilled water.

Example 3 cDNA amplification by PCR

dGH cDNA is specifically amplified using PCR using the following synthetic primers that were prepared by comparing DNA sequences of known, related growth hormones:
5′-primers: 5′-CTAGGAATTCCATGGCTGCAGGCCCCCGGACCTC-3 ′ and 3′-primers: 5′-GGAAGCTTCTAGAAGGCACAGCTGGCCTCCCCGAA-3 ′.

Can also be used as a 3'-primer 5'-TGCATGCAAAGCTTTTTTTTTTTTTTTTT-3 '.  

After isolation of the total RNA-cDNA hybrids, the dGH cDNA is specifically amplified using the polymerase chain reaction. 50 .mu.l of a reaction mixture, consisting of 38.7 .mu.l of distilled water, 5 .mu.l amplification buffer (100 mM Tris.HCl pH 8.3, 15 mM MgCl ₂ , 500 mM KCl and 1 mg / ml gelatin) are prepared in a tube. 2 µl total RNA-cDNA hybrid, 1.3 µl dNTP's 8 mM, 1 µl 5'-primer 2.5 µM, 1 µl 3'-primer 2.5 µM and 1 µl Taq pol (5 U / 41 ). As a negative control, the amplification is carried out under the same amplification conditions with the same reagents, but without an RNA-cDNA hybrid. The reaction mixture is carried out for 30 amplification cycles, each cycle comprising 3 steps:

First step: denaturation of the cDNA, 95 ° C, 1.5 minutes,
Second step: coupling of the cDNA with the primers, 55 ° C, 1.5 minutes
Third step: extension of the cDNA, 72 ° C, 2 minutes.

After the reaction is over, you check that Amplification product by adding 5 µl of it to a 1% Applies agarose gel, electrophoresed, with ethidium bromide colors and makes visible with UV light. Under these conditions a cDNA band can be obtained with the expected size that the dGH cDNA corresponds, observe.

Example 4 Insertion of the amplified dGH cDNA into bacteriophages M13, Cloning, sequencing and restriction enzyme mapping

Because the primers at their 5′-ends are artificial, unique Have restriction sites, the amplified product treated with the appropriate restriction endonucleases. The dGH cDNA obtained in this way is inserted into the EcoRI-HindIII treated plasmids M13mp18 and M13mp19. The recombinant Clones are characterized using restriction enzymes and  sequences the dGH cDNA using the enzyme method [Sanger et al., 1977].

The ssDNA- required for sequencing the dGH cDNA Templates are made from the bacteriophage particles that is present in the culture medium of such bacterial clones in which dGH cDNA in the recombinant M13 replicative form (double-stranded) could be demonstrated.

This template DNA is made as follows: A fresh, exponential culture of the host Escherichia coli (JM101) is diluted 1: 100 in 1.5 ml of 2 × YT medium and infected with a purified recombinant M13 plaque from a ligation Transformation agar plate, incubated for 5-8 hours Shake at 37 ° C, transferred to a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes. The phage The supernatant containing is carefully separated and placed in a transferred fresh vessel and with 1/9 volume 40% PEG-8000 and 1/9 volume of 5 M sodium acetate, pH 7 added. The contents of the vessel mix carefully and incubate for 15 minutes at 4 ° C Precipitation of the phage. After centrifugation for 10 minutes carefully remove the liquid supernatant with a Pipette and clean the inside wall of the jar. As much PEG as possible is removed. The precipitation resuspended in 0.2 ml of TE buffer and extracted with 0.2 ml of phenol (equilibrated with 0.05 M Tris-HCl pH 7.5 before use): Chloroform (3: 1) by shaking (vortex shaker) and centrifuged to separate the layers. The the upper aqueous layer is carefully lifted off, the phenolic layer and the intermediate layer in the vessel remain. This extraction step is repeated twice and performs a final extraction step with 0.2 ml Chloroform through.

0.1 is added to the aqueous phase finally obtained Volume 3 M sodium acetate and 2.5 volumes 100% ethanol. The The vessel is cooled to -20 ° C. and centrifuged for at least 1 hour 10 minutes at 10,000 rpm to isolate precipitated DNA. The  DNA pellet is washed with 70% cold ethanol, centrifuged, removes ethanol and dries the pellet through Evaporation Then the ssDNA M13 in conventional Sequenced way.

Example 5 Subcloning and expression

The expression vectors of the pMAL series (NEB) produce one Fusion protein from maltose binding protein and Lac Z.

E. coli strain TB1 is used for expression. The transformed cells are grown for 2 hours at 37 ° C. 2x YT Medium [Sambrock et al., 1989] Sambrook, J., Fritsch, E.F. and Maniatis, T .: Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor: Laboratory Press, New York, 1989]. After the first incubation phase (O.D. at 600 nm = 0.4) there is a further 5-15 hour incubation with 0.3 mM IPTG.

Whole cell lysate for electrophoresis is prepared by centrifuging 1 ml of bacterial culture, resuspending in lysing buffer and boiling for 4 minutes. The samples are analyzed by SDS-polyacrylamide gel electrophoresis, stained with Coomassie Brillant Blue or an immunodetection is carried out after blotting ( FIG. 6).

Immunoblotting: After gel electrophoresis, the Proteins transferred electrophoretically to nitrocellulose, with Rabbit anti-bovine GH serum incubated with biotinylated second antibody and with streptavidin-biotin Peroxidase complex developed.

For electrophoretic transfer to nitrocellulose, the SDS-polyacrylamide gel with the separated bacterial proteins is placed in an electrotransfer device, the transfer buffers (39 mM glycine, 48 mM Tris base pH 8.3, 0.037% SDS and 20% methanol) contains. A voltage of 100 volts is applied to the system for 2-3 hours. After the transfer is complete, the nitrocellulose filter is incubated in a blocking solution [5% defatted dry milk in TBST [0.05% Tween 10, 50 mM Tris.HCl pH 7.4 and 150 mM NaCl)] and incubated with gentle stirring for 2 hours at room temperature . The blocking solution is discarded. Then add 15-20 ml of a first antibody solution (rabbit antiserum against bovine growth hormone (anti-BGH) in a dilution of 1: 10,000 TBST plus 5% defatted dry milk) and incubate with gentle stirring for 2-3 hours Room temperature. Discard the solution and wash the filter four times (10 minutes each) with 15-20 ml TBST. In the next step, the filter is incubated with 15-20 ml of the second antibody solution (anti-rabbit IgG, covalently coupled with alkaline phosphatase in a 1: 1000 dilution in TBST plus 5% defatted dry milk) and stirred gently for 1 hour at room temperature. The solution is discarded and the filter is washed four times with 15-20 ml of TBST (10 minutes each). This is followed by two further washing steps with PA solution (0.1 M Tris-HCl pH 9.5 and 0.1 M NaCl). The PA solution is then replaced by the chromogenic reaction mixture [10 ml PA, 10 μl MgCl ₂ , 4.9 M, 40 μl NBT (nitro blue tetrazolium on 50% vadium dimethylformamide), 40 μl BCIP (5-bromo-4-chlorine -3-indolyl phosphate diluted to 50% in dimethylformamide)]. Incubate with gentle stirring at room temperature until bands appear. When they have reached the desired intensity, wash the nitrocellulose filter extensively in water to stop the reaction.

Example 6 Characterization of the expression product

100 ml of 2 × YT medium (100 µg / ml ampicillin) are inoculated with 1 ml a culture of a bacterial clone grown overnight, who carries the plasmid pMALcdGH and incubates with vigorous Stir at 37 ° C to an absorbance of 0.5 at 600 nm to reach. Isopropylthiogalactosidpyranose (IPTG) is given added (final concentration 0.1 mM) and incubated for 8-12 hours.  

The cells are centrifuged off at 5000 rpm for 10 minutes. The cell pellet is resuspended in 0.5 ml of 10 mM Tris-HCl, pH 7.8, 0.1 mM EDTA and 25% sucrose. A 1 mg / ml lysozyin is added and the suspension is incubated in ice for 1.5 hours. MgCl ₂ and DNAse I are added in concentrations of 20 mM or 50 µg / ml. After a further 1.5 hour incubation, 6 ml of lysing buffer (20 mM Tris-HCl pH 7.8, 0.2 M NaCl, 1% deoxycholic acid, 1.6% Nonidet P-40, 2 mM EDTA and 0, 5 mM dithiothreitol) and centrifuged the lysate for 10 minutes at 6000 rpm. The pellet, which contains MBP-dGH fusion protein, is resuspended in 0.5% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol and centrifuged off. This washing step is repeated four times. The pellet obtained in this way is dissolved at 4 ° C. in 2 ml of 9 M urea, 20 mM Tris-HCl, pH 7.8 and 20 mM dithiothreitol. The buffer solution is dialyzed against 500 ml of X _a buffer (0.1 M NaCl, 20 mM Tris-HCl, pH 7.8) after 12 hours at 4 ° C. and twice changing the buffer. After dialysis against factor X _a buffer, 15 μl (1 μg / μl) of the fusion protein (MBP-dGH) are cleaved with 1.5 μl (200 μg / ml) factor X _a . The reaction mixture is incubated at 25 ° C. for 2 days.

A negative control is carried out under the same conditions and with the same reagents, but without factor X _a . To check the cleavage of MBP-dGH by factor X _a , an 8 µl sample of the reaction mixture is applied to an SDS polyacrylamide gel. The proteins are transferred to nitrocellulose filters and immunologically detected dGH with anti-bGH serum.

As expected, a dGH band of low insensitivity was observed at around 25 kd, from which an effective proteolytic cleavage of the MBP-dGH fusion protein can be concluded ( FIG. 6).

Claims (16)

1. Dog growth hormone (dGH), characterized by an amino acid sequence which is essentially shown in Table I and extends from amino acid residue +1 to amino acid residue +190, and the precursor polypeptide thereof, which additionally has an amino-terminal amino acid sequence which is essentially in Table I is shown and ranges from amino acid residue -26 to amino acid residue -1, and the functional equivalents thereof with growth hormone activity. 2. ^{Dog growth hormone} according to claim 1, characterized in that Val ⁺¹⁸⁴ is replaced by Gly ⁺¹⁸⁴ and Ser ⁺¹⁸⁶ is replaced by Ala ⁺¹⁸⁶ . 3. DNA for a polypeptide according to claim 1 or 2 encoded. 4. DNA according to claim 3, characterized by a nucleotide Sequence from nucleotide +1 to nucleotide +659 or from Nucleotide +90 to nucleotide +659 according to Table I, where the partial sequence from nucleotide +640 to nucleotide +646, TGGAAAG, optionally replaced by the partial sequence GGGAGGC. 5. Transfer vector for the expression of a polypeptide with Growth hormone activity in a eukaryotic or prokaryotic host, characterized in that the Vector a DNA sequence coding for dGH according to claim 3 or 4 includes. 6. Vector according to claim 5, characterized in that the Vector is a plasmid of the pMAL series, in which those for dGH coding DNA sequence for controlled expression is inserted in E. coli.   7. Plasmid according to claim 6 deposited with the American type Culture Collection under the deposit number ATCC 69176. 8. A process for the preparation of a DNA molecule which codes for a polypeptide with growth hormone activity, characterized in that

• a) total mRNA extracted from the pituitary gland of dogs,
• b) produces cDNA single strands;
• c) the cDNA hybridizes with primer oligonucleotides specific for the growth hormone, and the cDNA is amplified using the PCR technique; and
• d) the amplified cDNA is isolated.

9. The method according to claim 8, characterized in that the PCR reaction is carried out with the 5'-primer:
5′-CTAGGAATTCCATGGCTGCAGGCCCCCGGACCTG-3 ′ and with the 3′-primer. 5′-GGAAGCTTCTAGAAGGCACAGCTGGCCTCCCCGAA-3 ′ or 5'-TGCATGCAAAGCTTTTTTTTTTTTTTTTT-3 '. 10. A method for producing a polypeptide with growth hormone activity, characterized in that

• a) transformed eukaryotic or prokaryotic host cells with a vector according to one of claims 5 or 6;
• b) the expression of the exogenous DNA sequence coding for dGH is induced;
• c) the expression product is separated from the cells; and
• d) optionally separating non-dGH peptide fragments and / or the dGH singal peptide sequence from the expression product and optionally renaturing the product obtained.

11. A pharmaceutical composition comprising an effective amount of dGH or a functional equivalent thereof Claim 1 or 2, optionally in combination with one pharmaceutically acceptable carrier or Diluent. 12. Use of dGH or a functional equivalent of which for the treatment of infertility, chronic renal Insufficiency, growth hormone deficiency, atrophy, Burns, aging, osteoporosis and obesity. 13. Use of dGH or a functional equivalent of which in a veterinary composition for Promote animal growth, to increase Feed conversion efficiency, to stimulate the Milking and reducing meat fat. 14. Use according to claim 13 for the treatment of dogs or Pigs. 15. Microorganism transformed with a vector according to one of claims 5 to 7.