WO2023125739A1 - 一种病毒样颗粒及其制备方法和应用 - Google Patents

一种病毒样颗粒及其制备方法和应用 Download PDF

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WO2023125739A1
WO2023125739A1 PCT/CN2022/143081 CN2022143081W WO2023125739A1 WO 2023125739 A1 WO2023125739 A1 WO 2023125739A1 CN 2022143081 W CN2022143081 W CN 2022143081W WO 2023125739 A1 WO2023125739 A1 WO 2023125739A1
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virus
seq
mouth disease
amino acid
acid sequence
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French (fr)
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郭慧琛
孙世琪
董虎
穆素雨
白满元
何融泽
张韵
吴金恩
丁耀忠
冯霞
尹双辉
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中国农业科学院兰州兽医研究所
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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
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • 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
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention belongs to the technical field of agricultural science, animal husbandry and veterinary medicine, and in particular relates to a virus-like particle and its preparation method and application.
  • Swine vesicular disease virus is a contagious viral disease of pigs. It causes vesicular lesions that are indistinguishable from those observed with FMD. Infection with SVDV can lead to viremia within 1 day and clinical signs can appear 2 days after pigs come into contact with infected pigs or virus-contaminated environments. 3 to 5 hours after infection, the virus can be detected by immunohistochemistry.
  • Virus-like particles are a new form of genetically engineered subunits that have emerged in recent years.
  • the conformation-dependent antigenic epitope can be presented correctly; it can bind to cell surface receptors and enter cells like natural virus particles, thereby inducing a strong immune response; on the basis of not affecting the structure of VLPs, some can be inserted or deleted as needed
  • the amino acid sequence is artificially modified to realize the pleiotropic characteristics of simultaneous immunity to multiple viruses. Therefore, VLPs, as a vaccine, is considered to be the best candidate vaccine form that can replace the whole virus vaccine at present.
  • the object of the present invention is to provide a virus-like particle and its preparation method and application, which have the advantages of high yield and good immunogenicity.
  • the invention provides a method for preparing virus-like particles, comprising the following steps:
  • VPO gene The viral structural protein gene VPO gene, VP1 gene and VP3 gene were respectively fused with the small ubiquitin-like modification protein gene sequence, and the obtained fusion genes were respectively cloned into the pET-28a vector to obtain pET/VP0-VP1 and pET/VP3;
  • Said virus comprises porcine vesicular disease virus and/or South African type foot-and-mouth disease virus;
  • step 2) replacing the kanamycin resistance gene in pET/VP3 described in step 1) with the ampicillin resistance gene to obtain pETa/VP3;
  • step 4) Digest the fusion proteins of the three small ubiquitinated proteases described in step 3) to remove small ubiquitinated tagged proteins, collect the recombinant proteins VPO, VP1 and VP3, and assemble them in vitro to obtain virus-like particles.
  • nucleotide sequence of the small ubiquitin-like modifier protein gene in step 1) is shown in SEQ ID NO:1.
  • the multiple cloning sites of the pET-28a vector described in step 1) are Sal I/BamH I and Hind III/Xho I restriction sites.
  • the invention provides the virus-like particle prepared by the preparation method, and the virus-like particle is composed of virus structural proteins VP1, VPO and VP3.
  • the amino acid sequence of VPO is as shown in SEQ ID NO:18; the amino acid sequence of VP1 is as shown in SEQ ID NO:19; the amino acid sequence of VP3 is as shown in SEQ ID NO:20 Show.
  • the virus is South African type foot-and-mouth disease virus
  • the South African type foot-and-mouth disease virus includes one or more of the following serotypes of foot-and-mouth disease virus: SAT1 type foot-and-mouth disease virus, SAT2 type foot-and-mouth disease virus and SAT3 type foot-and-mouth disease virus;
  • the amino acid sequence of the SAT1 type foot-and-mouth disease virus VPO is shown in SEQ ID NO:21; the amino acid sequence of VP1 is shown in SEQ ID NO:22; the amino acid sequence of VP3 is shown in SEQ ID NO:23;
  • amino acid sequence of the SAT2 type foot-and-mouth disease virus VP0 is shown in SEQ ID NO:24; the amino acid sequence of VP1 is shown in SEQ ID NO:25; the amino acid sequence of VP3 is shown in SEQ ID NO:26;
  • the amino acid sequence of the SAT3 type foot-and-mouth disease virus VPO is shown in SEQ ID NO:27; the amino acid sequence of VP1 is shown in SEQ ID NO:28; the amino acid sequence of VP3 is shown in SEQ ID NO:29.
  • the invention provides the application of the virus-like particles in the preparation of vaccines for preventing and controlling viral diseases, and the viral diseases include porcine vesicular disease and/or foot-and-mouth disease.
  • the invention provides a virus-like particle vaccine for preventing and controlling viral diseases, comprising the virus-like particle and an adjuvant.
  • the preparation method of the virus-like particles provided by the invention uses small ubiquitin-like modified proteins as tag proteins to promote the correct folding of the structural proteins VPO, VP1 and VP3 of porcine vesicular disease virus and/or South African type foot-and-mouth disease virus, while ensuring the structure of the protein Stability, to achieve a large amount of expression of structural proteins, after self-assembly of three structural proteins in vitro, a large number of virus-like particles with properties similar to those of natural porcine vesicular disease virus were obtained.
  • the virus-like particle prepared by the invention has high immunogenicity, and can be used as a reserve vaccine for the prevention and control of porcine vesicular disease virus transmission.
  • Fig. 1 is the SDS-PAGE detection result of SVDV recombinant capsid protein, swimming lane 1: fusion tag protein, swimming lane 2: SVDV capsid protein;
  • Fig. 2 is the electron micrograph (a) of SVDV recombinant virus reported in the literature, the electron micrograph (b) of the SVDV virus-like particle prepared by the present invention
  • Fig. 3 is the particle size distribution situation of the SVDV virus-like particle prepared by the present invention.
  • Fig. 4 is the fluorescent signal picture that adopts IFA method to identify virus-like particles
  • Fig. 5 is the detection result of ELISA antibody level in serum of pigs immunized with SVDV virus-like particles
  • Fig. 6 is the SDS-PAGE detection result of SAT1, SAT2, SAT3 type foot-and-mouth disease capsid protein
  • Fig. 7 is the particle size distribution situation of the SAT type foot-and-mouth disease virus-like particle prepared by the present invention.
  • Fig. 8 is the electron micrograph of the SAT type foot-and-mouth disease virus-like particle prepared by the present invention.
  • Fig. 9 is the detection of antibodies in guinea pig immune serum of SAT-type foot-and-mouth disease virus-like particles prepared by the present invention by IFA.
  • the invention provides a method for preparing virus-like particles, comprising the following steps:
  • VPO gene The viral structural protein gene VPO gene, VP1 gene and VP3 gene were respectively fused with the small ubiquitin-like modification protein gene, and the resulting fusion gene was cloned into the pET-28a vector to obtain pET/VP0-VP1 and pET/VP3;
  • Said virus comprises porcine vesicular disease virus and/or South African type foot-and-mouth disease virus;
  • step 1) co-transforming the pET/VPO-VP1 described in step 1) and the pETa/VP3 described in step 2) into a prokaryotic expression system, and then recombinantly expressed to obtain three small ubiquitination-modified protease-cleaved fusion proteins;
  • step 4) Digest the fusion proteins of the three small ubiquitinated proteases described in step 3) to remove small ubiquitinated tagged proteins, collect the recombinant proteins VPO, VP1 and VP3, and assemble them in vitro to obtain virus-like particles.
  • the virus structural protein gene VPO gene, VP1 gene and VP3 gene are respectively fused with the small ubiquitin-like modified protein sequence, respectively cloned into the pET-28a vector to obtain pET/VPO-VP1 and pET/VP3;
  • the viruses include Porcine vesicular disease virus and/or South African foot-and-mouth disease virus.
  • the fusion is preferably a seamless fusion, and the fusion gene is formed in the sequence of small ubiquitin-like modifier protein gene sequence-structural protein gene.
  • the nucleotide sequence of the small ubiquitin-like modified protein is shown in SEQ ID NO:1, and the amino acid sequence of the small ubiquitin-like modified protein is shown in SEQ ID NO:30.
  • the multiple cloning sites of the pET-28a vector are preferably Sal I/BamH I (VPO or VP3) and Hind III/Xho I (VP1) restriction sites.
  • the present invention has no special limitation on the cloning method, and cloning methods well known in the art can be used. After cloning, the resulting vectors were validated.
  • the present invention has no special limitation on the verification method of the vector, and the verification method of the recombinant vector well known in the art can be used.
  • the present invention has no special limitation on the source of the pET-28a vector, and the well-known pET-28a vector in the art can be used.
  • the pET-28a vector was purchased from Qingke Biotechnology Co., Ltd.
  • the multiple cloning site of VPO gene and VP3 gene in pET-28a carrier is preferably Sal I/BamH I, and the multiple cloning site of VP1 gene fragment in pET-28a carrier is preferably Hind III/Xho I .
  • the present invention has no special limitation on the cloning method, and the cloning method well known in the art can be used. After cloning, the obtained vector is preferably validated.
  • the present invention has no special limitation on the verification method of the vector, and the verification method of the recombinant vector well known in the art can be used.
  • the present invention replaces the kanamycin resistance gene in the pET/VP3 with the ampicillin resistance gene to obtain the vector pETa/VP3.
  • the method for replacing the kanamycin resistance gene in the pET/VP3 with the ampicillin resistance gene is preferably as follows: design 2 pairs of homologous recombination primers, wherein the ampicillin resistance gene primer: F: CAGTAATACAAGGGGTGTTATGTTACCAATGCTTAATCAGTGAGG (SEQ ID NO: 14); R: ATCCGCTCATGAATTAATTCTTAATGAGTATTCAACATTTCCGTG (SEQ ID NO: 15); PET28a linearization primer: F: CGGAAATGTTGAATACTCATTAAGAATTAATTCATGAGCGGATAC (SEQ ID NO: 16), R: CCTCACTGATTAAGCATTGGTAACATAACCCCCTT GTATTACTG (SEQ ID NO: 17).
  • the function of replacing the resistance gene is to carry out double resistance screening to ensure the uniform expression of capsid proteins VPO, VP1, and VP3.
  • the present invention co-transforms the pET/VPO-VP1 and the pETa/VP3 into a prokaryotic expression system, expresses them recombinantly, and separates three small ubiquitination-modified proteases Digest the fusion protein.
  • the present invention has no special limitation on the prokaryotic expression system, and the method of the prokaryotic expression system well-known in the art can be used.
  • the prokaryotic expression system is BL21(DE3).
  • the copy number ratio of pET/VPO-VP1 and pETa/VP3 is preferably 1-3:1-3, more preferably 1.5:1.
  • the transformed prokaryotic expression system is screened for positive clones by kanamycin and ampicillin, and positive clones are picked for double-antibody culture. After the cultured two bacterial solutions are mixed, high-density fermentation culture and induced expression are carried out.
  • the temperature of the cultivation is preferably 36-38° C., more preferably 37° C.; the shaking speed of the cultivation is preferably 800 rpm.
  • the OD 600nm of the bacterial solution is about 40 and enters into the induced expression.
  • the temperature for inducing expression is optionally 20-37°C, more preferably 30°C.
  • the final concentration of IPTG is preferably 1 mM when the expression is induced.
  • the separation is preferably centrifuged, broken cells, supernatant collected and mixed with Ni-NTA His ⁇ Bind Resins, eluted to obtain small ubiquitinated modified protease digestion fusion expression protein.
  • the present invention digests the three small ubiquitinated modified proteases to remove the small ubiquitinated modified tag proteins, collects the recombinant proteins of VPO, VP1 and VP3, and in vitro Assembled to obtain virus-like particles.
  • the method for removing the small ubiquitination-modified tag protein preferably includes enzymatic cleavage with a small ubiquitination modification protease.
  • the enzyme digestion system is preferably mixed with 100 ml of enzyme digestion buffer per 1 mg of the fusion protein.
  • the post-digestion solution preferably uses HisTrap HP chromatographic column to remove small ubiquitinated tagged proteins.
  • the composition of the solution for eluting is preferably 20mM Tris-HCl, 300mM NaCl, pH8.0.
  • the mass ratio of VPO, VP1 and VP3 recombinant proteins is 1:1:1.
  • the composition of the assembly buffer used is preferably 20 mM Tris-HCl, 500 mM NaCl, pH 8.0.
  • the temperature of the in vitro assembly is preferably 3-5°C, more preferably 4°C.
  • the time for the in vitro assembly is preferably 10-14 hours, more preferably 12 hours.
  • an ultrafiltration tube to concentrate the assembled solution and collect the retentate.
  • the molecular weight cut-off of the ultrafiltration tube is preferably 100KD.
  • sucrose density gradient separation is used to separate virus-like particles: 1.0ml of liquid containing virus-like particles (mutated and unmutated) is placed on the top layer of sucrose gradient with a concentration of 15% to 45%, and centrifuged at 38000rpm and 4°C 3.5h. Samples from 20 layers were collected for testing. The collected samples were detected in an ultraviolet detector (259nm) by continuous sampling method, and the 13th tube sample was taken for DLS detection to obtain the particle size of the virus-like particles.
  • the virus-like particles prepared by the preparation method provided by the present invention are composed of virus structural proteins VP1, VP0 and VP3, the diameters are mainly distributed between 20-30nm, the shape is complete, and the size is similar to natural virus particles resemblance.
  • the amino acid sequence of VP0 is shown in SEQ ID NO:18
  • the amino acid sequence of VP1 is shown in SEQ ID NO:19
  • the amino acid sequence of VP3 is shown in SEQ ID NO:20.
  • the virus is South African type foot-and-mouth disease virus; the South African type foot-and-mouth disease virus includes one or more serotypes of foot-and-mouth disease virus: SAT1 type foot-and-mouth disease virus, SAT2 type foot-and-mouth disease virus and SAT3 type foot-and-mouth disease virus.
  • the amino acid sequence of the SAT1 type foot-and-mouth disease virus VPO is shown in SEQ ID NO: 21; the amino acid sequence of VP1 is shown in SEQ ID NO: 22; the amino acid sequence of VP3 is shown in SEQ ID NO: 23; the SAT2 type foot-and-mouth disease
  • the amino acid sequence of virus VPO is shown in SEQ ID NO:24;
  • the amino acid sequence of VP1 is shown in SEQ ID NO:25;
  • the amino acid sequence of VP3 is shown in SEQ ID NO:26;
  • the amino acid sequence of the SAT3 type foot-and-mouth disease virus VP0 As shown in SEQ ID NO:27; the amino acid sequence of VP1 is shown in SEQ ID NO:28; the amino acid sequence of VP3 is shown in SEQ ID NO:29.
  • the present invention provides the application of the virus-like particles in the preparation of vaccines for the prevention and control of viral diseases.
  • the viral disease preferably comprises foot and mouth disease or swine vesicular disease.
  • the invention provides a virus-like particle vaccine for preventing and controlling viral diseases, comprising the virus-like particle and an adjuvant.
  • the content of said adjuvant is preferably 50% V/V.
  • the content of porcine vesicular virus-like particles is 100 ⁇ g/head.
  • the present invention has no special limitation on the preparation method of the vaccine, and the preparation method of the vaccine well known in the art can be adopted.
  • the vaccine is used for immunogenicity detection of each virus.
  • the viral disease preferably comprises foot and mouth disease or swine vesicular disease.
  • virus-like particle provided by the present invention and its preparation method and application are described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.
  • the nucleotide sequence (SEQ ID NO:2) of the SVDV structural protein VPO gene, the nucleotide sequence (SEQ ID NO:3) of the VP1 gene, the nucleotide sequence (SEQ ID NO:3) of the VP3 gene were obtained by GenScript :4) were seamlessly fused with the small ubiquitin-modified protein gene (SEQ ID NO: 1), cloned into the pET-28a vector through Sal I/BamH I and Hind III/Xho I endonucleases respectively, to obtain pET/ VP0-VP1 and pET/VP3;
  • the constructed pET/VPO-VP1 and pETa/VP3 plasmids were co-transformed into BL21(DE3) Escherichia coli competent cells, and a single colony was obtained through ampicillin and kanamycin double resistance selection.
  • step b Inoculate 50ml of the strain containing the SVDV plasmid in step a into 50ml of double-resistant LB medium containing kanamycin and ampicillin, culture at 37°C and 230rpm for 12h, and then transfer to 500ml of LB culture containing the same resistance culture medium at 37°C to prepare seeds for fermentation.
  • Small ubiquitination modified protease enzyme digestion of fusion protein according to the following methods and ratios: 1mg of the fusion protein purified above, 100mL enzyme digestion buffer (50mM Tris-HCl, 150mM NaCl, pH8.0, 0.2% Igepal (NP -40), 1mM DTT), 10 ⁇ L small ubiquitin-modifying protease (1U/ ⁇ L), digest at 37°C for 30min.
  • step b Add 10 ⁇ L of the peak sample in step b to a 200-mesh copper grid, absorb at room temperature for 10 minutes, blot the remaining liquid on the copper grid with filter paper and stain with 3% phosphotungstic acid, and observe VLPs with Hitachi, H-7100FA transmission electron microscope form.
  • the diameters of virus-like particles assembled under this condition are mainly distributed between 20 and 30 nm (b in Figure 2), and their shape and size are similar to those of SVDV recombinant viruses reported in the literature (a in Figure 2 ).
  • the present invention uses the IFA method to identify whether the specific antibody against VP1 is produced.
  • the specific operation is as follows: Synthesize the SVDV VP1 capsid protein gene into the pCMV-N-Flag vector by the method of gene synthesis, and name it as pCMV-N-Flag-VP1.
  • Example 1 the recombinant vectors of three kinds of foot-and-mouth disease virus VPO, VP1, and VP3 genes fused with small ubiquitinated modified proteins were constructed.
  • nucleotide sequences of SAT1, SAT2, and SAT3 FMD VP0, VP1, and VP3 are as follows:
  • VPO nucleotide sequence of SAT1 type foot-and-mouth disease is shown in sequence table SEQ ID NO:5;
  • VP1 nucleotide sequence of SAT1 type foot-and-mouth disease is shown in sequence table SEQ ID NO:6;
  • VP3 nucleotide sequence of SAT1 type foot-and-mouth disease is shown in sequence table SEQ ID NO:7;
  • VPO nucleotide sequence of SAT2 type foot-and-mouth disease is shown in sequence table SEQ ID NO:8;
  • VP1 nucleotide sequence of SAT2 type foot-and-mouth disease is shown in sequence table SEQ ID NO:9;
  • VP3 nucleotide sequence of SAT2 type foot-and-mouth disease is shown in sequence table SEQ ID NO:10;
  • VPO nucleotide sequence of SAT3 type foot-and-mouth disease is shown in sequence table SEQ ID NO:11;
  • VP1 nucleotide sequence of SAT3 type foot-and-mouth disease is shown in sequence table SEQ ID NO:12;
  • the VP3 nucleotide sequence of SAT3 type foot-and-mouth disease is shown in the sequence table SEQ ID NO: 13.
  • SAT foot-and-mouth disease was co-transformed with pET/VPO-VP1 and pETa/VP3 plasmids into BL21(DE3) Escherichia coli competent cells, and a single colony was obtained by ampicillin and kanamycin double resistance screening.
  • step b Inoculate 50ml of the strain containing the SAT plasmid in step a into 50ml of double-resistant LB medium containing kanamycin and ampicillin, culture at 37°C and 230rpm for 12 hours, and then transfer to 500ml of LB culture with the same resistance culture medium at 37°C to prepare seeds for fermentation.
  • Small ubiquitination modified protease enzyme digestion of fusion protein according to the following methods and ratios: 1mg of the fusion protein purified above, 100mL enzyme digestion buffer (50mM Tris-HCl, 150mM NaCl, pH8.0, 0.2% Igepal (NP -40), 1mM DTT), 10 ⁇ L small ubiquitin-modifying protease (1U/ ⁇ L), digest at 37°C for 30min.
  • step c Add 10 ⁇ L of the peak sample in step b to a 200-mesh copper grid, absorb at room temperature for 10 minutes, blot the remaining liquid on the copper grid with filter paper and stain with 3% phosphotungstic acid, and observe VLPs with Hitachi, H-7100FA transmission electron microscope form. As shown in Figure 8, the diameters of the virus-like particles assembled under this condition are mainly distributed between 20-30 nm.
  • the IFA method is used to identify whether specific antibodies are produced.
  • the specific operation is as follows: by gene synthesis, the VP1 capsid protein gene of SAT1, SAT2, SAT3 type FMDV is synthesized into pCMV-N-Flag vector, named pCMV-N-Flag-VP1.

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Abstract

提供了一种病毒样颗粒及其制备方法和应用，该方法以小泛素样修饰蛋白作为标签蛋白促进病毒结构蛋白VP0、VP1和VP3的正确折叠，同时保证了蛋白的结构稳定性，实现结构蛋白的大量表达，经过3种结构蛋白体外自组装后，获得大量的与天然病毒性能相似的病毒样颗粒。经过免疫原性检测，所制备的病毒样颗粒具有较高的免疫原性，可作为储备疫苗在防控猪水泡病或口蹄疫病毒传播中应用。

Description

一种病毒样颗粒及其制备方法和应用
本申请要求于2021年12月29日提交中国专利局、申请号为202111641645.X、发明名称为“一种病毒样颗粒及其制备方法和应用”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
本发明属于农业科学畜牧兽医科学技术领域，具体涉及一种病毒样颗粒及其制备方法和应用。
背景技术
随着国际间的交流与合作日渐频繁，当今世界日渐成为一个“地球村”。在这种大环境中，如非典型性肺炎(2002年)，新型冠状病毒(2019年)等疫病很容易发展成全球性大流行。我国作为养猪大国，猪、牛、羊等家畜疫病防控责任重大，为避免其他疫区和国家疫病传入，或旧病新发，对邻近周边国家流行性疾病进行研究和疫苗研发势在必行。口蹄疫(Foot-and-mouth disease,FMD)主要有7种血清型，目前我国主要以A型、O型FMD流行为主。近年来，南非型(SAT)FMD不仅对撒哈拉以南非洲国家造成经济损失，已逐渐传入欧洲等国威胁当地畜牧业发展。猪水泡病(Swine vesicular disease virus，SVDV)是一种猪传染性病毒性疾病。它会引起水泡性病变，与观察到的口蹄疫难以区分。感染SVDV可在1天内导致病毒血症，并可在猪接触受感染的猪或受病毒污染的环境后2天出现临床症状。感染后3～5小时，用免疫组织化学方法可以检测到病毒。对该病毒的理化分析表明，它不同于口蹄疫、水泡性口炎和水泡性疹病毒，与小核糖核酸科肠道病毒属的病毒非常相似。
病毒样颗粒(Virus-like particles,VLPs)是近年来新出现的一种基因工程亚单位形式，其优点包括不含病毒复制所必须的遗传物质，生物安全性优越；可以模拟病毒的天然结构而使构象依赖型抗原表位得以正确呈现；能像自然病毒粒子一样与细胞表面受体结合进入细胞，从而诱导较强的免疫反应；在不影响VLPs结构的基础上可以根据需要插入或删除某些氨基酸序列，对其进行人工改造，从而实现对多种病毒同时免疫的多效性特点等。因此，VLPs作为疫苗，被认为是目前能够替代全病毒疫苗的最 佳候选疫苗形式。然而目前还未有关于制备病毒样颗粒的方法的报道。
发明内容
有鉴于此，本发明的目的在于提供一种病毒样颗粒及其制备方法和应用，具有产量高、免疫原性好的优点。
本发明提供了一种病毒样颗粒的制备方法，包括以下步骤：
1)将病毒结构蛋白基因VP0基因、VP1基因和VP3基因分别与小泛素样修饰蛋白基因序列融合，所得融合基因分别克隆至pET-28a载体中，得到pET/VP0-VP1和pET/VP3；
所述病毒包括猪水泡病病毒和/或南非型口蹄疫病毒；
2)将步骤1)中所述pET/VP3中卡那霉素抗性基因替换为氨苄青霉素抗性基因，得到pETa/VP3；
3)将步骤1)中所述pET/VP0-VP1和步骤2)中所述pETa/VP3共转化至原核表达系统中，经重组表达，分离得到3种小泛素化修饰蛋白酶酶切融合蛋白；
4)将步骤3)中所述3种小泛素化修饰蛋白酶酶切融合蛋白分别去除小泛素化修饰标签蛋白，收集得到VP0、VP1和VP3重组蛋白，经体外组装，得到病毒样颗粒。
优选的，步骤1)中小泛素样修饰蛋白基因的核苷酸序列如SEQ ID NO:1所示。
优选的，步骤1)中所述pET-28a载体的多克隆位点为Sal I/BamH I和Hind III/Xho I酶切位点。
本发明提供了所述制备方法制备得到的病毒样颗粒，所述病毒样颗粒由病毒结构蛋白VP1、VP0和VP3组成。
优选的，所述病毒为猪水泡病病毒时，VP0的氨基酸序列如SEQ ID NO:18所示；VP1的氨基酸序列如SEQ ID NO:19所示；VP3的氨基酸序列如SEQ ID NO:20所示。
优选的，所述病毒为南非型口蹄疫病毒；
所述南非型口蹄疫病毒包括以下一种或几种血清型的口蹄疫病毒：SAT1型口蹄疫病毒、SAT2型口蹄疫病毒和SAT3型口蹄疫病毒；
优选的，所述SAT1型口蹄疫病毒VP0的氨基酸序列如SEQ ID NO:21所示；VP1的氨基酸序列如SEQ ID NO:22所示；VP3的氨基酸序列如SEQ ID NO:23所示；
所述SAT2型口蹄疫病毒VP0的氨基酸序列如SEQ ID NO:24所示；VP1的氨基酸序列如SEQ ID NO:25所示；VP3的氨基酸序列如SEQ ID  NO:26所示；
所述SAT3型口蹄疫病毒VP0的氨基酸序列如SEQ ID NO:27所示；VP1的氨基酸序列如SEQ ID NO:28所示；VP3的氨基酸序列如SEQ ID NO:29所示。
本发明提供了所述病毒样颗粒在制备防控病毒病的疫苗中的应用，所述病毒病包括猪水泡病和/或口蹄疫。
本发明提供了一种用于防控病毒病的病毒样颗粒疫苗，包括所述病毒样颗粒和佐剂。
本发明提供的病毒样颗粒的制备方法，以小泛素样修饰蛋白作为标签蛋白促进猪水泡病病毒和/或南非型口蹄疫病毒结构蛋白VP0、VP1和VP3的正确折叠，同时保证了蛋白的结构稳定性，实现结构蛋白的大量表达，经过3种结构蛋白体外自组装后，获得大量的与天然猪水泡病病毒性能相似的病毒样颗粒。经过免疫原性检测，本发明制备的病毒样颗粒具有较高的免疫原性，可作为储备疫苗，在防控猪水泡病病毒传播中应用。
附图说明
图1为SVDV重组衣壳蛋白的SDS-PAGE检测结果，泳道1：融合标签蛋白，泳道2：SVDV衣壳蛋白；
图2为文献报道的SVDV重组毒电镜照片(a)，本发明制备的SVDV病毒样颗粒的电镜照片(b)；
图3为本发明制备的SVDV病毒样颗粒的粒径分布情况；
图4为采用IFA法鉴定病毒样颗粒的荧光信号图片；
图5为SVDV病毒样颗粒免疫猪血清ELISA抗体水平检测结果；
图6为SAT1、SAT2、SAT3型口蹄疫衣壳蛋白的SDS-PAGE检测结果；
图7为本发明制备的SAT型口蹄疫病毒样颗粒的粒径分布情况；
图8为本发明制备的SAT型口蹄疫病毒样颗粒的电镜照片；
图9为采用IFA检测本发明制备的SAT型口蹄疫病毒样颗粒豚鼠免疫血清抗体。
具体实施方式
本发明提供了一种病毒样颗粒的制备方法，包括以下步骤：
1)将病毒结构蛋白基因VP0基因、VP1基因和VP3基因分别与小泛素样修饰蛋白基因融合，所得融合基因克隆至pET-28a载体中，得到pET/VP0-VP1和pET/VP3；
所述病毒包括猪水泡病病毒和/或南非型口蹄疫病毒；
2)将所述pET/VP3中卡那霉素抗性基因替换为氨苄青霉素抗性基因，得到pETa/VP3；
3)将步骤1)所述pET/VP0-VP1和步骤2)所述pETa/VP3共转化至原核表达系统中，经重组表达，分离得到3种小泛素化修饰蛋白酶酶切融合蛋白；
4)将步骤3)中所述3种小泛素化修饰蛋白酶酶切融合蛋白分别去除小泛素化修饰标签蛋白，收集得到VP0、VP1和VP3重组蛋白，经体外组装，得到病毒样颗粒。
本发明将病毒结构蛋白基因VP0基因、VP1基因和VP3基因分别与小泛素样修饰蛋白序列融合，分别克隆至pET-28a载体中，得到pET/VP0-VP1和pET/VP3；所述病毒包括猪水泡病病毒和/或南非型口蹄疫病毒。
在本发明中，所述融合优选为无缝融合，以小泛素样修饰蛋白基因序列-结构蛋白基因的顺序形成融合基因。小泛素样修饰蛋白的核苷酸序列如SEQ ID NO:1所示，小泛素样修饰蛋白的氨基酸序列如SEQ ID NO:30所示。pET-28a载体的多克隆位点优选为Sal I/BamH I(VP0或VP3)和Hind III/Xho I(VP1)酶切位点。本发明对所述克隆方法没有特殊限制，采用本领域所熟知的克隆方法即可。克隆后，得到的载体进行验证。本发明对所述载体的验证方法没有特殊限制，采用本领域所熟知的重组载体的验证方法即可。本发明对pET-28a载体的来源没有特殊限制，采用本领域所熟知的pET-28a载体即可。在本发明实施例中，所述pET-28a载体购自擎科生物科技有限公司。
在本发明中，VP0基因和VP3基因在pET-28a载体中的多克隆位点优选为Sal I/BamH I，VP1基因片段在pET-28a载体中的多克隆位点优选为Hind III/Xho I。本发明对所述克隆的方法没有特殊限制，采用本领域所熟知的隆的方法即可。克隆后，优选对获得的载体进行验证。本发明对所述载体的验证方法没有特殊限制，采用本领域所熟知的重组载体的验证方法即可。
得到pET/VP3后，本发明将所述pET/VP3中卡那霉素抗性基因替换为氨苄青霉素抗性基因，得到载体pETa/VP3。
在本发明中，将所述pET/VP3中卡那霉素抗性基因替换为氨苄青霉素抗性基因的方法优选如下：设计2对同源重组引物，其中氨苄青霉素抗性基因引物：F：CAGTAATACAAGGGGTGTTATGTTACCAATGCTTAATCAGTGAGG(SEQ ID NO:14)；R：ATCCGCTCATGAATTAATTCTT AATGAGTATTCAACATTTCCGTG(SEQ ID NO:15)；PET28a线性化引物：F：CGGAAATGTTGAATACTCATTAAGAATTAATTCATGAGCGGATAC(SEQ ID NO:16)，R：CCTCACTGATTAAGCATTGGTAACATAACACCCCTTGTATTACTG(SEQ ID NO:17)。替换抗性基因作用是进行双抗性筛选，确保衣壳蛋白VP0、VP1、VP3均一表达。
得到pET/VP0-VP1和pETa/VP3后，本发明将所述pET/VP0-VP1和所述pETa/VP3共转化至原核表达系统中，经重组表达，分离得到3种小泛素化修饰蛋白酶酶切融合蛋白。
本发明对所述原核表达系统没有特殊限制，采用本领域所熟知的原核表达系统的方法即可，例如本发明实施例中，所述原核表达系统为BL21(DE3)。所述共转化时，所述pET/VP0-VP1和pETa/VP3的拷贝数比优选为1～3：1～3，更优选为1.5：1。所述重组表达的方法，优选将转化后的原核表达系统分别经卡那霉素和氨苄青霉素筛选阳性克隆，挑取阳性克隆进行双抗培养。培养后的两种菌液混合后进行高密度发酵培养和诱导表达。所述培养的温度优选为36～38℃，更优选为37℃；所述培养的振荡速度优选为800rpm。在所述诱导表达前，菌液的OD 600nm为40左右时进入诱导表达。所述诱导表达的温度有选为20～37℃，更优选为30℃。所述诱导表达时IPTG的终浓度优选为1mM。所述分离优选离心菌体、破碎菌体细胞，收集上清与Ni-NTA His·Bind Resins混合，洗脱后得到小泛素化修饰蛋白酶酶切融合表达蛋白。
得到小泛素化修饰蛋白酶酶切融合表达蛋白后，本发明将所述3种小泛素化修饰蛋白酶酶切去除小泛素化修饰标签蛋白，收集得到VP0、VP1和VP3重组蛋白，经体外组装，得到病毒样颗粒。
在本发明中，去除小泛素化修饰标签蛋白的方法优选包括用小泛素化修饰蛋白酶进行酶切。所述酶切的体系优选为每1mg所述融合蛋白与100ml酶切缓冲液混合。酶切后溶液优选采用HisTrap HP色谱柱去除小泛素化修饰标签蛋白。所述洗脱用溶液的成分优选为20mM Tris-HCl，300mM NaCl，pH8.0。
在本发明中，所述体外组装时，VP0、VP1和VP3重组蛋白的质量比为1:1:1。所用组装缓冲液成分优选为20mM Tris-HCl，500mM NaCl，pH 8.0。所述体外组装的温度优选为3～5℃，更优选为4℃。所述体外组装的时间优选为10～14h，更优选为12h。体外组装结束后，优选采用超滤管浓缩组装后溶液，收集截留物。超滤管的截留分子量优选为100KD。
在本发明中，采用蔗糖密度梯度分离，分离病毒样颗粒：将1.0ml 含病毒样颗粒的液体(突变和未突变)置于15％～45％浓度的蔗糖梯度顶层，以38000rpm、4℃离心3.5h。收集20层的样本用于检测。将收集的样本用连续进样的方法在紫外检测仪(259nm)检测，取第13管样本进行DLS检测，得到病毒样颗粒的粒径大小。
本发明提供的所述制备方法制备得到的病毒样颗粒，所述病毒样颗粒由病毒结构蛋白VP1、VP0和VP3组成，直径主要分布在20～30nm之间，形态完整，大小和自然的病毒粒子相似。所述病毒为猪水泡病病毒时，VP0的氨基酸序列如SEQ ID NO:18所示；VP1的氨基酸序列如SEQ ID NO:19所示；VP3的氨基酸序列如SEQ ID NO:20所示。所述病毒为南非型口蹄疫病毒；所述南非型口蹄疫病毒包括以下一种或几种血清型的口蹄疫病毒：SAT1型口蹄疫病毒、SAT2型口蹄疫病毒和SAT3型口蹄疫病毒。所述SAT1型口蹄疫病毒VP0的氨基酸序列如SEQ ID NO:21所示；VP1的氨基酸序列如SEQ ID NO:22所示；VP3的氨基酸序列如SEQ ID NO:23所示；所述SAT2型口蹄疫病毒VP0的氨基酸序列如SEQ ID NO:24所示；VP1的氨基酸序列如SEQ ID NO:25所示；VP3的氨基酸序列如SEQ ID NO:26所示；所述SAT3型口蹄疫病毒VP0的氨基酸序列如SEQ ID NO:27所示；VP1的氨基酸序列如SEQ ID NO:28所示；VP3的氨基酸序列如SEQ ID NO:29所示。
鉴于病毒样颗粒具有较高的免疫原性，本发明提供了所述病毒样颗粒在制备防控病毒病的疫苗中的应用。所述病毒病优选包括口蹄疫或猪水泡病。
本发明提供了一种用于防控病毒病的病毒样颗粒疫苗，包括所述病毒样颗粒和佐剂。所述佐剂的含量优选为50％V/V。猪水泡病毒样颗粒的含量为100μg/头份。本发明对所述疫苗的制备方法没有特殊限制，采用本领域所熟知的疫苗的制备方法即可。所述疫苗用于各病毒的免疫原性检测。所述病毒病优选包括口蹄疫或猪水泡病。
为了进一步说明本发明，下面结合实施例对本发明提供的一种病毒样颗粒及其制备方法和应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
(1)融合小泛素化修饰蛋白的猪水泡病毒VP0、VP1、VP3基因的重组载体构建：
a.根据已公布的SVDV的序列(GenBank登陆号：D16364.1)，参考 Hoover等的方法(Hoover DM1,Lubkowski J.DNAWorks:an automated method for designing oligonucleotides for PCR-based gene synthesis.Nucl.Acids Res.(2002)30(10):e43.)进行密码子优化。由金斯瑞公司将SVDV结构蛋白VP0基因的核苷酸序列(SEQ ID NO:2)、VP1基因的核苷酸序列(SEQ ID NO:3)、VP3基因的核苷酸序列(SEQ ID NO:4)分别与小泛素化修饰蛋白基因(SEQ ID NO:1)无缝融合，分别经Sal I/BamH I，Hind III/Xho I内切酶克隆至pET-28a载体中，得到pET/VP0-VP1和pET/VP3；
(2)SVDV抗原蛋白的表达及纯化：
a.将构建的pET/VP0-VP1和pETa/VP3质粒共转化至BL21(DE3)大肠杆菌感受态细胞中，经氨苄青霉素和卡那霉素双抗性筛选获得单菌落。挑取单克隆至双抗性的LB培养集中，于37℃、230rpm培养12h，分装后加入甘油LB培养基于-80℃冷冻保存，获得种子库。
b.将步骤a中含SVDV质粒的菌种接种50ml含卡那霉素和氨苄青霉素双抗性LB培养基中,于37℃、230rpm培养12h后，转接入500ml含相同抗性的LB培养基中，37℃培养制备发酵用种子。
c.使用30L发酵罐，配制10L培养基于发酵罐中，121℃，灭菌30min后，待培养基温度降至37℃将500ml种子接入发酵罐中，待菌液浓度达到OD 600值约为40左右时，加入0.5g IPTG诱导培养6h后终止培养，离心收集菌体。
d.冰浴裂解溶液(20mM Tris-HCl，500mM NaCl，20mM咪唑，pH7.4)重悬菌体，高压均质机以1000bar压力破碎4次，10,000×g离心30min，取上清，弃沉淀，上清进行色谱纯化，目标蛋白经SDS-PAGE电泳检测，-70℃保存(图1)。
(3)SVDV病毒样颗粒的体外组装
a.小泛素化修饰蛋白酶酶切融合蛋白，按照如下方法和比例进行：1mg上述纯化的融合蛋白，100mL酶切缓冲液(50mM Tris-HCl，150mM NaCl，pH8.0,0.2％Igepal(NP-40)，1mM DTT)，10μL小泛素化修饰蛋白酶(1U/μL)，37℃酶切30min。将酶切混合物通过HisTrap HP除去小泛素化修饰标签蛋白，收集含有VP0、VP1和VP3的流穿液在组装缓冲液(20mM Tris-HCl，500mM NaCl，pH 8.0)中，4℃过夜组装VLPs，样品经SDS-PAGE电泳检测(图1)。
b.将1ml上述制备的含病毒样颗粒的样品置于15％～45％线性蔗糖梯度顶层，以38000rpm、4℃离心3.5h，收集20份样品，500μl/份，然后用连续进样的方法经紫外检测仪(280nm)检测，并绘制图谱，随后取峰 值样本进行DLS进行水和粒径大小的检测。结果如图3所示，本发明制备的病毒样颗粒的粒径主要分布在10～30nm。
c.将步骤b中的峰值样品10μL加到200目的铜网上，室温吸附10min，用滤纸吸干铜网上剩余的液体后用3％的磷钨酸染色，用Hitachi，H-7100FA透射电镜观察VLPs形态。如图2所示，在此条件下组装的病毒样颗粒直径主要分布在20～30nm之间(图2中b)，其形态，大小与文献中报道的SVDV重组毒大小相似(图2中a)。
实施例2
SVDV病毒样颗粒免疫原性分析
选取SVDV，FMDV，SVA抗体为阴性的20kg左右的健康猪8头，随机分成3组，实验组3头/组，对照组2头，其中，1组为本发明实施例1制备的猪水泡病毒样颗粒疫苗免疫组，2组为猪水泡病毒VP1蛋白免疫组，3组为PBS对照组。100μg抗原与等体积的206佐剂乳化后颈部肌肉注射免疫，采集免疫前、免疫后第7天，14天，21天，28天血液，并分离血清。
由于国内无SVDV阳性血清以及抗原，本发明采用IFA法鉴定是否产生抗VP1的特异性抗体。具体操作如下：通过基因合成的方法，将SVDV VP1衣壳蛋白基因合成到pCMV-N-Flag载体中，命名为pCMV-N-Flag-VP1。随后将质粒转染进入BHK细胞，36h后收取细胞样品，经4％多聚甲醛(PFA)室温固定15min；PBST(PBS+0.1％Tween-20)洗3遍；0.1％TritonX-100(PBS+0.1％TritonX-100)室温处理细胞15min，以破坏细胞膜；PBST(PBS+0.1％Tween-20)洗3遍；5％NBS(PBS+5％NBS)37℃恒温培养箱中孵育1h，以封闭非特异性结合；稀释于5％NBS中的一抗(1：100)孵育细胞(一抗使用SVDV VLPs多抗)，37℃下处理1h(或4℃过夜)；PBST(PBS+0.1％Tween-20)洗5遍；稀释于5％NBS中的荧光标记二抗(1：400)孵育细胞(抗小鼠的FITC抗体)，37℃1h(或4℃过夜)；PBST(PBS+0.1％Tween-20)洗5遍；荧光显微镜下观察荧光强度。
具体检测结果如图4。转染pCMV-N-Flag-VP1(含SVDV VP1基因)质粒的细胞有明显的荧光信号，说明SVDV VLPs免疫猪产生了SVDV特异性的抗体。
采用仕诺达生物公司生产的猪水疱病病毒抗体ELISA检测试剂盒进行抗体水平检测。结果见图5所示：病毒样颗粒免疫组猪血清抗体水平显著高于VP1蛋白免疫组，且在免疫后28天抗体水平最高。该结果表明， 衣壳蛋白的组装更有利于抗原递呈，增强免疫反应。
实施例3
SAT1、SAT2、SAT3型口蹄疫病毒样颗粒的制备方法
(1)根据已公布的SAT(1、2、3)型口蹄疫病毒的序列(GenBank登陆号：SAT1：KR108962.1；SAT2：AJ251473；SAT3:MK415736.1)，对结构蛋白基因VP0，VP3和VP1分别进行密码子优化。
按照实施例1记载的方法融合小泛素化修饰蛋白的3种口蹄疫病毒VP0、VP1、VP3基因的重组载体构建。
SAT1、SAT2、SAT3型口蹄疫VP0、VP1、VP3的核苷酸序列分别如下：
SAT1型口蹄疫的VP0核苷酸序列如序列表SEQ ID NO:5所示；
SAT1型口蹄疫的VP1核苷酸序列如序列表SEQ ID NO:6所示；
SAT1型口蹄疫的VP3核苷酸序列如序列表SEQ ID NO:7所示；
SAT2型口蹄疫的VP0核苷酸序列如序列表SEQ ID NO:8所示；
SAT2型口蹄疫的VP1核苷酸序列如序列表SEQ ID NO:9所示；
SAT2型口蹄疫的VP3核苷酸序列如序列表SEQ ID NO:10所示；
SAT3型口蹄疫的VP0核苷酸序列如序列表SEQ ID NO:11所示；
SAT3型口蹄疫的VP1核苷酸序列如序列表SEQ ID NO:12所示；
SAT3型口蹄疫的VP3核苷酸序列如序列表SEQ ID NO:13所示。
(2)SAT1、SAT2、SAT3型口蹄疫衣壳蛋白的表达及纯化
a.分别将SAT口蹄疫的表达载pET/VP0-VP1和pETa/VP3质粒共转化至BL21(DE3)大肠杆菌感受态细胞中，经氨苄青霉素和卡那霉素双抗性筛选获得单菌落。挑取单克隆至双抗性的LB培养集中，于37℃、230rpm培养12h，分装后加入甘油LB培养基于-80℃冷冻保存，获得SAT1，SAT2，SAT3甘油菌种子库。
b.将步骤a中含SAT质粒的菌种接种50ml含卡那霉素和氨苄青霉素双抗性LB培养基中,于37℃、230rpm培养12h后，转接入500ml含相同抗性的LB培养基中，37℃培养制备发酵用种子。
c.使用30L发酵罐，配制10L培养基于发酵罐中，121℃，灭菌30min后，待培养基温度降至37℃将500ml种子接入发酵罐中，待菌液浓度达到OD 600值约为40左右时，加入0.5g IPTG诱导培养6h后终止培养，离心收集菌体。
d.冰浴裂解溶液(20mM Tris-HCl，500mM NaCl，20mM咪唑，pH7.4)重悬菌体，高压均质机以1000bar压力破碎4次，10,000×g离心 30min，取上清，弃沉淀，上清进行色谱纯化，目标蛋白经SDS-PAGE电泳检测，-70℃保存。
(3)SAT1、SAT2、SAT3型口蹄疫病毒样颗粒的体外组装
a.小泛素化修饰蛋白酶酶切融合蛋白，按照如下方法和比例进行：1mg上述纯化的融合蛋白，100mL酶切缓冲液(50mM Tris-HCl，150mM NaCl，pH8.0,0.2％Igepal(NP-40)，1mM DTT)，10μL小泛素化修饰蛋白酶(1U/μL)，37℃酶切30min。将酶切混合物通过HisTrap HP除去小泛素化修饰标签蛋白，收集含有VP0、VP1和VP3的流穿液在组装缓冲液(20mM Tris-HCl，500mM NaCl，pH 8.0)中，4℃过夜组装VLPs，样品经SDS-PAGE电泳检测(图6)。
b.将1ml上述制备的含病毒样颗粒的样品置于15％～45％线性蔗糖梯度顶层，以38000rpm、4℃离心3.5h，收集20份样品，500μl/份，然后用连续进样的方法经紫外检测仪(280nm)检测，并绘制图谱，随后取峰值样本进行DLS进行水和粒径大小的检测。结果如图7所示，本发明制备的病毒样颗粒的粒径主要分布在10～30nm。
c.将步骤b中的峰值样品10μL加到200目的铜网上，室温吸附10min，用滤纸吸干铜网上剩余的液体后用3％的磷钨酸染色，用Hitachi,H-7100FA透射电镜观察VLPs形态。如图8所示，在此条件下组装的病毒样颗粒直径主要分布在20～30nm之间。
(4)SAT1、SAT2、SAT3型口蹄疫病毒样颗粒免疫小鼠血清鉴定
(a)将获得的SAT1、SAT2、SAT3 FMDV VLPs免疫Babl/c小鼠，并在两周后加强免疫，采集免疫后28天的小鼠血液，分离血清冻存于-20℃冰箱备用，分别命名为SAT1、SAT2、SAT3型FMDV VLPs多抗。
(b)由于国内无SAT1、SAT2、SAT3型口蹄疫病原，采用IFA法鉴定是否产生特异性抗体。具体操作如下：通过基因合成的方法，将SAT1、SAT2、SAT3型FMDV的VP1衣壳蛋白基因合成到pCMV-N-Flag载体中，命名为pCMV-N-Flag-VP1。随后将质粒转染进入BHK细胞，36h后收取细胞样品，经4％多聚甲醛(PFA)室温固定15min；PBST(PBS+0.1％Tween-20)洗3遍；0.1％TritonX-100(PBS+0.1％TritonX-100)室温处理细胞15min，以破坏细胞膜；PBST(PBS+0.1％Tween-20)洗3遍；5％NBS(PBS+5％NBS)37℃恒温培养箱中孵育1h，以封闭非特异性结合；稀释于5％NBS中的一抗(1：100)孵育细胞(一抗使用SAT1、SAT2、SAT3 FMDV VLPs多抗)，37℃下处理1h(或4℃过夜)；PBST(PBS+0.1％Tween-20)洗5遍；稀释于5％NBS中的荧光标记二抗(1：400)孵育 细胞(抗小鼠的FITC抗体)，37℃1h(或4℃过夜)；PBST(PBS+0.1％Tween-20)洗5遍；之后再在荧光显微镜下观察荧光数量。
结果如图9所示。结果表明：分别转染pCMV-N-Flag-VP1(含SAT1或SAT2或SAT3 FMDV VP1基因)质粒的细胞都有明显的荧光信号，说明对应VLPs免疫猪分别产生了SAT1、SAT2、SAT3型FMDV特异性的抗体。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (12)

  1. 一种病毒样颗粒的制备方法，其特征在于，包括以下步骤：
    1)将病毒结构蛋白基因VP0基因、VP1基因和VP3基因分别与小泛素样修饰蛋白基因序列融合，所得融合基因分别克隆至pET-28a载体中，得到pET/VP0-VP1和pET/VP3；
    所述病毒包括猪水泡病病毒和/或南非型口蹄疫病毒；
    2)将步骤1)中所述pET/VP3中卡那霉素抗性基因替换为氨苄青霉素抗性基因，得到pETa/VP3；
    3)将步骤1)中所述pET/VP0-VP1和步骤2)中所述pETa/VP3共转化至原核表达系统中，经重组表达，分离得到3种小泛素化修饰蛋白酶酶切融合蛋白；
    4)将步骤3)中所述3种小泛素化修饰蛋白酶酶切融合蛋白分别去除小泛素化修饰标签蛋白，收集得到VP0、VP1和VP3重组蛋白，经体外组装，得到病毒样颗粒。
  2. 根据权利要求1所述制备方法，其特征在于，步骤1)中小泛素样修饰蛋白基因的核苷酸序列如SEQ ID NO:1所示。
  3. 根据权利要求1所述制备方法，其特征在于，步骤1)中所述pET-28a载体的多克隆位点为SalI/BamHI和/或HindIII/Xho I酶切位点。
  4. 权利要求1～3任意一项所述制备方法制备得到的病毒样颗粒，其特征在于，所述病毒样颗粒由病毒结构蛋白VP1、VP0和VP3组成。
  5. 根据权利要求4所述病毒样颗粒，其特征在于，所述病毒为猪水泡病病毒时，VP0的氨基酸序列如SEQ ID NO:18所示；VP1的氨基酸序列如SEQ ID NO:19所示；VP3的氨基酸序列如SEQ ID NO:20所示。
  6. 根据权利要求4所述病毒样颗粒，其特征在于，所述病毒为南非型口蹄疫病毒时，所述南非型口蹄疫病毒包括以下一种或几种血清型的口蹄疫病毒：SAT1型口蹄疫病毒、SAT2型口蹄疫病毒和SAT3型口蹄疫病毒。
  7. 根据权利要求6所述病毒样颗粒，其特征在于，所述SAT1型口蹄疫病毒的VP0的氨基酸序列如SEQ ID NO:21所示；VP1的氨基酸序列如SEQ ID NO:22所示；VP3的氨基酸序列如SEQ ID NO:23所示。
  8. 根据权利要求6所述病毒样颗粒，其特征在于，所述SAT2型口蹄 疫病毒VP0的氨基酸序列如SEQ ID NO:24所示；VP1的氨基酸序列如SEQ ID NO:25所示；VP3的氨基酸序列如SEQ ID NO:26所示。
  9. 根据权利要求6所述病毒样颗粒，其特征在于，所述SAT3型口蹄疫病毒VP0的氨基酸序列如SEQ ID NO:27所示；VP1的氨基酸序列如SEQ ID NO:28所示；VP3的氨基酸序列如SEQ ID NO:29所示。
  10. 权利要求5～9任意一项所述病毒样颗粒在制备防控病毒病疫苗中的应用；所述病毒病包括猪水泡病和/或口蹄疫。
  11. 一种用于防控病毒病的病毒样颗粒疫苗，其特征在于，包括权利要求5～8任意一项所述病毒样颗粒和佐剂。
  12. 权利要求11所述疫苗在病毒病中的应用，所述病毒病包括猪水泡病和/或口蹄疫。
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