WO2020147015A1 - 口蹄疫病毒样颗粒抗原、及其疫苗组合物、制备方法和应用 - Google Patents

口蹄疫病毒样颗粒抗原、及其疫苗组合物、制备方法和应用 Download PDF

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WO2020147015A1
WO2020147015A1 PCT/CN2019/071813 CN2019071813W WO2020147015A1 WO 2020147015 A1 WO2020147015 A1 WO 2020147015A1 CN 2019071813 W CN2019071813 W CN 2019071813W WO 2020147015 A1 WO2020147015 A1 WO 2020147015A1
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Prior art keywords
foot
type
mouth disease
disease virus
antigen
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PCT/CN2019/071813
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English (en)
French (fr)
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田克恭
肖燕
逄文强
孙进忠
张许科
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普莱柯生物工程股份有限公司
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Priority to JP2021532196A priority Critical patent/JP7303306B2/ja
Priority to PCT/CN2019/071813 priority patent/WO2020147015A1/zh
Priority to US17/421,541 priority patent/US20220096620A1/en
Publication of WO2020147015A1 publication Critical patent/WO2020147015A1/zh

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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
    • A61K39/135—Foot- and mouth-disease virus
    • 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
    • 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
    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32123—Virus like particles [VLP]
    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention belongs to a viral antigen and a pharmaceutical preparation of the antigen. Specifically, the present invention relates to a foot-and-mouth disease virus-like particle antigen, a vaccine composition prepared therefrom, and a preparation method and application thereof.
  • Foot-and-mouth disease (foot-and-mouth disease, FMD) is an acute, highly contagious animal disease that can quickly spread over long distances. It is the most infectious disease in mammals. Among them, cloven-hoofed animals will cause more disease. Significant economic losses worldwide. Animals suffering from foot-and-mouth disease include cattle, sheep and pigs.
  • the causative agent is Foot-and-Mouth Disease Virus (FMDV), which is an aphthous virus belonging to the picornavirus family. The virus is divided into 7 serotypes (types A, O, C, Asia, SAT1, SAT2 and SAT3), among which type O foot-and-mouth disease virus is the most widespread.
  • FMDV Foot-and-Mouth Disease Virus
  • CATHAY type Choinese type
  • ME-SA type Middle East-South Asia type
  • SEA type Southeast Asian type
  • VLPs virus-like particles
  • Vaccines based on virus-like particles are an ideal form of vaccine.
  • the present invention provides a type O foot-and-mouth disease virus-like particle antigen, wherein the type O foot-and-mouth disease virus-like particle antigen is CATHAY type Type O foot-and-mouth disease virus-like particle antigen, the CATHAY type O foot-and-mouth disease virus-like particle antigen is assembled from CATHAY type O foot-and-mouth disease virus VP0, VP3, and VP1 antigen proteins.
  • the foot-and-mouth disease virus-like particle antigen has good immunogenicity, and only one immunization can provide complete protection against foot-and-mouth disease virus.
  • the foot-and-mouth disease virus-like particle antigen has good stability. After being placed at 4°C for 3 months, it can be seen that the virus-like particles are still full without aggregation after phosphotungstic acid negative staining and electron microscope observation.
  • the gene of the CATHAY type O foot-and-mouth disease virus VP0 antigen protein is shown in SEQ ID No. 1 or its degenerate sequence;
  • the gene of the CATHAY type O foot-and-mouth disease virus VP3 antigen protein is shown in SEQ ID No. 2 or its degenerate sequence;
  • the gene of the CATHAY type O foot-and-mouth disease virus VP1 antigen protein is shown in SEQ ID No. 3 or its degenerate sequence The sequence is shown.
  • the CATHAY type O foot-and-mouth disease virus-like particle antigen is derived from the current epidemic strain and can produce complete protection against the current epidemic wild virus strain.
  • the present invention also provides a type O foot-and-mouth disease virus-like particle vaccine, wherein the type O foot-and-mouth disease virus-like particle vaccine comprises an immunological amount of the CATHAY type O foot-and-mouth disease virus-like particle antigen and a pharmaceutically acceptable carrier.
  • the O-type foot-and-mouth disease virus-like particle vaccine of the present invention can reach an antibody titer of more than 1:128 on the 14th day after immunization, and can maintain a long-term high-titer antibody titer, and can protect against the entire fattening period.
  • the antigen content of the CATHAY type O foot-and-mouth disease virus-like particle is 160-240 g/ml.
  • the antigen content of CATHAY type O foot-and-mouth disease virus-like particles can be selected from 160 ⁇ g/ml, 170 ⁇ g/ml, 180 ⁇ g/ml, 190 ⁇ g/ml, 200 ⁇ g/ml, 210 ⁇ g/ml, 220 ⁇ g/ml, 230 ⁇ g/ml, 240 ⁇ g/ml.
  • the antibody titer of more than 1:128 can be reached on the 14th day after immunization, and the antibody titer of high titer can be maintained for a long time.
  • the antigen content of the CATHAY type O foot-and-mouth disease virus-like particle is 200 ⁇ g/ml.
  • the pharmaceutically acceptable carrier includes an adjuvant
  • the adjuvant includes: (1) white oil, aluminum gum adjuvant (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) acrylic acid or methacrylic acid polymer, maleic anhydride and chain Copolymer of alkenyl derivatives; and RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314.
  • the content of the adjuvant is 5%-60% V/V, preferably from 30%-60% V/V, more preferably 50% V/V.
  • the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, coloring agents, volatile oils, buffers, dispersants, propellants and preservatives
  • the immunostimulants include ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and interleukin-2 (IL2).
  • GM-CSF granulocyte macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • IL2 interleukin-2
  • the type O foot-and-mouth disease virus-like particle vaccine further contains an immune amount of SEA type O foot-and-mouth disease virus-like particle antigen.
  • the vaccine of the present invention adds SEA type O foot-and-mouth disease virus-like particle antigen, which can produce immune protection against CATHAY type O foot-and-mouth disease and SEA type O foot-and-mouth disease.
  • the SEA type O foot-and-mouth disease virus-like particle antigen is composed of SEA type O foot-and-mouth disease virus VP4, VP2, VP3, VP1 antigen The protein is assembled.
  • the SEA type O foot-and-mouth disease virus-like particle antigen of the present invention has good immunogenicity and can reach a SEA type O foot-and-mouth disease antibody titer of more than 1:128 on the 14th day after vaccine immunization.
  • the SEA type O foot-and-mouth disease virus-like particle antigen has good stability. After being placed at 4°C for 3 months, it can be seen that the virus-like particles are still full without aggregation after phosphotungstic acid negative staining and electron microscope observation.
  • the gene of the SEA type O foot-and-mouth disease virus VP4 antigen protein is shown in SEQ ID No. 4 or its degenerate sequence;
  • the gene of the SEA type O foot-and-mouth disease virus VP2 antigen protein is shown in SEQ ID No. 5 or its degenerate sequence;
  • the gene of the SEA type O foot-and-mouth disease virus VP3 antigen protein is shown in SEQ ID No. 6 or its degenerate sequence
  • the sequence is shown;
  • the gene of the SEA type O foot-and-mouth disease virus VP1 antigen protein is shown in SEQ ID No. 7 or its degenerate sequence.
  • the antigen content of the SEA type O foot-and-mouth disease virus-like particle is 160-240 ⁇ g/ml.
  • the antigen content of SEA type O foot-and-mouth disease virus-like particles can be selected from 160 ⁇ g/ml, 170 ⁇ g/ml, 180 ⁇ g/ml, 190 ⁇ g/ml, 200 ⁇ g/ml, 210 ⁇ g/ml, 220 ⁇ g/ml, 230 ⁇ g/ml, 240 ⁇ g/ml.
  • the antigen content of the SEA type O foot-and-mouth disease virus-like particle is 200 ⁇ g/ml.
  • the present invention also relates to a method for preparing the type O foot-and-mouth disease virus-like particle vaccine, wherein the method comprises: step (1) respectively amplifying the genes of CATHAY type O foot-and-mouth disease virus VP0, VP3, VP1 antigen protein, Cloned into the same tandem expression vector to obtain a recombinant expression vector containing CATHAY type O foot-and-mouth disease virus VP0, VP3, VP1 antigen protein genes; step (2) the step (1) obtained by the step (1) contains CATHAY type O foot-and-mouth disease virus VP0, The recombinant expression vector of VP3, VP1 antigen protein gene transforms or transduces the host to obtain a recombinant containing the recombinant expression vector; step (3) cultivating the recombinant obtained in step (2), and expressing the CATHAY type O in series Type foot-and-mouth disease virus VP0, VP3, VP1 antigen proteins, the expressed CATHAY type O foot-and-mouth disease virus
  • the present invention can obtain stable self-assembled virus-like particles, and its immune effect can realize complete protection of pigs.
  • the present invention expresses type O foot-and-mouth disease virus VP0, VP3, VP1 antigen proteins in series, and the expressed active protein self-assembles into CATHAY type O foot-and-mouth disease virus-like particle antigen, which facilitates subsequent antigen purification and separation.
  • the gene of the CATHAY type O foot-and-mouth disease virus VPO antigen protein in the step (1) is as shown in SEQ ID No. 1 or its degenerate sequence.
  • the gene of the CATHAY type O foot-and-mouth disease virus VP3 antigen protein is shown in SEQ ID No. 2 or its degenerate sequence;
  • the gene of the CATHAY type O foot-and-mouth disease virus VP1 antigen protein is shown in SEQ ID No. 3 or its The degenerate sequence is shown;
  • the host in the step (2) is E. coli;
  • the CATHAY type O foot-and-mouth disease virus VP0, VP3, and VP1 antigen proteins expressed in the step (3) are intracellular soluble proteins.
  • the invention utilizes the E. coli expression system to produce foot-and-mouth disease virus-like particles, and has the advantages of high yield, low production cost, good immunogenicity, no biological safety risk, and the like.
  • the virus-like particle vaccine composition prepared by the present invention can not only provide protective activity against type O foot-and-mouth disease, but also compared with the existing commercialized whole virus inactivated vaccines, it not only produces fast antibody, but also has a high level of antibody production, and the duration of immunity is significant Growth can maintain immune protection for a longer time.
  • the present invention also relates to the application of the O-type foot-and-mouth disease virus-like particle vaccine in preparing medicines for preventing and/or treating O-type foot-and-mouth disease.
  • the subject of administration of the medicine for preventing and/or treating foot-and-mouth disease virus infection of the present invention includes pigs.
  • “Foot-and-mouth disease virus” belongs to the Picornaviridae family and belongs to the genus of foot-and-mouth disease virus.
  • the virus has 7 sera of O, A, C, SAT1, SAT2, SAT3 (that is, South African foot-and-mouth disease virus 1, 2, 3) and Asia1 (Asia 1) Type, there is no cross-protection reaction between each type, and there are multiple subtypes within each type.
  • At the center of the virus is a single-stranded positive-strand RNA consisting of approximately 8000 bases, which is the basis of infection and heredity; the surrounding protein determines the antigenicity, immunity and serological response of the virus; the outer shell of the virus is Symmetrical 20-sided body.
  • Foot-and-mouth disease virus is the pathogen of foot-and-mouth disease, a highly contagious disease of cloven-hoofed animals.
  • the International Bureau of Ophthalmology listed foot-and-mouth disease as the first in the "A list of infectious diseases of animals", and China listed it as "an infectious disease of entry animal quarantine.” , China's prevention and treatment of foot-and-mouth disease is mainly through vaccination, and if foot-and-mouth disease occurs, it is killed.
  • Antigen refers to a substance that can induce an immune response in the body, that is, it can be specifically recognized and bound by the antigen receptor (TCR/BCR) on the surface of T/B lymphocytes to activate T/B cells and make them proliferate Substances that differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.
  • TCR/BCR antigen receptor
  • VLPs are particles assembled from one or more viral structural proteins, which have similar external structure and antigenicity to virus particles, but do not contain virus genes.
  • FMDV structural protein precursor protein P1 is catalyzed by protease 3C and processed into VP0, VP1 and VP3. These three proteins self-assemble into an icosahedral virus capsid.
  • VP0 protein is the intermediate of P1 after protease 3C cleavage. In the final stage of virus particle formation, VP0 matures and cleaves into VP2 and VP4.
  • vaccine and "vaccine composition” used in the present invention refer to a pharmaceutical composition containing foot-and-mouth disease virus-like particle antigens, which can induce, stimulate or enhance the immune response of pigs against foot-and-mouth disease.
  • immune amount should be understood as an "immune effective amount”, also known as an immune protective amount or an effective amount for generating an immune response, which is an antigen amount that can effectively induce an immune response in the recipient's body, and the amount is sufficient to prevent or improve the signs of disease Or symptoms, including adverse health effects or their complications.
  • the immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications caused by infections caused by pathogens. Humoral immunity or cell-mediated immunity or both can be induced.
  • the immune response of the animal to the immunogenic composition can be assessed indirectly by, for example, measuring antibody titer, lymphocyte proliferation analysis, or directly by monitoring signs or symptoms after challenge with a wild-type strain, and the vaccine provided Protective immunity can be evaluated by measuring, for example, the subject's clinical signs such as mortality, reduction in morbidity, temperature values, the subject's overall physiological condition, and overall health and performance.
  • the immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
  • pharmaceutically acceptable carrier refers to all ingredients in the vaccine composition of the present invention except for the foot-and-mouth disease virus antigen, a carrier or diluent that does not stimulate the body and does not hinder the biological activity and characteristics of the compound used, preferably Adjuvant.
  • adjuvant may include aluminum gel adjuvant; saponin, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsion; Oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; compounds selected from copolymers of maleic anhydride and alkenyl derivatives.
  • saponin such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL)
  • water-in-oil emulsion oil-in-water emulsions
  • water-in-oil-in-water emulsions polymers of acrylic acid or methacrylic acid; compounds selected from copolymers of maleic anhydride and alkenyl derivatives.
  • emulsion may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil (isoprenoid oil) produced by oligomerization of olefins, such as squalane or squalene oil , Especially isobutylene or decene; linear alkyl-containing esters of acids or alcohols, more especially vegetable oils, ethyl oleate, propylene glycol di-(caprylate/decanoate), glycerol tri-(caprylate/decanoate) Esters) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearates.
  • light liquid paraffin oil European Pharmacopea type
  • isoprenoid oil isoprenoid oil
  • isoprenoid oil produced by oligomerization of olefins, such as squalane or squalene oil , Especially isobutylene or decen
  • Emulsifiers are preferably nonionic surfactants, especially sorbitan esters, mannide esters (such as anhydrous mannide oleate), aliphatic glycol esters, polyglycerol ( polyglycerol), propylene glycol and oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxy Ethylene block copolymers, especially Pluronic products, especially L121. See “Theory and practical application of adjuvants" (Ed.
  • acrylic acid or methacrylic acid polymer is preferably a crosslinked acrylic or methacrylic acid polymer, especially crosslinked with polyalkenyl ethers or polyols of sugar.
  • Carbomer Carbomer, trade name Carbopol
  • Carbopol trade name Carbopol
  • Those skilled in the art can also refer to US Patent No. 2,09,462, which describes this type of acrylic polymer which is cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably no more than 8, of which at least 3 hydroxyl groups
  • the hydrogen atom of is replaced by an unsaturated aliphatic radical with at least 2 carbon atoms.
  • Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups.
  • the unsaturated group itself may contain other substituents, such as methyl.
  • Carbopol BF Goodrich, Ohio, USA
  • Carbopol 974P, 934P and 971P can be mentioned, and Carbopol 971P is most preferably used.
  • the term "copolymer of maleic anhydride and alkenyl derivatives" can also consider the copolymer of maleic anhydride and ethylene, EMA (Monsanto). These polymers are dissolved in water to produce an acidic solution. After neutralization, preferably It is neutralized to physiological pH in order to produce an adjuvant solution into which the immunogenic, immunogenic or vaccine composition itself can be incorporated.
  • adjuvant also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A) lipid A), Avridine lipid-amine adjuvant, E. coli heat labile enterotoxin (recombinant or other), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, etc.
  • the adjuvant includes white oil, aluminum gum adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, maleic anhydride and Copolymer of alkenyl derivatives, RIBI adjuvant system, Blockco-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera One or more of toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206 or Gel adjuvant.
  • “Degenerate sequence” In molecular biology, the phenomenon that the same amino acid has two or more codons is called codon degeneracy (degeneracy), such a sequence is called a degenerate sequence.
  • Gene recombination refers to the recombination of genes that control different traits. Modern genetic engineering technology implements genetic recombination in test tubes according to artificial design, also known as recombinant DNA. The purpose is to transfer the genetic gene in one individual cell to the DNA molecule in another individual cell with different traits to cause genetic variation. . After the target gene from the donor is transferred into the recipient bacteria, the gene product can be expressed, thereby obtaining products that are difficult to obtain by general methods.
  • Transformation refers to the acquisition of new genetic phenotypes for cells or cultured recipient cells through automatic acquisition or artificial supply of foreign DNA.
  • Transduction refers to the transfer of DNA and genes between the donor cell and the recipient cell when the virus is released from an infected (donor) cell and reinfects another (recipient) cell. Recombination is transduction.
  • prevention and/or treatment when referring to foot-and-mouth disease virus infection refers to inhibiting the replication of foot-and-mouth disease virus, inhibiting the spread of foot-and-mouth disease virus, or preventing foot-and-mouth disease virus from colonizing its host, and reducing the symptoms of diseases or symptoms of foot-and-mouth disease virus infection. If the viral load is reduced, the disease is reduced, and/or food intake and/or growth is increased, then the treatment can be considered to have achieved a therapeutic effect.
  • the chemical reagents used in the embodiments of the present invention are all analytically pure and purchased from Sinopharm Group.
  • the above-mentioned recombinant plasmid pET28a-VP0-VP3-VP1 inserted into the CATHAY type O foot-and-mouth disease virus VP0, VP3, VP1 gene was transformed into 40 ⁇ l of competent E. coli BL21 (DE3) prepared by the calcium chloride method, and then coated on Kanamya Solid LB medium resistant to steroids, stand at 37°C for 10-12 hours until a single colony is clearly visible, pick a single colony into a test tube containing 4ml of kanamycin-resistant liquid LB medium, 37°C, 230 Incubate with shaking for 12 hours, and then take 1ml of bacterial solution and freeze-dry it at -80°C for storage.
  • the VP1 gene fragment shown in ID NO.7 was used to construct an E. coli expression strain with the recombinant plasmid pET28a-VP4-VP2-VP3-VP1, which can express the SEA type O foot-and-mouth disease virus VP4, VP2, VP3, and VP1 genes in series. Freeze-dried storage at -80°C.
  • the fermenter used was a 50L fermentor of Shanghai Baoxing Biological Company, and 30L of culture medium was prepared into the fermentor, and sterilized at 121°C for 30 minutes. On the next day, 5L of seed liquid was connected to the fermentor. When the concentration of the culture liquid reached about 10 OD 600 , the culture temperature was lowered to 25°C, and 4g IPTG was added to induce the culture for 12 hours. The final concentration is about 40 (OD 600 ). Remove the fermentor and centrifuge to collect the bacteria.
  • the four SEA-type O-type foot-and-mouth disease virus antigens expressed in tandem in the bacteria were separated, purified and identified according to the preparation conditions similar to the above-mentioned CATHAY-type O-type foot-and-mouth disease virus antigen.
  • the SEA-type O-type foot-and-mouth disease virus-like particles were observed by phosphotungstic acid negative staining and electron microscope.
  • Vaccine composition containing CATHAY type O foot-and-mouth disease virus-like particle antigen
  • Adjuvants suitable for use in the present invention may be adjuvants known to those skilled in the art.
  • the adjuvant ISA 206 France Sepic is selected.
  • Vaccine composition containing CATHAY type O foot-and-mouth disease virus-like particle antigen and SEA type O foot-and-mouth disease virus-like particle antigen
  • the prepared CATHAY type O foot-and-mouth disease virus-like particle antigen and SEA type O foot-and-mouth disease virus-like particle antigen are prepared, and the vaccine composition is prepared according to the method of preparing a vaccine composition containing CATHAY type O foot-and-mouth disease virus-like particle antigen.
  • Adjuvants suitable for use in the present invention may be adjuvants known to those skilled in the art. In the present invention, the adjuvant ISA 206 (France Sepic) is selected.
  • the immunogenicity of the antigen in the vaccine composition was detected by the ELISA antibody level of the antibody in the pig serum after immunization.
  • the ELISA antibody level of the antibody in the pig serum after immunization is used to detect the duration of immunity of the antigen in the vaccine composition.
  • the commercial inactivated vaccine (O/Mya98/XJ/2010 strain + O/GX/09-7 strain) immunized group was used as the control group.
  • the immunization route was 2ml injection into the neck muscle.
  • the blank control group was immunized with the same amount of PBS and vaccine Blood was collected from each pig before immunization, blood was collected on the 21st day after immunization, and the second immunization was performed. After the second immunization, blood was collected on the 7, 14, 56, 84, and 112 days.
  • the immunogenicity of the antigen in the vaccine composition was detected by the ELISA antibody level of the antibody in the pig serum after immunization.
  • a vaccine composition containing CATHAY type O foot-and-mouth disease virus-like particle antigen and SEA type O foot-and-mouth disease virus-like particle antigen prepared by selecting healthy and susceptible pigs weighing about 40 kg with negative antigens and antibodies of type O foot-and-mouth disease virus.
  • the immunization route is neck muscle injection of 1ml
  • the control group is immunized with a vaccine composition containing CATHAY type O foot-and-mouth disease virus-like particle antigen, or a vaccine composition containing SEA type O foot-and-mouth disease virus-like particle antigen
  • the immunization route is neck muscle 2ml was injected, and the blank control group was immunized with 2ml PBS. Blood was collected from each pig before immunization, and blood was collected on the 7, 14, 21, and 28 days after immunization.
  • the bacterial cells expressing the antigen protein were resuspended and detected by SDS-PAGE electrophoresis. At this time, the expression levels of the three tandemly expressed proteins in the supernatant were all about 20%. SDS-PAGE electrophoresis of the purified protein showed that the target protein was purified and enriched.
  • Groups 1-3 are respectively the vaccine 1, vaccine 2, and vaccine 3 immunized groups prepared in Example 2 of the present invention, and the fourth group is a blank control group.
  • the immunization route of the immunization group was injection of 2ml into the neck muscle, and the control group was immunized with the same amount of PBS.
  • the antibody titer results showed that the antibodies of all pigs were negative before the vaccine immunization, and they could reach more than 1:128 on the 14th day after the first immunization.
  • the antibody of the blank control group was negative and there was no change.
  • the specific results are shown in Table 2.
  • virus-like particles prepared by the present invention can quickly form high-level specific antibodies, even if the antigen content is 160 ⁇ g/ml, it can play a very good immune protection against CATHAY type O foot-and-mouth disease on the 14th day after immunization.
  • Example 4 Comparative test of immunogenicity of vaccine composition containing CATHAY type O foot-and-mouth disease virus-like particle antigen
  • the fifth group is the vaccine 2 immunization group prepared in Example 2 of the present invention
  • the seventh group is the commercial inactivated vaccine (O/Mya98/XJ/2010 strain + O/GX/09-7 strain) immunization group
  • the sixth group is the control group.
  • the immunization route of the fifth group was intramuscular injection of 2ml in the neck, and the sixth group was immunized with the same amount of PBS, and each pig was immunized once.
  • the virus-like particle vaccine composition prepared by the present invention compared with the commercialized whole virus inactivated vaccine, not only produces fast antibodies to the CATHAY type O foot-and-mouth disease virus-like particle antigen, but also has a high antibody level, which can be achieved with only one immunization. Very good immune protection, and the duration of immunity is significantly increased, which can maintain immune protection for a longer time.
  • the bacterial cells expressing protein antigens were resuspended and detected by SDS-PAGE electrophoresis. At this time, the expression levels of the four serially expressed proteins in the supernatant were all about 20%. SDS-PAGE electrophoresis of the purified protein showed that the target protein was purified and enriched.
  • the SEA-type O-type foot-and-mouth disease protein formed virus-like particles, and the formed virus-like particles were full, with high assembly efficiency and no aggregation.
  • the negative staining with phosphotungstic acid and electron microscopy showed that the virus-like particles were still full without aggregation. It shows that the foot-and-mouth disease protein prepared by the sequence screened in the present invention forms a stable virus-like particle.
  • the adjuvant ISA 206 France Sepic is selected.
  • Example 70 Immunogenicity test of the vaccine composition of type O foot-and-mouth disease virus-like particles
  • Groups 9-13 are respectively the vaccine 4, vaccine 5, vaccine 6, and vaccine 7 prepared in Example 6 of the present invention, and the vaccine 2 prepared in Example 2 of the present invention.
  • Group 14 is a blank control group.
  • the 9th, 10th, and 11th groups are immunized by intramuscular injection of 1ml in the neck, and the 12th group (vaccine 7 immunization group) and 13 (vaccine 2 immunization group) )
  • the route of immunization was 2ml intramuscular injection in the neck, and the control group was immunized with 2ml PBS. Blood was collected from each pig before immunization, and blood was collected on the 7, 14, 21, and 28 days after immunization.
  • the sera collected from the 9th, 10th, 11th, 13th and 14th groups were tested for related antibodies using CATHAY type O foot-and-mouth disease antibody ELISA test kit.
  • the results showed that the antibodies of all pigs were negative before the vaccine immunization, and it could reach more than 1:128 on the 14th day after one immunization; the bivalent type O foot-and-mouth disease virus-like particle vaccine composition was immunized in 1ml (conventional dosage of 2ml).
  • Half The antibody level still meets or exceeds the antibody level of the monovalent type O foot-and-mouth disease virus-like particle vaccine composition in the immune volume of 2 ml.
  • the antibody of the blank control group was negative and there was no change.
  • Table 5 The specific results are shown in Table 5.
  • the sera collected from the 9th, 10th, 11th, 12th and 14th groups were tested for related antibodies using the SEA type O foot-and-mouth disease antibody ELISA test kit.
  • the results showed that the antibodies of all pigs were negative before the vaccine immunization, and it could reach more than 1:128 on the 14th day after one immunization; the bivalent type O foot-and-mouth disease virus-like particle vaccine composition was immunized in 1ml (conventional dosage of 2ml).
  • Half The antibody level still meets or exceeds the antibody level of the monovalent type O foot-and-mouth disease virus-like particle vaccine composition in the immune volume of 2 ml.
  • the antibody of the blank control group was negative and there was no change.
  • Table 6 The specific results are shown in Table 6.
  • the bivalent O-type foot-and-mouth disease virus-like particles prepared by the present invention can quickly form high-level specific antibodies, and can play a good immune protection against CATHAY-type O-type foot-and-mouth disease and SEA-type O-type foot-and-mouth disease; at the same time, it also It shows that the bivalent type O foot-and-mouth disease virus-like particles can initiate an immune response faster and better than the monovalent type O foot-and-mouth disease virus-like particles, and can achieve a better immune response with a lower immune dose (half the conventional dose or less than half the dose). Good immune effect.

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Abstract

提供了一种O型口蹄疫病毒样颗粒抗原，其中，所述O型口蹄疫病毒样颗粒抗原为CATHAY型O型口蹄疫病毒样颗粒抗原，所述CATHAY型O型口蹄疫病毒样颗粒抗原由CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白组装而成。所述O型口蹄疫病毒样颗粒抗原免疫原性好，制备的疫苗一次免疫，免疫后第14日就能产生对O型口蹄疫病毒的完全保护，产生的抗体效价较商业灭活疫苗滴度更高，且免疫保护期可维持至少133天。还提供了制备的疫苗组合物、及其制备方法和应用。

Description

口蹄疫病毒样颗粒抗原、及其疫苗组合物、制备方法和应用 技术领域
本发明属于病毒性抗原及该抗原的医药配制品。具体地，本发明涉及一种口蹄疫病毒样颗粒抗原、其制备的疫苗组合物及其制备方法和应用。
背景技术
口蹄疫(foot-and-mouth disease,FMD)为一种急性、高度接触传染性和可快速远距离传播的动物疾病，是哺乳类动物中感染性最强的疾病，其中偶蹄类动物染病更会造成全球重大的经济损失。遭受口蹄疫危害的动物包括牛、羊和猪。致病因子为口蹄疫病毒(FMDV)，是小核糖核酸病毒属家族的一种口疮病毒。该病毒分为7个血清型(A、O、C、Asia l、SAT1、SAT2和SAT3型)，其中O型口蹄疫病毒流行最为广泛。按遗传分类，我国目前主要流行三种遗传拓扑型(Topotype)的O型口蹄疫病毒，分别属于CATHAY型(中国型)、ME-SA型(中东-南亚型)和SEA型(东南亚型)。疫苗免疫是控制此病、保护家畜免受危害的有效措施。
然而，O型口蹄疫病毒灭活抗原抗原性弱，现有的O型口蹄疫病毒灭活苗往往要经两次免疫才能产生保护。病毒样颗粒(VLPs)是一种在体外和/或体内表达时能够自主包装成病毒外壳结构的类病毒粒子，它们是具有病毒的相似外壳结构但不具有病毒复制能力的假病毒。VLPs疫苗能有效地激发机体产生抗感染，抗肿瘤免疫，基于病毒样颗粒设计的疫苗是一种较为理想的疫苗形式。然而，目前还未有关于O型口蹄疫病毒样颗粒疫苗实际应用的报道。
另外我国流行的CATHAY型变异株，其抗原发生较大变异，原有疫苗对其保护力明显下降，加之自身免疫原性较弱，不能诱导动物机体 产生足够的免疫力。因此，筛选理想的毒株序列制备病毒样颗粒是当务之急，也符合国家提出的有效防控重大动物疫病，保障畜牧业健康可持续发展的需要。
发明内容
为解决现有技术中的O型口蹄疫病毒灭活疫苗免疫原性不强的缺陷，本发明提供了一种O型口蹄疫病毒样颗粒抗原，其中，所述O型口蹄疫病毒样颗粒抗原为CATHAY型O型口蹄疫病毒样颗粒抗原，所述CATHAY型O型口蹄疫病毒样颗粒抗原由CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白组装而成。
所述口蹄疫病毒样颗粒抗原具有良好的免疫原性，仅一次免疫就能提供对口蹄疫病毒的完全保护。所述口蹄疫病毒样颗粒抗原具有良好的稳定性，在4℃下放置3个月，再通过磷钨酸负染及电镜观察仍可见病毒样颗粒依旧饱满，无聚集现象。
作为本发明的一种实施方式，本发明所述的O型口蹄疫病毒样颗粒抗原中，所述CATHAY型O型口蹄疫病毒VP0抗原蛋白的基因如SEQ ID No.1或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP3抗原蛋白的基因如SEQ ID No.2或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP1抗原蛋白的基因如SEQ ID No.3或其简并序列所示。
所述CATHAY型O型口蹄疫病毒样颗粒抗原来源于现在的流行株，能针对现在的流行野毒株产生完全保护。
本发明还提供一种O型口蹄疫病毒样颗粒疫苗，其中，所述O型口蹄疫病毒样颗粒疫苗包含免疫量的所述的CATHAY型O型口蹄疫病毒样颗粒抗原和药学上可以接受的载体。
本发明的O型口蹄疫病毒样颗粒疫苗免疫后第14日即能达到1:128以上的抗体效价，且能维持长时期高滴度抗体效价，能针对整个育肥期产生保护。
作为本发明的一种实施方式，本发明所述的O型口蹄疫病毒样颗 粒疫苗中，所述的CATHAY型O型口蹄疫病毒样颗粒抗原含量为160-240μg/ml。
CATHAY型O型口蹄疫病毒样颗粒抗原含量可选自160μg/ml、170μg/ml、180μg/ml、190μg/ml、200μg/ml、210μg/ml、220μg/ml、230μg/ml、240μg/ml。
即使当CATHAY型O型口蹄疫病毒样颗粒抗原含量仅为160μg/ml，免疫后第14日也能达到1:128以上的抗体效价，且能维持长时期的高滴度的抗体效价。
作为本发明的一种优选实施方式，本发明所述的O型口蹄疫病毒样颗粒疫苗中，所述的CATHAY型O型口蹄疫病毒样颗粒抗原含量为200μg/ml。
作为本发明的一种实施方式，本发明所述的O型口蹄疫病毒样颗粒疫苗中，所述药学上可以接受的载体包括佐剂，所述佐剂包括：(1)白油、铝胶佐剂、皂苷、阿夫立定、DDA；(2)油包水乳剂、水包油乳剂、水包油包水乳剂；或(3)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的共聚物；以及RIBI佐剂系统、Block co-polymer、SAF-M、单磷酰脂质A、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素、霍乱毒素、IMS 1314、胞壁酰二肽、Montanide ISA 206、Gel佐剂中的一种或几种；优选地，皂苷为Quil A、QS-21、GPI-0100。
所述佐剂含量为5％-60％V/V，优选从30％-60％V/V，更优选50％V/V。
作为本发明的一种实施方式，所述的药学上可接受的载体包括药物、免疫刺激剂、抗氧化剂、表面活性剂、着色剂、挥发性油、缓冲剂、分散剂、推进剂和防腐剂；所述免疫刺激剂包括α-干扰素、β-干扰素、γ-干扰素、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)和白介素2(IL2)。
为了制备这样的组合物，可以使用本领域公知的方法。
作为本发明的一种实施方式，本发明所述的O型口蹄疫病毒样颗粒疫苗中，所述O型口蹄疫病毒样颗粒疫苗还包含免疫量的SEA型O 型口蹄疫病毒样颗粒抗原。
本发明的所述疫苗中加入SEA型O型口蹄疫病毒样颗粒抗原，能对CATHAY型O型口蹄疫和SEA型O型口蹄疫产生免疫保护。
作为本发明的一种优选实施方式，本发明所述的O型口蹄疫病毒样颗粒疫苗中，所述SEA型O型口蹄疫病毒样颗粒抗原由SEA型O型口蹄疫病毒VP4、VP2、VP3、VP1抗原蛋白组装而成。
本发明的SEA型O型口蹄疫病毒样颗粒抗原具有良好的免疫原性，疫苗免疫后第14日即能达到1:128以上的SEA型O型口蹄疫抗体效价。所述SEA型O型口蹄疫病毒样颗粒抗原具有良好的稳定性，在4℃下放置3个月，再通过磷钨酸负染及电镜观察仍可见病毒样颗粒依旧饱满，无聚集现象。
作为本发明的一种实施方式，本发明所述的O型口蹄疫病毒样颗粒疫苗中，所述SEA型O型口蹄疫病毒VP4抗原蛋白的基因如SEQ ID No.4或其简并序列所示；所述SEA型O型口蹄疫病毒VP2抗原蛋白的基因如SEQ ID No.5或其简并序列所示；所述SEA型O型口蹄疫病毒VP3抗原蛋白的基因如SEQ ID No.6或其简并序列所示；所述SEA型O型口蹄疫病毒VP1抗原蛋白的基因如SEQ ID No.7或其简并序列所示。
作为本发明的一种实施方式，本发明所述的O型口蹄疫病毒样颗粒疫苗中，所述的SEA型O型口蹄疫病毒样颗粒抗原含量为160-240μg/ml。
SEA型O型口蹄疫病毒样颗粒抗原含量可选自160μg/ml、170μg/ml、180μg/ml、190μg/ml、200μg/ml、210μg/ml、220μg/ml、230μg/ml、240μg/ml。
作为本发明的一种优选实施方式，本发明所述的O型口蹄疫病毒样颗粒疫苗中，所述的SEA型O型口蹄疫病毒样颗粒抗原含量为200μg/ml。
本发明还涉及一种制备所述O型口蹄疫病毒样颗粒疫苗的方法，其中，所述方法包括：步骤(1)将CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白的基因分别扩增、克隆到同一串联表达载体，得到 含有CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白基因的重组表达载体；步骤(2)将所述步骤(1)得到的含有CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白基因的重组表达载体转化或转导宿主，得到含有所述重组表达载体的重组子；步骤(3)培养所述步骤(2)得到的重组子，串联表达所述CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白，所述表达的CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白自我组装，形成所述CATHAY型O型口蹄疫病毒样颗粒抗原；以及步骤(4)纯化所述步骤(3)得到的所述CATHAY型O型口蹄疫病毒样颗粒抗原，加入佐剂，得到所述O型口蹄疫病毒样颗粒疫苗。
本发明通过表达CATHAY型O型口蹄疫病毒的VP0、VP3、VP1抗原蛋白，能够得到稳定的自我组装的病毒样颗粒，其免疫效果能够实现对猪只的完全保护。本发明通过串联表达O型口蹄疫病毒VP0、VP3、VP1抗原蛋白，表达的活性蛋白自我组装成CATHAY型O型口蹄疫病毒样颗粒抗原，利于后续的抗原纯化和分离。
作为本发明的一种实施方式，本发明所述的方法中，所述步骤(1)中的所述CATHAY型O型口蹄疫病毒VP0抗原蛋白的基因如SEQ ID No.1或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP3抗原蛋白的基因如SEQ ID No.2或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP1抗原蛋白的基因如SEQ ID No.3或其简并序列所示；所述步骤(2)中的宿主为E.coli；所述步骤(3)中所述表达的CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白为细胞内可溶性蛋白。
本发明利用大肠杆菌表达系统生产口蹄疫病毒样颗粒，具有产量高、生产成本低、免疫原性好、无生物安全风险等优点。本发明制备的病毒样颗粒疫苗组合物不仅能对O型口蹄疫提供保护活性，并且相比于现有的商品化全病毒灭活疫苗，不仅抗体产生快，抗体产生水平高，而且免疫持续期显著增长，能维持更长时间的免疫保护。
本发明还涉及所述的O型口蹄疫病毒样颗粒疫苗在制备预防和/或治疗O型口蹄疫的药物中的应用。
本发明所述的制备预防和/或治疗口蹄疫病毒感染的药物的施用对象包括猪。
具体实施方式
以下，对本发明的实施方式进行说明。
定义
“口蹄疫病毒”属于小RNA病毒科，口蹄疫病毒属，该病毒有O、A、C、SAT1、SAT2、SAT3(即南非口蹄疫病毒1、2、3型)和Asia1(亚洲1型)7个血清型，各型之间无交叉保护反应，每个类型内又有多个亚型。在病毒的中心为一条单链的正链RNA，由大约8000个碱基组成，是感染和遗传的基础；周围包裹着蛋白质决定了病毒的抗原性、免疫性和血清学反应能力；病毒外壳为对称的20面体。口蹄疫病毒是偶蹄类动物高度传染性疾病——口蹄疫的病原，国际兽疫局将口蹄疫列为“A类动物传染病名单”中的首位，中国把它列为“进境动物检疫一类传染病”，我国对口蹄疫的防治，预防主要通过疫苗注射接种，发生口蹄疫的则捕杀。
“抗原(Antigen)”是指能诱导机体发生免疫应答的物质，即能被T/B淋巴细胞表面的抗原受体(TCR/BCR)特异性识别与结合，活化T/B细胞，使之增殖分化，产生免疫应答产物(致敏淋巴细胞或抗体)，并能与相应产物在体内外发生特异性结合的物质。
“病毒样颗粒(virus-like particles，VLPs)”是由一种或多种病毒结构蛋白组装成的颗粒，具有与病毒颗粒相似的外部结构和抗原性，但不含病毒基因。
“口蹄疫病毒VP0、VP3、VP1抗原蛋白”：FMDV结构蛋白前体蛋白P1被蛋白酶3C催化加工成VP0、VP1和VP3，这3种蛋白质自我组装成二十面体的病毒衣壳。VP0蛋白是P1经蛋白酶3C裂解后的中间体，在病毒粒子形成的最后阶段，VP0成熟裂解为VP2和VP4。
本发明所用术语“疫苗”、“疫苗组合物”指含有口蹄疫病毒样颗粒抗原的药物组合物，该药物组合物可诱发、刺激或增强猪只针对口蹄疫的 免疫反应。
术语“免疫量”应当理解为“免疫有效量”，又称免疫保护量或产生免疫应答的有效量，为可在接受者体内有效诱导免疫应答的抗原量，该量足以预防或改善疾病的体征或症状，包括不利的健康影响或其并发症。所述免疫应答可能足以用于诊断目的或其它试验，或可能适合用于预防疾病的征兆或症状，包括由病原体引起的感染所造成的不利的健康结果或其并发症。体液免疫力或由细胞介导的免疫力或此二者均可被诱导。动物对免疫原性组合物的免疫应答可通过例如测量抗体效价、淋巴细胞增殖分析而间接评估，或在以野生型毒株攻击后通过监测征兆或症状来直接评估，而该由疫苗提供的保护性免疫力可通过测量例如受试者的临床征兆如死亡率、发病率的减少、温度数值、受试者总体生理状况及总体健康和表现来评估。所述免疫应答可包括但不限于诱导细胞性和/或体液免疫力。
术语“药学上可接受的载体”是指在本发明疫苗组合物中除口蹄疫病毒抗原之外的其它所有成分，不刺激机体不阻碍使用化合物的生物学活性和特性的载体或者稀释剂，优选为佐剂。术语“佐剂”可包括铝胶佐剂；皂苷(saponin)，如Quil A、QS-21(Cambridge Biotech Incorporation,Cambridge MA)、GPI-0100(Galenica Pharmaceuticals Incorporation，Birmingham AL)；油包水乳剂；水包油乳剂；水包油包水乳剂；丙烯酸或甲基丙烯酸的聚合物；顺丁烯二酸酐和链烯基(alkenyl)衍生物的共聚物选出的化合物。术语“乳剂”可尤其基于轻液体石蜡油(European Pharmacopea类型)；因烯烃寡聚产生的类异戊二烯油(isoprenoid oil)，如角鲨烷(squalane)或角鲨烯油(squalene oil)，尤其异丁烯或葵烯；酸或醇的含线性烷基的酯，更尤其植物油、油酸乙酯、丙二醇二-(辛酸酯/葵酸酯)、甘油三-(辛酸酯/葵酸酯)或丙二醇二油酸酯；支链脂肪酸或醇的酯，尤其异硬脂酸酯。油与乳化剂组合使用以便形成乳剂。乳化剂优选非离子表面活性剂，尤其山梨聚糖的酯、二缩甘露醇(mannide)的酯(如无水甘露醇油酸酯)、脂肪族二元醇(glycol)的酯、聚甘油(polyglycerol)的酯、丙二醇的酯以及油酸的酯、异硬脂酸 的酯、蓖麻油酸的酯或羟基硬脂酸的酯，它们任选乙氧基化，还有聚氧丙烯-聚氧乙烯嵌段共聚物，尤其Pluronic产品，特别是L121。参见Hunter等编写的《The theory and practical application of adjuvants》(Ed.by DES Stewart-Tull,John Wiley and Sons,New York,1995:51-94)和Todd等编写的《Vaccine》(1997,15:564-570)。例如，可使用Powell M和Newman M编写的《Vaccine design,the Subunit and adiuvant approach》(Plenum Press,1995)第147页描述的SPT乳剂及第183页描述的MF59乳剂。术语“丙烯酸或甲基丙烯酸的聚合物”优选为交联的丙烯酸或甲基丙烯酸聚合物，尤其是与糖(sugar)的聚链烯基醚或聚醇交联，这些化合物已知被称为卡波姆(Carbomer，商品名Carbopol)(Phameuropa,1996,8(2))。本领域技术人员还可参见美国专利US2909462，其描述了这类丙烯酸聚合物，其与聚羟基化的化合物交联，所述化合物具有至少3个羟基，优选不超过8个，其中至少3个羟基的氢原子被具有至少2个碳原子的不饱和脂烃基(aliphatic radical)取代。优选的基团是那些含有2-4个碳原子的基团，例如乙烯基、烯丙基和其它烯属不饱和基团(ethylenically unsaturated group)。所述不饱和基团自身可包含其它取代基，如甲基。这些产品以卡波普的名义出售，(BF Goodrich,Ohio,USA)特别合适。它们与烯丙基蔗糖或与烯丙基季戊四醇(allyl pentaerythritol)交联。这其中可提及卡波普974P、934P和971P，最优选使用卡波普971P。术语“顺丁烯二酸酐和链烯基衍生物的共聚物”也可考虑顺丁烯二酸酐与乙烯的共聚物EMA(Monsanto)，这些聚合物在水中溶解产生酸性溶液，经中和，优选中和至生理pH，以便产生佐剂溶液，能向其中掺入免疫原性、致免疫性或疫苗性组合物本身。术语“佐剂”还包括，但不限于，RIBI佐剂系统(Ribi Incorporation)、Block co-polymer(CytRx,Atlanta GA)、SAF-M(Chiron,Emeryville CA)、单磷酰脂质A(monophosphoryl lipid A)、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素(重组或其它)、霍乱毒素、IMS 1314、胞壁酰二肽、Gel佐剂等。优选地，所述佐剂包括白油、铝胶佐剂、皂苷、油包水乳剂、水包油乳剂、水包油包水乳剂、丙烯酸或甲基丙烯酸的聚合物、顺 丁烯二酸酐和链烯基(alkenyl)衍生物的共聚物、RIBI佐剂系统、Block co-polymer、SAF-M、单磷酰脂质A、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素、霍乱毒素、IMS 1314、胞壁酰二肽、Montanide ISA 206或Gel佐剂中的一种或几种。
“简并序列”：在分子生物学中，同一种氨基酸具有两个或更多个密码子的现象称为密码子的简并性(degeneracy)，这样的序列就叫简并序列。
“基因重组”：是指控制不同性状的基因重新组合。现代基因工程技术在试管内按人为的设计实施基因重组，也称为重组DNA，目的是将一个个体细胞内的遗传基因转移到另一个不同性状的个体细胞内DNA分子上，使之发生遗传变异。来自供体的目的基因被转入受体细菌后，可进行基因产物的表达，从而获得用一般方法难以获得的产品。
“转化(transformation)”是指通过自动获取或人为地供给外源DNA，使细胞或培养的受体细胞获得新的遗传表型。
“转导(transduction)”是指当病毒从被感染的(供体)细胞释放出来、再次感染另一(受体)细胞时，发生在供体细胞与受体细胞之间的DNA转移及基因重组即为转导。
术语“预防和/或治疗”在涉及口蹄疫病毒感染时是指抑制口蹄疫病毒的复制、抑制口蹄疫病毒的传播或防止口蹄疫病毒在其宿主体内定居，以及减轻口蹄疫病毒感染的疾病或病症的症状。若病毒荷载量减少、病症减轻和/或摄食量和/或生长增加，那么就可以认为所述治疗达到了治疗效果。
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
本发明实施例中所用到的化学试剂均为分析纯，购自国药集团。
为使本发明更加容易理解，下面结合具体实施例，进一步阐述本发明。本发明所述的实验方法，若无特殊说明，均为常规方法；所述的生物材料，若无特殊说明，均可从商业途径获得。
实施例
材料及方法
载体的构建和转化
含有CATHAY型O型口蹄疫病毒VP0、VP3、VP1基因的重组载体pET28a-VP0-VP3-VP1
由金唯智公司合成序列表SEQ ID NO.1所示的VP0基因片段、序列表SEQ ID NO.2所示的VP3基因片段、序列表SEQ ID NO.3所示的VP1基因片段，分别与pBLUE-T Vector载体连接，将连接成功的重组克隆分别经BamH I/EcoR I、Sac I/Sal I、Hind III/Xho I酶切消化后得到的片段，与经过相同的酶切的pET28a载体连接。
将连接产物转化到用CaCl 2制备的DH5α感受态细胞中，涂布于卡那霉素抗性的固体LB培养基上，等单克隆菌落清晰可见时，挑取单克隆至含有卡那霉素的LB液体培养基中，37℃230转/分钟，培养12小时过夜，提取重组质粒pET28a-VP0-VP3-VP1。
将上述的插入CATHAY型O型口蹄疫病毒VP0、VP3、VP1基因的重组质粒pET28a-VP0-VP3-VP1转化40μl以氯化钙法制备的感受态大肠杆菌BL21(DE3)，涂布于卡那霉素抗性的固体LB培养基，37℃静置培养10-12小时至单菌落清晰可见时，挑取单克隆至含4ml卡那霉素抗性的液体LB培养基的试管，37℃，230转/分振荡培养12小时，从中取1ml菌液于-80℃冻干保存。
含有SEA型O型口蹄疫病毒VP4、VP2、VP3、VP1基因的重组载体
由金唯智公司合成序列表SEQ ID NO.4所示的VP4基因片段、序列表SEQ ID NO.5所示的VP2基因片段、序列表SEQ ID NO.6所示的VP3基因片段、序列表SEQ ID NO.7所示的VP1基因片段，构建带有重组质粒pET28a-VP4-VP2-VP3-VP1、能串联表达SEA型O型口蹄疫 病毒VP4、VP2、VP3、VP1基因的大肠杆菌表达菌株，于-80℃冻干保存。
抗原蛋白的表达及O型口蹄疫病毒样颗粒粒子的鉴定
CATHAY型O型口蹄疫病毒抗原和病毒样颗粒粒子
从-80℃中取出带有重组质粒pET28a-VP0-VP3-VP1的大肠杆菌菌种接种卡那霉素抗性的50ml LB液体培养基，37℃，230转/分振荡培养12小时后，转接入1L LB液体培养基中，37℃培养，制备发酵用种子液。
使用发酵罐为上海保兴生物公司50L发酵罐，配制30L培养基装入发酵罐，121℃灭菌30分钟。第二天将5L种子液接入发酵罐，培养菌液浓度达到OD 600大约10左右时将培养温度降至25℃，加入4gIPTG诱导培养12小时。终浓度大约为40左右(OD 600)拆下发酵罐，离心收集菌体。
重悬菌体，采用均质机以800bar压力破碎菌体4次。13500rpm，离心40min，留取上清，通过15％SDS-PAGE电泳检测。采用硫酸铵分级沉淀法进行蛋白粗纯，随后进行色谱纯化，纯化后的蛋白经SDS-PAGE电泳。
通过磷钨酸负染及电镜观察CATHAY型O型口蹄疫病毒样颗粒。
SEA型O型口蹄疫病毒样颗粒抗原和病毒样颗粒粒子
从-80℃中取出带有重组质粒pET28a-VP4-VP2-VP3-VP1的大肠杆菌菌种接种卡那霉素抗性的50ml LB液体培养基，按上述CATHAY型O型口蹄疫病毒抗原相似的制备条件培养，再转接入1L LB液体培养基中，37℃培养。
使用50L发酵罐，按上述CATHAY型O型口蹄疫病毒抗原相似的制备条件大规模发酵、表达SEA型O型口蹄疫病毒抗原。
按上述CATHAY型O型口蹄疫病毒抗原相似的制备条件分离、纯化和鉴定菌体中的串联表达的四个SEA型O型口蹄疫病毒抗原。
通过磷钨酸负染及电镜观察SEA型O型口蹄疫病毒样颗粒。
O型口蹄疫病毒样颗粒疫苗组合物的制备
含CATHAY型O型口蹄疫病毒样颗粒抗原疫苗组合物
取制备的CATHAY型O型口蹄疫病毒样颗粒抗原缓缓加入到佐剂中，加的过程不断用转速为800rpm乳化机搅拌12min，混匀，4℃保存，即为含CATHAY型O型口蹄疫病毒样颗粒抗原的疫苗组合物。适用于本发明的佐剂可以为本领域技术人员公知的佐剂。在本发明中，选用佐剂ISA 206(法国赛比克公司)。
含CATHAY型O型口蹄疫病毒样颗粒抗原和SEA型O型口蹄疫病毒样颗粒抗原的疫苗组合物
取制备的CATHAY型O型口蹄疫病毒样颗粒抗原、SEA型O型口蹄疫病毒样颗粒抗原，按上述制备含CATHAY型O型口蹄疫病毒样颗粒抗原疫苗组合物的方法，制备疫苗组合物。适用于本发明的佐剂可以为本领域技术人员公知的佐剂。在本发明中，选用佐剂ISA 206(法国赛比克公司)。
O型口蹄疫病毒样颗粒疫苗组合物的免疫原性分析
CATHAY型O型口蹄疫病毒样颗粒疫苗组合物的免疫原性
采用免疫后猪血清中抗体的ELISA抗体水平检测疫苗组合物中抗原的免疫原性。
选取O型口蹄疫病毒抗原、抗体均为阴性的体重40kg左右的健康易感架子猪，免疫制备的含CATHAY型O型口蹄疫病毒样颗粒抗原的疫苗组合物，免疫途径为颈部肌肉注射2ml，空白对照组免疫等量的PBS。疫苗免疫前每头猪采血，免疫后分别于第7日、14日、21日、28日采血。对采集的血清使用CATHAY型O型口蹄疫抗体ELISA检测试剂盒进行抗体的检测。
CATHAY型O型口蹄疫病毒样颗粒疫苗组合物的免疫持续期实验
采用免疫后猪血清中抗体的ELISA抗体水平检测疫苗组合物中抗原的免疫持续期。
选取O型口蹄疫病毒抗原、抗体均为阴性的体重40kg左右的健康 易感架子猪，免疫制备的含CATHAY型O型口蹄疫病毒样颗粒抗原的疫苗组合物，免疫途径为颈部肌肉注射2ml，空白对照组免疫等量的PBS，均一次免疫，疫苗免疫前每头猪采血，免疫后21日、28日、35日、77日、105日、133日采血。
商品化灭活苗(O/Mya98/XJ/2010株+O/GX/09-7株)免疫组作为对照组，免疫途径为颈部肌肉注射2ml，其空白对照组免疫等量的PBS，疫苗免疫前每头猪采血，免疫后第21日采血并进行第2次免疫，2次免疫后分别于第7日、14日、56日、84日、112日采血。
含CATHAY型O型口蹄疫病毒样颗粒抗原和SEA型O型口蹄疫病毒样颗粒抗原的疫苗组合物中抗原免疫原性
采用免疫后猪血清中抗体的ELISA抗体水平检测疫苗组合物中抗原的免疫原性。
选取O型口蹄疫病毒抗原、抗体均为阴性的体重40kg左右的健康易感架子猪，免疫制备的含CATHAY型O型口蹄疫病毒样颗粒抗原和SEA型O型口蹄疫病毒样颗粒抗原的疫苗组合物，免疫途径为颈部肌肉注射1ml，对照组免疫含CATHAY型O型口蹄疫病毒样颗粒抗原的疫苗组合物，或免疫含SEA型O型口蹄疫病毒样颗粒抗原的疫苗组合物，免疫途径为颈部肌肉注射2ml，空白对照组免疫2ml的PBS。疫苗免疫前每头猪采血，免疫后分别于第7日、14日、21日、28日采血。
实施例1 CATHAY型O型口蹄疫病毒样颗粒
重悬表达抗原蛋白的菌体，通过SDS-PAGE电泳检测，此时上清中三个串联表达的蛋白其各自表达量均在20％左右。纯化后的蛋白经SDS-PAGE电泳，显示目的蛋白均得到了纯化和富集。
通过磷钨酸负染及电镜观察可见CATHAY型O型口蹄疫蛋白形成了病毒样颗粒，并且形成的病毒样颗粒饱满，组装效率高，没有聚集。通过将口蹄疫病毒样颗粒置于4℃下放置3个月，再通过磷钨酸负染及电镜观察可见病毒样颗粒依旧颗粒饱满，无聚集现象。说明本发明筛选的序列制备的口蹄疫蛋白形成了稳定的病毒样颗粒。
实施例2 CATHAY型O型口蹄疫病毒样颗粒疫苗组合物的制备
制备的疫苗中各组分的具体配比见表1。
表1 CATHAY型O型口蹄疫病毒样颗粒疫苗组合物成分配比
  疫苗1 疫苗2 疫苗3
口蹄疫抗原(μg/ml) 160 200 240
ISA 206佐剂(V/V％) 50％ 50％ 50％
实施例3含CATHAY型O型口蹄疫病毒样颗粒抗原的疫苗组合物的免疫原性试验
选取O型口蹄疫病毒抗原、抗体均为阴性的体重40kg左右的健康易感架子猪20头，随机分为4组，每组5头。1-3组分别为本发明实施例2制备的疫苗1、疫苗2、疫苗3免疫组，第4组为空白对照组。免疫组免疫途径为颈部肌肉注射2ml，对照组免疫等量的PBS。
抗体效价结果显示，疫苗免疫前所有猪只的抗体均为阴性，1次免疫后第14日均能达到1:128以上。空白对照组猪只抗体为阴性，无变化。具体结果见表2。
表2 CATHAY型O型口蹄疫ELISA抗体水平
Figure PCTCN2019071813-appb-000001
Figure PCTCN2019071813-appb-000002
表明本发明制备的病毒样颗粒能快速形成高水平的特异性抗体，即便抗原含量为160μg/ml，在免疫后第14天即能对CATHAY型O型口蹄疫起到很好的免疫保护作用。
实施例4含CATHAY型O型口蹄疫病毒样颗粒抗原的疫苗组合物的免疫原性对比试验
选取O型口蹄疫病毒抗原、抗体均为阴性的体重40kg左右的健康易感架子猪20头，随机分为4组，每组5头。第5组为本发明实施例2制备的疫苗2免疫组，第7组为商品化灭活苗(O/Mya98/XJ/2010株+O/GX/09-7株)免疫组，第6组、第8组为对照组。第5组免疫组免疫途径为颈部肌肉注射2ml，第6组对照组免疫等量的PBS，均一次免疫，疫苗免疫前每头猪采血，免疫后21日、28日、35日、77日、105日、133日采血；第7组免疫组免疫途径为颈部肌肉注射2ml，第8组对照组免疫等量的PBS，疫苗免疫前每头猪采血，免疫后第21日采血并进行第2次免疫，2次免疫后分别于第7日、14日、56日、84日、112日采血。
结果显示，疫苗免疫前所有猪只的抗体均为阴性，1次免疫后第21日疫苗2免疫组能达到1:128以上，商品苗免疫组不能达到1:128，商 品苗免疫组2次免疫后第7日才能达到1:128；疫苗2免疫组1次免疫133日，依然维持较高的抗体水平，而商品苗免疫组2次免疫后第112日，抗体水平接近1:128免疫保护临界值。对照组猪只抗体为阴性，无变化。具体结果见表3。
表3 CATHAY型O型口蹄疫ELISA抗体水平比较结果
Figure PCTCN2019071813-appb-000003
上述实验表明本发明制备的病毒样颗粒疫苗组合物与商品化全病毒灭活苗相比，对CATHAY型O型口蹄疫病毒样颗粒抗原不仅抗体产生快，抗体水平高，仅一次免疫就能起到很好的免疫保护作用，而且免疫持续期显著增长，能维持更长时间的免疫保护。
实施例5SEA型O型口蹄疫病毒样颗粒
重悬表达蛋白抗原的菌体，通过SDS-PAGE电泳检测，此时上清中四个串联表达的蛋白其各自表达量均在20％左右。纯化后的蛋白经SDS-PAGE电泳，显示目的蛋白均得到了纯化和富集。
通过磷钨酸负染及电镜观察可见SEA型O型口蹄疫蛋白形成了病毒样颗粒，并且形成的病毒样颗粒饱满，组装效率高，没有聚集。通过将口蹄疫病毒样颗粒置于4℃下放置3个月，再通过磷钨酸负染及电镜观察可见病毒样颗粒依旧颗粒饱满，无聚集现象。说明本发明筛选的序列制备的口蹄疫蛋白形成了稳定的病毒样颗粒。
实施例6O型口蹄疫病毒样颗粒疫苗组合物的制备
制备的疫苗中各组分的具体配比见表4。在本实施例中，选用佐剂ISA 206(法国赛比克公司)。
表4 O型口蹄疫病毒样颗粒疫苗组合物成分配比
Figure PCTCN2019071813-appb-000004
实施例7O型口蹄疫病毒样颗粒疫苗组合物的免疫原性试验
选取O型口蹄疫病毒抗原、抗体均为阴性的体重40kg左右的健康易感架子猪30头，随机分为6组，每组5头。9-13组分别为本发明实施例6制备的疫苗4、疫苗5、疫苗6、疫苗7以及实施例2制备的疫 苗2免疫组，第14组为空白对照组。第9组、第10组、第11组(疫苗4、疫苗5、疫苗6免疫组)免疫途径为颈部肌肉注射1ml，第12组(疫苗7免疫组)、第13组(疫苗2免疫组)免疫途径为颈部肌肉注射2ml，对照组免疫2ml的PBS。疫苗免疫前每头猪采血，免疫后分别于第7日、14日、21日、28日采血。
对采集的第9组、第10组、第11组、第13组和第14组血清使用CATHAY型O型口蹄疫抗体ELISA检测试剂盒进行相关抗体的检测。结果显示，疫苗免疫前所有猪只的抗体均为阴性，1次免疫后第14日均能达到1:128以上；二价O型口蹄疫病毒样颗粒疫苗组合物在免疫量1ml(常规用量2ml的一半)抗体水平依然达到或超过单价O型口蹄疫病毒样颗粒疫苗组合物在免疫量2ml抗体水平。空白对照组猪只抗体为阴性，无变化。具体结果见表5。
表5 CATHAY型O型口蹄疫ELISA抗体水平
Figure PCTCN2019071813-appb-000005
Figure PCTCN2019071813-appb-000006
对采集的第9组、第10组、第11组、第12组和第14组血清使用SEA型O型口蹄疫抗体ELISA检测试剂盒进行相关抗体的检测。结果显示，疫苗免疫前所有猪只的抗体均为阴性，1次免疫后第14日均能达到1:128以上；二价O型口蹄疫病毒样颗粒疫苗组合物在免疫量1ml(常规用量2ml的一半)抗体水平依然达到或超过单价O型口蹄疫病毒样颗粒疫苗组合物在免疫量2ml抗体水平。空白对照组猪只抗体为阴性，无变化。具体结果见表6。
表6 SEA型O型口蹄疫ELISA抗体水平
Figure PCTCN2019071813-appb-000007
Figure PCTCN2019071813-appb-000008
上述实验表明本发明制备的二价O型口蹄疫病毒样颗粒能快速形成高水平的特异性抗体，能对CATHAY型O型口蹄疫和SEA型O型口蹄疫起到很好的免疫保护作用；同时，也表明二价O型口蹄疫病毒样颗粒较单价O型口蹄疫病毒样颗粒能更快、更好的起免疫反应，以较低的免疫剂量(常规剂量一半的剂量或不到一半的剂量)能达到较好的免疫效果。
以上所述仅是本发明的优选实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以优选实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质 对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (15)

  1. 一种O型口蹄疫病毒样颗粒抗原，其中，所述O型口蹄疫病毒样颗粒抗原为CATHAY型O型口蹄疫病毒样颗粒抗原，所述CATHAY型O型口蹄疫病毒样颗粒抗原由CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白组装而成。
  2. 根据权利要求1所述的一种O型口蹄疫病毒样颗粒抗原，其中，所述CATHAY型O型口蹄疫病毒VP0抗原蛋白的基因如SEQ ID No.1或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP3抗原蛋白的基因如SEQ ID No.2或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP1抗原蛋白的基因如SEQ ID No.3或其简并序列所示。
  3. 一种O型口蹄疫病毒样颗粒疫苗，其中，所述O型口蹄疫病毒样颗粒疫苗包含免疫量的权利要求1～2任一项所述的CATHAY型O型口蹄疫病毒样颗粒抗原和药学上可以接受的载体。
  4. 根据权利要求3所述的O型口蹄疫病毒样颗粒疫苗，其中，所述的CATHAY型O型口蹄疫病毒样颗粒抗原含量为160-240μg/ml。
  5. 根据权利要求3所述的O型口蹄疫病毒样颗粒疫苗，其中，所述的CATHAY型O型口蹄疫病毒样颗粒抗原含量为200μg/ml。
  6. 根据权利要求3所述的O型口蹄疫病毒样颗粒疫苗，其中，所述药学上可以接受的载体包括佐剂，所述佐剂包括：(1)白油、铝胶佐剂、皂苷、阿夫立定、DDA；(2)油包水乳剂、水包油乳剂、水包油包水乳剂；或(3)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的共聚物；以及RIBI佐剂系统、Block co-polymer、SAF-M、单磷酰脂质A、Avridine脂质-胺佐剂、大肠杆菌不耐热肠毒素、霍乱毒素、IMS 1314、胞壁酰二肽、Montanide ISA 206、Gel佐剂中的一种或几种；
    所述佐剂含量为5％-60％V/V。
  7. 根据权利要求6所述的O型口蹄疫病毒样颗粒疫苗，其中，皂苷为Quil A、QS-21、GPI-0100；所述佐剂含量为30％-60％V/V。
  8. 根据权利要求6所述的O型口蹄疫病毒样颗粒疫苗，其中，所 述佐剂含量50％V/V。
  9. 根据权利要求3所述的O型口蹄疫病毒样颗粒疫苗，其中，所述O型口蹄疫病毒样颗粒疫苗还包含免疫量的SEA型O型口蹄疫病毒样颗粒抗原，所述SEA型O型口蹄疫病毒样颗粒抗原由SEA型O型口蹄疫病毒VP4、VP2、VP3、VP1抗原蛋白组装而成。
  10. 根据权利要求9所述的O型口蹄疫病毒样颗粒疫苗，其中，所述SEA型O型口蹄疫病毒VP4抗原蛋白的基因如SEQ ID No.4或其简并序列所示；所述SEA型O型口蹄疫病毒VP2抗原蛋白的基因如SEQ ID No.5或其简并序列所示；所述SEA型O型口蹄疫病毒VP3抗原蛋白的基因如SEQ ID No.6或其简并序列所示；所述SEA型O型口蹄疫病毒VP1抗原蛋白的基因如SEQ ID No.7或其简并序列所示。
  11. 根据权利要求9所述的O型口蹄疫病毒样颗粒疫苗，其中，所述的SEA型O型口蹄疫病毒样颗粒抗原含量为160-240μg/ml。
  12. 根据权利要求9所述的O型口蹄疫病毒样颗粒疫苗，其中，所述的SEA型O型口蹄疫病毒样颗粒抗原含量为200μg/ml。
  13. 一种制备权利要求3所述O型口蹄疫病毒样颗粒疫苗的方法，其中，所述方法包括：
    步骤(1)将CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白的基因分别扩增、克隆到同一串联表达载体，得到含有CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白基因的重组表达载体；
    步骤(2)将所述步骤(1)得到的含有CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白基因的重组表达载体转化或转导宿主，得到含有所述重组表达载体的重组子；
    步骤(3)培养所述步骤(2)得到的重组子，串联表达所述CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白，所述表达的CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白自我组装，形成所述CATHAY型O型口蹄疫病毒样颗粒抗原；以及
    步骤(4)纯化所述步骤(3)得到的所述CATHAY型O型口蹄疫病毒样颗粒抗原，加入佐剂，得到所述O型口蹄疫病毒样颗粒疫苗。
  14. 根据权利要求13所述的方法，其中，所述步骤(1)中的所述CATHAY型O型口蹄疫病毒VP0抗原蛋白的基因如SEQ ID No.1或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP3抗原蛋白的基因如SEQ ID No.2或其简并序列所示；所述CATHAY型O型口蹄疫病毒VP1抗原蛋白的基因如SEQ ID No.3或其简并序列所示；所述步骤(2)中的宿主为E.coli；所述步骤(3)中所述表达的CATHAY型O型口蹄疫病毒VP0、VP3、VP1抗原蛋白为细胞内可溶性蛋白。
  15. 根据权利要求3～12任一项所述的O型口蹄疫病毒样颗粒疫苗在制备预防和/或治疗O型口蹄疫的药物中的应用。
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