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

Abstract

The present invention provides an African swine fever virus subunit vaccine composition, which is composed of an immunizing amount of the following components: African swine fever virus p72 protein, p30 protein, p54 protein, African swine fever virus nucleocapsid assembly protein, African swine fever virus replication protein, African swine fever virus adsorption protein, and African swine fever virus virulence protein; and the African swine fever virus subunit vaccine composition also includes a pharmaceutically acceptable carrier. The present invention also provides a combination of African swine fever virus protein antigens. The combination of the African swine fever virus subunit vaccine composition of the present invention and the African swine fever virus protein antigen can provide good immune efficacy for pigs. The protein antigens have a synergistic effect, which can provide better immune efficacy under conditions of low total protein content.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and specifically relates to an African swine fever virus subunit vaccine composition, a combination of African swine fever virus protein antigens, a preparation method and applications thereof. Background Art

[0002] African swine fever (ASF) is an acute, severe, and highly contagious disease caused by the African swine fever virus (ASFV). It carries a high morbidity rate and can reach a mortality rate of 100%. my country classifies it as a Category I animal disease. The disease was first confirmed in Kenya, Africa, in 1921. Since 2007, ASF has occurred, spread, and become endemic in numerous countries worldwide, reaching my country in 2018, causing significant direct and indirect economic losses. Despite being discovered nearly a century ago, there are currently no approved vaccines or effective treatments globally.

[0003] The difficulty in developing an African swine fever vaccine lies in the fact that the viral genome is large, contains many proteins, has a complex structure, and the functions of most genes are unknown. The mechanism of interaction between the virus and host cells is unclear, making it difficult to culture on a large scale. It is prone to mutation during passage, and the mechanism of viral immune escape is unclear. Therefore, there has been no substantial progress in conventional inactivated vaccines and live attenuated vaccines so far, and improper use of live attenuated vaccines poses biosafety risks of reversion to virulence and spread of the virus.

[0004] Therefore, there is an urgent need for an African swine fever vaccine clinically, which is required to have good immune effects, effectively protect pigs, and pose no biosafety risks. Summary of the Invention

[0005] To this end, the present invention uses modern biological methods to recombinantly express the genes and proteins of the pathogen, and tests their immune protection capabilities to develop subunit vaccines, effectively solving the problem of unsatisfactory immune effects of various current vaccines.

[0006] The present invention provides an African swine fever virus subunit vaccine composition, wherein the protein antigen of the African swine fever virus subunit vaccine composition is composed of multi-component protein antigens, which can provide good immune efficacy for pigs.

[0007] The present invention provides an African swine fever virus subunit vaccine composition. The multi-component protein antigens of the African swine fever virus subunit vaccine composition have a synergistic effect and can provide better immune efficacy under the condition of low total protein content.

[0008] The present invention provides an application of the African swine fever virus subunit vaccine composition, and the application refers to an application in the preparation of a drug for preventing African swine fever virus infection.

[0009] The present invention provides a combination of African swine fever virus protein antigens that can effectively immunize and protect pigs.

[0010] The present invention provides a combination of African swine fever virus protein antigens, wherein the multi-component protein antigens have a synergistic effect and can provide better immune efficacy under the condition of low total protein dosage.

[0011] The present invention provides an application of a combination of African swine fever virus protein antigens, wherein the application refers to an application in the preparation of a drug for preventing African swine fever virus infection.

[0012] The African swine fever virus subunit vaccine of the present invention adopts multi-component African swine fever virus protein antigens. The multi-component African swine fever virus protein antigens are administered together to synergistically enhance the efficacy and have a good immune effect, effectively solving the problem of poor immunogenicity faced by the current African swine fever virus vaccine; the vaccine composition can be expressed in large quantities through genetic engineering means, which is not only time-saving but also convenient for large-scale production and has no biosafety risks. DETAILED DESCRIPTION

[0013] Hereinafter, embodiments of the present invention will be described.

[0014] definition

[0015] The term "African swine fever virus" refers to the African swine fever virus (ASFV), the only species in the Asfarviridae family. It is contagious and highly pathogenic. ASFV is an icosahedron approximately 200 nanometers in diameter and composed of multiple layers: a central protein nucleocapsid containing a nucleoid, followed by a lipid envelope and a protein capsid. The capsid is composed of 8,280 major capsid proteins, p72, and 60 pentaproteins. In addition, at least three other proteins maintain capsid structure stability by binding to adjacent proteins. Acute clinical symptoms are characterized by high fever, a short course of illness, high mortality, extensive bleeding in internal organs, and respiratory and neurological dysfunction. As of 2020, there is still no specific vaccine or antiviral drug for ASFV that can effectively control its spread during an outbreak.

[0016] The term "African swine fever virus p72 protein" refers to a protein produced in the late stage of viral infection and encoded by the ORF B646L gene. It is an important antigenic protein of African swine fever virus, the main component of the viral icosahedron, and is crucial for the formation of the viral capsid.

[0017] The term "African swine fever virus p30 protein" refers to the early membrane protein expressed by African swine fever virus, which is encoded by the ORFCP204L gene. It is usually produced 2-4 hours after infection and is continuously expressed throughout the infection. It is related to the virus invading host cells and is an important structural protein.

[0018] The term "African swine fever virus p54 protein" refers to the early membrane protein expressed by African swine fever virus, which is encoded by the OR FE183L gene, contains a transmembrane domain, and is located in the endoplasmic reticulum-derived inner membrane precursor. It plays an important role in the virus adsorption to susceptible cells and invasion process and is an important structural protein.

[0019] The term "African swine fever virus p34 protein" refers to a polyprotein called p220, which is encoded by the African swine fever virus. This polyprotein, p220, is present in the nucleocapsid of mature virions, comprising approximately 30% of the total viral protein, and plays a crucial role in viral assembly and infection. The p220 polyprotein is sequentially cleaved by proteases into p150, p34, p37, and p14. These three proteins, p150, p34, p37, and p14, play crucial roles in viral capsid assembly. Among them, p34 is a key structural protein within p220, playing a crucial role in the packaging of the viral nucleocapsid protein.

[0020] The term "African swine fever virus p62 protein" refers to a polyprotein encoded by African swine fever virus, present in the nucleocapsid, and encoded by the ORF CP530R gene.

[0021] The term "African swine fever virus p17 protein" refers to a late membrane protein expressed by African swine fever virus, encoded by the ORF D117L gene, and a transmembrane protein located in the inner membrane of the virus.

[0022] The term "African swine fever virus E165R protein" refers to dUTP pyrophosphatase (dUTPase), a key enzyme in DNA synthesis.

[0023] The term "African swine fever virus pS273R protein" refers to a protease encoded by African swine fever virus that is responsible for polyprotein cleavage.

[0024] The term "African swine fever virus A104R protein" refers to a DNA-binding protein that is involved in viral transcription, DNA replication and genome packaging, also known as "pA104R protein".

[0025] The term "African swine fever virus C129R protein" refers to manganese-dependent superoxide dismutase.

[0026] The term "African swine fever virus p12 protein" refers to an adhesion protein encoded by African swine fever virus, which is involved in viral entry.

[0027] The term "African swine fever virus p22 protein" refers to the transmembrane domain of African swine fever virus, which is located on the outside of the virus particle.

[0028] The term "African swine fever virus DP96R protein" refers to a protein encoded by African swine fever virus that can inhibit type I IFN expression and NF-κB activation, also known as "pDP96R protein".

[0029] The term "African swine fever virus DP71L protein" refers to a protein encoded by African swine fever virus that can inhibit cGAS+MITA or MITA-induced IFNβ activity.

[0030] The term "degenerate sequence" refers to the phenomenon that the same amino acid has two or more codons. Such a sequence is called a degenerate sequence.

[0031] The term "antigen" refers to a substance that can induce an immune response in the body, that is, a substance that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to corresponding products in vivo and in vitro.

[0032] The terms "vaccine" and "vaccine composition" used in the present invention refer to a pharmaceutical composition containing African swine fever virus protein antigens, which can induce, stimulate or enhance the immune response of pigs against African swine fever.

[0033] The term "protein combination" used in the present invention refers to a pharmaceutical composition containing more than one African swine fever virus protein antigen, which can induce, stimulate or enhance the immune response of pigs against African swine fever.

[0034] The term "immunizing amount" should be understood as an "immunologically effective amount", also known as an immunoprotective amount or an effective amount to produce an immune response, which is the amount of antigen that can effectively induce an immune response in the recipient, and this amount is sufficient to prevent or improve the signs or symptoms of the disease, including adverse health effects or its complications. The immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of the disease, including adverse health consequences or complications caused by infection caused by pathogens. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be indirectly assessed by, for example, measuring antibody titers, lymphocyte proliferation assays, or directly assessed by monitoring signs or symptoms after challenge with a wild-type strain, and 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, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, inducing cellular and / or humoral immunity.

[0035] The term "pharmaceutically acceptable carrier" refers to all other ingredients in the vaccine composition of the present invention except the African swine fever virus protein antigen, a carrier or diluent that does not stimulate the body and does not hinder the biological activity and properties of the compound used, preferably an adjuvant. The term "adjuvant" may include alumina adjuvants; saponins (saponin), such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; copolymers of maleic anhydride and alkenyl derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from olefin oligomerization, such as squalane or squalene oil, in particular isobutene or deuterene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / deuterate), glycerol tri-(caprylate / deuterate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearates. The oil is used in combination with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, esters of mannide (e.g. anhydrous mannitol oleate), esters of fatty glycols, esters of polyglycerols, esters of propylene glycol, and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which may be ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic products, in particular L 121. See Hunter et al., The theory and practical application of adjuvants (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and Todd et al., Vaccine (1997, 15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of “Vaccine design, the Subunit and adiuvant approach” edited by Powell M and Newman M (Plenum Press, 1995) can be used.The term "polymer of acrylic acid or methacrylic acid" preferably refers to a cross-linked polymer of acrylic acid or methacrylic acid, in particular cross-linked with a polyalkenyl ether of sugar or a polyol, compounds known as carbomers (trade name Carbopol) (Phameuropa, 1996, 8 (2)). A person skilled in the art may also refer to US Pat. No. 2,909,462, which describes such acrylic acid polymers cross-linked 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 replaced by unsaturated 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. The unsaturated groups themselves may contain other substituents, such as methyl. These products are sold under the name Carbopol (BF Goodrich, Ohio, USA) and are particularly suitable. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among these, Carbopol 974P, 934P and 971P may be mentioned, with Carbopol 971P being most preferred. The term "copolymers of maleic anhydride and alkenyl derivatives" also contemplates copolymers of maleic anhydride and ethylene, such as EMA (Monsanto), which dissolve in water to produce an acidic solution that is neutralized, preferably to physiological pH, to produce an adjuvant solution into which the immunogenic, immunogenic or vaccine 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, Avridine lipid-amine adjuvant, Escherichia 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, aluminum gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, 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 ISA 206 or Gel adjuvant.

[0036] The term "lyoprotectant" refers to an ingredient, other than excipients, that protects the efficacy of the active ingredient during the freeze-drying process and storage after freeze-drying.

[0037] The term "dosage form" refers to the physical form of a drug preparation. It also refers to a finished drug (finished drug) formulated based on the drug's properties, the disease it is intended to treat, and the prescription it requires. The appropriate dosage form is designed to maximize the drug's efficacy, minimize toxic side effects, and facilitate use, storage, and transportation.

[0038] The term "injection" refers to sterile solutions (including emulsions and suspensions) made of drugs for injection into the body, as well as sterile powders or concentrated solutions for preparation into solutions or suspensions before use. It can be an injection of water (the solvent is water) or an injection of oil (the solvent is oil); there are also injections using other solvents, such as ethanol (the solvent of hydrocortisone injection is ethanol), glycerol, propylene glycol (PEG), etc.

[0039] The term "powder for injection" refers to the process of freezing the drug solution under a sterile environment, "mixing" the raw drug with certain excipients or dissolving it in certain solvents, and then processing it into preparations in different forms.

[0040] The term "prevention" when referring to African swine fever virus infection means inhibiting the replication of African swine fever virus, inhibiting the spread of African swine fever virus or preventing African swine fever virus from taking root in its host, and alleviating the symptoms of diseases or conditions caused by African swine fever virus infection. Detailed Description of the Invention

[0042] The present invention provides an African swine fever virus subunit vaccine composition, wherein the protein antigens of the African swine fever virus subunit vaccine composition are composed of immune amounts of the following components: African swine fever virus p72 protein, p30 protein, p54 protein, African swine fever virus nucleocapsid assembly protein, African swine fever virus replication protein, African swine fever virus adsorption protein, and African swine fever virus virulence protein; wherein the African swine fever virus nucleocapsid assembly protein is at least one of p34, p62, and p17 proteins, the African swine fever virus replication protein is at least one of E165R, S273R, A104R, and C129R proteins, the African swine fever virus adsorption protein is at least one of p12 and p22 proteins, and the African swine fever virus virulence protein is at least one of DP96R and DP71L proteins; and the African swine fever virus subunit vaccine composition also includes a pharmaceutically acceptable carrier.

[0043] As an embodiment of the present invention, in the African swine fever virus subunit vaccine composition of the present invention, the African swine fever virus subunit vaccine composition contains African swine fever virus p72 protein, p30 protein, and p54 protein; the African swine fever virus nucleocapsid assembly protein is p34, p17 protein or p62, p17 protein; the African swine fever virus replication protein is E165R, A104R, C129R protein or E165R, S273R, A104R, C129R protein or E165R, A104R protein; the African swine fever virus adsorption protein is p22 protein or p12 protein; the African swine fever virus virulence protein is DP96R protein or DP71L protein. As one embodiment of the present invention, in the African swine fever virus subunit vaccine composition of the present invention, the protein antigens of the African swine fever virus subunit vaccine composition are composed of immune amounts of the following components: the African swine fever virus p72, p30 and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, the African swine fever virus replication proteins E165R, A104R and C129R proteins, the African swine fever virus adsorption protein p12 protein, and the African swine fever virus virulence protein DP71L protein. As one embodiment of the present invention, in the African swine fever virus subunit vaccine composition of the present invention, the protein antigens of the African swine fever virus subunit vaccine composition are composed of immune amounts of the following components: the African swine fever virus p72, p30 and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p62 and p17 proteins, the African swine fever virus replication proteins E165R, S273R, A104R and C129R proteins, the African swine fever virus adsorption protein p22 protein, and the African swine fever virus virulence protein DP71L protein. As one embodiment of the present invention, in the African swine fever virus subunit vaccine composition of the present invention, the protein antigens of the African swine fever virus subunit vaccine composition are composed of an immunizing amount of the following components: the African swine fever virus p72, p30 and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, the African swine fever virus replication proteins E165R and A104R, the African swine fever virus adsorption protein p12 protein, and the African swine fever virus virulence protein DP96R protein. As one embodiment of the present invention, in the African swine fever virus subunit vaccine composition of the present invention, the protein antigens of the African swine fever virus subunit vaccine composition are composed of an immunizing amount of the following components: the African swine fever virus p72, p30, and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, the African swine fever virus replication proteins E165R, A104R and C129R proteins, the African swine fever virus adsorption protein p22 protein, and the African swine fever virus virulence protein DP96R protein.

[0044] As one embodiment of the present invention, in the African swine fever virus subunit vaccine composition of the present invention, the African swine fever virus p72 protein is encoded by SEQ ID NO.1 or its degenerate sequence; the African swine fever virus p30 protein is encoded by SEQ ID NO.2 or its degenerate sequence; the African swine fever virus p54 protein is encoded by SEQ ID NO.3 or its degenerate sequence; the African swine fever virus p34 protein is encoded by SEQ ID NO.4 or its degenerate sequence; the African swine fever virus p62 protein is encoded by SEQ ID NO.5 or its degenerate sequence; the African swine fever virus p17 protein is encoded by SEQ ID NO.6 or its degenerate sequence; the African swine fever virus E165R protein is encoded by SEQ ID NO.7 or its degenerate sequence; the African swine fever virus S273R protein is encoded by SEQ ID NO.8 or its degenerate sequence; the African swine fever virus A104R protein is encoded by SEQ ID NO.9 or its degenerate sequence; the African swine fever virus C129R protein is encoded by SEQ ID NO.10 or its degenerate sequence; the African swine fever virus p12 protein is encoded by SEQ ID NO.11 or its degenerate sequence; the African swine fever virus p22 protein is encoded by SEQ ID NO.12 or its degenerate sequence; the African swine fever virus DP96R protein is encoded by SEQ ID NO.13 or its degenerate sequence; and the African swine fever virus DP71L protein is encoded by SEQ ID NO.14 or its degenerate sequence.

[0045] According to a preferred embodiment, those skilled in the art can select other degenerate sequences encoding the African swine fever virus p72, p30, p54, p34, p62, p17, E165R, S273R, A104R, C129R, p12, p22, DP96R, and DP71L proteins based on conventional technical knowledge. Other degenerate sequences can be selected based on the preferred codons of the specific expression system.

[0046] According to a preferred embodiment, the African swine fever virus p30, p54, p34, p62, E165R, S273R, A104R, C129R, p12, DP96R, and DP71L proteins are expressed by an Escherichia coli expression system, and their coding sequences are selected according to the preferred codons of the Escherichia coli expression system.

[0047] According to a preferred embodiment, the African swine fever virus p72 protein is expressed by the sf9 insect cell expression system, and its coding sequence is selected according to the preferred codons of the sf9 insect cell expression system.

[0048] According to a preferred embodiment, the African swine fever virus p17 and p22 proteins are expressed by a CHO cell expression system, and their coding sequences are selected according to the preferred codons of the CHO cell expression system.

[0049] According to a preferred embodiment, in the African swine fever virus subunit vaccine composition of the present invention, the African swine fever virus p72 protein is encoded by SEQ ID NO.1; the African swine fever virus p30 protein is encoded by SEQ ID NO.2; the African swine fever virus p54 protein is encoded by SEQ ID NO.3; the African swine fever virus p34 protein is encoded by SEQ ID NO.4; the African swine fever virus p62 protein is encoded by SEQ ID NO.5; the African swine fever virus p17 protein is encoded by SEQ ID NO.6; the African swine fever virus E165R protein is encoded by SEQ ID NO.7; the African swine fever virus S273R protein is encoded by SEQ ID NO.8; the African swine fever virus A104R protein is encoded by SEQ ID NO.9; the African swine fever virus C129R protein is encoded by SEQ ID NO.10; the African swine fever virus p12 protein is encoded by SEQ ID NO.11; the African swine fever virus p22 protein is encoded by SEQ ID NO.12; the African swine fever virus DP96R protein is encoded by SEQ ID NO. NO.13 encoding; and the African swine fever virus DP71L protein is encoded by SEQ ID NO.14.

[0050] As an embodiment of the present invention, in the African swine fever virus subunit vaccine composition of the present invention, the content of the African swine fever virus p72 protein is ≥25 μg / ml; the content of the African swine fever virus p30 protein is ≥25 μg / ml; the content of the African swine fever virus p54 protein is ≥25 μg / ml; the content of the African swine fever virus p34 protein is ≥25 μg / ml; the content of the African swine fever virus p62 protein is ≥25 μg / ml; the content of the African swine fever virus p17 protein is ≥25 μg / ml; the content of the African swine fever virus E165R protein is ≥25 μg / ml; The white content of the African swine fever virus is ≥25μg / ml; the African swine fever virus S273R protein content is ≥25μg / ml; the African swine fever virus A104R protein content is ≥25μg / ml; the African swine fever virus C129R protein content is ≥25μg / ml; the African swine fever virus p12 protein content is ≥25μg / ml; the African swine fever virus p22 protein content is ≥25μg / ml; the African swine fever virus DP96R protein content is ≥25μg / ml; the African swine fever virus DP71L protein content is ≥25μg / ml.

[0051] According to a preferred embodiment, the African swine fever virus p72 protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml l, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml , 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The African swine fever virus p30 protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The African swine fever virus p54 protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml.The African swine fever virus p34 protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The African swine fever virus p62 protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The African swine fever virus p17 protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml.The content of the African swine fever virus E165R protein is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The content of the African swine fever virus S273R protein is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The content of the African swine fever virus A104R protein is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml.The content of the African swine fever virus C129R protein is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The content of the African swine fever virus p12 protein is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The African swine fever virus p22 protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml.The African swine fever virus DP96R protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml. The African swine fever virus DP71L protein content is selected from 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 45μg / ml, 50μg / ml, 55μg / ml, 60μg / ml, 65μg / ml, 70μg / ml, 75μg / ml, 80μg / ml, 85μg / ml, 90μg / ml, 95μg / ml, 100μg / ml, 105μg / ml, 110μg / ml, 115μg / ml, 120μg / ml, 125μg / ml, 130μg / ml, 135μg / ml, 140μg / ml, 145μg / ml, 150μg / ml, 155μg / ml, 160μg / ml, 165μg / ml, 170μg / ml or 175μg / ml.

[0052] According to a preferred embodiment, the African swine fever virus p72 protein, p30 protein, p54 protein, p34 protein, p62 protein, p17 protein, E165R protein, S273R protein, A104R protein, C129R protein, p12 protein, p22 protein, DP96R protein, and DP71L protein are all selected to have lower content. Even if the total amount of African swine fever virus protein antigen is lower than that of a single component antigen, a higher antibody titer can be obtained, thereby ensuring a better immune effect.

[0053] According to a preferred embodiment, in the African swine fever virus subunit vaccine composition of the present invention, the content of the African swine fever virus p72 protein is 25-175 μg / ml; the content of the African swine fever virus p30 protein is 25-175 μg / ml; the content of the African swine fever virus p54 protein is 25-175 μg / ml; the content of the African swine fever virus p34 protein is 25-175 μg / ml; the content of the African swine fever virus p62 protein is 25-175 μg / ml; the content of the African swine fever virus p17 protein is 25-175 μg / ml; the content of the African swine fever virus E165R protein is 25-175 μg / ml; 25~175μg / ml; the content of the African swine fever virus S273R protein is 25~175μg / ml; the content of the African swine fever virus A104R protein is 25~175μg / ml; the content of the African swine fever virus C129R protein is 25~175μg / ml; the content of the African swine fever virus p12 protein is 25~175μg / ml; the content of the African swine fever virus p22 protein is 25~175μg / ml; the content of the African swine fever virus DP96R protein is 25~175μg / ml; the content of the African swine fever virus DP71L protein is 25~175μg / ml.

[0054] According to a preferred embodiment, in the African swine fever virus subunit vaccine composition of the present invention, the content of the African swine fever virus p72 protein is 75-125 μg / ml; the content of the African swine fever virus p30 protein is 75-125 μg / ml; the content of the African swine fever virus p54 protein is 75-125 μg / ml; the content of the African swine fever virus p34 protein is 75-125 μg / ml; the content of the African swine fever virus p62 protein is 75-125 μg / ml; the content of the African swine fever virus p17 protein is 75-125 μg / ml; the content of the African swine fever virus E165R protein is 75-125 μg / ml; The content of the African swine fever virus S273R protein is 75-125 μg / ml; the content of the African swine fever virus A104R protein is 75-125 μg / ml; the content of the African swine fever virus C129R protein is 75-125 μg / ml; the content of the African swine fever virus p12 protein is 75-125 μg / ml; the content of the African swine fever virus p22 protein is 75-125 μg / ml; the content of the African swine fever virus DP96R protein is 75-125 μg / ml; the content of the African swine fever virus DP71L protein is 75-125 μg / ml.

[0055] According to a preferred embodiment, the African swine fever virus subunit vaccine composition is composed of the following components: 25 μg / ml of the African swine fever virus p72 protein, 25 μg / ml of the African swine fever virus p30 protein, 25 μg / ml of the African swine fever virus p54 protein, 25 μg / ml of the African swine fever virus p34 protein, 25 μg / ml of the African swine fever virus p17 protein, 25 μg / ml of the African swine fever virus E165R protein, 25 μg / ml of the African swine fever virus A104R protein, 25 μg / ml of the African swine fever virus C129R protein, 25 μg / ml of the African swine fever virus p12 protein, and 25 μg / ml of the African swine fever virus DP71L protein.

[0056] According to a preferred embodiment, the African swine fever virus subunit vaccine composition is composed of the following components: 75 μg / ml of the African swine fever virus p72 protein, 75 μg / ml of the African swine fever virus p30 protein, 75 μg / ml of the African swine fever virus p54 protein, 75 μg / ml of the African swine fever virus p62 protein, 75 μg / ml of the African swine fever virus p17 protein, 75 μg / ml of the African swine fever virus E165R protein, 75 μg / ml of the African swine fever virus S273R protein, 75 μg / ml of the African swine fever virus A104R protein, 75 μg / ml of the African swine fever virus C129R protein, 75 μg / ml of the African swine fever virus p22 protein, and 75 μg / ml of the African swine fever virus DP71L protein.

[0057] According to a preferred embodiment, the African swine fever virus subunit vaccine composition is composed of the following components: 125 μg / ml of the African swine fever virus p72 protein, 125 μg / ml of the African swine fever virus p30 protein, 125 μg / ml of the African swine fever virus p54 protein, 125 μg / ml of the African swine fever virus p34 protein, 125 μg / ml of the African swine fever virus p17 protein, 125 μg / ml of the African swine fever virus E165R protein, 125 μg / ml of the African swine fever virus A104R protein, 125 μg / ml of the African swine fever virus p12 protein, and 125 μg / ml of the African swine fever virus DP96R protein.

[0058] According to a preferred embodiment, the African swine fever virus subunit vaccine composition is composed of the following components: 125 μg / ml of the African swine fever virus p72 protein, 125 μg / ml of the African swine fever virus p30 protein, 125 μg / ml of the African swine fever virus p54 protein, 125 μg / ml of the African swine fever virus p34 protein, 125 μg / ml of the African swine fever virus p17 protein, 125 μg / ml of the African swine fever virus E165R protein, 125 μg / ml of the African swine fever virus A104R protein, 125 μg / ml of the African swine fever virus C129R protein, 125 μg / ml of the African swine fever virus p22 protein, and 125 μg / ml of the African swine fever virus DP96R protein.

[0059] According to a preferred embodiment, the African swine fever virus subunit vaccine composition is composed of the following components: 175 μg / ml of the African swine fever virus p72 protein, 175 μg / ml of the African swine fever virus p30 protein, 175 μg / ml of the African swine fever virus p54 protein, 175 μg / ml of the African swine fever virus p34 protein, 175 μg / ml of the African swine fever virus p17 protein, 175 μg / ml of the African swine fever virus E165R protein, 175 μg / ml of the African swine fever virus A104R protein, 175 μg / ml of the African swine fever virus C129R protein, 175 μg / ml of the African swine fever virus p22 protein, and 175 μg / ml of the African swine fever virus DP96R protein.

[0060] According to a preferred embodiment, in the African swine fever virus subunit vaccine composition of the present invention, the total content of African swine fever virus protein antigen is 250-1750 μg / ml.

[0061] The total content of African swine fever virus protein can be selected from 250μg / ml, 300μg / ml, 350μg / ml, 400μg / ml, 450μg / ml, 500μg / ml, 550μg / ml, 600μg / ml, 650μg / ml, 700μg / ml, 750μg / ml, 800μg / ml, 850μg / ml, 900μg / ml, 950μg / ml, 1000μg / ml, g / ml, 1050μg / ml, 1100μg / ml, 1150μg / ml, 1200μg / ml, 1250μg / ml, 1300μg / ml, 1350μg / ml, 14 00μg / ml, 1450μg / ml, 1500μg / ml, 1550μg / ml, 1600μg / ml, 1650μg / ml, 1700μg / ml, 1750μg / ml.

[0062] According to a preferred embodiment, the pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes: (1) mineral oil, aluminum gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; and one or more of the following: 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 ISA 206, 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.

[0063] According to a preferred embodiment, the adjuvant is Montanide ISA 206 adjuvant, and the adjuvant content is 50% V / V.

[0064] According to a preferred embodiment, the pharmaceutically acceptable carrier comprises a lyoprotectant, and the lyoprotectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

[0065] According to a preferred embodiment, the pharmaceutically acceptable carrier includes drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants and preservatives; the immunostimulants include α-interferon, β-interferon, γ-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and interleukin 2 (IL2).

[0066] To prepare such a composition, methods known in the art can be used.

[0067] According to a preferred embodiment, the dosage form of the African swine fever virus subunit vaccine composition is selected from a solution injection, a suspension injection, a powder for injection, a sustained-release microsphere preparation, a controlled-release microsphere preparation, or a sustained-release and controlled-release implant.

[0068] According to a preferred embodiment, the dosage form of the African swine fever virus subunit vaccine composition is a solution injection, a suspension injection, or a powder for injection.

[0069] According to a preferred embodiment, the administration method of the African swine fever virus subunit vaccine composition is selected from subcutaneous injection, oral administration, buccal administration, sublingual administration, nasal administration, pulmonary administration, colon administration, rectal administration, or transdermal administration.

[0070] According to a more preferred embodiment, the African swine fever virus subunit vaccine composition is administered by subcutaneous injection.

[0071] According to a preferred embodiment, the dosage form of the African swine fever virus subunit vaccine composition is a solution injection or a suspension injection.

[0072] The present invention provides an application of the African swine fever virus subunit vaccine composition, wherein the application refers to an application in the preparation of a drug for preventing African swine fever virus infection.

[0073] According to a preferred embodiment, the African swine fever virus subunit vaccine composition can exert a synergistic immune protection effect and have a better immune effect on pigs.

[0074] The present invention provides a combination of African swine fever virus protein antigens, wherein the African swine fever virus protein antigens are composed of immune amounts of the following components: African swine fever virus p72 protein, p30 protein, p54 protein, African swine fever virus nucleocapsid assembly protein, African swine fever virus replication protein, African swine fever virus adsorption protein, and African swine fever virus virulence protein; wherein the African swine fever virus nucleocapsid assembly protein is p34, p17 protein or p62, p17 protein; the African swine fever virus replication protein is E165R, A104R, C129R protein or E165R, S273R, A104R, C129R protein or E165R, A104R protein; the African swine fever virus adsorption protein is p22 protein or p12 protein; the African swine fever virus virulence protein is DP96R protein or DP71L protein; wherein the African swine fever virus p72 protein is SEQ ID NO.1 or its degenerate sequence; the African swine fever virus p30 protein is encoded by SEQ ID NO.2 or its degenerate sequence; the African swine fever virus p54 protein is encoded by SEQ ID NO.3 or its degenerate sequence; the African swine fever virus p34 protein is encoded by SEQ ID NO.4 or its degenerate sequence; the African swine fever virus p62 protein is encoded by SEQ ID NO.5 or its degenerate sequence; the African swine fever virus p17 protein is encoded by SEQ ID NO.6 or its degenerate sequence; the African swine fever virus E165R protein is encoded by SEQ ID NO.7 or its degenerate sequence; the African swine fever virus S273R protein is encoded by SEQ ID NO.8 or its degenerate sequence; the African swine fever virus A104R protein is encoded by SEQ ID NO.9 or its degenerate sequence; the African swine fever virus C129R protein is encoded by SEQ ID NO.10 or its degenerate sequence; the African swine fever virus p12 protein is encoded by SEQ ID NO.11 or its degenerate sequence; the African swine fever virus p22 protein is encoded by SEQ ID NO. NO.12 or its degenerate sequence encoding; the African swine fever virus DP96R protein is encoded by SEQ ID NO.13 or its degenerate sequence encoding; and the African swine fever virus DP71L protein is encoded by SEQ ID NO.14 or its degenerate sequence encoding.

[0075] According to a preferred embodiment, those skilled in the art can administer the various components of the combination of African swine fever virus protein antigens simultaneously or intermittently.

[0076] As one embodiment of the present invention, in the combination of African swine fever virus protein antigens described in the present invention, the combination of African swine fever virus protein antigens is composed of immune amounts of the following components: the African swine fever virus p72, p30 and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, the African swine fever virus replication proteins E165R, A104R and C129R proteins, the African swine fever virus adsorption protein p12 protein, and the African swine fever virus virulence protein DP71L protein.

[0077] As one embodiment of the present invention, in the combination of African swine fever virus protein antigens described in the present invention, the combination of African swine fever virus protein antigens is composed of immune amounts of the following components: the African swine fever virus p72, p30 and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p62 and p17 proteins, the African swine fever virus replication proteins E165R, S273R, A104R and C129R proteins, the African swine fever virus adsorption protein p22 protein, and the African swine fever virus virulence protein DP71L protein.

[0078] As one embodiment of the present invention, in the combination of African swine fever virus protein antigens described in the present invention, the combination of African swine fever virus protein antigens is composed of immune amounts of the following components: the African swine fever virus p72, p30 and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, the African swine fever virus replication proteins E165R and A104R, the African swine fever virus adsorption protein p12 protein, and the African swine fever virus virulence protein DP96R protein.

[0079] As one embodiment of the present invention, in the combination of African swine fever virus protein antigens described in the present invention, the combination of African swine fever virus protein antigens is composed of immune amounts of the following components: the African swine fever virus p72, p30 and p54 proteins, the African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, the African swine fever virus replication proteins E165R, A104R and C129R proteins, the African swine fever virus adsorption protein p22 protein, and the African swine fever virus virulence protein DP96R protein.

[0080] According to a preferred embodiment, those skilled in the art can select other degenerate sequences encoding the African swine fever virus p72, p30, p54, p34, p62, p17, E165R, S273R, A104R, C129R, p12, p22, DP96R, and DP71L proteins based on conventional technical knowledge. Other degenerate sequences can be selected based on the preferred codons of the specific expression system.

[0081] According to a preferred embodiment, the African swine fever virus p30, p54, p34, p62, E165R, S273R, A104R, C129R, p12, DP96R, and DP71L proteins are expressed by an Escherichia coli expression system, and their coding sequences are selected according to the preferred codons of the Escherichia coli expression system.

[0082] According to a preferred embodiment, the African swine fever virus p72 protein is expressed by the sf9 insect cell expression system, and its coding sequence is selected according to the preferred codons of the sf9 insect cell expression system.

[0083] According to a preferred embodiment, the African swine fever virus p17 and p22 proteins are expressed by a CHO cell expression system, and their coding sequences are selected according to the preferred codons of the CHO cell expression system.

[0084] According to a preferred embodiment, in the combination of African swine fever virus protein antigens of the present invention, the African swine fever virus p72 protein is encoded by SEQ ID NO.1; the African swine fever virus p30 protein is encoded by SEQ ID NO.2; the African swine fever virus p54 protein is encoded by SEQ ID NO.3; the African swine fever virus p34 protein is encoded by SEQ ID NO.4; the African swine fever virus p62 protein is encoded by SEQ ID NO.5; the African swine fever virus p17 protein is encoded by SEQ ID NO.6; the African swine fever virus E165R protein is encoded by SEQ ID NO.7; the African swine fever virus S273R protein is encoded by SEQ ID NO.8; the African swine fever virus A104R protein is encoded by SEQ ID NO.9; the African swine fever virus C129R protein is encoded by SEQ ID NO.10; the African swine fever virus p12 protein is encoded by SEQ ID NO.11; the African swine fever virus p22 protein is encoded by SEQ ID NO.12; the African swine fever virus DP96R protein is encoded by SEQ ID NO. NO.13 encoding; and the African swine fever virus DP71L protein is encoded by SEQ ID NO.14.

[0085] As an embodiment of the present invention, the combination of African swine fever virus protein antigens further comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier comprises an adjuvant, and the adjuvant comprises: (1) mineral oil, aluminum gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; 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; or the pharmaceutically acceptable carrier includes a lyoprotectant, and the lyoprotectant is selected from a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

[0086] As an embodiment of the present invention, in the combination of African swine fever virus protein antigens, the dosage form of the p72, p30, p54, p34, p62, p17, E165R, S273R, A104R, C129R, p12, p22, DP96R or DP71L protein is a solution injection, a suspension injection, or a powder for injection.

[0087] According to a preferred embodiment, the combined administration of the African swine fever virus protein antigen is selected from subcutaneous injection, oral administration, buccal administration, sublingual administration, nasal administration, pulmonary administration, colon administration, rectal administration, or transdermal administration.

[0088] According to a more preferred embodiment, the combination of African swine fever virus protein antigens is administered by subcutaneous injection.

[0089] As an embodiment of the present invention, in the combination of African swine fever virus protein antigens, the dosage form of the p72, p30, p54, p34, p62, p17, E165R, S273R, A104R, C129R, p12, p22, DP96R or DP71L protein is a solution injection or a suspension injection; the content of the African swine fever virus p72 protein is ≥25 μg / ml; the content of the African swine fever virus p30 protein is ≥25 μg / ml; the content of the African swine fever virus p54 protein is ≥25 μg / ml; the content of the African swine fever virus p34 protein is ≥25 μg / ml; the content of the African swine fever virus p62 protein is ≥25 μg / ml. / ml; the content of the African swine fever virus p17 protein is ≥25μg / ml; the content of the African swine fever virus E165R protein is ≥25μg / ml; the content of the African swine fever virus S273R protein is ≥25μg / ml; the content of the African swine fever virus A104R protein is ≥25μg / ml; the content of the African swine fever virus C129R protein is ≥25μg / ml; the content of the African swine fever virus p12 protein is ≥25μg / ml; the content of the African swine fever virus p22 protein is ≥25μg / ml; the content of the African swine fever virus DP96R protein is ≥25μg / ml; and the content of the African swine fever virus DP71L protein is ≥25μg / ml.

[0090] According to a preferred embodiment, the content of the African swine fever virus p72 protein is 25-175 μg / ml; the content of the African swine fever virus p30 protein is 25-175 μg / ml; the content of the African swine fever virus p54 protein is 25-175 μg / ml; the content of the African swine fever virus p34 protein is 25-175 μg / ml; the content of the African swine fever virus p62 protein is 25-175 μg / ml; the content of the African swine fever virus p17 protein is 25-175 μg / ml; the content of the African swine fever virus E165R protein is 25-175 μg / ml ; The content of the African swine fever virus S273R protein is 25~175μg / ml; the content of the African swine fever virus A104R protein is 25~175μg / ml; the content of the African swine fever virus C129R protein is 25~175μg / ml; the content of the African swine fever virus p12 protein is 25~175μg / ml; the content of the African swine fever virus p22 protein is 25~175μg / ml; the content of the African swine fever virus DP96R protein is 25~175μg / ml; the content of the African swine fever virus DP71L protein is 25~175μg / ml.

[0091] According to a preferred embodiment, the content of the African swine fever virus p72 protein is 75-125 μg / ml; the content of the African swine fever virus p30 protein is 75-125 μg / ml; the content of the African swine fever virus p54 protein is 75-125 μg / ml; the content of the African swine fever virus p34 protein is 75-125 μg / ml; the content of the African swine fever virus p62 protein is 75-125 μg / ml; the content of the African swine fever virus p17 protein is 75-125 μg / ml; the content of the African swine fever virus E165R protein is 75-125 μg / ml. ; The content of the African swine fever virus S273R protein is 75-125 μg / ml; the content of the African swine fever virus A104R protein is 75-125 μg / ml; the content of the African swine fever virus C129R protein is 75-125 μg / ml; the content of the African swine fever virus p12 protein is 75-125 μg / ml; the content of the African swine fever virus p22 protein is 75-125 μg / ml; the content of the African swine fever virus DP96R protein is 75-125 μg / ml; the content of the African swine fever virus DP71L protein is 75-125 μg / ml.

[0092] The present invention provides an application of the combination of African swine fever virus protein antigens, wherein the application refers to an application in the preparation of a drug for preventing African swine fever virus infection.

[0093] According to a preferred embodiment, the combination of African swine fever virus protein antigens can exert a synergistic immune protection effect and have a better immune effect on pigs.

[0094] The present invention also provides a method for preparing an African swine fever virus subunit vaccine composition, the method comprising:

[0095] Step (1) synthesizing the African swine fever virus protein gene, cloning and recombining it into a cloning vector;

[0096] Step (2) amplifying the African swine fever virus protein gene on the cloning vector of step (1) and the expression vector, cutting them with enzymes, and recombining them to obtain an expression vector containing the African swine fever virus protein gene;

[0097] (3) introducing the expression vector containing the African swine fever virus protein gene into a host, expressing the African swine fever virus protein, and identifying the expressed African swine fever virus protein to obtain the African swine fever virus protein;

[0098] (4) The obtained African swine fever virus protein is mixed with a pharmaceutically acceptable carrier to obtain an African swine fever virus subunit vaccine composition.

[0099] As one embodiment of the present invention, the African swine fever virus protein described in the present invention can be prepared through a prokaryotic expression system, or through a eukaryotic expression system, a cell expression system or a chemical synthesis method.

[0100] According to a preferred embodiment, the expression vector of the African swine fever virus p30, p54, p34, p62, E165R, S273R, A104R, C129R, p12, DP96R, and DP71L protein genes is an Escherichia coli expression system cloning vector.

[0101] According to a preferred embodiment, the expression vector of the African swine fever virus p72 protein gene is an sf9 insect cell expression system expression vector.

[0102] According to a preferred embodiment, the expression vector of the African swine fever virus p17 and p22 protein genes is a CHO cell expression system expression vector.

[0103] The present invention also provides an expression vector containing the gene sequence of the African swine fever virus protein.

[0104] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0105] All chemical reagents used in the examples of the present invention were of analytical grade and purchased from Sinopharm Group. The experimental methods described in the present invention, unless otherwise specified, are conventional methods; the biological materials described, unless otherwise specified, can be obtained from commercial channels.

[0106] Example 1 Prokaryotic expression of African swine fever virus p30, p54, p34, p62, E165R, S273R, A104R, C129R, p12, DP96R, and DP71L proteins

[0107] The nucleotide sequence of the expression vector shown in SEQ ID NO. 15 in the corresponding coding sequence table was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and named as pNVC1 vector.

[0108] The nucleotide sequences shown in SEQ ID NO.2 to SEQ ID NO.5 in the sequence list, synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., correspondingly encode p30, p54, p34, and p62 proteins; the nucleotide sequences shown in SEQ ID NO.7 to SEQ ID NO.11, correspondingly encode E165R, S273R, A104R, C129R, and p12 proteins; the nucleotide sequence shown in SEQ ID NO.13, correspondingly encodes DP96R protein; and the nucleotide sequence shown in SEQ ID NO.14, correspondingly encodes DP96R protein. The nucleotide sequence shown in NO.14 encodes the DP71L protein, and the synthesized nucleotides were cloned into the pNVC1 vector and named as recombinant plasmids pNVC1-p30, pNVC1-p54, pNVC1-p34, pNVC1-p62, pNVC1-E165R, pNVC1-S273R, pNVC1-A104R, pNVC1-C129R, pNVC1-p12, pNVC1-DP96R, and pNVC1-DP71L.

[0109] The above-mentioned recombinant plasmids pNVC1-p30, pNVC1-p54, pNVC1-p34, pNVC1-p62, pNVC1-E165R, pNVC1-S273R, pNVC1-A104R, pNVC1-C129R, pNVC1-p12, pNVC1-DP96R and pNVC1-DP71L were transformed into competent Escherichia coli BL21 (DE3) to construct expression strains. 50 ml of kanamycin-resistant LB liquid medium was inoculated and cultured with shaking at 37°C and 230 rpm for 12 hours. After that, the strains were transferred into 1 L of LB liquid medium and cultured at 37°C to prepare seed liquid for fermentation.

[0110] The fermenter used was a 50L fermenter from Shanghai Baoxing Biotechnology Co., Ltd. 30L of culture medium was prepared and loaded into the fermenter, which was sterilized at 121°C for 30 minutes. The next day, 3L of seed solution was added to the fermenter. When the culture reached an OD600 of approximately 10, the temperature was lowered to 25°C, and IPTG was added to a final concentration of 0.5mM for induction for 12 hours. The culture was stopped when the fermentation density reached approximately 40 (OD600), and the cells were collected by centrifugation.

[0111] Resuspend the cells and disrupt them three times using a homogenizer at 800 bar. Centrifuge at 13,500 rpm for 40 minutes, and collect the supernatant for analysis by 12% SDS-PAGE. Protein was crudely purified using ammonium sulfate fractional precipitation, followed by chromatographic purification. SDS-PAGE electrophoresis of the purified protein confirmed the purification and enrichment of the target protein.

[0112] Example 2 Eukaryotic expression of African swine fever virus p72 protein

[0113] The nucleotide sequence shown in SEQ ID NO.1 in the sequence list was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., which correspondingly encodes the p72 protein. The synthesized nucleotide was cloned into the pFB vector and named as the recombinant plasmid pFB-p72.

[0114] The recombinant plasmid pFB-p72 was transformed into DH10Bac competent cells. After blue-white screening, single white colonies were picked and incubated overnight in liquid LB medium containing kanamycin, tetracycline, and gentamicin resistance. The recombinant bacmid was extracted according to the Bac-to-Bac operating instructions and named Bac-p72.

[0115] Refer to the instructions for the Cellfectin II Reagent Transfection Kit and transfect sf9 insect cells with the recombinant bacmid Bac-p72. Culture in a 27°C incubator for approximately 72 hours. When cytopathic effects are evident, harvest the cell supernatant. This is the P1 recombinant baculovirus and label it as rBac-p72. Add the P1 recombinant baculovirus to the sf9 cell shaker flask at a volume ratio of 1:20-1:40. Continue incubating at 27°C until cytopathic effects are evident after approximately 72 hours. Harvest the supernatant and label it as the P2 recombinant baculovirus. Inoculate 1 L of sf9 cells with the P2 recombinant baculovirus at a volume ratio of 1:100. Harvest the cells 48-72 hours after inoculation and collect the infected cells by centrifugation at 1000 × g for 10 minutes.

[0116] The cell pellet was lysed with cell lysis buffer (25 mM NaHCO3, pH 8.3) for 30 minutes and centrifuged at 10,000 × g at 4°C for 10 minutes to obtain the supernatant. Expression of the target protein was confirmed by Western blotting. The protein was crudely purified by nickel affinity chromatography and then purified by molecular sieve purification. SDS-PAGE electrophoresis of the purified protein showed that the target protein was purified and enriched.

[0117] Example 3 Expression of African swine fever virus p17 and p22 proteins in CHO cells

[0118] The nucleotide sequences shown in SEQ ID NO.6 and SEQ ID NO.12 in the sequence listing were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., encoding p17 and p22 proteins respectively. The synthesized nucleotides were cloned into the pCHO1.0 vector and named as recombinant plasmids pCHO-p17 and pCHO-p22, respectively.

[0119] Plasmid pCHO-p17 was extracted according to the instructions of the endotoxin-free extraction kit and linearized with Nru I. The treated plasmid was co-transfected into CHO cells using Free Style MAX reagent. After 48 hours, the cell suspension was collected by centrifugation and resuspended in two 150 cm2 square flasks containing CHO complete medium containing a 1:100 dilution of an anti-clumping agent. The cells were then cultured in two CD Forti complete medium containing different concentrations of MTX and puromycin. Cell growth was observed. When cells began to show signs of recovery, they were transferred to 125 ml shake flasks and supplemented with appropriate amounts of puromycin and MTX to maintain selection pressure. The two cell pools obtained from the screening were designated A and B. Cell pools A and B were then cultured in two CD Forti complete medium containing different concentrations of MTX and puromycin, respectively, for a second round of pressure screening. After new resistant cell clones grew, four cell pools were obtained, named A1, A2, B1, and B2, respectively. They were cultured using the fed-batch method for 10 days. The expression levels of different cell pools were compared by dot blot hybridization, and the cell pool with the highest yield was selected for limiting dilution cloning.

[0120] Take the two cell pools with higher yields obtained from the two-stage screening and carry out serial dilution inoculation into 96-well plates (0.5 cells / well). Each cell pool is plated with 20 96-well plates, and a total of 40 96-well plates are plated. After 12 days of inoculation, when the cells in the 96-well plate form monoclonal colonies, samples are taken from 524 wells that form single colonies, and protein expression is identified by dot hybridization, and 100 clones are selected. The 100 clones are expanded from 96-well plates to 6-well plates, and the expression yield of each 6-well plate cell is identified by dot hybridization. The 20 clones with the highest efficiency are selected for fed-batch culture. Samples on the 12th day of culture are collected to identify the expression levels of different clones. The harvested samples are identified by SDS-PAGE for expression yield, and finally 5 high-yield clones are selected for the second round of limiting dilution.

[0121] The 5 clones obtained in the first round of limiting dilution screening were subjected to the second round of limiting dilution cloning isolation, and serial dilutions were inoculated into 96-well plates (0.5 cells / well). Each clone was plated with 10 96-well plates, for a total of 50 plates. 12 days after inoculation, when the cells in the 96-well plates formed monoclonal colonies, samples were taken from 584 wells that formed single colonies, and protein expression was identified by dot hybridization, and 100 clones were selected. The 100 clones were expanded from 96-well plates to 6-well plates, and the expression yield of each 6-well plate cell was identified by dot hybridization. The 20 clones with the highest efficiency were selected for fed-batch culture, the expression levels of different clones were identified, and the harvested samples were subjected to expression yield identification by SDS-PAGE, and finally 5 high-yield clones were screened out.

[0122] Five high-yielding clones, isolated and screened through two rounds of limiting dilution cloning, were revived and subcultured in 125-ml culture flasks for 30 generations. Based on daily measurements of viable cell counts and cell viability, growth curves were plotted for each clone over 30 generations, demonstrating stable proliferation characteristics for all five cell lines. Fed-batch cultures were performed at the 10th, 20th, and 30th generations, and samples harvested 10 days later were analyzed by SDS-PAGE and Western blotting to compare target protein production. A single clone with stable proliferation characteristics and the highest yield was identified from these five clones and designated rCHO-p17.

[0123] The recombinant plasmid pCHO-p22 was screened according to the above method to obtain a clone with stable proliferation characteristics and the highest yield, which was named rCHO-p22.

[0124] The constructed and screened rCHO-p17 and rCHO-p22 cell lines were inoculated into bioreactors containing Dynamis medium at a density of 3×10 5 Viable cells / mL. Parameters were set at pH 7.1-7.2, dissolved oxygen 40%, temperature 37°C, and agitation at 130 rpm. Samples were collected daily starting on day 3 to measure glucose and lactate concentrations and perform cell counts. When glucose levels fell below 2 g / L, glucose was fed to 6 g / L.

[0125] When the cell viability dropped to about 80%, the cell culture was harvested, and the supernatant obtained by centrifugation was subjected to Western Blot to confirm that the target proteins African swine fever virus p17 and p22 proteins were expressed.

[0126] Example 4 Preparation of African swine fever virus subunit vaccine composition

[0127] The p30, p54, p34, p62, E165R, S273R, A104R, C129R, p12, DP96R, DP71L proteins prepared in Example 1, the p72 protein prepared in Example 2, and the p17 and p22 proteins prepared in Example 3 were added to the adjuvant, and the addition process was continuously stirred for 12 minutes with an emulsifier at a speed of 800 rpm, mixed, and stored at 4 ° C., which is a subunit vaccine composition containing multi-component protein antigens of African swine fever virus. The adjuvant suitable for the present invention can be an adjuvant known to those skilled in the art. In the present invention, the adjuvant is selected as a biphasic adjuvant (water-in-oil-in-water emulsion), for example, it can be adjuvant ISA 206 (France Seppic Company). The specific ratio of each component in the prepared vaccine is shown in Table 1.

[0128] Table 1 Composition ratio of African swine fever virus subunit vaccine composition of the present invention

[0129] Components Vaccine 1 Vaccine 2 Vaccine 3 Vaccine 4 Vaccine 5 p72 (μg / ml) 25 75 125 125 175 p30 (μg / ml) 25 75 125 125 175 p54 (μg / ml) 25 75 125 125 175 p34 (μg / ml) 25 - 125 125 175 p62 (μg / ml) - 75 - - - p17 (μg / ml) 25 75 125 125 175 E165R (μg / ml) 25 75 125 125 175 S273R (μg / ml) - 75 - - - A104R (μg / ml) 25 75 125 125 175 C129R (μg / ml) 25 75 - 125 175 p12 (μg / ml) 25 - 125 - - p22 (μg / ml) - 75 - 125 175 DP96R (μg / ml) - - 125 125 175 DP71L (μg / ml) 25 75 - - - Biphasic adjuvant (V / V%) 50％ 50％ 50％ 50％ 50％

[0130] Example 5 Immunogenicity Test of African Swine Fever Virus Subunit Vaccine Composition

[0131] Thirty healthy, susceptible piglets weighing approximately 20 kg, negative for both African swine fever virus antigen and antibody, were randomly divided into six groups of five pigs each. Group 1 received vaccine 1, Group 2 received vaccine 2, Group 3 received vaccine 3, Group 4 received vaccine 4, and Group 5 received vaccine 5. Group 6 served as a blank control group. The immunized groups received 4 ml of vaccine via intramuscular injection into the neck, while the control group received an equivalent volume of PBS plus adjuvant. Two immunizations were administered, with a 14-day interval between each. Blood was collected before the first immunization and 14 days after the second.

[0132] The ELISA plate was coated with inactivated African swine fever virus antigen (purchased from the European Union African swine fever reference laboratory (URL-ASF)) at 4°C overnight; the coating solution was discarded, and the plate was washed with detergent; blocking solution (50 g of sucrose was weighed, 200 mL of newborn calf serum, 1 mL of Proclin 300, and PBS (0.01 mol / L, pH 7.4) was added to make the volume 1000 mL) and blocked at 2-8°C for 16-24 hours. The blocking solution was discarded, dried, sealed, and stored at 2-8°C for later use.

[0133] Sample diluent: Dissolve 8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 0.2 g of potassium chloride, 600 mL of purified water, 1 mL of Proclin 300, 200 mL of newborn calf serum, and 0.028 g of PUR dye completely. Dilute to 1000 mL with purified water, mix well, filter through 0.22 μm, and aliquot aseptically. Store at 2-8°C.

[0134] Washing solution: Dissolve 160g of sodium chloride, 58g of disodium hydrogen phosphate, 4.8g of potassium dihydrogen phosphate, 4g of potassium chloride, 800mL of ultrapure water, and 10mL of Tween 20. Dilute completely and dilute to 1000mL with purified water. Filter through a 0.22μm filter membrane and aliquot aseptically. Dilute 20-fold with distilled water before use.

[0135] Secondary antibody: enzyme-linked goat anti-pig secondary antibody, diluted 1:2000 when used.

[0136] Color developing solution: Disodium hydrogen phosphate (14.7 g), citric acid (9.3 g), and carbamide peroxide (0.3 g) are dissolved in purified water, the volume is adjusted to 1000 mL, mixed, filtered, and aseptically packaged to obtain Color Developer A. Dissolve tetramethylbenzyl diamine (TMB) (0.2 g) and anhydrous ethanol (10 mL) in purified water, the volume is adjusted to 1000 mL, mixed, filtered, and aseptically packaged to obtain Color Developer B.

[0137] Stop solution: 2M H2SO4.

[0138] During the test, 90 μl of sample diluent was first added to the enzyme-labeled reaction well, followed by 10 μl of serum to be tested, negative and positive controls, and incubated at 37°C for 30 minutes. The plate was washed three times with washing solution, and 100 μl / well of goat anti-pig enzyme-labeled antibody was added to the reaction plate, incubated at 37°C for 30 minutes, washed three times with washing solution, and 50 μl / well of color developer A and color developer B were added. The reaction was developed at 37°C for 10 minutes, and 50 μl / well of stop solution was added to terminate the reaction. The absorbance OD was read using a microplate reader. 450nm , make a judgment based on the judgment result.

[0139] Judgment criteria: OD value ≥ 0.19 is positive, OD value < 0.19 is negative.

[0140] The test results are shown in Table 2.

[0141] Table 2 Results of immunogenicity test of African swine fever virus subunit vaccine composition

[0142]

[0143] The results showed that the African swine fever virus subunit vaccine compositions all had good immunogenicity, and the antibody tests were all positive. This shows that the African swine fever virus subunit vaccine of the present invention uses multi-component African swine fever virus protein antigens and has a good immune effect.

[0144] Example 6 African swine fever virus protein immunogenicity verification test

[0145] 1. Preparation of African swine fever virus single-component subunit vaccine composition

[0146] The p30, p54, p34, p62, E165R, S273R, A104R, C129R, p12, DP96R, DP71L proteins prepared in Example 1, the p72 protein prepared in Example 2, and the p17 and p22 proteins prepared in Example 3 were added to the adjuvant respectively, and the addition process was continuously stirred for 12 minutes with an emulsifier at a speed of 800 rpm, mixed, and stored at 4 ° C., which is a subunit vaccine composition containing a single-component protein antigen of African swine fever virus. The adjuvant suitable for the present invention can be an adjuvant known to those skilled in the art. In the present invention, the adjuvant is selected as a biphasic adjuvant (water-in-oil-in-water emulsion), for example, it can be adjuvant ISA 206 (France Seppic Company). The specific ratios of the components in the prepared vaccine are shown in Tables 3, 4, and 5.

[0147] Table 3 Composition ratio of African swine fever virus single-component subunit vaccine composition 1

[0148] Components Vaccine 6 Vaccine 7 Vaccine 8 Vaccine 9 Vaccine 10 p30 (μg / ml) 1250 - - - - p54 (μg / ml) - 1250 - - - p34 (μg / ml) - - 1250 - - p62 (μg / ml) - - - 1250 - E165R (μg / ml) - - - - 1250 Biphasic adjuvant (V / V%) 50％ 50％ 50％ 50％ 50％

[0149] Table 4 Composition ratio of African swine fever virus single-component subunit vaccine 2

[0150] Components Vaccine 11 Vaccine 12 Vaccine 13 Vaccine 14 Vaccine 15 S273R (μg / ml) 1250 - - - - A104R (μg / ml) - 1250 - - - C129R (μg / ml) - - 1250 - - P12 (μg / ml) - - - 1250 - DP96R (μg / ml) - - - - 1250 Biphasic adjuvant (V / V%) 50％ 50％ 50％ 50％ 50％

[0151] Table 5 Composition ratio of African swine fever virus single-component subunit vaccine composition 3

[0152] Components Vaccine 16 Vaccine 17 Vaccine 18 Vaccine 19 DP71L (μg / ml) 1250 - - - p72 (μg / ml) - 1250 - - p17 (μg / ml) - - 1250 - p22 (μg / ml) - - - 1250 Biphasic adjuvant (V / V%) 50％ 50％ 50％ 50％

[0153] 2. Immunogenicity test of African swine fever virus single-component subunit vaccine composition

[0154] Seventy-five healthy, susceptible piglets weighing approximately 20 kg, negative for both African swine fever virus antigen and antibody, were randomly divided into 15 groups of 5 pigs each. Groups 7 through 20 were immunized with Vaccine 6 through Vaccine 19, respectively. Group 21 served as a blank control group. The immunized groups received 4 ml of the vaccine intramuscularly via neck injection, while the control group received an equivalent volume of PBS plus adjuvant. Immunizations were repeated twice, 14 days apart. Blood samples were collected before the first immunization and 14 days after the second.

[0155] Referring to the preparation method of the African swine fever virus antibody detection kit in Example 5, the African swine fever virus p30 protein, p54 protein, p34 protein, p62 protein, E165R protein, S273R protein, A104R protein, C129R protein, p12 protein, DP96R protein, DP71L0 protein, p72 protein, p17 protein, and p22 protein were coated on an ELISA plate to prepare an African swine fever virus antibody detection kit.

[0156] The test results are shown in Tables 6 to 19.

[0157] Table 6 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 1

[0158]

[0159] Table 7 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 2

[0160]

[0161] Table 8 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 3

[0162]

[0163] Table 9 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 4

[0164]

[0165] Table 10 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 5

[0166]

[0167] Table 11 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 6

[0168]

[0169] Table 12 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 7

[0170]

[0171] Table 13 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 8

[0172]

[0173] Table 14 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 9

[0174]

[0175] Table 15 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 10

[0176]

[0177] Table 16 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 11

[0178]

[0179] Table 17 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 12

[0180]

[0181] Table 18 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 13

[0182]

[0183] Table 19 Results of immunogenicity test of African swine fever virus single-component subunit vaccine composition 14

[0184]

[0185] The results showed that all single-component subunit vaccine compositions of African swine fever virus elicited varying degrees of immune responses, with all testing positive for antibodies. This suggests that the African swine fever virus protein antigens used in the subunit vaccine of the present invention are all immunogenic, and that the multi-component subunit vaccine of African swine fever virus prepared with them exhibits better immune efficacy and higher antibody levels.

[0186] Comparing the OD values detected in Table 2 and Tables 6 to 19, when the total amount of antigen used in the African swine fever virus subunit vaccine composition of the present invention (i.e., the sum of the content of each antigen protein in the African swine fever virus subunit vaccine composition of the present invention with an immunization dose of 4 ml) is only 1 mg (vaccine 1) or 3.3 mg (vaccine 2) or 4.5 mg (vaccine 3) or 5 mg (vaccine 4), the OD values detected are 450nm The values were 2.684, 3.064, 3.282 and 3.286 respectively, while the antigen dosage of the African swine fever virus single-component subunit vaccine composition tested in Tables 6 to 19 (i.e., the antigen protein content in the African swine fever virus single-component subunit vaccine composition with an immunization dose of 4 ml) was 5 mg, which was higher or equivalent to the total antigen dosage of vaccine 1 or vaccine 2 or vaccine 3 or vaccine 4. The OD 450nm The values were much lower than the antibody OD in serum after immunization with vaccine 1, vaccine 2, vaccine 3 or vaccine 4. 450nm The value indicates that there is a synergistic effect between the antigen components in the African swine fever virus subunit vaccine composition of the present invention, which can produce a stronger immune response and a higher antibody titer.

[0187] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the present profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention. SEQUENCE LISTING <110> Plike Bioengineering Co., Ltd. <120> African swine fever virus subunit vaccine composition, combination and application of African swine fever virus protein antigen <160> 15 <170> PatentIn version 3.3 <210> 1 <211> 1965 <212> DNA <213> African swine fever virus <400> 1 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> 2 <211> 354 <212> DNA <213> African swine fever virus <400> 2 atgattctgc atgttctgtt tgaagaagaa accgaaagca gtgcaagcag tgaaaatatt catgaaaga atgacaacga gaccaatga tgtaccagca gctttgaac cctgtttga caggaaccga gcagcgaagt gccgaaagat agcaaactgt atatgctggc ccagaaaacc gttcagcata ttgaacagta tggcaaagca ccggatttta ataaggttat tcgtgcccat aatttcattc agaccatcta tggtaccccg ctgaaagaag aagaaaaaga agttgttcgt 300. ctgatggtta ttagctgct gaaaaagatt agtttcttcc tgacctatat ctaa <210> 3 <211> 399 <212> DNA <213> Antimicrobial agent(African swine fever virus) <400> 3 agtagcgca aaaagaaagc agccgcaatt gaagaagaag atattcagtt tatcaacccg tatcaggatc agcagtgggt tgaagtgacc ccgcagccgg gcaccagtaa accggcaggt gccaccaccg caagtgtggg taaccggtt accggtcgcc cggcaccaa tcgtccggca accaacaaac cggtgaccga taatccggtg accgaccgtc tggttatggc caccggtggc ccggccgctg cacctgcagc agcatcagca ccggcacatc cggcagaacc gtataccacc 300 gtgaccaccc agaataccgc cagccagacc atgagtgcca ttgaaaatct gcgccagcgt 360 aatacctata cccataaaga tctggaaaat agcctgtaa 399 <210> 4 <211> 975 <212> DNA <213> African swine fever virus <400> 4 ggtgacaaaa acccggttca gcacatcaaa gactaccaca tcgactctgt ttcttctaaa 60 gctaaactgc gtatcatcga aggtatcatc cgtgctatcg ctaaaatcgg tttcaaagtt 120 gacaccaaac agccgatcga agacatcctg aaagacatca aaaaacagct gccggacccg 180 cgtgctggtt ctaccttcgt taaaaacgct gaaaaacagg aaaccgtttg caaaatgatc 240 gctgacgcta tcaaccagga attcatcgac ctgggtcagg acaaactgat cgacaccacc 300 gaaggtgctg cttctatctg ccgtcagatc gttctgtaca tcaactctct gacccacggt 360 ctgcgtgctg aatacctgga cgttcacggt tctatcgaaa acaccctgga aaacatcaaa 420 ctgctgaacg acgctatcaa acagctgcac gaacgtatgg ttaccgaagt taccaaagct 480 gctccgaacg aagaagttat caacgctgtt accatgatcg aagctgttta ccgtcgtctg 540 ctgaacgaac agaacctgca gatcaacatc ctgaccaact tcatcgacaa catcctgacc 600 ccgacccaga aagaactgga caaactgcag accgacgaag ttgacatcat caaactgctg 660 aacgacacca actctgttct gggtaccaaa aacttcggta aagttctgtc ttacaccctg 720 tgcaacctgg gtatcgctgc ttctgttgct aacaaaatca acaaagctct gcagaaagtt 780 ggtctgaaag ttgaacagta cctgcagtct aaaaactggg ctgaattcga caaagaactg 840 gacctgaaac gtttctctgg tctggtttct gctgaaaaca tcgctgaatt cgaaaaagct 900 gttaacctgc tgcgtcagac cttcaacgaa cgtcacaaaa tcctggaaaa ctcttgcgct 960 aaaaaaggtg gttaa 975 <210> 5 <211> 1593 <212> DNA <213> African swine fever virus <400> 5 atgccgtcta acatgaaaca gttctgcaaa atctctgttt ggctgcagca gcacgacccg 60 gacctgctgg aaatcatcaa caacctgtgc atgctgggta acctgtctgc tgctaaatac 120 aaacacggtg ttaccttcat ctacccgaaa caggctaaaa tccgtgacga aatcaaaaaa 180 cacgcttact ctaacgaccc gtctcaggct atcaaaaccc tggaatctct gatcctgccg 240 ttctacatcc cgaccccggc tgaattcacc ggtgaaatcg gttcttacac cggtgttaaa 300 ctggaagttg aaaaaaccga agctaacaaa gttatcctga aaaacggtga agctgttctg 360 gttccggctg ctgacttcaa accgttcccg gaccgtcgtc tggctgtttg gatcatggaa 420 tctggttcta tgccgctgga aggtccgccg tacaaacgta aaaaagaagg tggtggtaac 480 gacccgccgg ttccgaaaca catctctccg tacaccccgc gtacccgtat cgctatcgaa 540 gttgaaaaag ctttcgacga ctgcatgcgt cagaactggt gctctgttaa caacccgtac 600 ctggctaaat ctgtttctct gctgtctttc ctgtctctga accacccgac cgaattcatc 660 aaagttctgc cgctgatcga cttcgacccg ctggttacct tctacctgct gctggaaccg 720 tacaaaaccc acggtgacga cttcctgatc ccggaaacca tcctgttcgg tccgaccggt 780 tggaacggta ccgacctgta ccagtctgct atgctggaat tcaaaaaatt cttcacccag 840 atcacccgtc agaccttcat ggacatcgct gactctgcta ccaaagaagt tgacgttccg 900 atctgctact ctgacccgga aaccgttcac tcttacgcta accacgttcg taccgaaatc 960 ctgcaccaca acgctgttaa caaagttacc accccgaacc tggttgttca ggcttacaac 1020 gaactggaac agaccaacac catccgtcac tacggtccga tcttcccgga atctaccatc 1080 aacgctctgc gtttctggaa aaaactgtgg caggacgaac agcgtttcgt tatccacggt 1140 ctgcaccgta ccctgatgga ccagccgacc tacgaaacct ctgaattcgc tgaaatcgtt 1200 cgtaacctgc gtttctctcg tccgggtaac aactacatca acgaactgaa catcacctct 1260 ccggctatgt acggtgacaa acacaccacc ggtgacatcg ctccgaacga ccgtttcgct 1320 atgctggttg ctttcatcaa ctctaccgac ttcctgtaca ccgctatccc ggaagaaaaa 1380 gttggtggta acgaaaccca gacctcttct ctgaccgacc tggttccgac ccgtctgcac 1440 tctttcctga accacaacct gtctaaactg aaaatcctga accgtgctca gcagaccgtt 1500 cgtaacatcc tgtctaacga ctgcctgaac cagctgaaac actacgttaa acacaccggt 1560 aaaaacgaaa tcctgaaact gctgcaggaa taa 1593 <210> 6 <211> 315 <212> DNA <213> African swine fever virus <400> 6 atggacaccg agacaagccc tctgctgtct cacaacctgt ctaccagaga gggcatcaaa 60 cagtccaccc agggcctcct ggctcacacc atcgccaagt atcctggcac cggcggcggc 120 ggatccggag gaggaggatc taacagaacc atcgattgca agtcctccat ccctaagcct 180 cctccatcct actacgtcca acaacctgag cctcaccacc actttcctgt gttcttccgc 240 aagagaaaga actccacctc cctgcagagc cacatcccct ccgacgagca gctggccgag 300 ctggcccact cctga 315 <210> 7 <211> 498 <212> DNA <213> African swine fever virus <400> 7 atggctacca acttcttcat ccagccgatc accgaagaag ctgaagctta ctacccgccg 60 tctgttatca ccaacaaacg taaagacctg ggtgttgacg tttactgctg ctctgacctg 120 gttctgcagc cgggtctgaa catcgttcgt ctgcacatca aagttgcttg cgaacacatg 180 ggtaaaaaat gcggtttcaa aatcatggct cgttcttcta tgtgcaccca cgaacgtctg 240 ctgatcctgg ctaacggtat cggtctgatc gacccgggtt acgttggtga actgatgctg 300 aaaatcatca acctgggtga caccccggtt cagatctggg ctaaagaatg cctggttcag 360 ctggttgctc agggtgacca cgttccggac cacatcaaca tcctgaaacg taaccagatc 420 ttcccgctgt tcgctccgac cccgcgtggt gaaggtcgtt tcggttctac cggtgaagct 480 ggtatcatgc gtacctaa 498 <210> 8 <211> 822 <212> DNA <213> African swine fever virus <400> 8 atgtctatcc tggaaaaaat cacctcttct ccgtctgaat gcgctgaaca cctgaccaac 60 aaagactctt gcctgtctaa aaaaatccag aaagaactga cctctttcct ggaaaaaaaa 120 gaaaccctgg gttgcgactc tgaatcttgc gttatcaccc acccggctgt taaagcttac 180 gctcagcaga aaggtctgga cctgtctaaa gaactggaaa cccgtttcaa agctccgggt 240 ccgcgtaaca acaccggtct gctgaccaac ttcaacatcg acgaaaccct gcagcgttgg 300 gctatcaaat acaccaaatt cttcaactgc ccgttctcta tgatggactt cgaacgtgtt 360 cactacaaat tcaaccaggt tgacatggtt aaagtttaca aaggtgaaga actgcagtac 420 gttgaaggta aagttgttaa acgtccgtgc aacaccttcg gttgcgttct gaacaccgac 480 ttctctaccg gtaccggtaa acactgggtt gctatcttcg ttgacatgcg tggtgactgc 540 tggtctatcg aatacttcaa ctctaccggt aactctccgc cgggtccggt tatccgttgg 600 atggaacgtg ttaaacagca gctgctgaaa atccaccaca ccgttaaaac cctggctgtt 660 accaacatcc gtcaccagcg ttctcagacc gaatgcggtc cgtactctct gttctacatc 720 cgtgctcgtc tggacaacgt ttcttacgct cacttcatct ctgctcgtat caccgacgaa 780 gacatgtaca aattccgtac ccacctgttc cgtatcgctt aa 822 <210> 9 <211> 315 <212> DNA <213> African swine fever virus <400> 9 atgtctacca aaaaaaaacc gaccatcacc aaacaggaac tgtactctct ggttgctgct 60 gacacccagc tgaacaaagc tctgatcgaa cgtatcttca cctctcagca gaaaatcatc 120 cagaacgctc tgaaacacaa ccaggaagtt atcatcccgc cgggtatcaa attcaccgtt 180 gttaccgtta aagctaaacc ggctcgtcag ggtcacaacc cggctaccgg tgaaccgatc 240 cagatcaaag ctaaaccgga acacaaagct gttaaaatcc gtgctctgaa accggttcac 300 gacatgctga actaa 315 <210> 10 <211> 390 <212> DNA <213> African swine fever virus <400> 10 atggaacacc cgtctaccaa ctacaccccg gaacagcagc acgaaaaact gaaacactac 60 gttctgatcc cgaaacacct gtggtcttac atcaaatacg gtacccacgt tcgttactac 120 accacccaga acgttttccg tgttggtggt ttcgttctgc agaacccgta cgaagctgtt 180 atcaaaaacg aagttaaaac cgctatccgt ctgcagaact ctttcaacac caaagctaaa 240 ggtcacgtta cctgggctgt tccgtacgac aacatctcta aactgtacgc taaaccggac 300 gctatcatgc tgaccatcca ggaaaacgtt gaaaaagctc tgcacgctct gaaccagaac 360 gttctgaccc tggcttctaa aatccgttaa <210> 11 <211> 186 <212> DNA <213> Antimicrobial agent(African swine fever virus) <400> 11 atggctctgg acggttcttc tggtggtggt tctaacgttg aaaccctgct gatcgttgct atcatcgttg ttatcatggc tatcatgctg tactacttct ggtggatgcc gcgtcagcag 180. aaaaaatgct ctaaagctga agaatgcacc tgcaacaacg gttcttgctc tctgaaaacc tcttaa <210> 12 <211> 450 <212> DNA <213> Antimicrobial agent(African swine fever virus) <400> 12 atgaagaagc agcagcctcc aaagaaagtt tgtaaggtgg acaaggactg cggatccggc gagcattgcg tgcggggcag ctgtagctct ctgtcctgcc tggacgccgt gaagatggat 180. aagcggaaca ttaagatcga ctccaaaatc tccagttgcg agttcacacc taacttctac cggttcaccg ataccgctgc cgacgaacag caggagttcg gcaagaccag acacccaatc aagatcaccc ctagcccctc tgagtcccat tctcctcagg aagtgtgtga aaagtactgc 300 tcttggggca ccgacgactg caccggctgg gagtacgtgg gcgacgagaa agaaggcaca 360 tgctacgtgt acaacaaccc ccaccacccc gtgctgaagt acggcaagga tcacatcatc 420 gccctgccta gaaatcacaa gcacgcctga 450 <210> 13 <211> 291 <212> DNA <213> African swine fever virus <400> 13 atgtctaccc acgactgctc tctgaaagaa aaaccggttg acatgaacga catctctgaa 60 aaatctgttg ttgttgacaa cgctccggaa aaaccggctg gtgctaacca catcccggaa 120 aaatctgctc gtgaaatgac ctcttctgaa tggatcgctg aatactggaa aggtatcaaa 180 cgtggtaacg acgttccgtg ctgctgcccg cgtaaaatga cctctgctga caaaaaattc 240 tctgttttcg gtaaaggttc tctgatgcgt tctatccaga aaaacaacta a 291 <210> 14 <211> 213 <212> DNA <213> African swine fever virus <400> 14 atgggtcgtc gtcgtaaaaa acgtaccaac gacgctaaac acgttcactt cgctaccgct 60 gttgaagttt gggaagctga cgacatcgaa cgtaaaggtc cgtgggaaca ggttgctgtt 120 gaccgtttcc gtttccagcg tcgtatcgct tctgttgaag aactgctgtc tgctgttctg 180 ctgcgtcaga aaaaactgct ggaacagcag taa 213 <210> 15 <211> 5369 <212> DNA <213> Escherichia coli <400> 15 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 tttattttc taaatacatt caatatgta 540 tccgctcatg aattaatttct tagaaaact catcgagcat caatgaac tgcaatttat 600 tcatatcagg attatcaata ccatatttt gaaaaagccg ttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctgta tcggtctgcg attccgactc 720 gtccaacatc atacaacct attaatttcc cctcgtcaaa ataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gatccggtg agaatggcaa aagtttagc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaccaac 900 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 attackacaac aggaatcgaa tgcaccggc gcaggacc tgccagcgca tcacaat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgtttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt agccagttt 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 tcccgctgttt 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 aaaggaatggt 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 attaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040 ttttgtttaa cttaagaag 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 antigens of the African swine fever virus subunit vaccine composition are composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus p72, p30 and p54 proteins, African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, African swine fever virus replication proteins E165R, A104R and C129R proteins, African swine fever virus adsorption protein p12 protein, and African swine fever virus virulence protein DP71L protein; or The protein antigens of the African swine fever virus subunit vaccine composition are composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus p72, p30, and p54 proteins, African swine fever virus nucleocapsid assembly proteins p62 and p17 proteins, African swine fever virus replication proteins E165R, S273R, A104R and C129R proteins, African swine fever virus adsorption protein p22 protein, and African swine fever virus virulence protein DP71L protein; or The protein antigens of the African swine fever virus subunit vaccine composition are composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus p72, p30 and p54 proteins, African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, African swine fever virus replication proteins E165R and A104R, African swine fever virus adsorption protein p12 protein, and African swine fever virus virulence protein DP96R protein; or The protein antigens of the African swine fever virus subunit vaccine composition are composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus proteins p72, p30 and p54 proteins, African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, African swine fever virus replication proteins E165R, A104R and C129R proteins, African swine fever virus adsorption protein p22 protein, and African swine fever virus virulence protein DP96R protein; as well as The African swine fever virus subunit vaccine composition further comprises a pharmaceutically acceptable carrier; The African swine fever virus p72 protein is encoded by SEQ ID NO.1 or its degenerate sequence; the African swine fever virus p30 protein is encoded by SEQ ID NO.2 or its degenerate sequence; the African swine fever virus p54 protein is encoded by SEQ ID NO.3 or its degenerate sequence; the African swine fever virus p34 protein is encoded by SEQ ID NO.4 or its degenerate sequence; the African swine fever virus p62 protein is encoded by SEQ ID NO.5 or its degenerate sequence; the African swine fever virus p17 protein is encoded by SEQ ID NO.6 or its degenerate sequence; the African swine fever virus E165R protein is encoded by SEQ ID NO.7 or its degenerate sequence; the African swine fever virus S273R protein is encoded by SEQ ID NO.8 or its degenerate sequence; the African swine fever virus A104R protein is encoded by SEQ ID NO.9 or its degenerate sequence; the African swine fever virus C129R protein is encoded by SEQ ID NO.10 or its degenerate sequence; the African swine fever virus p12 protein is encoded by SEQ ID NO. NO.11 or its degenerate sequence encoding; the African swine fever virus p22 protein is encoded by SEQ ID NO.12 or its degenerate sequence encoding; the African swine fever virus DP96R protein is encoded by SEQ ID NO.13 or its degenerate sequence encoding; and the African swine fever virus DP71L protein is encoded by SEQ ID NO.14 or its degenerate sequence encoding.

2. The African swine fever virus subunit vaccine composition according to claim 1, wherein The content of the African swine fever virus p72 protein is 75-125 μg / ml; the content of the African swine fever virus p30 protein is 75-125 μg / ml; the content of the African swine fever virus p54 protein is 75-125 μg / ml; the content of the African swine fever virus p34 protein is 75-125 μg / ml; the content of the African swine fever virus p62 protein is 75-125 μg / ml; the content of the African swine fever virus p17 protein is 75-125 μg / ml; the content of the African swine fever virus E165R ...54 protein is 75-125 μg / ml; the content of the African swine fever virus p34 protein is 75-125 μg / ml; the content of the African swine fever virus p62 protein is 75-125 μg / ml; the content of the African swine fever virus p17 protein is 75-125 μg / ml; the content of the African swine fever virus E165R protein is 75-125 μg / ml; the content of the African swine fever virus p The content of the S273R protein of the classical swine fever virus is 75 to 125 μg / ml; the content of the A104R protein of the African swine fever virus is 75 to 125 μg / ml; the content of the C129R protein of the African swine fever virus is 75 to 125 μg / ml; the content of the p12 protein of the African swine fever virus is 75 to 125 μg / ml; the content of the p22 protein of the African swine fever virus is 75 to 125 μg / ml; the content of the DP96R protein of the African swine fever virus is 75 to 125 μg / ml; and the content of the DP71L protein of the African swine fever virus is 75 to 125 μg / ml.

3. The African swine fever virus subunit vaccine composition according to claim 1, wherein The pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes: (1) mineral oil, aluminum gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; 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, IMS1314, muramyl dipeptide, Montanide ISA206, and Gel adjuvant; The adjuvant content is 5%-60% V / V; or The pharmaceutically acceptable carrier comprises a lyoprotectant, which is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

4. The African swine fever virus subunit vaccine composition according to claim 3, wherein The saponins are QuilA, QS-21, and GPI-0100.

5. The African swine fever virus subunit vaccine composition according to claim 3, wherein The adjuvant content is 30%-60% V / V.

6. The African swine fever virus subunit vaccine composition according to claim 5, wherein The adjuvant content is 50% V / V.

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

8. Use of the African swine fever virus subunit vaccine composition according to any one of claims 1 to 7, wherein: The application refers to the application in the preparation of drugs for preventing African swine fever virus infection.

9. A combination of African swine fever virus protein antigens, wherein: The African swine fever virus protein antigen is composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus p72, p30 and p54 proteins, African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, African swine fever virus replication proteins E165R, A104R and C129R proteins, African swine fever virus adsorption protein p12 protein, and African swine fever virus virulence protein DP71L protein; or The African swine fever virus protein antigen is composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus p72, p30 and p54 proteins, African swine fever virus nucleocapsid assembly proteins p62 and p17 proteins, African swine fever virus replication proteins E165R, S273R, A104R and C129R proteins, African swine fever virus adsorption protein p22 protein, and African swine fever virus virulence protein DP71L protein; or The African swine fever virus protein antigen is composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus p72, p30 and p54 proteins, African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, African swine fever virus replication proteins E165R and A104R, African swine fever virus adsorption protein p12 protein, and African swine fever virus virulence protein DP96R protein; or The African swine fever virus protein antigen is composed of the following components with protein contents of 25 to 175 μg / ml respectively: African swine fever virus p72, p30 and p54 proteins, African swine fever virus nucleocapsid assembly proteins p34 and p17 proteins, African swine fever virus replication proteins E165R, A104R and C129R proteins, African swine fever virus adsorption protein p22 protein, and African swine fever virus virulence protein DP96R protein; wherein the African swine fever virus p72 protein is encoded by SEQ ID NO.1 or its degenerate sequence; the African swine fever virus p30 protein is encoded by SEQ ID NO.2 or its degenerate sequence; the African swine fever virus p54 protein is encoded by SEQ ID NO.3 or its degenerate sequence; the African swine fever virus p34 protein is encoded by SEQ ID NO.4 or its degenerate sequence; the African swine fever virus p62 protein is encoded by SEQ ID NO.5 or its degenerate sequence; the African swine fever virus p17 protein is encoded by SEQ ID NO.6 or its degenerate sequence; the African swine fever virus E165R protein is encoded by SEQ ID NO.7 or its degenerate sequence; the African swine fever virus S273R protein is encoded by SEQ ID NO.8 or its degenerate sequence; the African swine fever virus A104R protein is encoded by SEQ ID NO.9 or its degenerate sequence; the African swine fever virus C129R protein is encoded by SEQ ID NO.10 or its degenerate sequence; the African swine fever virus p12 protein is encoded by SEQ ID NO. NO.11 or its degenerate sequence encoding; the African swine fever virus p22 protein is encoded by SEQ ID NO.12 or its degenerate sequence encoding; the African swine fever virus DP96R protein is encoded by SEQ ID NO.13 or its degenerate sequence encoding; and the African swine fever virus DP71L protein is encoded by SEQ ID NO.14 or its degenerate sequence encoding.

10. The combination of African swine fever virus protein antigens according to claim 9, wherein The combination of African swine fever virus protein antigens further comprises a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises an adjuvant, wherein the adjuvant comprises: (1) mineral oil, aluminum gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a polymer of acrylic acid or methacrylic acid, a copolymer of 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, IMS1314, muramyl dipeptide, Montanide ISA 206, and Gel adjuvant; The adjuvant content is 5%-60% V / V; or The pharmaceutically acceptable carrier comprises a lyoprotectant, which is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

11. The African swine fever virus subunit vaccine composition according to claim 10, wherein The saponins are QuilA, QS-21, and GPI-0100.

12. The African swine fever virus subunit vaccine composition according to claim 10, wherein The adjuvant content is 30%-60% V / V.

13. The African swine fever virus subunit vaccine composition according to claim 12, wherein The adjuvant content is 50% V / V.

14. The combination of African swine fever virus protein antigens according to claim 9, wherein The dosage form of the p72, p30, p54, p34, p62, p17, E165R, S273R, A104R, C129R, p12, p22, DP96R or DP71L protein is a solution injection, a suspension injection or a powder for injection.

15. The combination of African swine fever virus protein antigens according to claim 9, wherein: The dosage form of the p72, p30, p54, p34, p62, p17, E165R, S273R, A104R, C129R, p12, p22, DP96R or DP71L protein is a solution injection or a suspension injection; The content of the African swine fever virus p72 protein is 75-125 μg / ml; the content of the African swine fever virus p30 protein is 75-125 μg / ml; the content of the African swine fever virus p54 protein is 75-125 μg / ml; the content of the African swine fever virus p34 protein is 75-125 μg / ml; the content of the African swine fever virus p62 protein is 75-125 μg / ml; the content of the African swine fever virus p17 protein is 75-125 μg / ml; the content of the African swine fever virus E165R ...54 protein is 75-125 μg / ml; the content of the African swine fever virus p34 protein is 75-125 μg / ml; the content of the African swine fever virus p62 protein is 75-125 μg / ml; the content of the African swine fever virus p17 protein is 75-125 μg / ml; the content of the African swine fever virus E165R protein is 75-125 μg / ml; the content of the African swine fever virus p The content of the S273R protein of the classical swine fever virus is 75 to 125 μg / ml; the content of the A104R protein of the African swine fever virus is 75 to 125 μg / ml; the content of the C129R protein of the African swine fever virus is 75 to 125 μg / ml; the content of the p12 protein of the African swine fever virus is 75 to 125 μg / ml; the content of the p22 protein of the African swine fever virus is 75 to 125 μg / ml; the content of the DP96R protein of the African swine fever virus is 75 to 125 μg / ml; and the content of the DP71L protein of the African swine fever virus is 75 to 125 μg / ml.

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