African swine fever virus p34-p14 subunit protein and its applications and products

Abstract

The application relates to the technical field of veterinary biological products, and provides an African swine fever virus p34-p14 subunit protein and application and products thereof. The amino acid sequence of the African swine fever virus p34-p14 subunit protein is shown as SEQ ID NO. 4. The protein serves as an antigen and can provide good immunological efficacy for pigs, and can provide better immunological efficacy under the condition of low total protein content.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, specifically to the p34-p14 subunit protein of African swine fever virus and its applications and products. Background Technology

[0002] African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease caused by the African swine fever virus (ASFV). It has a high morbidity rate and a mortality rate that can reach 100%. In my country, it is classified as a Class A animal disease. The disease was first confirmed in Kenya, Africa in 1921. Since 2007, African swine fever has occurred, spread, and become prevalent in many countries worldwide. Although the disease has been known for nearly a century, there are currently no approved vaccines or effective treatments globally.

[0003] The difficulty in developing African swine fever vaccines lies in the fact that the virus 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 large-scale culture difficult. The virus 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 to date. Furthermore, improper use of live attenuated vaccines poses a biosafety risk of virulence reversion and virus shedding.

[0004] Therefore, there is an urgent clinical need for the research and development and production of new vaccines to prevent African swine fever. Summary of the Invention

[0005] The primary objective of this invention is to provide a p34-p14 subunit protein of African swine fever virus.

[0006] A second objective of this invention is to provide biomaterials related to the p34-p14 subunit protein of African swine fever virus.

[0007] A third objective of this invention is to provide the application of African swine fever virus p34-p14 subunit proteins or biological materials.

[0008] The fourth objective of this invention is to provide an African swine fever virus subunit vaccine composition and its preparation method.

[0009] The technical solution adopted in this invention is as follows:

[0010] The amino acid sequence of the African swine fever virus p34-p14 subunit protein provided by the present invention is shown in SEQ ID NO.4.

[0011] Biomaterials related to the p34-p14 subunit protein of African swine fever virus, including any of the following:

[0012] (a) Nucleic acid molecules encoding the above-mentioned African swine fever virus p34-p14 subunit proteins;

[0013] (b) An expression cassette containing the nucleic acid molecule described in (a);

[0014] (c) A recombinant vector containing the nucleic acid molecule described in (a) or the expression cassette described in (b);

[0015] (d) Recombinant cells containing the vector described in (c).

[0016] The application of the above-mentioned African swine fever virus p34-p14 subunit proteins or biological materials in the preparation of drugs for preventing African swine fever virus infection.

[0017] An African swine fever virus subunit vaccine composition comprising African swine fever virus p34-p14 subunit proteins and a pharmaceutically acceptable vector.

[0018] The above-mentioned method for preparing the African swine fever virus subunit vaccine composition involves constructing a recombinant vector using a nucleic acid molecule encoding the African swine fever virus p34-p14 subunit protein, introducing the recombinant vector into expression cells for expression, purifying the African swine fever virus p34-p14 subunit protein, and then mixing it with a pharmaceutically acceptable vector to obtain the African swine fever virus subunit vaccine composition.

[0019] The beneficial effects of this invention are as follows:

[0020] The amino acid sequence of the African swine fever virus p34-p14 subunit protein provided by this invention is shown in SEQ ID NO.4. This protein, as an antigen, exhibits synergistic effects and good immunogenicity. It can provide better immunogenicity even with low total protein content, effectively solving the problem of poor immunogenicity currently faced by African swine fever virus vaccines. It can be expressed in large quantities through genetic engineering, which is not only time-efficient but also facilitates large-scale production without biosafety risks. Related biomaterials can be used directly as biological modules for protein production. Detailed Implementation

[0021] The specific embodiments of the present invention will now be described in detail.

[0022] The following explanations are provided for the relevant terms used in this invention:

[0023] The term "African swine fever virus" refers to African swine fever virus (ASFV), the only species in the family Asfarviridae. It is highly contagious and extremely pathogenic. ASFV has an icosahedral morphology, approximately 200 nanometers in diameter, and is composed of multiple layers: a central protein nucleoid shell, followed by a lipid envelope and a protein capsid. The capsid consists of 8280 major capsid proteins (p72) and 60 p72 proteins. In addition, at least three other proteins maintain the capsid structure's stability by adhering to adjacent proteins. Acute cases are characterized by high fever, short disease course, high mortality, widespread internal organ hemorrhage, and respiratory and nervous system dysfunction. As of 2020, there was still no specific vaccine or antiviral drug for ASFV to effectively control its spread during outbreaks.

[0024] The terms "African swine fever virus p34 protein" and "African swine fever virus p14 protein" refer to a polyprotein called p220 encoded by the African swine fever virus. This p220 polyprotein is present in the nucleocapsid of mature viral particles, accounting for approximately 30% of the total viral protein, and plays a crucial role in viral assembly and infection. Under the action of proteases, the p220 polyprotein can be orderly cleaved into p150, p34, p37, and p14. p150, p34, p37, and p14 play a vital role in the assembly of the viral capsid. Among them, p34 and p14 are important structural proteins within p220, playing a crucial role in the packaging of viral core proteins.

[0025] The term "antigen" refers to a substance that can induce an immune response in the body. That is, it can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0026] As used in this invention, the terms "vaccine" and "vaccine composition" refer to pharmaceutical compositions containing African swine fever virus protein antigens, which can induce, stimulate, or enhance the immune response of pigs against African swine fever.

[0027] The term "immune dose" should be understood as "immunely effective dose," also known as immune protective dose or effective dose to elicit an immune response. It refers to the amount of antigen that can effectively induce an immune response in a recipient, sufficient to prevent or improve the signs or symptoms of disease, including adverse health effects or complications thereof. This immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications of infection caused by a pathogen. Humoral immunity or cell-mediated immunity, or both, can be induced. An animal's immune response to an immunogenic composition can be indirectly assessed, for example, by measuring antibody titers, lymphocyte proliferation analysis, or directly assessed by monitoring signs or symptoms after challenge with a wild-type strain. The protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs in the subject such as mortality, reduction in morbidity, temperature values, overall physiological status, and overall health and performance. The immune response may include, but is not limited to, the induction of cellular and / or humoral immunity.

[0028] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent, preferably an adjuvant, that does not irritate the body or impede the biological activity and properties of the compound, except for the African swine fever virus p34-p14 subunit protein antigen, in the vaccine composition of the present invention.

[0029] The term "adjuvant" may include aluminum gel adjuvants; saponins, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), and 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; and compounds selected from copolymers of maleic anhydride and alkenyl derivatives.

[0030] The term "emulsion" may be particularly based on light liquid paraffin oils (European Pharmacopea type); isoprenoid oils resulting from olefin oligomerization, such as squalane or squalene oils, especially isobutylene or decanene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / decanoate), glyceryl tri-(octanoate / decanoate), or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearates. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably nonionic surfactants, especially esters of sorbitan, esters of mannitol (such as anhydrous mannitol oleate), esters of aliphatic glycols, esters of polyglycerol, esters of propylene glycol, and esters of oleic acid, isostearic acid, castor oil, or hydroxystearic acid. These may be ethoxylated, as well as polyoxypropylene-polyoxyethylene block copolymers, especially Pluronic products, particularly L121. 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 (Plenum Press, 1995) by Powell M and Newman M can be used.

[0031] The term "polymer of acrylic acid or methacrylic acid" preferably refers to crosslinked acrylic acid or methacrylic acid polymers, especially those crosslinked with polyolefin ethers or polyols of sugar, compounds known as Carbomer (trade name Carbopol) (Phameuropa, 1996, 8(2)). Those skilled in the art may also refer to U.S. Patent US2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least three hydroxyl groups, preferably no more than eight, wherein the hydrogen atoms of at least three hydroxyl groups are replaced by unsaturated aliphatic radicals having at least two carbon atoms. Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl, and other ethylenically unsaturated groups. These unsaturated groups may themselves contain other substituents, such as methyl groups. These products are sold under the name Carbopol (BF Goodrich, Ohio, USA), which is particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, Capop 974P, 934P and 971P can be mentioned, with Capop 971P being the preferred choice.

[0032] The term "copolymer of maleic anhydride and alkenyl derivatives" may also be considered as a copolymer of maleic anhydride and ethylene, EMA (Monsanto), which dissolves in water to produce an acidic solution, which is then neutralized, preferably to physiological pH, to produce an adjuvant solution into which immunogenic, immunizing, or vaccine compositions can be incorporated.

[0033] 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 other), cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvant, etc.

[0034] In a preferred embodiment, the adjuvant includes one or more of the following: mineral oil, aluminum gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin of Escherichia coli, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, or gel adjuvant.

[0035] The term "lyophilization protectant" refers to a component, other than excipients, that protects the efficacy of a drug's active ingredient during the freeze-drying process and its subsequent storage.

[0036] The term "dosage form" refers to the physical form of a pharmaceutical preparation. It also refers to a finished drug product (prepared medicine) made according to the properties of the drug and the requirements of treatment and prescription. A suitable dosage form is chosen to maximize the therapeutic effect of the drug, minimize side effects, and facilitate use, storage, and transportation.

[0037] The term "injection" refers to a sterile solution (including emulsions and suspensions) of a drug prepared for injection into the body, as well as a sterile powder or concentrated solution prepared into a solution or suspension before use. It can be a water-based injection (with water as the solvent) or an oil-based injection (with oil as the solvent). There are also injections that use other solvents, such as ethanol (the solvent for hydrocortisone injection is ethanol), glycerin, propylene glycol (PEG), etc.

[0038] The term "powder for injection" refers to preparations made by freezing the drug solution in a sterile environment, "mixing" the active pharmaceutical ingredient with certain excipients or dissolving it in certain solvents, and then processing it into different forms.

[0039] The term "prevention" in relation to African swine fever virus infection refers to inhibiting the replication of African swine fever virus, inhibiting the spread of African swine fever virus, or preventing African swine fever virus from settling in its host, as well as alleviating the symptoms of disease or illness caused by African swine fever virus infection.

[0040] The term "expression cassette" refers to a construct containing regulatory elements (such as promoters and polyadenylation sites), purification tags, screening tags, etc., necessary for the expression of at least the contained nucleic acid molecules in the cell.

[0041] The amino acid sequence of the African swine fever virus p34-p14 subunit protein provided by the present invention is shown in SEQ ID NO.4.

[0042] GDKNPVQHIKDYHIDSVSSKAKLRIIEGIIRAIAKIGFKVDTKQPIEDILKDIKKQLPDPRAGSTFVKNAEKQETVCKMIADAINQEFIDLGQDKLIDTTEGAASICRQIVLYINSLTHG LRAEYLDVHGSIENTLENIKLLNDAIKQLHERMVTEVTKAAPNEEVINAVTMIEAVYRRLLNEQNLQINILTNFIDNILTPTQKELDKLQTDEVDIIKLLNDTNSVLGTKNFGKVLSYTLC NLGIAASVANKINKALQKVGLKVEQYLQSKNWAEFDKELDLKRFSGLVSAENIAEFEKAVNLLRQTFNERHKILENSCAKKGGDEEKTPLDRRIEAQRLDRKHILMEFLNKSTQAYNDFL ENVKKIGIKLVKEIALTPNITRRLRDALSRINDMGTIALDLSLIGFYTNAAAREERETFLTQLTLVKNVLEEISKTDPNFKNLYDSCSRLLQIIDFYTDIVQKKYGGEEDCECTRVGG (SEQ ID NO.4).

[0043] African swine fever virus p34-p14 subunit proteins can be prepared using prokaryotic expression systems, eukaryotic expression systems, cell expression systems, or chemical synthesis methods.

[0044] The present invention also provides related biological materials for the above-mentioned African swine fever virus p34-p14 subunit protein, including a nucleic acid molecule encoding the African swine fever virus p34-p14 subunit protein, an expression cassette containing the nucleic acid molecule, a recombinant vector and recombinant cells.

[0045] In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3, the backbone vector used is the pNVC1 vector, the nucleotide sequence of which is shown in SEQ ID NO.5, and the expression system used is the Escherichia coli expression system.

[0046]

[0047] All of the above-mentioned biomaterials can be used directly as biomodules in the preparation of African swine fever virus p34-p14 subunit proteins, thereby enabling the preparation of drugs to prevent African swine fever virus infection, such as vaccines.

[0048] An African swine fever virus subunit vaccine composition comprising African swine fever virus p34-p14 subunit proteins and a pharmaceutically acceptable vector.

[0049] In a preferred embodiment, the African swine fever p34-p14 subunit protein content is ≥100 μg / ml, preferably 100~300 μg / ml. The African swine fever p34-p14 subunit protein content is selected from 100 μg / ml, 105 μg / ml, 110 μg / ml, 115 μg / ml, 120 μg / ml, 125 μg / ml, 130 μg / ml, 135 μg / ml, 140 μg / ml, 145 μg / ml, 150 μg / ml, 155 μg / ml, 160 μg / ml, 165 μg / ml, 170 μg / ml, 175 μg / ml, 180 μg / ml, 185 μg / ml, 190 μg / ml, 195 μg / ml, etc. μg / ml, 200μg / ml, 205μg / ml, 210μg / ml, 215μg / ml, 220μg / ml, 225μg / ml, 230μg / ml, 235μg / ml, 240μg / ml, 245μg / ml, 2 50μg / ml, 255μg / ml, 260μg / ml, 265μg / ml, 270μg / ml, 275μg / ml, 280μg / ml, 285μg / ml, 290μg / ml, 295μg / ml or 300μg / ml.

[0050] In a preferred embodiment, the pharmaceutically acceptable carrier includes at least one of adjuvants, lyophilization protectants, drugs, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.

[0051] Furthermore, the adjuvant includes: (1) mineral oil, aluminum gel adjuvant, saponin, avrididine, 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 at least one of the following: RIBI adjuvant system, Block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, and Gel adjuvant; the saponin is preferably Quil A, QS-21, or GPI-0100; the adjuvant content is 5%-60% V / V, preferably 30%-60% V / V, and more preferably 50% V / V. The adjuvant is preferably 50% V / V Montanide ISA 206 adjuvant.

[0052] Furthermore, the freeze-drying protectant is preferably selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

[0053] Furthermore, immunostimulants include alpha-interferon, beta-interferon, gamma-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and interleukin-2.

[0054] In a preferred embodiment, the dosage form of the African swine fever virus subunit vaccine composition is selected from solution injection, suspension injection, injection powder, sustained-release microsphere formulation, controlled-release microsphere formulation, or sustained-release implant, preferably solution injection, suspension injection, or injection powder, and more preferably solution injection or suspension injection.

[0055] In a preferred embodiment, the African swine fever virus subunit vaccine composition is administered via a route selected from subcutaneous injection, oral administration, oral administration, sublingual administration, nasal administration, pulmonary administration, colonic administration, rectal administration, or transdermal administration, preferably subcutaneous injection.

[0056] The present invention also provides a method for preparing the above-mentioned African swine fever virus subunit vaccine composition: a recombinant vector is constructed using a nucleic acid molecule encoding the African swine fever virus p34-p14 subunit protein, the recombinant vector is introduced into expression cells for expression, the African swine fever virus p34-p14 subunit protein is obtained after purification, and then mixed with a pharmaceutically acceptable vector to obtain the African swine fever virus subunit vaccine composition.

[0057] 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 with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0058] All chemical reagents used in the embodiments of this invention are of analytical grade and were purchased from Sinopharm Group. Unless otherwise specified, the experimental methods described in this invention are conventional methods; and the biological materials described, unless otherwise specified, are commercially available. Example 1: Prokaryotic expression of African swine fever virus p34, p14, and p34-p14 proteins

[0059] The nucleotide sequence of the expression vector shown in SEQ ID NO.5 of the corresponding coding sequence listing was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and named pNVC1 vector.

[0060] The nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.3 were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., which encode the p34, p14, and p34-p14 proteins respectively. The synthesized nucleotides were cloned into the pNVC1 vector and named recombinant plasmids pNVC1-p34, pNVC1-p14, and pNVC1-p34-p14 respectively.

[0061] The recombinant plasmids pNVC1-p34, pNVC1-p14, and pNVC1-p34-p14 were transformed into competent Escherichia coli BL21(DE3) to construct expression strains. After inoculating with 50 ml of kanamycin-resistant LB liquid medium and shaking at 37°C and 230 rpm for 12 hours, the strains were transferred to 1 L of LB liquid medium and cultured at 37°C to prepare seed culture for fermentation.

[0062] A 50L fermenter from Shanghai Baoxing Biotechnology Co., Ltd. was used. 30L of culture medium was prepared and added to the fermenter, then sterilized at 121℃ for 30 minutes. The next day, 3L of seed culture was inoculated into the fermenter. When the bacterial concentration reached approximately OD600 of 10, the culture temperature was lowered to 25℃, and IPTG was added to a final concentration of 0.5mM for induction culture for 12 hours. The culture was stopped when the fermentation density reached approximately 40 (OD600), and the bacterial cells were collected by centrifugation.

[0063] Resuspend the bacterial cells at a ratio of 10 mL of lysis buffer (20 mmol / L Tris buffer (pH 7.0), 0.5 mol / L NaCl) per gram of wet bacterial weight, and homogenize the cells three times at 800 bar. Centrifuge at 13500 rpm for 40 min and collect the supernatant.

[0064] Add 0.02 mol / L imidazole to the supernatant, filter, and then perform protein affinity chromatography purification using a protein chromatography purification system. The chromatography medium was Ni Sepharose 6 Fast Flow, and the system flow rate was 90 cm / h. Before loading the sample, the chromatography column was equilibrated with equilibration buffer (0.02 mol / L Tris(pH 7.0), 0.02 mol / L imidazole, 0.5 mol / L NaCl). After loading the sample, elute the column with buffer solution (0.02 mol / L Tris(pH 7.0), 0.05 mol / L imidazole, 0.5 mol / L NaCl) to remove contaminating proteins, and then elute the target protein with buffer solution (0.02 mol / L Tris(pH 7.0), 0.5 mol / L imidazole, 0.5 mol / L NaCl). Collect the elution products. The purified protein was subjected to SDS-PAGE electrophoresis. After staining, clear bands of the target protein should be visible. The gel was scanned using a gel imaging system, and the purity of the target protein was analyzed by software and found to be above 85%. The protein content of p34, p14, and p34-p14 was determined by the BCA method to be 2.4 g / L, 2.5 g / L, and 2.4 g / L, respectively.

[0065] Example 2: Preparation of African swine fever virus subunit vaccine composition

[0066] The p34-p14 protein prepared in Example 1 was added to the adjuvant, and the mixture was continuously stirred in an emulsifier at 800 rpm for 12 minutes during the addition process. After thorough mixing, the mixture was stored at 4°C to obtain a subunit vaccine composition containing African swine fever virus protein antigen. The adjuvant suitable for this invention can be any adjuvant known to those skilled in the art. In this invention, a biphasic adjuvant (water-in-oil-in-water emulsion) was selected, for example, adjuvant ISA 206 (Sebic, France). The specific proportions of each component in the prepared vaccine are shown in Table 1.

[0067] Table 1. Composition ratio of African swine fever virus subunit vaccine composition

[0068]

[0069] Example 3: Immunogenicity test of African swine fever virus subunit vaccine composition

[0070] Twenty healthy, susceptible piglets weighing approximately 20 kg and negative for both African swine fever virus antigen and antibodies were randomly divided into four groups of five piglets each. Group 1 was immunized with vaccine 1, Group 2 with vaccine 2, Group 3 with vaccine 3, and Group 4 served as a blank control group. The immunization routes for the groups were intramuscular injection of 4 ml of PBS in the neck, while the control group was immunized with an equal volume of PBS plus adjuvant. Two immunizations were administered, with a 14-day interval between the two immunizations. Blood samples were collected before the first immunization and 14 days after the second immunization.

[0071] The ELISA plate was coated with inactivated whole African swine fever virus antigen (purchased from the European Union Reference Laboratory for African Swine Fever, URL-ASF) and incubated overnight at 4°C. The coating solution was discarded, and the plate was washed with washing buffer. Blocking buffer (50g of sucrose, 200mL of newborn calf serum, 1mL of Proclin 300, and PBS (0.01mol / L, pH 7.4) were added to bring the volume to 1000mL) and the plate was blocked at 2-8°C for 16-24 hours. The blocking buffer was discarded, the plate was dried, sealed, and stored at 2-8°C for later use.

[0072] Sample dilution solution: Take 8g sodium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 0.2g potassium chloride, 600mL purified water, 1mL Proclin 300, 200mL newborn calf serum, and 0.028g PUR dye. After completely dissolving, bring the volume to 1000mL with purified water. After mixing, filter through a 0.22μm filter and aseptically dispense. Store at 2-8℃.

[0073] Washing solution: Dissolve 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, 800mL ultrapure water, and 10mL Tween 20 completely. Adjust the volume to 1000mL with purified water, filter through a 0.22μm filter membrane, and aseptically dispense. Dilute 20 times with distilled water before use.

[0074] Secondary antibody: Enzyme-labeled goat anti-pig secondary antibody, diluted 1:2000 before use.

[0075] Colorimetric reagent A: Dissolve 14.7g of disodium hydrogen phosphate, 9.3g of citric acid, and 0.3g of urea peroxide in purified water, bring the volume to 1000mL, mix well, filter, and aseptically dispense. Colorimetric reagent B: Dissolve 0.2g of tetramethylbenzidine (TMB) and 10mL of anhydrous ethanol in purified water, bring the volume to 1000mL, mix well, filter, and aseptically dispense.

[0076] Termination solution: 2M H2SO4.

[0077] During the test, first add 90 μl of sample diluent to each well of the enzyme-labeled reaction, then add 10 μl of the serum to be tested, along with negative and positive controls. Incubate at 37°C for 30 minutes, then wash the plate three times with washing buffer. Add 100 μl / well of goat anti-pig enzyme-labeled antibody to the reaction plate, incubate at 37°C for 30 minutes, wash the plate three times with washing buffer, then add 50 μl / well each of chromogenic reagent A and chromogenic reagent B, incubate at 37°C for 10 minutes, and finally add 50 μl / well of stop solution to terminate the reaction. Read the absorbance (OD450nm) using an enzyme-labeled microplate reader and make a judgment based on the results.

[0078] Judgment criteria: OD value ≥ 0.19 is positive, OD value < 0.19 is negative.

[0079] The test results are shown in Table 2.

[0080] Table 2. Immunogenicity test results of African swine fever virus subunit vaccine compositions

[0081]

[0082] The results showed that all African swine fever virus subunit vaccine compositions exhibited good immunogenicity, and antibody tests were all positive. This indicates that the African swine fever virus subunit vaccine of the present invention, using African swine fever virus p34-p14 protein antigens, has good immunizing efficacy.

[0083] Example 4: Comparative Immunogenicity Test of African Swine Fever Virus Subunit Vaccine Compositions

[0084] 1. Preparation of African swine fever virus comparative vaccine composition

[0085] The p34 and p14 proteins prepared in Example 1 were added to the adjuvant, and the mixture was continuously stirred for 12 minutes using an emulsifier at 800 rpm during the addition process. After thorough mixing, the mixture was stored at 4°C to obtain the control vaccine composition containing African swine fever virus protein antigen. The adjuvant suitable for this invention can be any adjuvant known to those skilled in the art. In this invention, a biphasic adjuvant (water-in-oil-in-water emulsion) was selected, for example, adjuvant ISA 206 (Sebic, France). The specific proportions of each component in the prepared vaccine are shown in Table 3.

[0086] Table 3. Composition ratio of African swine fever virus comparative vaccine compositions

[0087] 2. Comparative study on the immunogenicity of African swine fever virus subunit vaccines

[0088] Twenty healthy, susceptible piglets weighing approximately 20 kg and negative for African swine fever virus antigen and antibodies were randomly divided into four groups of five piglets each. Groups 5 and 6 were immunized with vaccine 4 and vaccine 5, respectively, while group 7 was immunized with vaccine 1. Group 8 served as the blank control group. The immunization routes for the groups were intramuscular injection of 4 ml of PBS in the neck, while the control group was immunized with an equal volume of PBS plus adjuvant. Two immunizations were administered, 14 days apart. Blood samples were collected before the first immunization and 14 days after the second immunization.

[0089] Antibody detection was performed using the African swine fever virus antibody detection kit from Example 3, and the results are shown in Table 4.

[0090] Table 4. Results of comparative immunogenicity tests of African swine fever virus subunit vaccine compositions

[0091] The results showed that the African swine fever virus p34, p14, and p34-p14 protein subunit control vaccines all induced immune responses of varying degrees, and antibody tests were positive in all cases. The African swine fever virus p34-p14 protein subunit vaccine showed better immune efficacy and higher antibody levels.

[0092] Comparing the OD values ​​detected in Tables 2 and 4, the African swine fever virus p34-p14 protein subunit vaccine composition of the present invention, when the total antigen dosage (i.e., the total antigen protein content in a 4ml immunization dose) is only 0.4mg (vaccine 1), 0.8mg (vaccine 2), or 1.2mg (vaccine 3), has detected OD450nm values ​​of 2.018 or 2.021, 2.452, and 2.691, respectively. In contrast, the African swine fever virus p34 protein subunit vaccine composition detected in Table 4, when the total antigen dosage (i.e., the total antigen protein content in a 4ml immunization dose) is... The total antigen dosage of the African swine fever virus p14 protein subunit vaccine composition (vaccine 4) is 1.2 mg (vaccine 5), which is higher or equivalent to the total antigen dosage of vaccine 1, vaccine 2, or vaccine 3. The detected OD450nm values ​​are much lower than the antibody OD450nm values ​​in the serum after immunization with vaccine 1, vaccine 2, or vaccine 3. This indicates that the African swine fever virus p34-p14 protein subunit vaccine composition of the present invention has a synergistic effect, can produce a stronger immune response, and has a higher antibody titer.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. SEQUENCE LISTING <110> Pulike Biotechnology Co., Ltd. <120> African swine fever virus p34-p14 subunit proteins and their applications and products <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 972 <212> DNA <213> Artificial sequence <400> 1 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 gt 972 <210> 2 <211> 462 <212> DNA <213> Artificial sequence <400> 2 gacgaagaaa aaaccccgct ggaccgtcgt atcgaagctc agcgtctgga ccgtaaacac 60 atcctgatgg aattcctgaa caaatctacc caggcttaca acgacttcct ggaaaacgtt 120 aaaaaaatcg gtatcaaact ggttaaagaa atcgctctga ccccgaacat cacccgtctg 180 cgtgacgctc tgtctcgtat caacgacatg ggtaccatcg ctctggacct gtctctgatc 240 ggtttctaca ccaacgctgc tgctcgtgaa gaacgtgaaa ccttcctgac ccagctgacc 300 ctggttaaaa acgttctgga agaaatctct aaaaccgacc cgaacttcaa aaacctgtac 360 gactcttgct ctcgtctgct gcagatcatc gacttctaca ccgacatcgt tcagaaaaaa 420 tacggtggtg aagaagactg cgaatgcacc cgtgttggtg gt 462 <210> 3 <211> 1434 <212> DNA <213> Artificial sequence <400> 3 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 tctggttct gctgaaaaca tcgctgaatt cgaaaaagct 900 gttaacctgc tgcgtcagac cttcaacgaa cgtcacaaaa tctggaaa ctcttgcgct 960 aaaaaaggtg gtgacgaaga aaaaaccccg ctggaccgtc gtatcgaagc tcagcgtctg 1020 gaccgtaaac acatcctgat ggaattcctg aacaaatcta cccaggctta caacgacttc 1080 ctggaaaacg ttaaaaaaat cggtatcaaa ctggttaaag aaatcgctct gaccccgaac 1140 atcacccgtc tgcgtgacgc tctgtctcgt atcaacgaca tgggtaccat cgctctggac 1200 ctgtctctga tcggtttcta caccaacgct gctgctcgtg aagaacgtga aaccttcctg 1260 acccagctga ccctggttaa aaacgttctg gaagaaatct ctaaaaccga cccgaacttc 1320 aaaaacctgt acgactcttg ctctcgtctg ctgcagatca tcgacttcta caccgacatc 1380 gttcagaaaa aatacggtgg tgaagaagac tgcgaatgca cccgtgttgg tggt 1434 <210> 4 <211> 478 <212> PRT <213> Artificial sequence <400> 4 Gly Asp Lys Asn Pro Val Gln His Ile Lys Asp Tyr His Ile Asp Ser 1 5 10 15 Val Ser Ser Lys Ala Lys Leu Arg Ile Ile Glu Gly Ile Ile Arg Ala 20 25 30 Ile Ala Lys Ile Gly Phe Lys Val Asp Thr Lys Gln Pro Ile Glu Asp 35 40 45 Ile Leu Lys Asp Ile Lys Lys Gln Leu Pro Asp Pro Arg Ala Gly Ser 50 55 60 Thr Phe Val Lys Asn Ala Glu Lys Gln Glu Thr Val Cys Lys Met Ile 65 70 75 80 Ala Asp Ala Ile Asn Gln Glu Phe Ile Asp Leu Gly Gln Asp Lys Leu 85 90 95 Ile Asp Thr Thr Glu Gly Ala Ala Ser Ile Cys Arg Gln Ile Val Leu 100 105 110 Tyr Ile Asn Ser Leu Thr His Gly Leu Arg Ala Glu Tyr Leu Asp Val 115 120 125 His Gly Ser Ile Glu Asn Thr Leu Glu Asn Ile Lys Leu Leu Asn Asp 130 135 140 Ala Ile Lys Gln Leu His Glu Arg Met Val Thr Glu Val Thr Lys Ala 145 150 155 160 Ala Pro Asn Glu Glu Val Ile Asn Ala Val Thr Met Ile Glu Ala Val 165 170 175 Tyr Arg Arg Leu Leu Asn Glu Gln Asn Leu Gln Ile Asn Ile Leu Thr 180 185 190 Asn Phe Ile Asp Asn Ile Leu Thr Pro Thr Gln Lys Glu Leu Asp Lys 195 200 205 Leu Gln Thr Asp Glu Val Asp Ile Ile Lys Leu Leu Asn Asp Thr Asn 210 215 220 Ser Val Leu Gly Thr Lys Asn Phe Gly Lys Val Leu Ser Tyr Thr Leu 225 230 235 240 Cys Asn Leu Gly Ile Ala Ala Ser Val Ala Asn Lys Ile Asn Lys Ala 245 250 255 Leu Gln Lys Val Gly Leu Lys Val Glu Gln Tyr Leu Gln Ser Lys Asn 260 265 270 Trp Ala Glu Phe Asp Lys Glu Leu Asp Leu Lys Arg Phe Ser Gly Leu 275 280 285 Val Ser Ala Glu Asn Ile Ala Glu Phe Glu Lys Ala Val Asn Leu Leu 290 295 300 Arg Gln Thr Phe Asn Glu Arg His Lys Ile Leu Glu Asn Ser Cys Ala 305 310 315 320 Lys Lys Gly Gly Asp Glu Glu Lys Thr Pro Leu Asp Arg Arg Ile Glu 325 330 335 Ala Gln Arg Leu Asp Arg Lys His Ile Leu Met Glu Phe Leu Asn Lys 340 345 350 Ser Thr Gln Ala Tyr Asn Asp Phe Leu Glu Asn Val Lys Lys Ile Gly 355 360 365 Ile Lys Leu Val Lys Glu Ile Ala Leu Thr Pro Asn Ile Thr Arg Leu 370 375 380 Arg Asp Ala Leu Ser Arg Ile Asn Asp Met Gly Thr Ile Ala Leu Asp 385 390 395 400 Leu Ser Leu Ile Gly Phe Tyr Thr Asn Ala Ala Ala Arg Glu Glu Arg 405 410 415 Glu Thr Phe Leu Thr Gln Leu Thr Leu Val Lys Asn Val Leu Glu Glu 420 425 430 Ile Ser Lys Thr Asp Pro Asn Phe Lys Asn Leu Tyr Asp Ser Cys Ser 435 440 445 Arg Leu Leu Gln Ile Ile Asp Phe Tyr Thr Asp Ile Val Gln Lys Lys 450 455 460 Tyr Gly Gly Glu Glu Asp Cys Glu Cys Thr Arg Val Gly Gly 465 470 475 <210> 5 <211> 5369 <212> DNA <213> Artificial sequence <400> 5 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga cccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggtt tttcgccct tgacgttgg agtccacgtt 300 ctttaatagt ggacttgt tccaactgg aaacactc aaccctatct cggtctattc 360 ttttgattta taaggatt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attackttt acatttcag 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 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100 attaacgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 2160 caccgcatac aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 2220 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 2280 aatagcacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 2340 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 2400 tagaaaagat caaaggatct acgccagcaa cgcggccttt ttacggttcc tgggcttttg 2460 ctggcctttt gctcacatgt ctcttcgcga tgtacgggcc agatatacgc tgtgtcagag 2520 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760 acgagagagg atgctcacga tacgggttac tgatgatgaa cgttactccc acaggtgagc 2820 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggctcgtt 2880 tcttttctgt ggctgcgtga aagccttaaa gggctccggg agggcccttt gtgcgggggg 2940 gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg 3000 ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcgtgtgc 3060 gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg gggggctgcg aggggaacaa 3120 aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg 3180 gctgtaaccc ccccctgcac ccccctcccc gagttgctga gcacggcccg gcttcgggtg 3240 cggggctccg tgcggggcgt ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg 3300 tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg gaggggcgcg 3360 gcggccccgg agcgccggcg gctgtcgagg cgcggcgagc tggccaatgc cctggctcac 3420 aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt 3480 gagcatctga cttctggcta taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980 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 p34-p14 subunit protein, characterized in that, The amino acid sequence of the African swine fever virus p34-p14 subunit protein is shown in SEQ ID NO.

4.

2. A biomaterial related to the African swine fever virus p34-p14 subunit protein as described in claim 1, characterized in that, The biomaterial includes any one of the following: (a) A nucleic acid molecule encoding the p34-p14 subunit protein of the African swine fever virus as described in claim 1; (b) An expression cassette containing the nucleic acid molecule described in (a); (c) A recombinant vector containing the nucleic acid molecule described in (a) or the expression cassette described in (b); (d) Recombinant cells containing the vector described in (c).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule in (a) is shown in SEQ ID NO.

3.

4. The biomaterial according to claim 2, characterized in that, (c) The backbone of the recombinant vector is the pNVC1 vector, whose nucleotide sequence is shown in SEQ ID NO.

5.

5. The biomaterial according to claim 2, characterized in that, (d) The recombinant cells are Escherichia coli.

6. The use of the African swine fever virus p34-p14 subunit protein of claim 1 or the biological material of any one of claims 2 to 5 in the preparation of a medicament for preventing African swine fever virus infection.

7. An African swine fever virus subunit vaccine composition, characterized in that, It includes the African swine fever virus p34-p14 subunit protein as described in claim 1 and a pharmaceutically acceptable vector.

8. The African swine fever virus subunit vaccine composition according to claim 7, characterized in that, The content of the African swine fever p34-p14 subunit protein is ≥100μg / ml.

9. The African swine fever virus subunit vaccine composition according to claim 8, characterized in that, The content of the African swine fever p34-p14 subunit protein is 100~300μg / ml.

10. The African swine fever virus subunit vaccine composition according to claim 7, characterized in that, The pharmaceutically acceptable carriers include at least one of adjuvants, lyophilization protectants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.

11. The African swine fever virus subunit vaccine composition according to claim 7, characterized in that, Pharmaceutically acceptable carriers include immunostimulants.

12. The African swine fever virus subunit vaccine composition according to claim 10, characterized in that, The adjuvants include: (1) mineral oil, aluminum gel adjuvant, saponins, avrididine, 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 at least one of the following: RIBI adjuvant system, Block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, and gel adjuvant.

13. The African swine fever virus subunit vaccine composition according to claim 12, characterized in that, The saponins are QuilA, QS-21, and GPI-0100.

14. The African swine fever virus subunit vaccine composition according to claim 12, characterized in that, The adjuvant content is 5%-60% V / V.

15. The African swine fever virus subunit vaccine composition according to claim 14, characterized in that, The adjuvant content is 30%-60% V / V.

16. The African swine fever virus subunit vaccine composition according to claim 15, characterized in that, The adjuvant content is 50% V / V.

17. The African swine fever virus subunit vaccine composition according to claim 10, characterized in that, The adjuvant is 50% v / v Montanide ISA 206 adjuvant.

18. The African swine fever virus subunit vaccine composition according to claim 10, characterized in that, The freeze-drying protectant is selected from sugars, polyols, salts, amines, or amino acids.

19. The African swine fever virus subunit vaccine composition according to claim 11, characterized in that, The immunostimulants include α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and interleukin-2.

20. The African swine fever virus subunit vaccine composition according to any one of claims 7 to 19, characterized in that, The dosage form is selected from solution-type injections, suspension-type injections, powders for injection, sustained-release microspheres, controlled-release microspheres, or sustained-release implants.

21. The African swine fever virus subunit vaccine composition according to claim 20, characterized in that, The dosage form is selected from solution injection, suspension injection, or powder for injection.

22. The African swine fever virus subunit vaccine composition according to claim 21, characterized in that, The dosage form is selected from solution injection or suspension injection.

23. The African swine fever virus subunit vaccine composition according to claim 20, characterized in that, The route of administration can be selected from subcutaneous injection, oral administration, oral administration, nasal administration, pulmonary administration, colonic administration, rectal administration, or transdermal administration.

24. The African swine fever virus subunit vaccine composition according to claim 23, characterized in that, The administration method is subcutaneous injection.

25. The African swine fever virus subunit vaccine composition according to claim 23, characterized in that, The administration method is sublingual administration.

26. A method for preparing the African swine fever virus subunit vaccine composition according to any one of claims 7 to 25, characterized in that, A recombinant vector was constructed using a nucleic acid molecule encoding the African swine fever virus p34-p14 subunit protein as described in claim 1. The recombinant vector was introduced into expression cells for expression, and the African swine fever virus p34-p14 subunit protein was obtained after purification. The protein was then mixed with a pharmaceutically acceptable vector to obtain an African swine fever virus subunit vaccine composition.

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