WO2023131173A1 - 高产杆状病毒的草地贪夜蛾蛹卵巢细胞系及其构建和用途 - Google Patents

高产杆状病毒的草地贪夜蛾蛹卵巢细胞系及其构建和用途 Download PDF

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WO2023131173A1
WO2023131173A1 PCT/CN2023/070392 CN2023070392W WO2023131173A1 WO 2023131173 A1 WO2023131173 A1 WO 2023131173A1 CN 2023070392 W CN2023070392 W CN 2023070392W WO 2023131173 A1 WO2023131173 A1 WO 2023131173A1
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cell line
baculovirus
spodoptera frugiperda
cell culture
cell
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French (fr)
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张寰
佟岩
孟茜
秦启联
李瑄
张继红
王红托
苗麟
郭力
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中国科学院动物研究所
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    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
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  • the invention belongs to the field of biotechnology.
  • the present invention relates to an insect cell line, especially a cell line derived from insect ovary tissue and highly sensitive to baculovirus. applications in chemical growth.
  • insect cell lines since the first insect cell line was successfully established in 1965, more than 700 insect cell lines have been established in the past 60 years. They come from more than 170 kinds of insects including Lepidoptera, Diptera, Homoptera, Hymenoptera, Orthoptera and Coleoptera, most of which come from Lepidoptera and Diptera.
  • insect cell lines have always been an important tool for scientific research in physiology, developmental biology, cell biology, molecular biology and biochemistry, and insect cells, as an important part of the baculovirus expression vector system, express a large number of exogenous proteins of great economic or scientific significance; at the same time as a bioreactor to amplify insect baculoviruses for use as biopesticides, especially to amplify recombinant baculovirus insecticides containing exogenous genes, insects Cells also play an important role.
  • IPLB-Sf21-AE Sf-21
  • Sf-9 clone Sf-9 derived from Spodoptera frugiperda
  • source Tn-368 from Trichoplusia ni
  • Tn-5B1-4 trade name High Five
  • the present invention provides a Spodoptera frugiperda pupal ovary cell line with high yield of baculovirus derived from Spodoptera frugiperda.
  • the invention also provides the use of the high-yield baculovirus pupal ovary cell line of Spodoptera frugiperda in large-scale growth of baculovirus.
  • the present application relates to a Spodoptera frugiperda pupal ovary cell line with high yield of baculovirus, the name of the cell line is IOZCAS-Sf-1, and its preservation number is CGMCC No.21014.
  • the present application relates to a preparation method of Spodoptera frugiperda pupal ovary cell line with high yield of baculovirus, which comprises the following steps: 1) cultivating Spodoptera frugiperda ovary cells with cell culture medium; and 2) obtaining high-yield rods Spodoptera frugiperda ovary cell line of the virus.
  • the cell culture medium is a mixture of insect cell culture medium and antibiotics.
  • the antibiotics are penicillin and streptomycin.
  • the cell culture medium was also supplemented with fetal bovine serum.
  • the penicillin content is 100 U/mL; the streptomycin content is 100 g/mL.
  • the content of the fetal bovine serum is 10% by volume of the cell culture medium.
  • the cell culture fluid has a pH of 5.8-6.8.
  • the insect cell culture medium is selected from: Insect-XPRESS TM , TNM-FH, Grace's, Sf-900, TC-100, IPL-41 or Ex-Cell 400.
  • the present application relates to the use of a high-yielding baculovirus-producing Spodoptera frugiperda pupal ovary cell line in the production of baculovirus.
  • the baculovirus is Autographa californica nuclear polyhedrosis virus (AcMNPV).
  • the present application relates to the use of a high-yielding baculovirus-producing Spodoptera frugiperda pupal ovary cell line in the preparation of baculovirus insecticides.
  • the present application relates to the use of a high-yield baculovirus-producing Spodoptera frugiperda pupal ovary cell line in preparing a baculovirus expression vector system.
  • the Spodoptera frugiperda pupal ovary cell line with high yield of baculovirus can be used to replicate baculovirus for large-scale growth of baculovirus and large-scale production of baculovirus insecticide; It can be applied to the construction of a baculovirus expression vector system, which can express recombinant proteins with high commercial and scientific value.
  • Figure 1 is the adherent culture of the ovarian tissue of Spodoptera frugiperda during the primary culture process. It can be seen that the cells are dissociated from the tissue, which proves that the cells are derived from the ovarian tissue of Spodoptera frugiperda pupae, and the scale bar is 100 ⁇ m;
  • Fig. 2 is the cell morphology of Spodoptera frugiperda pupal ovary cell line IOZCAS-Sf-1 of the present invention, scale bar 50 ⁇ m;
  • Fig. 3 is the COI gene sequence of Spodoptera frugiperda pupal ovary cell line IOZCAS-Sf-1 of the present invention and the result of the sequence comparison of COI gene on Spodoptera frugiperda larva hemocyte, commercialized Sf9 cell line, NCBI;
  • Fig. 4 is the growth curve of Spodoptera frugiperda pupal ovary cell line IOZCAS-Sf-1 of the present invention
  • Fig. 5 is the flow cytometer detection chromosome ploidy of Spodoptera frugiperda pupal ovary cell line IOZCAS-Sf-1 of the present invention
  • Figure 6 shows that after IOZCAS-Sf-1 was infected with Autographa californica nuclear polyhedrosis virus (AcMNPV), a large number of viral polyhedrons were obtained, and the scale bar is 100 ⁇ m.
  • AcMNPV Autographa californica nuclear polyhedrosis virus
  • the high-yield baculovirus-producing Spodoptera frugiperda pupae ovary cell line IOZCAS-Sf-1, classified as Spodoptera frugiperda pupae ovary cell line, has been preserved in the General Committee of China Microorganism Culture Collection on November 25, 2020. Microbiology Center (CGMCC for short), address of depository unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, postcode: 100101, deposit number: CGMCC No.21014.
  • CGMCC Microbiology Center
  • the present application provides a Spodoptera frugiperda pupal ovary cell line with high production rate of baculovirus, the name of the cell line is IOZCAS-Sf-1, and its preservation number is CGMCC No.21014.
  • the taxonomically named Spodoptera frugiperda pupal ovary cell line has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on November 25, 2020.
  • Another aspect of the present application provides a method for preparing a high-yield baculovirus-producing Spodoptera frugiperda pupal ovary cell line, which comprises the following steps: 1) cultivating Spodoptera frugiperda ovary cells with cell culture fluid; and 2) obtaining high-yield Spodoptera frugiperda ovary cell line with baculovirus.
  • a method for preparing a high-yielding baculovirus-produced Spodoptera frugiperda pupal ovary cell line comprises the following steps: (1) Submerging the female Spodoptera frugiperda pupae in ethanol solution for 10-20 Minutes, carry out surface disinfection, then clean the insects with sterile distilled water, and then blot the surface of the insect body; (2) dissect the insects, and take out the complete Spodoptera frugiperda pupal ovary tissue; (3) wash the obtained in (2) with physiological saline The tissue was washed 2 to 3 times with cell culture solution.
  • the tissue was placed in a cell culture bottle containing 1 mL of cell culture solution and rinsed, tightly capped, and placed in a cell culture incubator at 27°C for 24 hours.
  • ( 5) Feed nitrogen into the three-gas incubator instead of oxygen, detect the oxygen concentration in the incubator by an oxygen sensor, control and reduce the oxygen content in the incubator to 5%; meanwhile, replace the airtight bottle cap of the cell culture bottle with a ventilating bottle cap, Make the gas in the cell culture bottle and the gas in the incubator circulate to achieve the effect of hypoxia, and carry out hypoxic culture on the primary insect cells at 27°C for 9 days;
  • the cell culture fluid used in the above-mentioned method of the present invention is the mixture of conventional insect cell culture fluid and penicillin, streptomycin and fetal bovine serum, and the pH of the cell culture fluid is 5.8-6.8 , for example, 5.8-5.9, 5.9-6.0, 6.0-6.1, 6.0-6.2, 6.0-6.3, 6.0-6.4, 6.0-6.5, 6.0-6.6, 6.0-6.7, 6.0-6.8.
  • the insect cell culture medium is commercial insect cell culture medium Insect-XPRESS TM , TNM-FH, Grace's, Sf-900, TC-100, IPL-41, or Ex-Cell 400.
  • the content of penicillin in the cell culture fluid is 10U/mL-500U/mL, preferably 100U/mL; the content of streptomycin is 10g/mL-500g/mL, preferably 100g/mL; the content of animal serum is the The volume ratio of 5%-20% of the cell culture fluid is preferably 10% of the volume ratio of the cell culture fluid.
  • Another aspect of the present application provides a use of a high-yield baculovirus-producing Spodoptera frugiperda pupal ovary cell line in the production of baculovirus.
  • said baculovirus is Autographa californica nuclear polyhedrosis virus (AcMNPV), Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV).
  • Another aspect of the present application provides a use of a high-yielding baculovirus-producing Spodoptera frugiperda pupal ovary cell line in the preparation of baculovirus insecticides.
  • Another aspect of the present application provides the use of a high-yielding baculovirus-producing Spodoptera frugiperda pupal ovary cell line in preparing a baculovirus expression vector system.
  • IOZCAS-Sf-1 was preserved in the China Committee for Microorganism Culture Collection on November 25, 2020. Microbiology Center, the deposit number is CGMCC No.21014.
  • the IOZCAS-Sf-1 cell line includes cells suspended in the culture medium and adherent cells. There are two types of cells: round and spindle. In IOZCAS-Sf-1, round cells accounted for 69.2%, with an average diameter of 7.98 ⁇ 0.105 (mean ⁇ standard deviation) ⁇ m; spindle cells accounted for 30.8%, with a length of 10.13 ⁇ 0.127 ⁇ m and a width of 6.50 ⁇ 0.0.101 ⁇ m (Fig. 2).
  • the 20th generation of IOZCAS-Sf-1 cell line is in the Insect-XPRESS TM medium containing 10% fetal bovine serum, 100U/mL of penicillin, and 100g/mL of streptomycin, and the doubling time of the 20th generation cell population is 46.47 hours . At present, the cell line has been passaged for more than 30 generations.
  • the cell line IOZCAS-Sf-1 is indeed derived from Spodoptera frugiperda, not the contamination of other cell lines and Sf9 cells.
  • the DNA extracted by IOZCAS-Sf-1 and the blood DNA of Spodoptera frugiperda were amplified by PCR, and the COI gene amplification primers used were (upstream primer: 5'-3'SEQ ID NO 1: TTCGAGCTGAATTAGGGACTC, downstream primer 5 '-3' SEQ ID NO 2: GATGTAAAATATGCTCGTGT) ( Figure 3).
  • Cryopreservation and resuscitation use the traditional cell cryopreservation method to cryopreserve the cells, preserve the seed material of the cells, and can be successfully resuscitated.
  • AcMNPV Autographa californica nuclear polyhedrosis virus

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Abstract

提供了一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系，该细胞系的名称为IOZCAS-Sf-1，其保藏号为CGMCC No. 21014。还公开了该细胞系的制备方法及该细胞系在杆状病毒规模化生长中的用途。该细胞系可以用来复制杆状病毒、用于杆状病毒杀虫剂的规模化生产及构建杆状病毒表达载体系统。

Description

高产杆状病毒的草地贪夜蛾蛹卵巢细胞系及其构建和用途 技术领域
本发明属于生物技术领域。本发明涉及一种昆虫细胞系，尤其是一种来源于昆虫卵巢组织，并且对杆状病毒高敏感的细胞系，本发明还涉及该细胞系的建立方法，以及该细胞系在杆状病毒规模化生长上的应用。
背景技术
据报道，在1965年第一株昆虫细胞系成功建立后至今，近60年的时间内，已经建立的昆虫细胞系超过700种。它们分别来源于鳞翅目、双翅目、同翅目、膜翅目、直翅目和鞘翅目等170多种昆虫，其中大部分来源于鳞翅目和双翅目昆虫。昆虫细胞系作为研究材料，一直是生理学、发育生物学、细胞生物学、分子生物学和生物化学等科学研究的重要工具，而且昆虫细胞作为杆状病毒表达载体系统的重要组成部分，表达了大量的具有重大经济意义或科学意义的外源蛋白质；同时作为生物反应器，扩增昆虫杆状病毒用作生物杀虫剂，特别是扩增含有外源基因的重组杆状病毒杀虫剂，昆虫细胞也发挥了重要的作用。
大量的来源于鳞翅目昆虫商品化的细胞系已得到人们广泛的应用，比如，来源于草地贪夜蛾Spodoptera frugiperda的IPLB-Sf21-AE(Sf-21)和其克隆株Sf-9，来源于粉纹夜蛾Trichoplusia ni的Tn-368和其克隆株Tn-5B1-4(商品名为High Five)等，已经成功的应用于表达和生产重组蛋白。
到目前为止，Cellosaurus数据库(https://web.expasy.org/cellosaurus)记录的原始建立的来自草地贪夜蛾Spodoptera frugiperda的细胞系共有约24株，其中商品化的Sf-21和其克隆株Sf-9由于其病毒产量高，增殖速度快等特点，在表达和生产重组蛋白等生产中得到了广泛的应用。但是，在蛋白表达过程中，仍然有一些种类的蛋白存在表达量低等问题。来源于不同种类昆虫的细胞系或来源于同一种昆虫不同组织部位的细胞系扩增病毒或重组蛋白的表 达能力各有不同。因此建立和筛选新的、更多的杆状病毒敏感细胞系(株)是一项非常有必要而且有理论和实践意义的工作。目前在各种生物学实验中仍对来自昆虫的细胞系存在很大的需求，将其用于构建更加高效的杆状病毒表达载体系统具有十分重要的意义。
发明内容
针对目前仍需进一步开发对杆状病毒敏感的昆虫细胞系的问题，本发明提供了一株草地贪夜蛾来源的高产杆状病毒的草地贪夜蛾蛹卵巢细胞系。本发明还提供了高产杆状病毒的草地贪夜蛾蛹卵巢细胞系在杆状病毒规模化生长中的用途。
具体来说，一方面，本申请涉及一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系，该细胞系的名称为IOZCAS-Sf-1，其保藏号为CGMCC No.21014。
一方面，本申请涉及一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系的制备方法，其包括以下步骤：1)用细胞培养液培养草地贪夜蛾卵巢细胞；和2)获得高产杆状病毒的草地贪夜蛾卵巢细胞系。
在一些实施方案中，所述细胞培养液是昆虫细胞培养液与抗菌素的混合物。所述抗菌素为青霉素和链霉素。所述细胞培养液还补充有胎牛血清。所述青霉素含量为100U/mL；所述链霉素含量为100g/mL。所述胎牛血清含量为细胞培养液的10％的体积比。所述细胞培养液具有5.8-6.8的pH。所述昆虫细胞培养液选自：Insect-XPRESS TM、TNM-FH、Grace’s、Sf-900、TC-100、IPL-41或Ex-Cell 400。
一方面，本申请涉及一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系在杆状病毒生产中的用途。
在一些实施方案中，所述杆状病毒是苜蓿银纹夜蛾核型多角体病毒(AcMNPV)。
一方面，本申请涉及一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系在制备杆状病毒杀虫剂中的用途。
一方面，本申请涉及一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系在制备杆状病毒表达载体系统中的用途。
本发明提供的高产杆状病毒的草地贪夜蛾蛹卵巢细胞系可以用来复制杆状病毒，用于杆状病毒的规模化生长和杆状病毒杀虫剂的规模化生产；该细胞系还可应用于构建杆状病毒表达载体系统，该系统可表达具有极高的商业和科研价值的重组蛋白。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为原代培养过程中，草地贪夜蛾卵巢组织贴壁培养，可见细胞从组织中游离出来，证明该细胞来源于草地贪夜蛾蛹的卵巢组织，标尺100μm；
图2为本发明的草地贪夜蛾蛹卵巢细胞系IOZCAS-Sf-1的细胞形态，标尺50μm；
图3为本发明的草地贪夜蛾蛹卵巢细胞系IOZCAS-Sf-1的COI基因序列与草地贪夜蛾幼虫血细胞、商品化的Sf9细胞株、NCBI上COI基因的序列比对结果；
图4为本发明的草地贪夜蛾蛹卵巢细胞系IOZCAS-Sf-1的生长曲线；
图5为本发明的草地贪夜蛾蛹卵巢细胞系IOZCAS-Sf-1的流式细胞仪检测染色体倍性；
图6为IOZCAS-Sf-1感染苜蓿银纹夜蛾核型多角体病毒(AcMNPV)后，获得大量的病毒多角体，标尺100μm。
生物材料的保藏
该高产杆状病毒的草地贪夜蛾蛹卵巢细胞系IOZCAS-Sf-1，分类命名为草地贪夜蛾蛹卵巢细胞系，已经于2020年11月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，保藏单位地址：北京市朝阳区北辰西路1号院3号，邮编：100101，保藏编号为：CGMCC No.21014。
具体实施方式
下面结合附图和具体实施例来详细说明本申请。应理解，以下实施例仅用于说明本申请而不用于限制本申请的范围。
本申请一方面提供了一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系，该细胞系的名称为IOZCAS-Sf-1，其保藏号为CGMCC No.21014。分类命名为草地贪夜蛾蛹卵巢细胞系，已经于2020年11月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心。
本申请另一方面提供了一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系的制备方法，其包括以下步骤：1)用细胞培养液培养草地贪夜蛾卵巢细胞；和2)获得高产杆状病毒的草地贪夜蛾卵巢细胞系。
在一个具体的实施方案中，一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系的制备方法，其包括以下步骤：(1)将草地贪夜蛾雌蛹浸没在乙醇溶液中10～20分钟，进行表面消毒，然后用无菌蒸馏水清洗昆虫，再吸干虫体表面；(2)解剖昆虫，取出完整草地贪夜蛾蛹卵巢组织；(3)用生理盐水清洗(2)中获得的组织2～3次，再用细胞培养液清洗，清洗后把该组织放入含有1mL细胞培养液润洗过的细胞培养瓶中，盖紧瓶盖，放入27℃的细胞培养箱中培养24小时；(4)加入适量的细胞培养液，使组织块完全或大部分浸没在该培养液中，在与步骤(3)同样条件下培养，培养约21天后可获得大量的原代细胞；(5)将三气培养箱通入氮气代替氧气，通过氧气传感器检测培养箱中氧气浓度，控制和降低培养箱内氧气含量至5％；同时将细胞培养瓶的密闭瓶盖更换为透气瓶盖，使细胞培养瓶内的气体与培养箱内的气体流通，达到低氧的效果，于27℃对昆虫原代细胞进行低氧培养9天；(6)更换细胞培养液及培养瓶瓶盖，于27℃，常氧培养10-30天，例如10天、11天、12天、13天、14天、15天、20天、25天或30天；(7)继续培养复氧后的细胞，使活着的细胞不断增殖至充满细胞培养瓶底，开始传代，从而成功建立草地贪夜蛾细胞系，得到本发明的高产杆状病毒的草地贪夜蛾的蛹卵巢细胞系；整个过程都是在无菌条件下进行的。
在一个具体的实施方案中，本发明上述方法中所使用细胞培养液是常规采用的昆虫细胞培养液与青霉素、链霉素和胎牛血清的混合物，配成的细胞培养液pH为5.8-6.8，例如，5.8-5.9、5.9-6.0、6.0-6.1、6.0-6.2、6.0-6.3、6.0-6.4、6.0-6.5、6.0-6.6、6.0-6.7、6.0-6.8。昆虫细胞培养液是商品化的昆虫细胞培养液Insect-XPRESS TM，TNM-FH，Grace’s，Sf-900，TC-100，IPL-41，或Ex-Cell 400。通常，细胞培养液中青霉素含量为10U/mL-500U/mL，优选为 100U/mL；链霉素含量为10g/mL-500g/mL，优选为100g/mL；动物血清含量为所述细胞培养液的5％-20％的体积比，优选为细胞培养液的10％的体积比。
本申请另一方面提供了一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系在杆状病毒生产中的用途。
在一个具体的实施方案中，所述杆状病毒是苜蓿银纹夜蛾核型多角体病毒(AcMNPV)，草地贪夜蛾核型多角体病毒(SfMNPV)。
本申请另一方面提供了一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系在制备杆状病毒杀虫剂中的用途。
本申请另一方面提供了一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系在制备杆状病毒表达载体系统中的用途。
实施例
实施例1.草地贪夜蛾蛹卵巢细胞系的建立
取发育中期的草地贪夜蛾蛹(中国科学院动物研究所农业虫鼠害综合治理国家重点实验室昆虫病毒学研究组饲养)浸没在体积分数为75％的乙醇溶液中10分钟，进行表面消毒。解剖该昆虫取出卵巢组织，操作时尽量保持其完整。用生理盐水清洗该组织2-3次，再用细胞培养液(Insect-XPRESS TM(BioWhittaker,Lonza,Walkersville,MD,USA,12-730Q)，含100U/mL的青霉素，100g/mL的链霉素和10％(v/v)的胎牛血清(BI,Beit Haemek,Israel,04-001-1)，pH＝6.2；清洗1-2次，放入使用1mL上述细胞培养液润洗过的25cm 2的细胞培养瓶中，放入摄氏27℃无光照的细胞培养箱中培养24小时。然后加入3mL上述细胞培养液，放入同样条件下培养。在此操作第2-3天后可以观察到组织周围游离出大量单个的细胞，并逐渐向外围扩展(图1)。在原代培养21天左右细胞逐渐增殖并充满整个培养瓶，将密闭瓶盖更换成透气瓶盖，放入三气培养箱中，通入氮气并控制氧气浓度为5％，在此低氧条件下培养9天后，更换新鲜培养液及密闭瓶盖，在复氧后的条件下培养30天左右把含有新增殖出的细胞，连同全部的细胞培养液吸出放入新培养瓶中，并加入2mL新的上述细胞培养液。细胞系建立初步成功。在细胞开始传代的第7天后开始第二次分瓶传代，以后传代时间逐渐缩短，传到第8代 时，细胞生长开始稳定，最终该细胞系被命名为IOZCAS-Sf-1。IOZCAS-Sf-1于2020年11月25日保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.21014。
实施例2.IOZCAS-Sf-1的生物学特性观察和测定
(1)形态特征：经显微镜观察，IOZCAS-Sf-1细胞系包含悬浮于培养液中的细胞及贴壁细胞，细胞的形状有2种类型：圆形和梭形。IOZCAS-Sf-1中圆形细胞占69.2％，平均直径7.98±0.105(平均值±标准差)μm；梭形细胞占30.8％，长度是10.13±0.127μm，宽度是6.50±0.0.101μm(图2)。
(2)细胞的生长：以2×10 5细胞/mL的浓度将IOZCAS-Sf-1接种到24孔板中，在27℃培养，按照McIntosh and Ignoffo,1989(McIntosh,A.H.and C.M.Ignoffo J.Invertebr.Pathol.1989,54:97～102)的方法，测定第20代细胞生长曲线和群体倍增时间。即，每24h测定细胞浓度，绘制生长曲线(图4)，并按公式T＝tlg2/[lg(N/N 0)]计算细胞群体倍增时间(其中，T＝在对数期平均增长一倍所需的时间；t＝接种到测定细胞数的时间；N 0＝接种时的细胞数；N＝时刻t所测定的细胞总数)。IOZCAS-Sf-1细胞系第20代在含10％胎牛血清、100U/mL的青霉素、100g/mL的链霉素的Insect-XPRESS TM培养液中，第20代细胞群体倍增时间为46.47小时。目前该细胞系已经传代超过30代。
(3)使用COI测序的方法鉴定：细胞系IOZCAS-Sf-1确是来源于草地贪夜蛾，而非其它细胞系及Sf9细胞的污染。由IOZCAS-Sf-1提取的DNA与草地贪夜蛾幼虫血DNA进行PCR扩增，所使用的COI基因扩增引物为(上游引物：5’-3’SEQ ID NO 1：TTCGAGCTGAATTAGGGACTC，下游引物5’-3’SEQ ID NO 2：GATGTAAAATATGCTCGTGT)(图3)。
(4)使用流式细胞仪检测细胞染色体倍数。以草地贪夜蛾幼虫血细胞作为二倍体对照。收集细胞，在4℃、70％乙醇中固定过夜。0.1mg/mL碘化丙啶(PI)(Solarbio,Beijing,CA1630-500T)于37℃下孵育30min，过滤后采用流式细胞仪(BD Biosciences,East Rutherford,NJ,USA,LSR Fortessa)检测。在相同电压下，以幼虫血细胞为二倍体对照，IOZCAS-Sf-1的第一个荧光强度峰值均幼虫血细胞的4倍，同时Sf9细胞为多倍体对照。因此，确定IOZCAS-Sf-1为8倍体(图5)。
(5)冻存和复苏：使用传统细胞冻存的方法对细胞进行冻存处理，保存细胞的种资，并可以成功复苏。
实施例3.IOZCAS-Sf-1对杆状病毒敏感性的测定
IOZCAS-Sf-1对苜蓿银纹夜蛾核型多角体病毒(AcMNPV)敏感：将AcMNPV出芽型病毒粒子BV(草地贪夜蛾幼虫取食AcMNPV多角体3天后，取幼虫血淋巴，去除血细胞后的上清溶液，由中国科学院动物研究所保藏)以MOI(感染复数，multiplicity of infection)＝1的浓度接种IOZCAS-Sf-1，培养7天后，可以收获细胞及病毒多角体，感染率约为56.4％，在倒置相差显微镜下，可以观察到典型的细胞病理特征，即细胞核增大，内含大量的病毒多角体颗粒(图6)。

Claims (13)

  1. 一种高产杆状病毒的草地贪夜蛾蛹卵巢细胞系，该细胞系的名称为IOZCAS-Sf-1，其保藏号为CGMCC No.21014。
  2. 根据权利要求1所述的卵巢细胞系的制备方法，其包括以下步骤：
    1)用细胞培养液培养草地贪夜蛾卵巢细胞；和
    2)获得高产杆状病毒的草地贪夜蛾卵巢细胞系。
  3. 如权利要求2所述的方法，其中所述细胞培养液是昆虫细胞培养液与抗菌素的混合物。
  4. 如权利要求3所述的方法，其中所述抗菌素为青霉素和链霉素。
  5. 如权利要求2所述的方法，其中所述细胞培养液还补充有胎牛血清。
  6. 如权利要求4所述的方法，其中，所述青霉素含量为10U/mL-500U/mL，优选为100U/mL；所述链霉素含量为10g/mL-500g/mL，优选为100g/mL。
  7. 如权利要求5所述的方法，其中，所述胎牛血清含量为所述细胞培养液的5％-20％的体积比，优选为所述细胞培养液的10％的体积比。
  8. 如权利要求2所述的方法，其中所述细胞培养液具有5.8-6.8的pH。
  9. 如权利要求2所述的方法，其中，所述昆虫细胞培养液选自：Insect-XPRESS TM、TNM-FH、Grace’s、Sf-900、TC-100、IPL-41或Ex-Cell 400。
  10. 一种权利要求1所述的卵巢细胞系在杆状病毒生产中的用途。
  11. 如权利要求4所述的用途，其中所述杆状病毒是苜蓿银纹夜蛾核型多角 体病毒(AcMNPV)和草地贪夜蛾核型多角体病毒(SfMNPV)。
  12. 一种权利要求1所述的卵巢细胞系在制备杆状病毒杀虫剂中的用途。
  13. 一种权利要求1所述的卵巢细胞系在制备杆状病毒表达载体系统中的用途。
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