CN115702928A

CN115702928A - African swine fever virus subunit vaccine composition, combination of African swine fever virus protein antigens and application thereof

Info

Publication number: CN115702928A
Application number: CN202110931295.4A
Authority: CN
Inventors: 田克恭; 逄文强; 谭菲菲; 张许科
Original assignee: Pulaike Biological Engineering Co Ltd
Current assignee: Pulaike Biological Engineering Co Ltd
Priority date: 2021-08-13
Filing date: 2021-08-13
Publication date: 2023-02-17

Abstract

The invention provides an African swine fever virus subunit vaccine composition, the protein antigen of the African swine fever virus subunit vaccine composition consists of proteins of P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72 and P30 of the African swine fever virus, and the subunit vaccine composition also comprises a pharmaceutically acceptable carrier. The subunit vaccine composition has synergistic effect of antigen components, and can produce powerful immunological reaction and high antibody titer. The invention also provides a combination of protein antigens of the African swine fever virus.

Description

African swine fever virus subunit vaccine composition, combination of African swine fever virus protein antigens and application thereof

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to a combination of an African swine fever virus subunit vaccine composition and an African swine fever virus protein antigen, a preparation method and application thereof.

Background

African Swine Fever (ASF) is an acute, virulent and highly contagious infectious disease caused by African Swine Fever Virus (ASFV), the incidence rate is high, and the death rate can reach 100 percent, so that the African swine fever is classified as an animal epidemic disease in China. The disease is confirmed to occur in kenia in africa in 1921 at the earliest time, and since 2007, african swine fever occurs, spreads and prevails in a plurality of countries around the world, and is introduced into China in 2018. The disease is found for nearly a hundred years till now, but no approved vaccine and specific medicine exist all over the world at present.

The research and development of the African swine fever vaccine have the difficulties that the virus genome is large, the protein is multiple, the structure is complex, most gene functions are unknown, the interaction mechanism of the virus and host cells is undefined and difficult to culture on a large scale, the passage is easy to be varied, and the immune escape mechanism of the virus is unclear, so that the conventional inactivated vaccine and the attenuated live vaccine have insubstantial progress so far, and the improper use of the attenuated live vaccine has the biological safety risks of strong toxicity return and virus dispersion.

Therefore, there is a need for an african swine fever vaccine in clinic, which has a good immune effect and can effectively protect pigs without the risk of biosafety.

Disclosure of Invention

Therefore, the invention analyzes the genes and proteins of the pathogens by a modern biological method, tests the immune protection capability of the pathogens, develops the subunit vaccine and effectively solves the problem that the immune effect of various vaccines is not ideal at present.

The invention provides an African swine fever virus subunit vaccine composition, wherein a protein antigen of the African swine fever virus subunit vaccine composition consists of multi-component protein antigens, and can provide good immune efficacy for pigs.

The invention also provides an application of the African swine fever virus subunit vaccine composition, and the application refers to an application in preparation of a medicine for preventing African swine fever virus infection.

The invention provides a combination of African swine fever virus protein antigens, wherein the African swine fever virus protein antigens consist of multi-component protein antigens. The combination of the African swine fever virus protein antigens is applied together, has synergistic effect and good immune effect, and effectively solves the problem that the immune effect of various vaccines in the prior art is not ideal.

The invention also provides application of the combination of the African swine fever virus protein antigens, wherein the application refers to application in preparing a medicament for preventing the African swine fever virus infection.

The combination of the African swine fever virus subunit vaccine and the protein antigen adopts a multi-component African swine fever virus protein antigen, has good immune effect, and effectively solves the problem of poor immunogenicity faced by the prior African swine fever virus vaccine; the vaccine composition can be subjected to mass expression through a genetic engineering means, not only is short in time consumption, but also is convenient for large-scale production, and has no biological safety risk.

Detailed Description

Hereinafter, embodiments of the present invention will be described.

Definition of

The term "African swine fever virus" means African Swine Fever Virus (ASFV), which is the only species of the African swine fever virus family (Asfarviridae), has infectivity and extremely high pathogenicity, is in the form of a regular icosahedron, about 200 nm in diameter, and is composed of multiple layers of material: the center is a protein nucleocapsid containing a nucleomimic, and a layer of lipid envelope and a layer of protein capsid are respectively arranged from inside to outside. The capsid is composed of 8280 major capsid proteins, p72 and 60 pentanes, and at least three proteins are responsible for the stability of the capsid structure through adhesion to adjacent proteins, with clinical symptoms in acute cases characterized primarily by high fever, short course, high mortality, generalized bleeding of internal organs, and dysfunction of the respiratory and nervous systems. No specific vaccine or antiviral medicine aiming at ASFV exists until 2020, and the virus can be effectively controlled to spread in time when epidemic outbreak occurs.

The terms "p 34 protein of African swine fever virus" and "p 14 protein of African swine fever virus" mean that the African swine fever virus encodes a polyprotein named p220, which is present in the nucleocapsid of the mature virion, accounting for about 30% of the total viral protein, and plays an important role in viral assembly and viral infection. The P220 polyprotein is orderly cleaved into P150, P34, P37 and P14 by protease. p150, p34, p37 and p14 play an important role in the assembly process of the viral capsid, wherein p34 and p14 belong to important structural proteins in p220 and play an important role in the packaging of viral nucleoprotein.

The term "African swine fever virus C129R protein" refers to manganese-dependent superoxide dismutase.

The term "African swine fever virus DP96R protein" refers to inhibition of type I IFN expression and NF- κ B activation, also known as "pDP96R protein".

The term "African swine fever virus A104R protein" refers to a DNA binding protein involved in virus transcription, DNA replication and genome packaging, also known as "pA104R protein".

The term "African swine fever virus p54 protein" refers to the early membrane protein expressed by African swine fever virus, which is encoded by OR FE183L gene, contains transmembrane domain, is located in endoplasmic reticulum-derived inner membrane precursor, plays an important role in virus adsorption to susceptible cells and invasion process, and is an important structural protein.

The term "p 17 protein of African swine fever virus" refers to the late membrane protein expressed by African swine fever virus, encoded by ORF D117L gene, located in the transmembrane protein of the virus inner membrane.

The term "african swine fever virus p22 protein" refers to the african swine fever virus transmembrane domain, located outside the viral particle.

The term "P72 protein of African swine fever virus" refers to an antigenic protein which is produced in the late stage of viral infection, encoded by the ORF B646L gene, is important for African swine fever virus, is the major component of the icosahedron virus, and is essential for the formation of the viral capsid.

The term "p 30 protein of African swine fever virus" refers to the early membrane protein expressed by African swine fever virus, encoded by the ORF CP204L gene, usually produced 2-4 hours after infection, and expressed continuously throughout the infection, which is an important structural protein in connection with virus invasion into host cells. The term "degenerate sequence" refers to the phenomenon of having two or more codons for the same amino acid, and such sequences are called degenerate sequences.

The term "Antigen" refers to a substance that induces an immune response in the body, i.e., a substance that is specifically recognized and bound by Antigen receptors (TCR/BCR) on the surface of T/B lymphocytes, activates T/B cells, proliferates and differentiates them, produces an immune response product (sensitized lymphocytes or antibodies), and specifically binds to the corresponding product in vitro or in vivo.

The term "vaccine", "vaccine composition" as used herein refers to a pharmaceutical composition comprising a protein antigen of African swine fever virus which induces, stimulates or enhances the immune response of pigs against African swine fever alone.

The term "immunizing amount" shall be understood as an "immunologically effective amount," also referred to as an immunoprotective amount or an amount effective to produce an immune response, of antigen effective to induce an immune response in a recipient, sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other testing, or may be suitable for use in preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by the pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs of the subject such as mortality, reduction in morbidity, temperature values, the subject's overall physiological status, and overall health and performance. The immune response may include, but is not limited to, inducing cellular and/or humoral immunity.

The term "pharmaceutically acceptable carrier" refers to all other components of the vaccine composition of the present invention other than the CSFV protein antigen, which do not stimulate the body and do not hinder the biological activity and properties of the compound, preferably an adjuvant. The term "adjuvant" may include an alumina gel adjuvant; saponins (saponin), such as Quil A, QS-21 (Cambridge Biotech Incorporation, cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, birmingham AL); a water-in-oil emulsion; an oil-in-water emulsion; an oil-in-water emulsion; polymers of acrylic acid or methacrylic acid; maleic anhydride and alkenyl (alkenyl) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils (isoprenoid oils) resulting from the oligomerization of olefins, such as squalane (squalane) or squalene oil (squalene oil), in particular isobutene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (such as, for example, anhydrous mannitol oleate), of aliphatic diols (glycols), of polyglycerols, of propylene glycol and of oleic acid, of isostearic acid, of ricinoleic acid or of hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See The "The term and practical application of adjuvants" by Hunter et al (Ed. By DES Stewart-Tull, john Wiley and Sons, new York, 1995. For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of Vaccine design, the Subunit and the adivant prophach, compiled by Powell M and Newman M (Plenum Press, 1995) can be used. The term "polymer of acrylic or methacrylic acid" is preferably a crosslinked polymer of acrylic or methacrylic acid, in particular a polyalkenyl ether or polyalcohol crosslinked with a sugar (sugar), these compounds being known as carbomers (Carbomer, trade name Carbopol) (Phameuropa, 1996,8 (2)). Those skilled in the art can also see US patent US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are substituted by unsaturated aliphatic (aliphatic) radicals having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups (ethylenically unsaturated groups). The unsaturated groups may themselves contain other substituents, such as methyl. These products are sold under the name carbopol (BF Goodrich, ohio, USA) and are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, carbopols 974P, 934P, and 971P may be mentioned, with carbopol 971P being most preferably used. The term "copolymers of maleic anhydride and alkenyl derivative" also contemplates the maleic anhydride and ethylene copolymers EMA (Monsanto), which are dissolved in water to give an acidic solution, neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccinal composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, the RIBI adjuvant system (Ribi Incorporation), block co-polymer (CytRx, atlanta GA), SAF-M (Chiron, emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), avridine lipoamine adjuvant, E.coli heat-labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of mineral oil, an alumina Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkenyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, monophosphoryl lipid a, an Avridine lipid-amine adjuvant, escherichia coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanide ISA206, or Gel adjuvant.

The term "lyoprotectant" refers to a composition that, in addition to excipients, protects the pharmaceutical effect of a pharmaceutical active ingredient given during the lyophilization process and during the storage period following lyophilization.

The term "dosage form" refers to the form of a pharmaceutical formulation. Also refers to the medicament (finished product) prepared according to the property of the medicament and the requirements of the treatment and prescription. The proper dosage form is to exert the best curative effect of the medicine, reduce toxic and side effects and facilitate the use, storage and transportation.

The term "injection" refers to a sterile solution (including emulsion and suspension) for injection into the body and a sterile powder or concentrated solution for preparing a solution or suspension before use, and can be injection solution (the solvent is water) or injection oil injection (the solvent is oil); other injection with other solvent, such as ethanol (the solvent of hydrocortisone injection is ethanol), glycerol, propylene glycol (PEG), etc. is also available.

The term "powder for injection" refers to a preparation prepared by freezing a liquid medicine in an aseptic environment, mixing a raw material medicine with certain auxiliary materials or dissolving the raw material medicine in certain solvent and preparing the mixture into different forms through certain processing. The term "preventing" when referring to an african swine fever virus infection means inhibiting the replication of, inhibiting the spread of, or preventing the colonization of an african swine fever virus in its host, and alleviating the symptoms of an african swine fever virus-infected disease or disorder.

Detailed Description

The invention provides an African swine fever virus subunit vaccine composition, wherein a protein antigen of the African swine fever virus subunit vaccine composition consists of proteins of P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72 and P30 of the African swine fever virus, and the African swine fever virus subunit vaccine composition further comprises a pharmaceutically acceptable carrier; wherein, the African swine fever virus p34 protein is coded by SEQ ID NO.1 or a degenerate sequence thereof; the African swine fever virus p14 protein is coded by SEQ ID NO.2 or a degenerate sequence thereof; the African swine fever virus C129R protein is SEQ ID NO.3 or a degenerate sequence code thereof; the African swine fever virus DP96R protein is SEQ ID NO.4 or a degenerate sequence code thereof; the African swine fever virus A104R protein is coded by SEQ ID NO.5 or a degenerate sequence thereof; the African swine fever virus p54 protein is coded by SEQ ID NO.6 or a degenerate sequence thereof; the African swine fever virus p17 protein is SEQ ID NO.7 or a degenerate sequence code thereof; the African swine fever virus p22 protein is coded by SEQ ID NO.8 or a degenerate sequence thereof; the African swine fever virus P72 protein is coded by SEQ ID NO.9 or a degenerate sequence thereof; and the African swine fever virus p30 protein is encoded by SEQ ID NO.10 or a degenerate sequence thereof.

According to a preferred embodiment, the African swine fever virus p34 protein is encoded by SEQ ID No. 1; the African swine fever virus p14 protein is coded by SEQ ID NO. 2; the African swine fever virus C129R protein is encoded by SEQ ID NO. 3; the African swine fever virus DP96R protein is encoded by SEQ ID NO. 4; the African swine fever virus A104R protein is coded by SEQ ID NO. 5; the African swine fever virus p54 protein is coded by SEQ ID NO. 6; the African swine fever virus p17 protein is encoded by SEQ ID NO. 7; the African swine fever virus p22 protein is coded by SEQ ID NO. 8; the African swine fever virus P72 protein is encoded by SEQ ID NO. 9; and the African swine fever virus p30 protein is coded by SEQ ID NO. 10.

According to a preferred embodiment, the person skilled in the art can select further degenerate sequences encoding the African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72, P30 proteins according to the general technical knowledge. Other degenerate sequences may be selected according to the preferred codon usage of the particular expression system.

According to a preferred embodiment, the African swine fever virus p34, p14, C129R, DP96R, A104R and p54 proteins are expressed by an Escherichia coli expression system, and the coding sequences are selected according to the preferred codons of the Escherichia coli expression system. According to a preferred embodiment, the African swine fever virus P17, P22, P72, P30 proteins are expressed by an sf9 insect cell expression system, and the coding sequence thereof is selected according to the preferred codon of the sf9 insect cell expression system.

According to a preferred embodiment, the dosage form of the African swine fever virus subunit vaccine composition is selected from the group consisting of solution type injection, suspension type injection, powder for injection, sustained release microsphere preparation, controlled release microsphere preparation and sustained and controlled release implant.

According to a preferred embodiment, the African swine fever virus subunit vaccine composition is in the form of a solution injection, a suspension injection, or a powder for injection.

According to a preferred embodiment, the administration of the african swine fever virus subunit vaccine composition is selected from the group consisting of subcutaneous injection, oral administration, buccal administration, sublingual administration, nasal administration, pulmonary administration, colonic administration, rectal administration, transdermal administration. According to a more preferred embodiment, the African swine fever virus subunit vaccine composition is administered by subcutaneous injection.

According to a preferred embodiment, the african swine fever virus subunit vaccine composition is in the form of a solution injection, a suspension injection; the content of the African swine fever virus p34 protein is more than or equal to 8 mu g/ml; the content of the African swine fever virus p14 protein is more than or equal to 8 mug/ml; the content of the African swine fever virus C129R protein is more than or equal to 8 mu g/ml; the content of the African swine fever virus DP96R protein is more than or equal to 8 mu g/ml; the protein content of the African swine fever virus A104R is more than or equal to 8 mu g/ml; the content of the African swine fever virus p54 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p17 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p22 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus P72 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p30 protein is more than or equal to 12 mu g/ml.

According to a preferred embodiment, the African swine fever virus p34 protein content is 8-24 mug/ml; the content of the African swine fever virus p14 protein is 8-24 mug/ml; the content of the African swine fever virus C129R protein is 8-24 mu g/ml; the African swine fever virus DP96R protein content is 8-24 mu g/ml; the protein content of the African swine fever virus A104R is 8-24 mug/ml; the content of the African swine fever virus p54 protein is 12-36 mu g/ml; the content of the African swine fever virus p17 protein is 12-36 mu g/ml; the content of the African swine fever virus p22 protein is 12-36 mug/ml; the content of the African swine fever virus P72 protein is 12-36 mu g/ml; the content of the African swine fever virus p30 protein is 12-36 mu g/ml.

According to a preferred embodiment, the African swine fever virus p34 protein content is selected from the group consisting of 8. Mu.g/ml, 9. Mu.g/ml, 10. Mu.g/ml, 11. Mu.g/ml, 12. Mu.g/ml, 13. Mu.g/ml, 14. Mu.g/ml, 15. Mu.g/ml, 16. Mu.g/ml, 17. Mu.g/ml, 18. Mu.g/ml, 19. Mu.g/ml, 20. Mu.g/ml, 21. Mu.g/ml, 22. Mu.g/ml, 23. Mu.g/ml, 24. Mu.g/ml. The African swine fever virus p14 protein content is selected from 8 μ g/ml, 9 μ g/ml, 10 μ g/ml, 11 μ g/ml, 12 μ g/ml, 13 μ g/ml, 14 μ g/ml, 15 μ g/ml, 16 μ g/ml, 17 μ g/ml, 18 μ g/ml, 19 μ g/ml, 20 μ g/ml, 21 μ g/ml, 22 μ g/ml, 23 μ g/ml, or 24 μ g/ml. The African swine fever virus C129R protein content is selected from 8 μ g/ml, 9 μ g/ml, 10 μ g/ml, 11 μ g/ml, 12 μ g/ml, 13 μ g/ml, 14 μ g/ml, 15 μ g/ml, 16 μ g/ml, 17 μ g/ml, 18 μ g/ml, 19 μ g/ml, 20 μ g/ml, 21 μ g/ml, 22 μ g/ml, 23 μ g/ml, or 24 μ g/ml. The African swine fever virus DP96R protein content is selected from 8 mu g/ml, 9 mu g/ml, 10 mu g/ml, 11 mu g/ml, 12 mu g/ml, 13 mu g/ml, 14 mu g/ml, 15 mu g/ml, 16 mu g/ml, 17 mu g/ml, 18 mu g/ml, 19 mu g/ml, 20 mu g/ml, 21 mu g/ml, 22 mu g/ml, 23 mu g/ml and 24 mu g/ml. The African swine fever virus A104R protein content is selected from 8 μ g/ml, 9 μ g/ml, 10 μ g/ml, 11 μ g/ml, 12 μ g/ml, 13 μ g/ml, 14 μ g/ml, 15 μ g/ml, 16 μ g/ml, 17 μ g/ml, 18 μ g/ml, 19 μ g/ml, 20 μ g/ml, 21 μ g/ml, 22 μ g/ml, 23 μ g/ml and 24 μ g/ml. The P54 protein content of the African swine fever virus is selected from 12 mu g/ml, 13 mu g/ml, 14 mu g/ml, 15 mu g/ml, 16 mu g/ml, 17 mu g/ml, 18 mu g/ml, 19 mu g/ml, 20 mu g/ml, 21 mu g/ml, 22 mu g/ml, 23 mu g/ml, 24 mu g/ml, 25 mu g/ml, 26 mu g/ml, 27 mu g/ml, 28 mu g/ml, 29 mu g/ml, 30 mu g/ml, 31 mu g/ml, 32 mu g/ml, 33 mu g/ml, 34 mu g/ml, 35 mu g/ml and 36 mu g/ml. The African swine fever virus p17 protein content is selected from 12 μ g/ml, 13 μ g/ml, 14 μ g/ml, 15 μ g/ml, 16 μ g/ml, 17 μ g/ml, 18 μ g/ml, 19 μ g/ml, 20 μ g/ml, 21 μ g/ml, 22 μ g/ml, 23 μ g/ml, 24 μ g/ml, 25 μ g/ml, 26 μ g/ml, 27 μ g/ml, 28 μ g/ml, 29 μ g/ml, 30 μ g/ml, 31 μ g/ml, 32 μ g/ml, 33 μ g/ml, 34 μ g/ml, 35 μ g/ml, 36 μ g/ml. The African swine fever virus p22 protein content is selected from 12 μ g/ml, 13 μ g/ml, 14 μ g/ml, 15 μ g/ml, 16 μ g/ml, 17 μ g/ml, 18 μ g/ml, 19 μ g/ml, 20 μ g/ml, 21 μ g/ml, 22 μ g/ml, 23 μ g/ml, 24 μ g/ml, 25 μ g/ml, 26 μ g/ml, 27 μ g/ml, 28 μ g/ml, 29 μ g/ml, 30 μ g/ml, 31 μ g/ml, 32 μ g/ml, 33 μ g/ml, 34 μ g/ml, 35 μ g/ml, or 36 μ g/ml. The African swine fever virus P72 protein content is selected from 12 μ g/ml, 13 μ g/ml, 14 μ g/ml, 15 μ g/ml, 16 μ g/ml, 17 μ g/ml, 18 μ g/ml, 19 μ g/ml, 20 μ g/ml, 21 μ g/ml, 22 μ g/ml, 23 μ g/ml, 24 μ g/ml, 25 μ g/ml, 26 μ g/ml, 27 μ g/ml, 28 μ g/ml, 29 μ g/ml, 30 μ g/ml, 31 μ g/ml, 32 μ g/ml, 33 μ g/ml, 34 μ g/ml, 35 μ g/ml, or 36 μ g/ml. The African swine fever virus p30 protein content is selected from 12. Mu.g/ml, 13. Mu.g/ml, 14. Mu.g/ml, 15. Mu.g/ml, 16. Mu.g/ml, 17. Mu.g/ml, 18. Mu.g/ml, 19. Mu.g/ml, 20. Mu.g/ml, 21. Mu.g/ml, 22. Mu.g/ml, 23. Mu.g/ml, 24. Mu.g/ml, 25. Mu.g/ml, 26. Mu.g/ml, 27. Mu.g/ml, 28. Mu.g/ml, 29. Mu.g/ml, 30. Mu.g/ml, 31. Mu.g/ml, 32. Mu.g/ml, 33. Mu.g/ml, 34. Mu.g/ml, 35. Mu.g/ml, or 36. Mu.g/ml.

According to a preferred embodiment, the African swine fever virus p34 protein content is 8-16 mug/ml; the content of the African swine fever virus p14 protein is 8-16 mu g/ml; the content of the African swine fever virus C129R protein is 8-16 mu g/ml; the African swine fever virus DP96R protein content is 8-16 mug/ml; the protein content of the African swine fever virus A104R is 8-16 mu g/ml; the content of the African swine fever virus p54 protein is 12-24 mug/ml; the content of the African swine fever virus p17 protein is 12-24 mu g/ml; the content of the African swine fever virus p22 protein is 12-24 mug/ml; the content of the African swine fever virus P72 protein is 12-24 mu g/ml; the content of the African swine fever virus p30 protein is 12-24 mu g/ml.

According to a preferred embodiment, the p34 protein, the p14 protein, the C129R protein, the DP96R protein, the A104R protein, the p54 protein, the p17 protein, the p22 protein and the p30 protein of the African swine fever virus are selected to be low in content, so that even if the total consumption of the African swine fever virus protein antigen is reduced compared with the consumption of a single component antigen, a higher antibody titer can be obtained, and a better immune effect can be ensured.

According to a preferred embodiment, the African swine fever virus p34 protein content is 8 μ g/ml; the content of the African swine fever virus p14 protein is 8 mug/ml; the content of the African swine fever virus C129R protein is 8 mu g/ml; the content of the African swine fever virus DP96R protein is 8 mu g/ml; the protein content of the African swine fever virus A104R is 8 mug/ml; the content of the P54 protein of the African swine fever virus is 12 mu g/ml; the content of the African swine fever virus p17 protein is 12 mug/ml; the content of the African swine fever virus p22 protein is 12 mu g/ml; the content of the P72 protein of the African swine fever virus is 12 mug/ml; the content of the African swine fever virus p30 protein is 12 mu g/ml.

According to a preferred embodiment, the African swine fever virus p34 protein content is 16 μ g/ml; the content of the African swine fever virus p14 protein is 16 mug/ml; the content of the African swine fever virus C129R protein is 16 mu g/ml; the content of the African swine fever virus DP96R protein is 16 mu g/ml; the content of the African swine fever virus A104R protein is 16 mug/ml; the content of the African swine fever virus p54 protein is 24 mug/ml; the content of the African swine fever virus p17 protein is 24 mu g/ml; the content of the African swine fever virus p22 protein is 24 mug/ml; the content of the African swine fever virus P72 protein is 24 mu g/ml; the content of the African swine fever virus p30 protein is 24 mu g/ml.

According to a preferable embodiment, in the African swine fever virus subunit vaccine composition, the protein antigen content of the African swine fever virus is 100-300 mug/ml.

The African swine fever virus protein content may be selected from 100. Mu.g/ml, 110. Mu.g/ml, 120. Mu.g/ml, 130. Mu.g/ml, 140. Mu.g/ml, 150. Mu.g/ml, 160. Mu.g/ml, 170. Mu.g/ml, 180. Mu.g/ml, 190. Mu.g/ml, 200. Mu.g/ml, 210. Mu.g/ml, 220. Mu.g/ml, 230. Mu.g/ml, 240. Mu.g/ml, 250. Mu.g/ml, 260. Mu.g/ml, 270. Mu.g/ml, 280. Mu.g/ml, 290. Mu.g/ml, 300. Mu.g/ml.

According to a preferred embodiment, the pharmaceutically acceptable carrier comprises an adjuvant comprising: (1) Mineral oil, alumina gel adjuvant, saponin, alfudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanide ISA206 and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; the adjuvant content is 5% -60% V/V, preferably from 30% -60% V/V, more preferably 50% V/V.

According to a preferred embodiment, the adjuvant is Montanide ISA206 adjuvant, the adjuvant content being 50% V/V.

According to a preferred embodiment, the pharmaceutically acceptable carrier comprises a lyoprotectant selected from the group consisting of a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

According to a preferred embodiment, the pharmaceutically acceptable carrier includes drugs, immune stimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants and preservatives; the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2 (IL 2).

To prepare such compositions, methods well known in the art may be used.

The invention also provides application of the African swine fever virus subunit vaccine composition, wherein the application refers to application in preparation of a medicine for preventing African swine fever virus infection. According to a preferred embodiment, the African swine fever virus subunit vaccine composition can play a synergistic immune protection role and has a better immune effect on pigs.

The invention also provides a preparation method of the African swine fever virus protein, which comprises the following steps:

(1) Artificially synthesizing the African swine fever virus protein gene, and connecting the African swine fever virus protein gene to a cloning vector, namely a cloning vector of the protein by a genetic engineering means;

(2) Carrying out enzyme digestion on the constructed cloning vector and the expression vector of the protein to construct an expression vector containing a protein gene, namely an expression vector of the protein;

(3) And introducing the expression vector of the protein into recipient bacteria for induction expression, and identifying the expressed protein to obtain the African swine fever virus protein.

As an embodiment of the invention, the African swine fever virus protein can be prepared by a prokaryotic expression system, or can be prepared by a eukaryotic expression system or a chemical synthesis method.

According to a preferred embodiment, the expression vector of the African swine fever virus p34, p14, C129R, DP96R, A104R and p54 protein genes is an Escherichia coli expression system cloning vector.

According to a preferred embodiment, the expression vector of the African swine fever virus P17, P22, P72 and P30 protein genes is an expression vector of an sf9 insect cell expression system. The invention also provides an expression vector containing the gene sequence of the African swine fever virus protein.

According to a preferred embodiment, the expression vector of the African swine fever virus P17, P22, P72 and P30 protein genes is an expression vector of an sf9 insect cell expression system.

The invention also provides a combination of African swine fever virus protein antigens, wherein the African swine fever virus protein antigen consists of African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72 and P30 proteins; wherein, the African swine fever virus p34 protein is coded by SEQ ID NO.1 or a degenerate sequence thereof; the African swine fever virus p14 protein is SEQ ID NO.2 or a degenerate sequence code thereof; the African swine fever virus C129R protein is coded by SEQ ID NO.3 or a degenerate sequence thereof; the African swine fever virus DP96R protein is encoded by SEQ ID NO.4 or a degenerate sequence thereof; the African swine fever virus A104R protein is coded by SEQ ID NO.5 or a degenerate sequence thereof; the African swine fever virus p54 protein is SEQ ID NO.6 or a degenerate sequence code thereof; the African swine fever virus p17 protein is SEQ ID NO.7 or a degenerate sequence code thereof; the African swine fever virus p22 protein is SEQ ID NO.8 or a degenerate sequence code thereof; the African swine fever virus P72 protein is coded by SEQ ID NO.9 or a degenerate sequence thereof; and the African swine fever virus p30 protein is encoded by SEQ ID NO.10 or a degenerate sequence thereof.

According to a preferred embodiment, the African swine fever virus p34, p14, C129R, DP96R, A104R and p54 proteins are expressed by an Escherichia coli expression system, and the coding sequences are selected according to the preferred codons of the Escherichia coli expression system. According to a preferred embodiment, the African swine fever virus P17, P22, P72, P30 proteins are expressed by sf9 insect cell expression system, and the coding sequences thereof are selected according to the preferred codons of the sf9 insect cell expression system.

According to a preferred embodiment, the African swine fever virus p34 protein is encoded by SEQ ID No. 1; the African swine fever virus p14 protein is coded by SEQ ID NO. 2; the African swine fever virus C129R protein is coded by SEQ ID NO. 3; the African swine fever virus DP96R protein is encoded by SEQ ID NO. 4; the African swine fever virus A104R protein is encoded by SEQ ID NO. 5; the African swine fever virus p54 protein is encoded by SEQ ID NO. 6; the African swine fever virus p17 protein is coded by SEQ ID NO. 7; the African swine fever virus p22 protein is encoded by SEQ ID NO. 8; the African swine fever virus P72 protein is coded by SEQ ID NO. 9; and the African swine fever virus p30 protein is encoded by SEQ ID NO. 10. According to a preferred embodiment, the combination of african swine fever virus protein antigens further comprises a pharmaceutically acceptable carrier comprising an adjuvant comprising: (1) Mineral oil, alumina gel adjuvant, saponin, alfudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a polymer of acrylic or methacrylic acid, maleic anhydride and a copolymer of an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanide ISA206 and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100; the adjuvant content is 5% -60% V/V, preferably from 30% -60% V/V, more preferably 50% V/V.

According to a preferred embodiment, the African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72 and P30 protein is in a dosage form selected from solution injection, suspension injection, powder for injection, sustained release microsphere preparation, controlled release microsphere preparation and sustained and controlled release implant.

According to a preferred embodiment, the dosage form of the African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72 and P30 protein is solution type injection, suspension type injection or powder for injection.

According to a preferred embodiment, the administration mode of the combination of african swine fever virus protein antigens is selected from the group consisting of subcutaneous injection, oral administration, buccal administration, sublingual administration, nasal administration, pulmonary administration, colonic administration, rectal administration, transdermal administration. According to a more preferred embodiment, the administration of the combination of African swine fever virus protein antigens is subcutaneous injection.

According to a preferred embodiment, the African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72, P30 protein is in the form of solution injection or suspension injection; the content of the African swine fever virus p34 protein is more than or equal to 8 mu g/ml; the content of the African swine fever virus p14 protein is more than or equal to 8 mug/ml; the content of the African swine fever virus C129R protein is more than or equal to 8 mu g/ml; the protein content of the African swine fever virus DP96R is more than or equal to 8 mug/ml; the protein content of the African swine fever virus A104R is more than or equal to 8 mug/ml; the content of the African swine fever virus p54 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p17 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p22 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus P72 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p30 protein is more than or equal to 12 mu g/ml.

According to a preferred embodiment, the African swine fever virus p34 protein content is 8-24 mug/ml; the content of the African swine fever virus p14 protein is 8-24 mug/ml; the content of the African swine fever virus C129R protein is 8-24 mu g/ml; the content of the African swine fever virus DP96R protein is 8-24 mu g/ml; the protein content of the African swine fever virus A104R is 8-24 mug/ml; the content of the African swine fever virus p54 protein is 12-36 mu g/ml; the content of the P17 protein of the African swine fever virus is 12-36 mu g/ml; the content of the African swine fever virus p22 protein is 12-36 mu g/ml; the content of the African swine fever virus P72 protein is 12-36 mu g/ml; the content of the African swine fever virus p30 protein is 12-36 mu g/ml.

According to a preferred embodiment, the content of the African swine fever virus p34 protein is 8-16 mug/ml; the content of the African swine fever virus p14 protein is 8-16 mu g/ml; the content of the African swine fever virus C129R protein is 8-16 mu g/ml; the African swine fever virus DP96R protein content is 8-16 mu g/ml; the protein content of the African swine fever virus A104R is 8-16 mug/ml; the content of the African swine fever virus p54 protein is 12-24 mug/ml; the content of the African swine fever virus p17 protein is 12-24 mu g/ml; the content of the African swine fever virus p22 protein is 12-24 mu g/ml; the content of the African swine fever virus P72 protein is 12-24 mu g/ml; the content of the African swine fever virus p30 protein is 12-24 mu g/ml.

According to a preferred embodiment, the p34 protein, the p14 protein, the C129R protein, the DP96R protein, the A104R protein, the p54 protein, the p17 protein, the p22 protein and the p30 protein of the African swine fever virus are selected to be low in content, so that even if the total dosage of the African swine fever virus protein antigen is reduced compared with the dosage of the single component antigen, higher antibody titer can be obtained, and a better immune effect is ensured. As the most preferred embodiment of the invention, the African swine fever virus p34 protein content is 8 μ g/ml; the content of the African swine fever virus p14 protein is 8 mug/ml; the content of the African swine fever virus C129R protein is 8 mug/ml; the content of the African swine fever virus DP96R protein is 8 mu g/ml; the protein content of the African swine fever virus A104R is 8 mug/ml; the content of the African swine fever virus p54 protein is 12 mu g/ml; the content of the African swine fever virus p17 protein is 12 mu g/ml; the content of the African swine fever virus p22 protein is 12 mu g/ml; the content of the African swine fever virus P72 protein is 12 mu g/ml; the content of the African swine fever virus p30 protein is 12 mu g/ml.

As the most preferred embodiment of the invention, the African swine fever virus p34 protein has a content of 16 μ g/ml; the content of the African swine fever virus p14 protein is 16 mug/ml; the content of the African swine fever virus C129R protein is 16 mu g/ml; the content of the African swine fever virus DP96R protein is 16 mu g/ml; the protein content of the African swine fever virus A104R is 16 mu g/ml; the content of the African swine fever virus p54 protein is 24 mug/ml; the content of the African swine fever virus p17 protein is 24 mu g/ml; the content of the African swine fever virus p22 protein is 24 mug/ml; the content of the African swine fever virus P72 protein is 24 mug/ml; the content of the African swine fever virus p30 protein is 24 mu g/ml.

The invention also provides application of the combination of the African swine fever virus protein antigens, wherein the application refers to application in preparation of drugs for preventing African swine fever virus infection.

According to a preferred embodiment, the combination of African swine fever virus antigens can play a synergistic immune protection role, and has a better immune effect on pigs.

The present invention will be further described with reference to specific embodiments, and advantages and features of the present invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.

Example 1 prokaryotic expression of P34, p14, C129R, DP96R, A104R, p54 proteins of African swine fever virus

The nucleotide sequence of the expression vector shown in the corresponding coding sequence table SEQ ID NO.11 is synthesized by Suzhou Jinzhi Biotechnology Limited and named as pNVC1 vector.

Nucleotide sequences encoding the p34, p14, C129R, DP96R, A104R and p54 proteins shown in the sequence tables SEQ ID NO. 1-SEQ ID NO.6 were synthesized by Jinzhi Biotechnology Ltd, suzhou, and the synthesized nucleotides were cloned into a pNVC1 vector, named recombinant plasmids pNVC1-p34, pNVC1-p14, pNVC1-C129R, pNVC1-DP96R, pNVC1-A104R and pNVC1-p54.

The recombinant plasmids pNVC1-p34, pNVC1-p14, pNVC1-C129R, pNVC1-DP96R, pNVC1-A104R and pNVC1-p54 were transformed into competent Escherichia coli BL21 (DE 3), respectively, to construct expression strains, inoculated with 50ml of LB liquid medium resistant to kanamycin, shake-cultured at 37 ℃ and 230 rpm for 12 hours, then transferred into 1L of LB liquid medium, cultured at 37 ℃ to prepare seed solutions for fermentation.

The fermentation tank was a 50L fermentation tank of Shanghai Baoxing Bio Inc., 30L of the medium was prepared and charged into the fermentation tank, and sterilized at 121 ℃ for 30 minutes. The next day, 3L of the seed solution was inoculated into a fermenter, and when the concentration of the culture solution reached about OD600, the culture temperature was lowered to 25 ℃ and IPTG was added to a final concentration of 0.5mM for induction culture for 12 hours. The culture was stopped at a fermentation density of about 40 (OD 600), and the cells were collected by centrifugation.

Resuspend the cells, crush the cells 3 times with a homogenizer at 800bar pressure. 13500rpm, centrifuged for 40min, the supernatant was retained and assayed by 12% SDS-PAGE electrophoresis. Coarse protein purification is carried out by adopting an ammonium sulfate fractional precipitation method, then chromatography purification is carried out, and the purified protein is subjected to SDS-PAGE electrophoresis, so that the target proteins are purified and enriched.

Example 2 eukaryotic expression of the P17, P22, P72, P30 proteins of African Swine fever Virus

Nucleotide sequences correspondingly encoding P17, P22, P72 and P30 proteins shown in sequence tables SEQ ID NO. 7-SEQ ID NO.10 are synthesized by Suzhou Jinzhi Biotechnology Co., ltd, and the synthesized nucleotides are cloned to a pFB vector to be named as recombinant plasmids pFB-P17, pFB-P22, pFB-P72 and pFB-P30.

The recombinant plasmids pFB-p17, pFB-p22, pFB-p72 and pFB-p30 are respectively transformed into DH10Bac competent cells, white-blue screening is carried out, white single bacterial colonies are selected to be put into a liquid LB culture medium containing three resistances of kanamycin, tetracycline and gentamicin for shaking for overnight, recombinant Bacmid is extracted according to the method on Bac-to-Bac operation instructions and is respectively named as Bac-p17, bac-p22, bac-p72 and Bac-p30.

Referring to the Cellfect II Reagent transfection kit operation instruction, the recombinant Bacmid Bac-P17, bac-P22, bac-P72 and Bac-P30 are used for transfecting sf9 insect cells, the cells are placed in a constant temperature incubator at 27 ℃ for about 72 hours to be cultured, after cytopathic effect is obvious, cell supernatants are harvested to be P1 generation recombinant baculovirus which are respectively marked as rBac-P17, rBac-P22, rBac-P72 and rBac-P30. Adding the P1 generation recombinant baculovirus into a cell shaking flask of sf9 according to the volume ratio of 1 to 1. Inoculating 1L volume of sf9 cells to the P2 generation recombinant baculovirus according to the volume ratio of 1.

With cell lysate (25 mM NaHCO) ₃ pH 8.3), cell pellet was lysed for 30 minutes, and the lysate supernatant was obtained by centrifugation at 10000 Xg and 4 ℃ for 10 minutes, and expression of the target protein was confirmed by Western Blot. And (3) performing protein crude purification by using nickel column affinity chromatography, then performing molecular sieve purification, and performing SDS-PAGE electrophoresis on the purified protein to show that the target proteins are purified and enriched.

Example 3 preparation of African Swine fever Virus subunit vaccine compositions

And adding the P34, P14, C129R, DP96R, A104R and P54 proteins prepared in the example 1 and the P17, P22, P72 and P30 proteins prepared in the example 2 into an adjuvant, continuously stirring for 12min by using an emulsifying machine with the rotating speed of 800rpm, uniformly mixing, and storing at 4 ℃ to obtain the subunit vaccine composition containing the African swine fever virus multi-component protein antigen. Adjuvants suitable for use in the present invention may be those known to those skilled in the art. In the present invention, the adjuvant is a biphasic adjuvant (water-in-oil-in-water emulsion), and may be, for example, adjuvant ISA206 (french seebeck). The specific proportions of the components in the prepared vaccine are shown in table 1.

TABLE 1 sub-unit vaccine composition for African swine fever virus

Components	Vaccine 1	Vaccine 2	Vaccine 3
				p34(μg/ml)	8	16	24
p14(μg/ml)	8	16	24
				C129R(μg/ml)	8	16	24
DP96R(μg/ml)	8	16	24
				A104R(μg/ml)	8	16	24
p54(μg/ml)	12	24	36
				p17(μg/ml)	12	24	36
p22(μg/ml)	12	24	36
				P72(μg/ml)	12	24	36
p30(μg/ml)	12	24	36
				ISA206 biphasic adjuvant (V/V%)	50％	50％	50％

Example 4 African Swine fever Virus subunit vaccine compositions immunogenicity assays

20 healthy susceptible piglets which are negative to African swine fever virus antigen and antibody and have the weight of about 20kg are randomly divided into 4 groups, and each group has 5 piglets. Group 1 immunization vaccine 1, group 2 immunization vaccine 2, group 3 immunization vaccine 3, group 4 blank control group. The immunization group had an immunization route of neck intramuscular injection of 2ml, and the control group had an immunization with an equivalent amount of PBS plus adjuvant. Immunizations were performed twice, with an interval of 14 days. Blood was collected before the 1 st immunization and 14 days after the 2 nd immunization.

Coating the ELISA plate with African SWINE FEVER whole virus inactivated antigen (purchased from European Union African SWINE FEVER REFERENCE LABORATORY (URL-ASF)) and coating overnight at 4 ℃; discarding the coating solution, and washing with a washing solution; adding blocking solution (weighing 50g of sucrose, adding 200mL of newborn bovine serum and 1mL of Proclin300, supplementing PBS (0.01 mol/L, pH value of 7.4) and fixing the volume to 1000 mL), blocking at 2-8 ℃ for 16-24 hours, removing the blocking solution, drying, sealing, and storing at 2-8 ℃ for later use.

Sample diluent: taking 8g of sodium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of monopotassium phosphate, 0.2g of potassium chloride, 600mL of purified water, 300 mL of Proclin, 200mL of newborn bovine serum and 0.028g of PUR dye, completely dissolving, then fixing the volume to 1000mL by using the purified water, uniformly mixing, filtering by 0.22 mu m, then carrying out aseptic subpackaging, and storing at 2-8 ℃.

Washing solution: taking 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of monopotassium phosphate, 4g of potassium chloride, 800mL of ultrapure water and 20 mL of Tween 0, completely dissolving, using purified water to fix the volume to 1000mL, filtering by using a 0.22 mu m filter membrane, and performing sterile subpackaging. It is diluted 20 times with distilled water.

Secondary antibody: an enzyme-labeled goat anti-porcine secondary antibody was used at 1.

Color development liquid: dissolving 14.7g of disodium hydrogen phosphate, 9.3g of citric acid and 0.3g of carbamide peroxide in purified water, metering volume to 1000mL, uniformly mixing, filtering, and performing aseptic subpackage to obtain the color developing agent A. Taking 0.2g of tetramethyl diphenyldiamine (TMB) and 10mL of absolute ethyl alcohol, dissolving in purified water to reach a constant volume of 1000mL, uniformly mixing, filtering, and performing sterile subpackage to obtain the color developing agent B.

Stopping liquid: 2M H ₂ SO ₄ 。

During detection, 90 mul of sample diluent is added into an enzyme-labeled reaction hole, 10 mul of serum to be detected is added, negative and positive controls are incubated for 30 minutes at 37 ℃, a plate is washed for three times by using washing liquid, the reaction plate is added into 100 mul/hole of goat anti-pig enzyme-labeled antibody, the incubation is carried out for 30 minutes at 37 ℃, the plate is washed for three times by using washing liquid, 50 mul/hole of each of a developer A and a developer B is added, the color development is carried out for 10 minutes at 37 ℃, 50 mul/hole of a stop solution is added to stop the reaction, and the OD (optical density) is read by an enzyme-labeling instrument _450nm And performing judgment according to the judgment result.

And (4) judging standard: the OD value is more than or equal to 0.19 positive, and the OD value is less than 0.19 negative.

The results are shown in Table 2.

TABLE 2 immunogenicity test results for African swine fever Virus subunit vaccine compositions

The result shows that the African swine fever virus subunit vaccine composition has good immunogenicity, and the antibody detection is positive. The African swine fever virus subunit vaccine provided by the invention adopts a multi-component African swine fever virus protein antigen, and the immune effect is good.

Example 5 African swine fever virus protein immunogenicity validation assay

1. Preparation of African swine fever virus single-component subunit vaccine composition

The P34, P14, C129R, DP96R, A104R and P54 proteins prepared in the example 1 and the P17, P22, P72 and P30 proteins prepared in the example 2 are respectively added into an adjuvant, and the mixture is stirred for 12min by an emulsifying machine with the rotating speed of 800rpm continuously in the adding process, uniformly mixed and stored at 4 ℃, thus obtaining the subunit vaccine composition containing the African swine fever virus single-component protein antigen. Adjuvants suitable for use in the present invention may be those known to those skilled in the art. In the present invention, the adjuvant is a biphasic adjuvant (water-in-oil-in-water emulsion), and may be, for example, adjuvant ISA206 (seebeck, france). The specific ratios of the components in the prepared vaccine are shown in tables 3 and 4.

TABLE 3 African swine fever virus monocomponent subunit vaccine composition ingredient ratio 1

TABLE 4 African swine fever virus monocomponent subunit vaccine composition ingredient ratio 2

2. Immunogenicity assay for African swine fever virus monocomponent subunit vaccine compositions

55 healthy susceptible piglets which are negative in African swine fever virus antigen and antibody and have the weight of about 20kg are randomly divided into 11 groups, and each group has 5 piglets. Groups 5-14 were immunized with vaccine 4-vaccine 13, respectively, and group 15 was a blank control. The immunization group was administered by intramuscular injection of 2ml into the neck, and the control group was immunized with the same amount of PBS + adjuvant. Immunizations were performed twice, with 14 days intervals. Blood was collected before the 1 st immunization and 14 days after the 2 nd immunization.

Referring to example 4, the african swine fever virus antibody detection kit is prepared by coating an elisa plate with the african swine fever virus P34 protein, P14 protein, C129R protein, DP96R protein, a104R protein, P54 protein, P17 protein, P22 protein, P72 protein, and P30 protein, respectively.

The results are shown in Table 5.

TABLE 5 African Swine fever Virus monocomponent subunit vaccine compositions immunogenicity test results 1

TABLE 6 immunogenicity test results of African swine fever Virus monocomponent subunit vaccine compositions 2

TABLE 7 immunogenicity test results of African Swine fever Virus monocomponent subunit vaccine compositions 3

TABLE 8 African swine fever virus monocomponent subunit vaccine composition immunogenicity test results 4

TABLE 9 African Swine fever Virus monocomponent subunit vaccine compositions immunogenicity test results 5

TABLE 10 African swine fever virus monocomponent subunit vaccine composition immunogenicity test results 6

TABLE 11 African swine fever virus monocomponent subunit vaccine composition immunogenicity test results 7

TABLE 12 immunogenicity test results of African swine fever Virus monocomponent subunit vaccine compositions 8

TABLE 13 African swine fever virus monocomponent subunit vaccine composition immunogenicity test results 9

TABLE 14 African swine fever virus monocomponent subunit vaccine composition immunogenicity test results 10

The results show that the African swine fever virus single-component subunit vaccine composition can cause immune reactions of different degrees, and antibody detection is positive. The African swine fever virus subunit vaccine prepared by the African swine fever virus multicomponent subunit vaccine has better immune effect and higher antibody level.

Comparing the OD values detected in tables 2 and 5-14, the detected OD of the African swine fever virus subunit vaccine composition is only 200 mug (vaccine 1) or 400 mug (vaccine 2) when the total dosage of the antigen (namely the sum of the content of each antigen protein in the African swine fever virus subunit vaccine composition with 2ml of immune dosage) is only 200 mug _400nm The values are 2.024 and 2.464 respectively, while the antigen dosage of the African swine fever virus single-component subunit vaccine composition detected in tables 5-14 (i.e. the antigen protein content in the African swine fever virus single-component subunit vaccine composition with 2ml of immunization dosage) is 400 mug, and the detected OD is higher than or equal to the total dosage of the vaccine 1 or the vaccine 2 antigen _400nm The values are all far smaller than the antibody OD in the serum after the immunization of the vaccine 1 or the vaccine 2 _400nm The value shows that the antigen components in the African swine fever virus subunit vaccine composition generate synergistic effect, can generate stronger immune response and has higher antibody titer.

Although the present invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the present invention.

SEQUENCE LISTING

<110> Puleco bioengineering GmbH

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<211> 384

<212> DNA

<213> African swine fever virus (African swine feber virus)

<400> 7

atggacactg aaacatctcc tctgctgtca cacaacctgt caactcgtga aggtatcaag 60

caatcaacac agggcctgct ggcccacacc atcgctaagt accctggtac caccgccatc 120

ctgctgggta tcctgatcct gctgatcatc atcctcatca tcgtggctat cgtgtactac 180

aaccgtacca tcgactgcaa gagcagcatc ccaaagcctc ccccttccta ctacgtgcag 240

cagccagaac cacaccacca cttccccgtg ttcttccgca agcgcaagaa cagcacctcc 300

ctgcagtccc acatccctag cgacgaacag ctggctgaac tggcccactc caacaaccac 360

caccatcacc accaccacca ctaa 384

<210> 8

<211> 594

<212> DNA

<213> African swine fever virus (African swine feber virus)

<400> 8

atgagatcct ccaagaagat caacaacaag aagaacatgt tcaacatcaa gatgactatc 60

tccactctgc tgatcgctct gatcatcctg ctgatcatta tcctggtggt gttcctgtac 120

tacaagaagc agcagccacc aaagaaggtg tgcaaggtgg acaaggactg cggttccggt 180

gagcactgcg tgagaggttc ctgctcctcc ctgtcctgcc tggacgctgt gaagatggac 240

aagagaaaca tcaagatcga ctccaagatc tcctcctgcg agttcactcc aaacttctac 300

agattcactg acactgctgc tgacgagcag caagagttcg gtaagactag acacccaatc 360

aagatcactc catccccatc cgagtcccac tccccacaag aggtgtgcga gaagtactgc 420

tcctggggta ctgacgactg cactggttgg gagtacgtgg gtgacgagaa ggagggtact 480

tgctacgtgt acaacaaccc acaccaccct gtgctgaagt acggtaagga ccacatcatc 540

gctctgccaa gaaaccacaa gcacgctcac caccatcacc accaccacca ctaa 594

<210> 9

<211> 1965

<212> DNA

<213> African swine fever virus (African swine feber virus)

<400> 9

atggcttctg gcggcgcctt ctgcctgatc gccaacgacg gtaaagctga caagatcatc 60

ctggcccaag acctgctgaa ctcacgcatc tccaacatca agaacgtcaa caagtcctac 120

ggcaagcctg acccagagcc tacattgtcc cagatcgagg aaacccacct cgtgcacttc 180

aacgctcact tcaagcccta cgttcctgtt ggcttcgagt acaacaaggt gcgccctcac 240

accggaaccc caacactggg caacaagctg actttcggca tccctcaata cggtgacttc 300

ttccacgaca tggtgggtca ccacatcctg ggcgcctgtc actccagctg gcaggacgcc 360

ccaatccagg gcacaagcca gatgggcgct cacggccagc tccagacttt ccctcgtaac 420

ggctacgact gggacaacca gacacccctg gagggagctg tgtacaccct cgtggaccca 480

ttcggacgcc caatcgtgcc tggaactaag aacgcctacc gtaacctcgt ctactactgc 540

gaataccctg gcgaacgtct ctacgaaaac gtgcgcttcg acgttaacgg taacagcctc 600

gacgaatact cttccgacgt gacaacactg gttaggaagt tctgcatccc cggagacaag 660

atgactggtt acaagcacct ggttggtcag gaagtgagcg tggaaggcac ctccggacca 720

ctcctctgca acatccacga cctgcacaag ccccaccaga gcaagcccat cctgactgac 780

gagaacgaca cacagcgcac ttgcagccac accaacccta agttcctctc ccagcacttc 840

ccagaaaact cccacaacat ccaaacagcc ggcaagcagg acatcacccc tatcaccgac 900

gccacctacc tcgacatccg ccgtaacgtt cactactcct gtaacggacc acaaacacct 960

aagtactacc aaccaccact ggctctctgg atcaagttgc gtttctggtt caacgagaac 1020

gttaacctcg ccatcccctc tgtgagcatc cccttcggcg aacgcttcat cacaatcaag 1080

ctcgcctcac agaaggacct ggttaacgag ttccctggcc tcttcgtgag gcagtcccgc 1140

ttcatcgctg gtcgccccag caggcgcaac atccgtttca agccttggtt catccccggc 1200

gtcatcaacg aaatcagcct gactaacaac gaactctaca tcaacaacct gttcgtgacc 1260

cctgagatcc acaacctgtt cgtgaagcgt gttaggttca gtctgatcag ggtccacaag 1320

actcaagtga cccacaccaa taacaaccac cacgacgaga agctgatgag cgctctgaag 1380

tggcccatcg aatacatgtt catcggtctg aagcccacct ggaacatctc cgaccagaac 1440

cctcaccagc accgtgattg gcacaagttc ggccacgtgg tcaacgctat catgcagcct 1500

actcaccacg ctgagatcag cttccaggac cgtgacacag ccctccctga cgcttgttct 1560

agcatctctg atatctctcc agtcacttac ccaatcaccc tgcctatcat caagaacatc 1620

tcagtcaccg cccacggtat taacctgatc gacaagttcc cttctaagtt ctgttcatcc 1680

tacatcccat tccactacgg aggtaacgcc atcaagacac ctgacgaccc tggtgctatg 1740

atgatcacct tcgctctcaa gccacgcgag gaataccagc cctccggtca catcaacgtt 1800

agccgcgctc gtgagttcta catcagctgg gacaccgact acgtgggtag catcactacc 1860

gctgacctcg tggtgtctgc ttccgccatc aacttcctgc tgctccagaa cggtagcgcc 1920

gtgctccgct actcaaccca ccaccaccac catcaccacc actaa 1965

<210> 10

<211> 630

<212> DNA

<213> African swine fever virus (African swine fever virus)

<400> 10

atggacttca tcctgaacat cagcatgaag atggaagtga tcttcaagac cgacctgcgt 60

agcagcagcc aggtggtgtt ccacgctggt agcctgtaca actggttcag cgtggagatc 120

atcaacagcg gtcgcatcgt gaccaccgct atcaagaccc tgctgagcac cgtgaagtac 180

gacatcgtga agtccgctcg tatctacgcc ggccagggtt acaccgagca ccaggcccag 240

gaagaatgga acatgatcct gcacgtgctg ttcgaagaag aaaccgagag ctccgcttcc 300

tccgaaaaca tccacgagaa gaacgacaac gaaaccaacg agtgtaccag ctccttcgaa 360

accctgttcg aacaggagcc ttcctccgag gtgcctaagg acagcaagct gtacatgctg 420

gcccagaaga ccgtgcagca catcgaacag tacggcaagg ctcctgactt caacaaggtt 480

atccgcgctc acaacttcat ccagaccatc tacggtaccc ctctgaagga agaggaaaag 540

gaagtggtgc gtctgatggt tatcaagctg ctgaagaaga tcagcttctt cctgacctac 600

atccaccacc accatcacca ccaccactaa 630

<210> 11

<211> 5369

<212> DNA

<213> Escherichia coli BL21 (DE 3) (E.coli BL21 (DE 3))

<400> 11

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 2160

caccgcatac aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 2220

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 2280

aatagcacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 2340

ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 2400

tagaaaagat caaaggatct acgccagcaa cgcggccttt ttacggttcc tgggcttttg 2460

ctggcctttt gctcacatgt ctcttcgcga tgtacgggcc agatatacgc tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa cgttactccc acaggtgagc 2820

gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggctcgtt 2880

tcttttctgt ggctgcgtga aagccttaaa gggctccggg agggcccttt gtgcgggggg 2940

gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg 3000

ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc 3060

gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg gggggctgcg aggggaacaa 3120

aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg 3180

gctgtaaccc ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg 3240

cggggctccg tgcggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg 3300

tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg 3360

gcggccccgg agcgccggcg gctgtcgagg cgcggcgagc tggccaatgc cctggctcac 3420

aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt 3480

gagcatctga cttctggcta taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100

agcagcggcc tggtgccgcg cggcagccat atggctagca tgactggtgg acagcaaatg 5160

ggtcgcggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg agcaccacca 5220

ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 5280

caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 5340

tttgctgaaa ggaggaacta tatccggat 5369

Claims

1. An African swine fever virus subunit vaccine composition, wherein the protein antigen of the African swine fever virus subunit vaccine composition consists of African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72, P30 proteins, and the African swine fever virus subunit vaccine composition further comprises a pharmaceutically acceptable carrier;

wherein, the African swine fever virus p34 protein is encoded by SEQ ID NO.1 or a degenerate sequence thereof; the African swine fever virus p14 protein is SEQ ID NO.2 or a degenerate sequence code thereof; the African swine fever virus C129R protein is SEQ ID NO.3 or a degenerate sequence code thereof; the African swine fever virus DP96R protein is SEQ ID NO.4 or a degenerate sequence code thereof; the African swine fever virus A104R protein is SEQ ID NO.5 or a degenerate sequence code thereof; the African swine fever virus p54 protein is coded by SEQ ID NO.6 or a degenerate sequence thereof; the African swine fever virus p17 protein is SEQ ID NO.7 or a degenerate sequence code thereof; the African swine fever virus p22 protein is SEQ ID NO.8 or a degenerate sequence code thereof; the African swine fever virus P72 protein is coded by SEQ ID NO.9 or a degenerate sequence thereof; and the African swine fever virus p30 protein is encoded by SEQ ID NO.10 or a degenerate sequence thereof.

2. The African swine fever virus subunit vaccine composition of claim 1, wherein the African swine fever virus subunit vaccine composition is in the form of a solution injection, a suspension injection, or a powder for injection.

3. The African swine fever virus subunit vaccine composition of claim 1, wherein the dosage form of the African swine fever virus subunit vaccine composition is solution injection, suspension injection; the content of the African swine fever virus p34 protein is more than or equal to 8 mug/ml; the content of the African swine fever virus p14 protein is more than or equal to 8 mug/ml; the content of the African swine fever virus C129R protein is more than or equal to 8 mug/ml; the protein content of the African swine fever virus DP96R is more than or equal to 8 mug/ml; the protein content of the African swine fever virus A104R is more than or equal to 8 mug/ml; the content of the African swine fever virus p54 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p17 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p22 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus P72 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p30 protein is more than or equal to 12 mu g/ml;

preferably, the content of the African swine fever virus p34 protein is 8-24 mug/ml; the content of the African swine fever virus p14 protein is 8-24 mug/ml; the content of the African swine fever virus C129R protein is 8-24 mug/ml; the protein content of the African swine fever virus DP96R is 8-24 mu g/ml; the protein content of the African swine fever virus A104R is 8-24 mu g/ml; the content of the African swine fever virus p54 protein is 12-36 mu g/ml; the content of the African swine fever virus p17 protein is 12-36 mu g/ml; the content of the African swine fever virus p22 protein is 12-36 mu g/ml; the content of the African swine fever virus P72 protein is 12-36 mu g/ml; the content of the African swine fever virus p30 protein is 12-36 mu g/ml;

more preferably, the content of the African swine fever virus p34 protein is 8-16 mug/ml; the content of the African swine fever virus p14 protein is 8-16 mug/ml; the content of the African swine fever virus C129R protein is 8-16 mug/ml; the protein content of the African swine fever virus DP96R is 8-16 mug/ml; the protein content of the African swine fever virus A104R is 8-16 mug/ml; the content of the African swine fever virus p54 protein is 12-24 mug/ml; the content of the African swine fever virus p17 protein is 12-24 mug/ml; the content of the African swine fever virus p22 protein is 12-24 mug/ml; the content of the African swine fever virus P72 protein is 12-24 mu g/ml; the content of the African swine fever virus p30 protein is 12-24 mu g/ml;

further preferably, the African swine fever virus p34 protein content is 8 μ g/ml; the content of the African swine fever virus p14 protein is 8 mug/ml; the content of the African swine fever virus C129R protein is 8 mug/ml; the content of the African swine fever virus DP96R protein is 8 mug/ml; the protein content of the African swine fever virus A104R is 8 mu g/ml; the content of the African swine fever virus p54 protein is 12 mu g/ml; the content of the African swine fever virus p17 protein is 12 mu g/ml; the content of the African swine fever virus p22 protein is 12 mu g/ml; the content of the African swine fever virus P72 protein is 12 mu g/ml; the content of the African swine fever virus p30 protein is 12 mu g/ml;

further preferably, the content of the African swine fever virus p34 protein is 16 mug/ml; the content of the African swine fever virus p14 protein is 16 mug/ml; the content of the African swine fever virus C129R protein is 16 mug/ml; the content of the African swine fever virus DP96R protein is 16 mu g/ml; the protein content of the African swine fever virus A104R is 16 mu g/ml; the content of the African swine fever virus p54 protein is 24 mug/ml; the content of the African swine fever virus p17 protein is 24 mu g/ml; the content of the African swine fever virus p22 protein is 24 mu g/ml; the content of the African swine fever virus P72 protein is 24 mug/ml; the content of the African swine fever virus p30 protein is 24 mug/ml.

4. The African swine fever virus subunit vaccine composition of claim 1, wherein the pharmaceutically acceptable carrier comprises an adjuvant comprising: (1) Mineral oil, alumina gel adjuvant, saponin, alfudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanideeISA 206 and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;

said adjuvant content is 5-60% V/V, preferably from 30-60% V/V, more preferably 50% V/V; or

The pharmaceutically acceptable carrier includes a lyoprotectant selected from the group consisting of a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

5. The use of the African swine fever virus subunit vaccine composition according to any one of claims 1 to 4, wherein the use is for the manufacture of a medicament for the prevention of African swine fever virus infection.

6. A combination of african swine fever virus protein antigens, wherein the african swine fever virus protein antigens consist of P34, P14, C129R, DP96R, a104R, P54, P17, P22, P72, P30 proteins of african swine fever virus;

wherein, the African swine fever virus p34 protein is coded by SEQ ID NO.1 or a degenerate sequence thereof; the African swine fever virus p14 protein is coded by SEQ ID NO.2 or a degenerate sequence thereof; the African swine fever virus C129R protein is SEQ ID NO.3 or a degenerate sequence code thereof; the African swine fever virus DP96R protein is SEQ ID NO.4 or a degenerate sequence code thereof; the African swine fever virus A104R protein is SEQ ID NO.5 or a degenerate sequence code thereof; the African swine fever virus p54 protein is coded by SEQ ID NO.6 or a degenerate sequence thereof; the African swine fever virus p17 protein is SEQ ID NO.7 or a degenerate sequence code thereof; the African swine fever virus p22 protein is SEQ ID NO.8 or a degenerate sequence code thereof; the African swine fever virus P72 protein is SEQ ID NO.9 or a degenerate sequence code thereof; and the African swine fever virus p30 protein is encoded by SEQ ID NO.10 or a degenerate sequence thereof.

7. The combination of African swine fever virus protein antigens according to claim 6, wherein the combination of African swine fever virus protein antigens further comprises a pharmaceutically acceptable carrier comprising an adjuvant comprising: (1) Mineral oil, alumina gel adjuvant, saponin, alfudine, DDA; (2) Water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanide ISA206, and Gel adjuvant; preferably, the saponin is QuilA, QS-21, GPI-0100;

said adjuvant content is 5-60% V/V, preferably from 30-60% V/V, more preferably 50% V/V;

or the pharmaceutically acceptable carrier comprises a lyoprotectant selected from the group consisting of a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

8. The combination of African swine fever virus protein antigens according to claim 6, wherein the African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72, P30 protein is in the form of solution type injection, suspension type injection, powder for injection.

9. The combination of African swine fever virus protein antigens according to claim 8, wherein the African swine fever virus P34, P14, C129R, DP96R, A104R, P54, P17, P22, P72, P30 proteins are in the form of solution injection, suspension injection;

the content of the African swine fever virus p34 protein is more than or equal to 8 mu g/ml; the content of the African swine fever virus p14 protein is more than or equal to 8 mu g/ml; the content of the African swine fever virus C129R protein is more than or equal to 8 mu g/ml; the protein content of the African swine fever virus DP96R is more than or equal to 8 mug/ml; the protein content of the African swine fever virus A104R is more than or equal to 8 mug/ml; the content of the African swine fever virus p54 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p17 protein is more than or equal to 12 mug/ml; the content of the African swine fever virus p22 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus P72 protein is more than or equal to 12 mu g/ml; the content of the African swine fever virus p30 protein is more than or equal to 12 mu g/ml;

preferably, the content of the African swine fever virus p34 protein is 8-24 mug/ml; the content of the African swine fever virus p14 protein is 8-24 mug/ml; the content of the African swine fever virus C129R protein is 8-24 mu g/ml; the protein content of the African swine fever virus DP96R is 8-24 mug/ml; the protein content of the African swine fever virus A104R is 8-24 mug/ml; the content of the African swine fever virus p54 protein is 12-36 mu g/ml; the content of the African swine fever virus p17 protein is 12-36 mu g/ml; the content of the African swine fever virus p22 protein is 12-36 mu g/ml; the content of the African swine fever virus P72 protein is 12-36 mu g/ml; the content of the African swine fever virus p30 protein is 12-36 mu g/ml;

more preferably, the content of the African swine fever virus p34 protein is 8-16 mug/ml; the content of the African swine fever virus p14 protein is 8-16 mug/ml; the content of the African swine fever virus C129R protein is 8-16 mu g/ml; the protein content of the African swine fever virus DP96R is 8-16 mu g/ml; the protein content of the African swine fever virus A104R is 8-16 mu g/ml; the content of the African swine fever virus p54 protein is 12-24 mug/ml; the content of the African swine fever virus p17 protein is 12-24 mu g/ml; the content of the African swine fever virus p22 protein is 12-24 mug/ml; the content of the African swine fever virus P72 protein is 12-24 mu g/ml; the content of the African swine fever virus p30 protein is 12-24 mu g/ml;

further preferably, the African swine fever virus p34 protein content is 8 μ g/ml; the content of the African swine fever virus p14 protein is 8 mug/ml; the content of the African swine fever virus C129R protein is 8 mug/ml; the content of the African swine fever virus DP96R protein is 8 mug/ml; the protein content of the African swine fever virus A104R is 8 mug/ml; the content of the African swine fever virus p54 protein is 12 mu g/ml; the content of the African swine fever virus p17 protein is 12 mu g/ml; the content of the African swine fever virus p22 protein is 12 mu g/ml; the content of the African swine fever virus P72 protein is 12 mu g/ml; the content of the African swine fever virus p30 protein is 12 mu g/ml;

further preferably, the content of the African swine fever virus p34 protein is 16 mug/ml; the content of the African swine fever virus p14 protein is 16 mug/ml; the content of the African swine fever virus C129R protein is 16 mu g/ml; the content of the African swine fever virus DP96R protein is 16 mu g/ml; the content of the African swine fever virus A104R protein is 16 mug/ml; the content of the African swine fever virus p54 protein is 24 mug/ml; the content of the African swine fever virus p17 protein is 24 mug/ml; the content of the African swine fever virus p22 protein is 24 mu g/ml; the content of the African swine fever virus P72 protein is 24 mu g/ml; the content of the African swine fever virus p30 protein is 24 mug/ml.

10. The use of a combination of African swine fever virus protein antigens according to claims 6 to 9, wherein the use is for the manufacture of a medicament for the prevention of African swine fever virus infection.