WO2020238458A1 - 用于表达e2蛋白的细胞株及其应用，e2蛋白及其应用 - Google Patents

用于表达e2蛋白的细胞株及其应用，e2蛋白及其应用 Download PDF

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WO2020238458A1
WO2020238458A1 PCT/CN2020/085026 CN2020085026W WO2020238458A1 WO 2020238458 A1 WO2020238458 A1 WO 2020238458A1 CN 2020085026 W CN2020085026 W CN 2020085026W WO 2020238458 A1 WO2020238458 A1 WO 2020238458A1
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protein
application
vaccine
swine fever
seq
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郭伟伟
刘大卫
向银辉
王玉红
窦小龙
刘蕾
范根成
杜元钊
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青岛易邦生物工程有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This application belongs to the technical field of veterinary biological products, and specifically relates to a cell strain for expressing E2 protein and its application, E2 protein and the prepared swine fever E2 protein subunit vaccine.
  • Swine fever is an acute, severe, and highly contagious infectious disease caused by Classical Swine Fever Virus (CSFV), causing significant losses to the world's swine industry.
  • CSFV Classical Swine Fever Virus
  • the attenuated live vaccine stimulates the body to produce antibodies, which is difficult to distinguish from the antibodies produced by wild virus-infected pigs, which is not conducive to the control of swine fever.
  • Popularity and purification The development of swine fever subunit vaccines can distinguish between vaccine-immunized animals and infected animals, and can facilitate the purification of swine fever virus.
  • the epidemic strains of Chinese swine fever can be divided into 2 gene groups and 4 gene subgroups, namely 2.1, 2.2, 2.3 and 1.1 gene subgroups. Studies have shown that there are large differences in antigen genes between the swine fever strains circulating in a large area in China, the traditional Shimen virulent strains and the attenuated vaccine strains.
  • the traditional Chinese lapinized attenuated vaccine strain belongs to the 1.1 gene subgroup, but the wild virus of this subgroup only accounts for a small proportion, and it mostly occurs in relatively remote free-range areas.
  • the main epidemic strains of Chinese swine fever virus are gene group 2.
  • the epidemiological investigation of swine fever virus found that the currently widely used swine fever traditional 1.1 gene subgroup attenuated vaccine cannot produce good immune protection against swine fever virus group 2 which is currently widespread.
  • This application provides a cell line for expressing E2 protein and its application, as well as an E2 protein and its application.
  • the swine fever E2 protein subunit vaccine prepared in this application can provide good immune protection for pigs.
  • the present application provides a cell line for expressing E2 protein, which is an E2-CHO cell line containing a nucleotide fragment encoding E2 protein; wherein, the E2 protein has amino acids
  • the sequence is SEQ ID NO: 3.
  • sequence of the nucleotide fragment is SEQ ID NO: 4.
  • this application provides an application of the E2-CHO cell strain in the recombinant expression of the E2 protein of classical swine fever virus.
  • this application provides an E2 protein whose amino acid sequence is SEQ ID NO: 3.
  • the sequence of the nucleotide fragment encoding the E2 protein is SEQ ID NO: 4.
  • the present application provides a subunit vaccine prepared from the E2 protein whose amino acid sequence is SEQ ID NO: 3.
  • the sequence of the nucleotide fragment encoding the E2 protein is SEQ ID NO: 4.
  • this application provides an application of the E2 protein in preparing vaccines.
  • this application provides a swine fever virus subunit vaccine, including an antigen and a vaccine adjuvant, and the antigen used is the E2 protein prepared in this application.
  • the subunit vaccine prepared from swine fever E2 protein in at least one embodiment of this application has the characteristics of high antigen stability, high purity, strong specificity, no other irrelevant antibodies, convenient and accurate detection methods, and can be used for the production of swine fever subunits.
  • Unit vaccines and diagnostic reagents have laid a solid foundation.
  • the E2 envelope glycoprotein is the main structural protein of the swine fever virus. It is the main determinant of the virulence and host range of the swine fever virus. It is located on the outer surface of the virus particle and produces virus neutralizing antibodies to protect immune pigs from swine fever virus.
  • the main antigen protein therefore, this application uses the swine fever virus E2 protein as an important protein molecule for the development of a new vaccine of swine fever virus.
  • the subunit vaccine developed by eukaryotic cells expressing recombinant E2 protein antigen is used to distinguish the immune response induced by natural virus infection and the immune response induced by subunit vaccine. This labeled vaccine will promote the purification of swine fever virus.
  • this application optimizes the E2 protein gene sequence of the isolated swine fever gene group 2 epidemic strains, so that the CHO cell (Chinese hamster ovary cell) expression system can be used to express the recombinant E2 protein; genetic engineering prepared by emulsification with vaccine adjuvants
  • the subunit vaccine has good safety and efficacy in immunizing pigs, has a reliable protective effect on epidemic strains, and can be used as a marker vaccine for swine fever virus purification.
  • a pig farm that had been injected with the existing swine fever virus vaccine (the vaccine is a 1.1-genotype whole virus live vaccine) showed typical symptoms of swine fever.
  • the spleens of dead pigs were collected aseptically, homogenized with sterile normal saline to prepare a suspension, centrifuged to obtain the supernatant, and sterilized, the supernatant was inoculated into ST (swine testis) cells. The virus supernatant was collected once in 96 hours, and the virus supernatant was collected twice after 72 hours.
  • the harvested virus liquid was purified to obtain a virus strain (referred to as a virus strain), which was analyzed and tested for virus characteristics in terms of virus content, immunogenicity, specificity, and purity. The results showed that the isolated virus strain and pig Plastivirus serum has a specific reaction without contamination by bacteria, mycoplasma and foreign viruses.
  • the traits of the strains screened in this example were tested, and the results showed that the strains belonged to swine fever virus.
  • the structural protein-E2 protein gene (referred to as E2 gene for short) of the screening and identification strains was sequenced.
  • the E2 gene is 1110 bp in length and encodes 370 amino acids. Its amino acid sequence is SEQ ID NO:1, the nucleotides of the coding gene The sequence is SEQ ID NO: 2; it is analyzed by genetic tree and nucleotide sequence homology analysis with the E2 gene sequence included in GenBank, and it is found that the E2 amino acid of the virus strain obtained in this example is similar to the currently used Shimen virulent The homology is about 87%, and the homology with the popular 2.1d genotype amino acid is about 98%. The results indicate that the swine fever virus has mutated, and the 1.1 type vaccine can no longer protect the popular swine fever strain.
  • the codon of the E2 gene is optimized, and the modified amino acid sequence is SEQ ID NO: 3, which can express the antigen site well.
  • the nucleotide sequence encoding the amino acid sequence SEQ ID NO: 3 is codon optimized, and the nucleotide sequence of the optimized gene is SEQ ID NO: 4.
  • sequence SEQ ID NO: 4 was synthesized by Shanghai Bioengineering Co., Ltd. and connected to the puc57 vector to obtain puc57-seq4.
  • the vectors in the table select pEE14.4 vector (plasmid), one of puc57-seq4 and puc57-seq1, and puc57-seq1 can be used as a control group. Enzyme digestion reactions were performed on the three vectors respectively.
  • step (2) Put the 1.5mL EP tube in step (1) in a 37°C constant temperature water bath for 2-3 hours; obtain three double enzyme digestion systems respectively.
  • the plasmid fragment and the optimized DNA fragment of the E2 gene can be obtained separately.
  • step (3) Add 600 ⁇ L extraction buffer to the 1.5 mL centrifuge tube in step (3), and place it in a 50°C water bath for about 5 minutes. During this time, gently turn the centrifuge tube up and down to ensure that the gel mass is fully dissolved and the first solution is obtained.
  • step (4) Add the first solution obtained in step (4) to the adsorption column, then centrifuge at 10,000 rpm for 30 s, discard the waste liquid in the collection tube, and then put the adsorption column into the collection tube.
  • step (10) Take out the centrifuge tube in step (10) from the centrifuge, discard the middle adsorption column, cover the centrifuge tube lid, and keep the DNA sample in the centrifuge tube.
  • step 11 Store the DNA sample in step 11 at 4°C, and prepare an agarose gel electrophoresis identification gel to recover DNA fragments.
  • the linearized vector pEE14.4 and the target gene can be obtained separately for subsequent ligation reactions.
  • the gel recovery kit was purchased from Hangzhou Bori Technology Co., Ltd.
  • the inserted DNA fragment in the table is the target gene obtained in step 2.2 (12).
  • reaction product of step (4) can be directly subjected to a transformation experiment, or it can be stored at -20°C, and defrosted for transformation when needed.
  • step (1) After step (1) is completed, take out the sample tube, place it in a 42°C water bath for 100 seconds, and then immediately take an ice bath for 2 minutes.
  • step (2) After step (2) is completed, take out the sample tube, add 600 ⁇ L of liquid LB medium to the sample tube in the ultra-clean workbench, and then place the sample tube on a constant temperature shaker at 37°C, 220 rpm, and incubate for 1 hour.
  • step (6) Place the plate in step (5) in a biochemical constant temperature incubator, and after incubating at 37°C for 1 hour, invert the transformation plate for 15 hours; thereby obtaining a single colony (ie a single colony).
  • step (3) Add 250 ⁇ L of plasmid extraction reagent P1 buffer to the EP tube in step (2) to completely suspend the bacteria.
  • step (3) Add 250 ⁇ L P2 buffer to the solution in step (3), and immediately mix by gently inverting the centrifuge tube 5-10 times. Let stand at room temperature for 2-4 min.
  • step (4) Add 350 ⁇ L N3 buffer to the solution in step (4), and immediately mix by gently inverting the centrifuge tube 5-10 times. Let stand at room temperature for 2-4 min.
  • step (6) Centrifuge the solution of step (5) at room temperature at 14,000 rpm for 10 min.
  • step (7) Move the supernatant in step (6) to the center of the adsorption column, centrifuge at room temperature, 12,000 rpm, 30 seconds, and discard the liquid in the collection tube.
  • a relatively pure recombinant plasmid (14.4-E2 recombinant plasmid), that is, the DNA solution in step (10), can be obtained for the subsequent transfection process.
  • the plasmid extraction kit was purchased from QIAGEN.
  • the extracted plasmid template described in the table is the relatively pure 14.4-E2 recombinant plasmid obtained after the recombinant plasmid extraction in 2.5.1.
  • the G418 gene stored in the laboratory and the 14.4-E2 recombinant plasmid with PCR positive identification were digested with SmaI and EcoRI respectively, and then the DNA fragments obtained respectively were ligated, transformed, and identified.
  • the method is the same as step 2 in this example to construct a 14.4-E2-G418 recombinant plasmid.
  • the transfection reagent was purchased from Thermo Fisher Scientific Co., Ltd.
  • Example 3 The cells selected in Example 3 (ie, positive cells) were cultured at 37° C. for 240 hours, and the protein solution was collected every 48 hours for a total of 5 times. The protein solution collected 5 times was subjected to SDS-PAGE protein analysis. It was found that the optimized E2 gene was not expressed in CHO cells, while the optimized E2 gene was expressed in CHO cells.
  • the supernatant of E2 protein solution collected in step 1 was subjected to affinity chromatography with GE's Ni Sepharose excel medium. The collected supernatant was centrifuged at 5000 rpm for 5 min and used as the loading solution.
  • the column can hang 4mg protein per ml.
  • the color was developed in the chemiDOC gel imaging system. The result was that the whole virus band was very weak, and the optimized nuclear The E2 protein band of the gene with the nucleotide sequence SEQ ID NO: 4 expressed in CHO cells is very bright, indicating that the expressed E2 protein is highly immunogenic.
  • Emulsification Take the same weight of oil phase and water phase, emulsify at 10000r/min for 5 minutes. After emulsification, take 10ml, centrifuge at 3000r/min for 15 minutes, and the water at the bottom of the tube should not exceed 0.5ml to obtain the vaccine.
  • mice Ten large-eared white rabbits of 2.5 kg were divided into two groups. Five rabbits in the immunization group were injected subcutaneously with 0.2ml subunit vaccine, and the other five rabbits were not immunized as a blank control. 21 days after immunization, blood was collected from ear vein, serum was separated, and ELISA antibody detection was performed. The results showed that the antibodies of the immune group were all positive, and the blocking rate was higher than 60%, while the antibodies of the control group were all negative, and the blocking rate was below 30% (see Table 1 for details).
  • the first group is the immunized whole virus group, each intramuscular injection of 1.0ml of vaccine, 21 days after the first immunization with the same vaccine, the same dose for the second immunization;
  • the second group is the vaccine group already in the immunization market, each intramuscular injection of 1.0ml vaccine 21 days after the first immunization, the same vaccine and the same dose were used for the second immunization;
  • the third group was not immunized as a control group.
  • the E2 subunit vaccine of the present application has the best protection against epidemic strains, indicating that the epidemic strains have mutated, and the current vaccines on the market can no longer protect against epidemic strains. Vaccines against epidemic strains are imminent, and the E2 protein subunit vaccine provided by this application is safe, stable, and has good protective effects, and has a good market prospect.

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Abstract

提供了一种用于表达E2蛋白的细胞株及其应用，E2蛋白及其制得的猪瘟E2蛋白亚单位疫苗，所述疫苗中氨基酸序列为SEQ ID NO:3的抗原蛋白是使用重组猪瘟E2-CHO细胞株表达制备的。

Description

用于表达E2蛋白的细胞株及其应用，E2蛋白及其应用
本申请要求在2019年05月25日提交中国专利局、申请号为201910443709.1、申请名称为“一种猪瘟E2蛋白亚单位疫苗”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请属于兽用生物制品技术领域，具体涉及一种用于表达E2蛋白的细胞株及其应用，E2蛋白及其制得的猪瘟E2蛋白亚单位疫苗。
背景技术
猪瘟是由猪瘟病毒(Classical Swine Fever Virus,CSFV)引起的一种急性、烈性、高度接触性传染病，给世界养猪业造成重大损失。现有技术中应用的猪瘟病毒弱毒疫苗虽然免疫效力好、安全性高，但是弱毒活疫苗刺激机体产生的抗体，很难和野毒感染猪产生的抗体区分开，这不利于控制猪瘟的流行与净化。研发猪瘟亚单位疫苗能够区分疫苗免疫动物和感染动物，能为猪瘟病毒净化提供便利。
***瘟的流行毒株可分为2个基因群，4个基因亚群，即2.1、2.2、2.3和1.1基因亚群。研究表明，中国较大范围内流行的猪瘟毒株与传统的石门强毒毒株和疫苗用兔化弱毒毒株在抗原基因上存在较大的差异。中国传统的兔化弱毒疫苗株属于1.1基因亚群，但该亚群的野毒只占很少的比例，且多发于相对偏远的散养地区。随着猪场集约化养殖的快速发展，***瘟病毒的主要流行毒株为基因2群。猪瘟病毒流行病学调查发现，目前广泛使用的猪瘟传统1.1基因亚群弱毒疫苗对目前广泛流行的基因2群猪瘟病毒不能产生良好的免疫保护。
发明内容
本申请提供一种用于表达E2蛋白的细胞株及其应用，以及一种E2蛋白及其应用。本申请所制得的猪瘟E2蛋白亚单位疫苗，可以为生猪提供良好的免疫保护。
作为第一种实施方式，本申请提供了一种用于表达E2蛋白的细胞株，为包含有编码E2蛋白的核苷酸片段的E2-CHO细胞株；其中，所述的E2蛋白，其氨基酸序列为SEQ ID NO:3。
所述的核苷酸片段，其序列为SEQ ID NO:4。
作为第二种实施方式，本申请提供了一种所述E2-CHO细胞株在重组表达猪瘟病毒E2蛋白中的应用。
作为第三种实施方式，本申请提供了一种E2蛋白，所述E2蛋白的氨基酸序列为SEQ ID NO:3。编码所述E2蛋白的核苷酸片段的序列为SEQ ID NO:4。
作为第四种实施方式，本申请提供了一种氨基酸序列为SEQ ID NO:3的E2蛋白制备的亚单位疫苗。编码所述E2蛋白的核苷酸片段的序列为SEQ ID NO:4。
作为第五种实施方式，本申请提供了一种所述E2蛋白在制备疫苗中的应用。
作为第六种实施方式，本申请提供了一种猪瘟病毒亚单位疫苗，包括有抗原和疫苗佐剂，其所用的抗原为本申请所制备的E2蛋白。
本申请至少一个实施方式中的猪瘟E2蛋白制备的亚单位疫苗具有抗原稳定性高，纯度高，特异性强，不产生其他不相关抗体，检测方法方便准确的特点，可为生产猪瘟亚单位疫苗和诊断试剂奠定了坚实的基础。
具体实施方式
E2囊膜糖蛋白是猪瘟病毒的主要结构蛋白，是猪瘟病毒毒力及宿主范围的主要决定性因素，位于病毒粒子外表面，是产生病毒中和抗体，保护免疫猪免受猪瘟病毒攻击的主要抗原蛋白。因此，本申请以猪瘟病毒E2蛋白作为开发猪瘟病毒新型疫苗的重要蛋白分子。以真核细胞表达重组E2蛋白抗原研制的亚单位疫苗，用以区分自然毒感染所诱导的免疫应答和亚单位疫苗所诱导的免疫应答，这种标记疫苗将促进猪瘟病毒的净化。
具体地，本申请优化了分离的猪瘟基因2群流行毒株的E2蛋白基因序列，从而可以使用CHO细胞(Chinese hamster ovary cell)表达***表达重组E2蛋白；与疫苗佐剂乳化制备的基因工程亚单位疫苗，免疫猪安全性和效力均良好，对流行毒株起到可靠的保护作用，并可作为猪瘟病毒净化的标记疫苗。
以下结合具体实施例对本申请的技术方案进行详实的阐述，然而应当理解，在没有进一步叙述的情况下，一个实施例的实现也可以采用本领域技术人员能够想到的其他方式或手段来实现，例如且不限于实验参数的变动，实验试剂的替换等。本申请所应用的方法可以采用疫苗制备领域中常用的方法，而不仅限于本申请实施例的具体记载，本领域的普通技术人员可以其它常规方法来实现本申请。
实施例1、猪瘟病毒E2蛋白的筛选
2018年某猪场的已注射过现有猪瘟病毒疫苗(该疫苗为1.1基因型的全病毒活疫苗)的猪群中出现了典型的猪瘟发病症状。为了分离病原体，无菌采集死亡猪的脾脏，用无菌生理盐水匀浆制成悬液，离心取上清液，除菌后将上清液接种至ST(swine testis)细胞。96小时一次收取病毒液上清，再过72小时 二次收取病毒液上清。收获的病毒液经纯化后得到病毒毒株(简称为毒株)，对其进行病毒含量、免疫原性、特异性及纯净性等方面的病毒特性的分析检测，结果表明分离的毒株与猪瘟病毒血清发生特异性反应，无细菌、支原体及外源病毒污染。
对于本实施例筛选的毒株的性状进行检测，结果表明，该株病毒属猪瘟病毒，攻毒实验结果表明筛选的该病毒可引起猪死亡，解剖发现内脏器官出血、坏死和梗死。
对筛选鉴定毒株的结构蛋白-E2蛋白的基因(简称为E2基因)进行了测序，E2基因全长1110bp，编码370个氨基酸，其氨基酸序列为SEQ ID NO:1，编码基因的核苷酸序列为SEQ ID NO:2；将其与GenBank中收录的E2基因序列进行遗传进化树及核苷酸序列同源性分析，发现本实施例获得毒株的E2氨基酸与目前使用的石门强毒的同源性为87％左右，与流行的2.1d基因型氨基酸的同源性为98％左右。该结果表明，猪瘟病毒已发生变异，而1.1型的疫苗已经不能保护流行的猪瘟毒株。
实施例2、表达E2基因的重组质粒的构建
1.E2基因的剪切、优化
将E2基因的pestivirus_E2结构域进行剪切，将N端23aa剪切掉，C端32aa剪切掉，帮助蛋白更好地折叠为三级结构，将E2蛋白的抗原位点更好地暴露出来。同时将E2基因的密码子进行优化，优化修饰后的氨基酸的序列为SEQ ID NO:3，能很好地表达出抗原位点。将编码氨基酸序列SEQ ID NO:3的核苷酸序列进行密码子优化，优化后的基因的核苷酸序列为SEQ ID NO:4。
将序列SEQ ID NO:4由上海生物工程有限公司合成，并连入puc57载体， 得到puc57-seq4。
2. 14.4-E2重组质粒的构建
2.1酶切反应
(1)标记好需要用到的1.5mL EP管，在1.5mL EP管中按照下表进行加样、混匀；反应体系为50μL，加样如下表所示：
加样成分名称 体积(μL)
载体 2μg
限制性内切酶BstI 2.5
限制性内切酶HindIII 2.5
10X buffer 5
dd H 2O 补充至50
每次酶切反应时，表中所述载体分别选择pEE14.4载体(质粒)，puc57-seq4和puc57-seq1中的一种，其中puc57-seq1可以作为对照组。分别对所述三种载体进行酶切反应。
(2)将步骤(1)中的1.5mL EP管置于37℃恒温水浴锅中，水浴2-3h；分别得到三种双酶切体系。
2.2双酶切产物胶回收
分别取出上述双酶切体系，进行琼脂糖凝胶电泳以回收其中的DNA片段。可以分别得到质粒片段和优化后E2基因的DNA片段(含目的基因)。
(1)标记好样品收集EP管、吸附柱以及收集管。
(2)称取标记好的空的EP管重量，并记录数值。
(3)将单一的目的DNA条带在切胶仪上从琼脂糖凝胶中用手术刀小心切下放入干净的1.5mL离心管中。
(4)向步骤(3)中的1.5mL离心管中加入600μL extraction buffer，50℃水浴放置5min左右，其间不断温和上下翻转离心管，以确保胶块充分溶解，得到第 一溶液。
(5)将步骤(4)所得的第一溶液加至吸附柱中，随后以10,000rpm，离心30s，倒掉收集管中的废液，再将吸附柱放入收集管中。
(6)将吸附柱内加入600μL extraction buffer，随后以10,000rpm，离心30s，倒掉收集管中的废液，再将吸附柱放入收集管中。
(7)向吸附柱中加入600μL漂洗液wash buffer，静置3min，然后离心10,000rpm，30s，倒掉收集管中的废液，将吸附柱放入收集管中。
(8)重复步骤(7)。
(9)空吸附柱离心，12,000rpm，2min，尽量除去漂洗液。将吸附柱置于室温放置10min，彻底晾干。
(10)将吸附柱放入收集管中，向吸附膜中间位置悬空滴加50μL Elution buffer(65℃预热)，静置3min，然后离心12,000rpm，2min。
(11)从离心机中取出步骤(10)中离心管，丢弃中间的吸附柱，盖上离心管盖子，保留离心管中的DNA样品。
(12)将步骤11中的DNA样品置于4℃保存，准备琼脂糖凝胶电泳鉴定胶回收DNA片段。从而可以分别得到线性化载体pEE14.4和目的基因，用于后续的连接反应。
本实施例中，所述胶回收试剂盒购至杭州博日科技有限公司。
2.3连接反应
(1)标记需要用到的0.2mL离心管。
(2)在标记完整的0.2mL离心管中按照下表的20μL反应体系进行加样：
加样成分名称 体积(μL)
线性化载体pEE14.4 50～200ng
***DNA片段 20～200ng
连接酶 1
10X buffer 2
dd H 2O 补充至20
表中所述***DNA片段即为步骤2.2中的(12)所得到的目的基因。
(3)完成加样后，用移液器轻轻吹打几次混匀各组分。
(4)将0.2mL离心管置于37℃反应30min，待反应完成后，立即将反应管置于冰水浴中冷却5min；得到含有反应产物的连接反应液，所述反应产物即为载有目的基因的质粒。
(5)步骤(4)的反应产物可直接进行转化实验，也可储存于-20℃，待需要时解冻转化。
2.4转化反应
(1)将10μL连接反应液快速加入位于样品管中的100μL感受态细胞(本实施例选用大肠杆菌DH5α感受态细胞)中，并吹打混匀，冰浴30min。
(2)步骤(1)完成后，取出样品管，置于42℃水浴100s，然后立即冰浴2min。
(3)步骤(2)完成后，取出样品管，在超净工作台中，向样品管中加入600μL液体LB培养基，然后将样品管置于37℃恒温摇床，220rpm，培养1h。
(4)制备转化平板，依据质粒抗性制备转化用LB抗性平板。
(5)涂板：取出步骤(3)中样品管，室温离心8,000rpm，2min，去掉600μL上清液，剩余上清液重悬管底部的菌体，将重悬的菌液放入相应的转化平板中心，用涂菌棒将转化平板中心的菌液均匀铺开。
(6)将步骤(5)平板正置于生化恒温培养箱中，37℃培养1h后，将转化平板倒置进行培养15h；从而得到单克隆(即单菌落)。
(7)观察记录转化结果。
2.5重组质粒抽提与PCR鉴定
2.5.1重组质粒抽提
(1)用10μL移液枪头从转化平板中挑取单克隆(含重组质粒)至5ml含氨苄抗性的LB液体培养基中，37℃，220rpm摇菌过夜。
(2)吸取菌液至1.5mL EP管中，室温离心，12,000rpm，2min，弃上清液。
(3)向步骤(2)中的EP管中加入250μL质粒提取试剂P1 buffer，彻底悬浮菌体。
(4)向步骤(3)溶液中加入250μL P2 buffer，立即温和颠倒离心管5-10次混匀。室温静置2-4min。
(5)向步骤(4)溶液中加入350μL N3 buffer，立即温和颠倒离心管5-10次混匀。室温静置2-4min。
(6)将步骤(5)溶液，室温离心，14,000rpm，10min。
(7)将步骤(6)中上清液移至吸附柱中心，室温离心，12,000rpm，30s，倒掉收集管中液体。
(8)向吸附柱中心加入500μL PE buffer，室温离心，12,000rpm，30s，倒掉收集管中液体。重复一次。
(9)空吸附柱，室温离心，12,000rpm，2min。
(10)将吸附柱放入一个干净的1.5ml离心管中，向吸附膜中心加入30μL Elution buffer，室温静置5min，室温离心，12,000rpm，2min，4℃保存管中DNA溶液。
在本步骤中，通过扩大培养以及重组质粒抽提，可以得到相对纯净的重组质粒(14.4-E2重组质粒)，即步骤(10)中的DNA溶液，以用于后续转染过程。
本实施例中，所述质粒提取试剂盒购至QIAGEN公司。
2.5.2 PCR鉴定
(1)标记好需要用到的PCR管，按照下表进行加样、混匀，反应体系为25μL：
加样成分名称 体积(μL)
上游引物(10μM) 0.5
下游引物(10μM) 0.5
提取的质粒模板 0.5
2X Mix 12.5
dd H 2O 补充至25
表中所述的提取的质粒模板即为2.5.1中重组质粒抽提后得到的相对纯净的14.4-E2重组质粒。
(2)PCR扩增程序：
Figure PCTCN2020085026-appb-000001
(3)测序：将pcr鉴定阳性的14.4-E2重组质粒送测序公司进行测序。测序结果证明：重组质粒中***的核苷酸与序列表SEQ ID NO:4一致，其翻译的氨基酸与序列表SEQ ID NO:3一致。
3 14.4-E2-G418重组质粒的构建
14.4-E2-G418重组质粒的构建方法同本实施例中的步骤2。
具体地，将实验室保存的G418基因、pcr鉴定阳性的14.4-E2重组质粒分别用SmaI和EcoRI进行酶切，然后对于分别得到的DNA片段进行连接、转化、 鉴定。方法同本实施例中的步骤2，从而构建得到14.4-E2-G418重组质粒。
实施例3：CHO-K1细胞的转染
(1)准备：生物安全柜紫外灭菌30min；F-12培养液置于37℃水浴锅预热至37℃。
(2)将2.5μg重组DNA(即14.4-E2-G418重组质粒)、P3000(转染试剂)加入到125μl OPTI-MEM培养液中，混匀。将3.75μl脂质体lipotectamine 3000加入到125μl OPTI-MEM培养液中，混匀。将脂质体与重组DNA混合，室温静止10min。
(3)从37℃培养箱中取出24小时前平铺的6孔板细胞(CHO-K1细胞)，弃去上清培养基，用预温的OPTI-MEM培养液洗细胞三次，并弃去OPTI-MEM培养液。
(4)每个细胞孔加入2ml 10％、1％双抗的胎牛血清的F-12培养液。
(5)将重组DNA与脂质体的混合物轻轻加入到每孔细胞中，轻轻混匀，在37℃，5％CO2细胞培养箱中培养。
(6)CHO-K1细胞转染后72小时，收取上清液，进行蛋白检测，同时将细胞加入G418进行加压筛选。
(7)144小时后，得到单克隆细胞；将单克隆细胞进行检测，将阳性的细胞(即重组CHO细胞)进行扩增。可用于大生产制备蛋白。
本实施例中，所述转染试剂购至赛默飞世尔科技有限公司。
实施例4：蛋白纯化与检测
1.重组E2蛋白在CHO细胞内的表达与鉴定
将实施例3挑选的细胞(即阳性的细胞)，37℃培养至240小时，每48 小时收集蛋白液，共收集5次，将5次收集的蛋白液进行SDS-PAGE蛋白分析。结果发现优化前的E2基因在CHO细胞中没有表达，而优化的E2基因在CHO细胞中有表达。
2.重组E2蛋白的层析纯化
将步骤1收集的E2蛋白液的上清液，用GE的Ni Sepharose excel介质进行亲和层析。收集的上清液，以5000rpm离心5min，作为上样液。
2.1用5倍柱体积的蒸馏水清洗介质。
2.2用5倍柱体积的平衡缓冲液清洗介质。
2.3上样，柱子每毫升可挂4mg蛋白。
2.4用20倍柱体积的洗涤缓冲液清洗介质。
2.5用5倍柱体积的洗脱缓冲液将蛋白洗脱。
3 Westernblot试验
将猪瘟全病毒和本实施例步骤2制备的蛋白同时进行SDS-PAGE，采用半干法20伏转印30min，将目的蛋白条带转移到PVDF(polyvinylidene fluoride)膜，转印膜用封闭液封闭过夜，磷酸盐吐温缓冲液(PBST)洗涤3次，1∶500稀释的猪瘟病毒阳性血清37℃作用1.5h，PBST洗3次，用1∶2000稀释的辣根过氧化物酶(HRP)标记的羊抗猪酶标抗体37℃作用1.5h，PBST洗涤3次，底物溶液作用5min，在chemiDOC凝胶成像***进行显色，结果全病毒条带很弱，而优化后的核苷酸序列为SEQ ID NO:4的基因在CHO细胞内表达的E2蛋白条带很亮，说明表达的E2蛋白免疫原性好。
4间接免疫荧光
对细胞(即重组CHO细胞)进行固定、透明、封闭等步骤之后，加入His 标签单抗，37℃孵育2h后，使用PBS洗涤3次，加入绿色荧光二抗37℃孵育1h。使用倒置荧光显微镜进行观察并拍照保存。结果转染优化前的E2基因的CHO细胞没有特异性荧光，转染优化后的E2基因的CHO细胞特异性荧光强。
实施例5疫苗的制备及效果检测
1、疫苗的制备
(1)将201佐剂116℃高压40分钟，进行灭菌。使用前37℃水浴至201佐剂温度维持在37℃左右。
(2)水相制备：将实施例4中步骤2得到的纯化的蛋白液与无菌生理盐水适当比例混合，使每0.2ml水相中含蛋白含量不低于10ug/ml。
(3)乳化：取相同重量的油相和水相，以10000r/min，乳化5分钟。乳化后，取10ml，以3000r/min离心15分钟，管底析出的水相应不超过0.5ml，从而得到疫苗。
2、疫苗的效力检验
1)对兔子的免疫检测法
将2.5kg的大耳白兔10只，分两组。免疫组5只兔，皮下注射0.2ml的亚单位疫苗，另5只不免疫做空白对照。免后21日，耳静脉采血，分离血清，进行ELISA抗体检测。结果显示，免疫组抗体均为阳性，阻断率均高于60％，而对照组抗体均为阴性，阻断率均在30％以下(详见表1)。
表1亚单位疫苗免疫兔后21日抗体检测
Figure PCTCN2020085026-appb-000002
Figure PCTCN2020085026-appb-000003
注：“/”指对照组未免疫；
2)对猪的免疫原性结果
用健康易感(猪瘟抗原、ELISA抗体均为阴性)猪15头，随机分为3组，每组5头。第1组为免疫全病毒组，各肌肉注射疫苗1.0ml，一免后21日用相同的疫苗，同等剂量进行二免；第2组为免疫市场已有的疫苗组，各肌肉注射疫苗1.0ml，一免后21日用相同的疫苗，同等剂量进行二免；第三组不免疫作为对照组。二免后14日，采血，检测猪瘟病毒ELISA抗体，然后所有猪各颈部肌肉注射实施例1分离的毒株1.0ml(含不少于105.0MLD)，观察16日。观察期内如有死亡猪，立即剖检，剩余猪试验结束时(攻毒后16日)全部剖检，记录试验结果。
结果表明亚单位组、市场疫苗组5头仔猪抗体均为阳性，阻断率分别为80％、60％、，对照组抗体均为阴性，阻断率均在30％以下。攻毒后，对照全部死亡，亚单位组均保护，而市场疫苗组3只保护，2只发病。(详见表3)。
表3：表达E2蛋白免疫原性试验猪一免后35日抗体检测结果
Figure PCTCN2020085026-appb-000004
Figure PCTCN2020085026-appb-000005
注：“1.0+1.0”指免疫组免疫两次，每次1.0ml/头；“/”指对照组未免疫。
综上，本申请的E2亚单位疫苗对于流行毒株的攻毒防护效果最好，表明流行毒株已发生了变异，目前市场的疫苗已经不能保护流行毒株的攻击。针对流行毒株的疫苗迫在眉睫，而本申请提供的E2蛋白亚单位疫苗安全、稳定、保护效果好，市场前景很好。
所述的实施方式仅仅是对本申请的优选实施方式进行描述，并非对本申请的范围进行限定，在不脱离本申请设计精神的前提下，本领域普通技术人员对本申请的技术方案作出的各种变形和改进，均应落入本申请权利要求书确定的保护范围内。
Figure PCTCN2020085026-appb-000006
Figure PCTCN2020085026-appb-000007
Figure PCTCN2020085026-appb-000008
Figure PCTCN2020085026-appb-000009
Figure PCTCN2020085026-appb-000010
Figure PCTCN2020085026-appb-000011

Claims (10)

  1. 一种用于表达E2蛋白的细胞株，其中，所述细胞株为包含有编码E2蛋白的核苷酸片段的E2-CHO细胞株；所述E2蛋白的氨基酸序列为SEQ ID NO:3。
  2. 根据权利要求1所述的细胞株，其中，所述核苷酸片段的序列为SEQ ID NO:4。
  3. 一种权利要求1所述的细胞株在重组表达猪瘟病毒E2蛋白中的应用。
  4. 根据权利要求3所述的应用，其中，编码所述E2蛋白的核苷酸片段的序列为SEQ ID NO:4。
  5. 一种E2蛋白，其中，所述的E2蛋白的氨基酸序列为SEQ ID NO:3。
  6. 根据权利要求5所述E2蛋白，其中，编码所述E2蛋白的核苷酸片段的序列为SEQ ID NO:4。
  7. 一种权利要求5所述的E2蛋白在制备亚单位疫苗中的应用。
  8. 根据权利要求7所述的应用，其中，编码所述E2蛋白的核苷酸片段的序列为SEQ ID NO:4。
  9. 一种猪瘟病毒亚单位疫苗，其中，所述的亚单位疫苗包含有抗原和疫苗佐剂，其所用的抗原为权利要求5所述的E2蛋白。
  10. 根据权利要求9所述的疫苗，其中，编码所述E2蛋白的核苷酸片段的序列为SEQ ID NO:4。
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