African swine fever virus subunit vaccine compositions, methods of making and use thereof

Abstract

This invention provides an African swine fever virus subunit vaccine composition, the protein antigen of which consists of African swine fever virus p34, p14, C129R, DP96R, A104R, p54, p22, p72, and p30 proteins. The subunit vaccine composition also includes a pharmaceutically acceptable carrier. The antigenic components of this subunit vaccine composition exhibit a synergistic effect, resulting in a stronger immune response and higher antibody titers.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, specifically relating to African swine fever virus subunit vaccine compositions, preparation methods, and their applications. Background Technology

[0002] 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.

[0003] Therefore, there is an urgent clinical need for an African swine fever vaccine that has good immunogenicity, can effectively protect pigs, and poses no biosecurity risks. Summary of the Invention

[0004] Therefore, this invention uses modern biological methods to analyze the genes and proteins of pathogens and test their immune protection capabilities, thereby developing subunit vaccines and effectively solving the problem of unsatisfactory immunization effects of various vaccines currently available.

[0005] This 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.

[0006] The present invention also provides the use of the African swine fever virus subunit vaccine composition, wherein the use refers to its use in the preparation of a medicament for the prevention of African swine fever virus infection.

[0007] The African swine fever virus subunit vaccine of the present invention uses multi-component African swine fever virus protein antigens. The multi-component African swine fever virus protein antigens are administered together, which synergistically enhances the effect and improves the immunization efficacy, effectively solving the problem of poor immunogenicity faced by current African swine fever virus vaccines. The vaccine composition can be expressed in large quantities through genetic engineering, which is not only time-saving but also facilitates large-scale production without biosafety risks. Detailed Implementation

[0008] The embodiments of the present invention will be described below.

[0009] definition

[0010] 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 nucleocapsid containing a nucleoid, 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 that could effectively control the spread of the virus during an outbreak.

[0011] 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.

[0012] The term "African swine fever virus C129R protein" refers to manganese-dependent superoxide dismutase.

[0013] The term "African swine fever virus DP96R protein" refers to the protein that inhibits type I IFN expression and NF-κB activation, also known as "pDP96R protein".

[0014] The term "African swine fever virus A104R protein" refers to a DNA-binding protein that participates in viral transcription, DNA replication, and genome packaging; it is also known as "pA104R protein".

[0015] The term "African swine fever virus p54 protein" refers to an early membrane protein expressed by African swine fever virus, encoded by the OR FE183L gene. It contains a transmembrane domain, is located in the endoplasmic reticulum-derived inner membrane precursor, and plays an important role in viral adsorption to susceptible cells and invasion. It is an important structural protein.

[0016] The term "African swine fever virus p22 protein" refers to the transmembrane domain of the African swine fever virus, located on the outside of the virus particle.

[0017] The term "African swine fever virus P72 protein" refers to an important antigenic protein of African swine fever virus produced in the late stage of viral infection, encoded by the ORF B646L gene. It is a major component of the icosahedron of the virus and is crucial for the formation of the viral capsid.

[0018] The term "African swine fever virus p30 protein" refers to the early membrane protein expressed by African swine fever virus, encoded by the ORFCP204L gene. It is usually produced 2-4 hours after infection and is continuously expressed throughout the infection period. It is involved in viral invasion of host cells and is an important structural protein.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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 in all components of the vaccine composition of the present invention, except for the African swine fever virus protein antigen. 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. 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.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. Carbopol 974P, 934P, and 971P may be mentioned, with Carbopol 971P being the most preferred. The term "copolymer of maleic anhydride and alkenyl derivatives" may also refer to the copolymer of maleic anhydride and ethylene, EMA (Monsanto), which dissolves in water to produce an acidic solution, which is neutralized, preferably to physiological pH, to produce an adjuvant solution into which immunogenic, immunizing, or vaccine compositions 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 other), cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvant, etc. Preferably, the adjuvant comprises 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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. Invention Details

[0030] This 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 African swine fever virus p34, p14, C129R, DP96R, A104R, p54, p22, p72, and p30 proteins, and the African swine fever virus subunit vaccine composition further includes a pharmaceutically acceptable vector; wherein the African swine fever virus p34 protein is encoded by SEQ ID NO.1 or its degenerate sequence; the African swine fever virus p14 protein is encoded by SEQ ID NO.2 or its degenerate sequence; the African swine fever virus C129R protein is encoded by SEQ ID NO.3 or its degenerate sequence; the African swine fever virus DP96R protein is encoded by SEQ ID NO.4 or its degenerate sequence; the African swine fever virus A104R protein is encoded by SEQ ID NO.5 or its degenerate sequence; the African swine fever virus p54 protein is encoded by SEQ ID NO.6 or its degenerate sequence; and the African swine fever virus p22 protein is encoded by SEQ ID NO. The African swine fever virus p72 protein is encoded by SEQ ID NO. 7 or its degenerate sequence; and the African swine fever virus p30 protein is encoded by SEQ ID NO. 9 or its degenerate sequence.

[0031] According to a preferred embodiment, the African swine fever virus p34 protein is encoded by SEQ ID NO.1; the African swine fever virus p14 protein is encoded by SEQ ID NO.2; the African swine fever virus C129R protein is encoded by SEQ ID NO.3; the African swine fever virus DP96R protein is encoded by SEQ ID NO.4; the African swine fever virus A104R protein is encoded by SEQ ID NO.5; the African swine fever virus p54 protein is encoded by SEQ ID NO.6; the African swine fever virus p22 protein is encoded by SEQ ID NO.7; the African swine fever virus P72 protein is encoded by SEQ ID NO.8; and the African swine fever virus p30 protein is encoded by SEQ ID NO.9.

[0032] According to a preferred embodiment, those skilled in the art can select other degenerate sequences encoding the African swine fever virus p34, p14, C129R, DP96R, A104R, p54, p22, p72, and p30 proteins based on conventional technical knowledge. Other degenerate sequences can be selected based on the preferred codons of the specific expression system.

[0033] According to a preferred embodiment, the African swine fever virus p34, p14, C129R, DP96R, A104R, and p54 proteins are expressed by an Escherichia coli expression system, and their coding sequences are selected according to the preferred codons of the Escherichia coli expression system.

[0034] According to a preferred embodiment, the African swine fever virus p22, p72, and p30 proteins are expressed by the sf9 insect cell expression system, and their coding sequences are selected according to the preferred codons of the sf9 insect cell expression system.

[0035] According to 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, and sustained-release implant.

[0036] 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 an injection powder.

[0037] According to a preferred embodiment, the African swine fever virus subunit vaccine composition can be administered via subcutaneous injection, oral administration, oral administration, sublingual administration, nasal administration, pulmonary administration, colonic administration, rectal administration, or transdermal administration.

[0038] According to a preferred embodiment, the African swine fever virus subunit vaccine composition is administered via subcutaneous injection.

[0039] According to a preferred embodiment, the African swine fever virus subunit vaccine composition is in the form of a solution injection or a suspension injection; the African swine fever virus p34 protein content is ≥8 μg / ml; the African swine fever virus p14 protein content is ≥8 μg / ml; the African swine fever virus C129R protein content is ≥8 μg / ml; the African swine fever virus DP96R protein content is ≥8 μg / ml; the African swine fever virus A104R protein content is ≥8 μg / ml; the African swine fever virus p54 protein content is ≥12 μg / ml; the African swine fever virus p22 protein content is ≥12 μg / ml; the African swine fever virus P72 protein content is ≥12 μg / ml; and the African swine fever virus p30 protein content is ≥12 μg / ml.

[0040] According to a preferred embodiment, the African swine fever virus (ASFV) p34 protein content is 8-24 μg / ml; the ASFV p14 protein content is 8-24 μg / ml; the ASFV C129R protein content is 8-24 μg / ml; the ASFV DP96R protein content is 8-24 μg / ml; the ASFV A104R protein content is 8-24 μg / ml; the ASFV p54 protein content is 12-36 μg / ml; the ASFV p22 protein content is 12-36 μg / ml; the ASFV P72 protein content is 12-36 μg / ml; and the ASFV p30 protein content is 12-36 μg / ml.

[0041] According to a preferred embodiment, the content of the African swine fever virus p34 protein is selected from 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, and 24 μg / ml. The African swine fever virus p14 protein content is selected from 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, or 24 μg / ml. The African swine fever virus C129R protein content is selected from 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, or 24 μg / ml. The African swine fever virus DP96R protein content was selected from 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, and 24 μg / ml. The A104R protein content of the African swine fever virus is selected from 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, and 24 μg / ml. The African swine fever virus p54 protein content was selected from 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml, 26 μg / ml, 27 μg / ml, 28 μg / ml, 29 μg / ml, 30 μg / ml, 31 μg / ml, 32 μg / ml, 33 μg / ml, 34 μg / ml, 35 μg / ml, and 36 μg / ml.The African swine fever virus p22 protein content is selected from 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml, 26 μg / ml, 27 μg / ml, 28 μg / ml, 29 μg / ml, 30 μg / ml, 31 μg / ml, 32 μg / ml, 33 μg / ml, 34 μg / ml, 35 μg / ml, or 36 μg / ml. The African swine fever virus P72 protein content is selected from 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml, 26 μg / ml, 27 μg / ml, 28 μg / ml, 29 μg / ml, 30 μg / ml, 31 μg / ml, 32 μg / ml, 33 μg / ml, 34 μg / ml, 35 μg / ml, or 36 μg / ml. The African swine fever virus p30 protein content is selected from 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, 21 μg / ml, 22 μg / ml, 23 μg / ml, 24 μg / ml, 25 μg / ml, 26 μg / ml, 27 μg / ml, 28 μg / ml, 29 μg / ml, 30 μg / ml, 31 μg / ml, 32 μg / ml, 33 μg / ml, 34 μg / ml, 35 μg / ml, or 36 μg / ml.

[0042] According to a preferred embodiment, the African swine fever virus (ASFV) p34 protein content is 8-16 μg / ml; the ASFV p14 protein content is 8-16 μg / ml; the ASFV C129R protein content is 8-16 μg / ml; the ASFV DP96R protein content is 8-16 μg / ml; the ASFV A104R protein content is 8-16 μg / ml; the ASFV p54 protein content is 12-24 μg / ml; the ASFV p22 protein content is 12-24 μg / ml; the ASFV P72 protein content is 12-24 μg / ml; and the ASFV p30 protein content is 12-24 μg / ml.

[0043] According to a preferred embodiment, the African swine fever virus p34 protein, p14 protein, C129R protein, DP96R protein, A104R protein, p54 protein, p22 protein, and p30 protein are all selected in lower amounts. Even if the total amount of African swine fever virus protein antigen is reduced compared to the amount of single-component antigen, a higher antibody titer can be obtained, ensuring a better immunization effect.

[0044] According to a preferred embodiment, the African swine fever virus (ASFV) p34 protein content is 8 μg / ml; the ASFV p14 protein content is 8 μg / ml; the ASFV C129R protein content is 8 μg / ml; the ASFV DP96R protein content is 8 μg / ml; the ASFV A104R protein content is 8 μg / ml; the ASFV p54 protein content is 12 μg / ml; the ASFV p22 protein content is 12 μg / ml; the ASFV P72 protein content is 12 μg / ml; and the ASFV p30 protein content is 12 μg / ml.

[0045] According to a preferred embodiment, the African swine fever virus (ASFV) p34 protein content is 16 μg / ml; the African swine fever virus (ASFV) p14 protein content is 16 μg / ml; the African swine fever virus (ASFV) C129R protein content is 16 μg / ml; the African swine fever virus (ASFV) DP96R protein content is 16 μg / ml; the African swine fever virus (ASFV) A104R protein content is 16 μg / ml; the African swine fever virus (ASFV) p54 protein content is 24 μg / ml; the African swine fever virus (ASFV) p22 protein content is 24 μg / ml; the African swine fever virus (ASFV) P72 protein content is 24 μg / ml; and the African swine fever virus (ASFV) p30 protein content is 24 μg / ml.

[0046] According to a preferred embodiment, the African swine fever virus subunit vaccine composition of the present invention contains an African swine fever virus protein antigen content of 88-264 μg / ml.

[0047] The African swine fever virus protein content can be selected from 88μg / ml, 90μg / ml, 100μg / ml, 110μg / ml, 120μg / ml, 130μg / ml, 140μg / ml, 150μg / ml, 160μg / ml, 170μg / ml, 180μg / ml, 190μg / ml, 200μg / ml, 210μg / ml, 220μg / ml, 230μg / ml, 240μg / ml, 250μg / ml, 260μg / ml, and 264μg / ml.

[0048] According to a preferred embodiment, the pharmaceutically acceptable carrier includes an adjuvant, which 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, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive 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.

[0049] According to a preferred embodiment, the adjuvant is Montanide ISA 206 adjuvant, and the adjuvant content is 50% V / V.

[0050] According to a preferred embodiment, the pharmaceutically acceptable carrier includes a lyophilization protectant selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

[0051] According to a preferred embodiment, the pharmaceutically acceptable carrier includes a drug, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative; the immunostimulant includes α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-2 (IL2).

[0052] To prepare such a composition, methods known in the art can be used.

[0053] The present invention also provides the application of the African swine fever virus subunit vaccine composition, wherein the application refers to its use in the preparation of a medicament for the prevention of African swine fever virus infection.

[0054] According to a preferred embodiment, the African swine fever virus subunit vaccine composition can exert a synergistic immune protective effect and has a better immune effect on pigs.

[0055] This invention also provides a method for preparing an African swine fever virus subunit vaccine composition, the method comprising:

[0056] (1) The African swine fever virus protein gene was artificially synthesized and linked to a cloning vector, i.e., a protein cloning vector, by genetic engineering means;

[0057] (2) The constructed protein cloning vector and expression vector are digested with enzymes to construct an expression vector containing the protein gene, i.e., the protein expression vector;

[0058] (3) The expression vector of the protein is introduced into the recipient bacteria for induced expression, and the expressed protein is identified to obtain African swine fever virus protein;

[0059] (4) Mix the obtained African swine fever virus protein with a pharmaceutically acceptable carrier to obtain an African swine fever virus subunit vaccine composition.

[0060] As one embodiment of the present invention, the African swine fever virus protein of the present invention can be prepared by a prokaryotic expression system, or by a eukaryotic expression system or by chemical synthesis method.

[0061] According to a preferred embodiment, the expression vector for the African swine fever virus p34, p14, C129R, DP96R, A104R, and p54 protein genes is an Escherichia coli expression system cloning vector.

[0062] According to a preferred embodiment, the expression vector for the African swine fever virus p22, p72, and p30 protein genes is an sf9 insect cell expression system expression vector.

[0063] The present invention also provides an expression vector containing the gene sequence of the African swine fever virus protein.

[0064] According to a preferred embodiment, the expression vector for the African swine fever virus p34, p14, C129R, DP96R, A104R, and p54 protein genes is an Escherichia coli expression system cloning vector.

[0065] According to a preferred embodiment, the expression vector for the African swine fever virus p22, p72, and p30 protein genes is an sf9 insect cell expression system expression vector.

[0066] 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.

[0067] 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.

[0068] Example 1: Prokaryotic expression of African swine fever virus p34, p14, C129R, DP96R, A104R, and p54 proteins.

[0069] The nucleotide sequence of the expression vector shown in SEQ ID NO.10 was synthesized by Suzhou Genewise Biotechnology Co., Ltd., and named pNVC1 vector.

[0070] The nucleotide sequences of the corresponding coding sequences p34, p14, C129R, DP96R, A104R, and p54 proteins shown in SEQ ID NO.1 to SEQ ID NO.6 were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the synthesized nucleotides were cloned into the pNVC1 vector and named recombinant plasmids pNVC1-p34, pNVC1-p14, pNVC1-C129R, pNVC1-DP96R, pNVC1-A104R, and pNVC1-p54.

[0071] The recombinant plasmids pNVC1-p34, pNVC1-p14, pNVC1-C129R, pNVC1-DP96R, pNVC1-A104R, and pNVC1-p54 were transformed into competent Escherichia coli BL21(DE3) to construct expression strains. After inoculating 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.

[0072] 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.

[0073] The bacterial cells were resuspended and homogenized three times at 800 bar. After centrifugation at 13500 rpm for 40 min, the supernatant was collected and analyzed by 12% SDS-PAGE electrophoresis. Crude protein purification was performed using ammonium sulfate fractionation, followed by chromatographic purification. SDS-PAGE electrophoresis of the purified proteins showed that the target proteins were purified and enriched.

[0074] Example 2: Eukaryotic expression of African swine fever virus p22, p72, and p30 proteins

[0075] The nucleotide sequences of the p22, p72, and p30 proteins shown in SEQ ID NO.7~SEQ ID NO.9 were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the synthesized nucleotides were cloned into the pFB vector and named recombinant plasmids pFB-p22, pFB-p72, and pFB-p30.

[0076] The recombinant plasmids pFB-p22, pFB-p72, and pFB-p30 were transformed into DH10Bac competent cells, respectively. After blue-white screening, single white colonies were picked and cultured overnight in liquid LB medium containing kanamycin, tetracycline, and gentamicin. Recombinant Bacmids were extracted according to the Bac-to-Bac instructions and named Bac-p22, Bac-p72, and Bac-p30, respectively.

[0077] Following the instructions of the Cellfectin II Reagent transfection kit, recombinant Bacmids Bac-p22, Bac-p72, and Bac-p30 were transfected into sf9 insect cells and incubated at 27 ℃ for approximately 72 h. After significant cytopathic effects were observed, the cell supernatant was harvested; this was identified as P1 generation recombinant baculovirus and labeled rBac-p22, rBac-p72, and rBac-p30, respectively. The P1 generation recombinant baculovirus was added to sf9 cell shake flasks at a volume ratio of 1:20–1:40, and the cells were incubated at 27 ℃ until approximately 72 h of cytopathic effects were observed. The supernatant was then harvested and labeled as P2 generation recombinant baculovirus. P2 generation recombinant baculovirus was inoculated into 1 L of sf9 cells at a volume ratio of 1:100. Cells were harvested 48–72 h after inoculation and collected by centrifugation at 1000 × g for 10 min.

[0078] Cell pellets were lysed with cell lysis buffer (25 mM NaHCO3, pH 8.3) for 30 minutes, centrifuged at 10000×g at 4°C for 10 minutes to obtain the lysate supernatant, and the expression of the target protein was confirmed by Western blotting. The protein was crudely purified by nickel affinity chromatography, followed by molecular sieve purification. SDS-PAGE electrophoresis of the purified protein showed that the target protein was purified and enriched.

[0079] Example 3: Preparation of African swine fever virus subunit vaccine composition

[0080] The p34, p14, C129R, DP96R, A104R, and p54 proteins prepared in Example 1, and the p22, p72, and p30 proteins prepared in Example 2, were added to the adjuvant. During the addition process, the mixture was continuously stirred in an emulsifier at 800 rpm for 12 minutes until homogeneous. The mixture was then stored at 4°C to obtain the subunit vaccine composition containing the multi-component protein antigen of African swine fever virus. 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 (Sepic, France). The specific proportions of each component in the prepared vaccine are shown in Table 1.

[0081] Table 1. Composition ratio of the African swine fever virus subunit vaccine composition of the present invention

[0082]

[0083] Example 4 Immunogenicity test of African swine fever virus subunit vaccine composition

[0084] 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 2 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.

[0085] 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.

[0086] 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℃.

[0087] 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.

[0088] Secondary antibody: Enzyme-labeled goat anti-pig secondary antibody, diluted 1:2000 before use.

[0089] 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.

[0090] Termination solution: 2M H2SO4.

[0091] For the assay, first add 90 μl of sample diluent to each well, then add 10 μl of 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, and wash the plate three times with washing buffer. Add 50 μl / well each of chromogenic reagent A and chromogenic reagent B, incubate at 37°C for 10 minutes, and then add 50 μl / well of stop solution to terminate the reaction. Read the absorbance (OD) using an ELISA reader. 450nm The determination will be made based on the judgment result.

[0092] Judgment criteria: OD value ≥ 0.19 is positive, OD value < 0.19 is negative.

[0093] The test results are shown in Table 2.

[0094] Table 2. Immunogenicity test results of African swine fever virus subunit vaccine compositions

[0095]

[0096] 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 multi-component African swine fever virus protein antigens, has good immunizing efficacy.

[0097] Example 5: Validation test of the immunogenicity of African swine fever virus protein

[0098] 1. Preparation of African swine fever virus single-component subunit vaccine composition

[0099] The p34, p14, C129R, DP96R, A104R, and p54 proteins prepared in Example 1, and the p22, p72, and p30 proteins prepared in Example 2, were added to the adjuvant. During the addition process, the mixture was continuously stirred in an emulsifier at 800 rpm for 12 minutes until homogeneous. The mixture was then stored at 4°C to obtain the subunit vaccine composition containing single-component protein antigens of African swine fever virus. 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) is selected, for example, adjuvant ISA 206 (Sepic, France). The specific proportions of each component in the prepared vaccine are shown in Tables 3 and 4.

[0100] Table 3. Composition Ratio of African Swine Fever Virus Single-Component Subunit Vaccine Composition 1

[0101]

[0102] Table 4. Composition Ratio of African Swine Fever Virus Single-Component Subunit Vaccine Composition 2

[0103]

[0104] 2. Immunogenicity test of African swine fever virus single-component subunit vaccine composition

[0105] Fifty healthy, susceptible piglets weighing approximately 20 kg and negative for both African swine fever virus antigen and antibody were randomly divided into 10 groups of 5 piglets each. Groups 5-13 were immunized with vaccines 4-12, respectively, while group 14 served as a blank control group. The immunization routes for the groups were intramuscular injection of 2 ml 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.

[0106] Referring to the preparation method of the African swine fever virus antibody detection kit in Example 4, enzyme-linked immunosorbent assay (ELISA) plates were coated with African swine fever virus p34 protein, p14 protein, C129R protein, DP96R protein, A104R protein, p54 protein, p22 protein, p72 protein, and p30 protein to prepare the African swine fever virus antibody detection kit.

[0107] The test results are shown in Table 5.

[0108] Table 5. Immunogenicity test results of African swine fever virus single-component subunit vaccine compositions 1

[0109]

[0110] Table 6. Immunogenicity test results of African swine fever virus single-component subunit vaccine compositions 2

[0111]

[0112] Table 7. Immunogenicity test results of African swine fever virus single-component subunit vaccine compositions 3

[0113]

[0114] Table 8. Immunogenicity test results of African swine fever virus single-component subunit vaccine compositions 4

[0115]

[0116] Table 9. Immunogenicity test results of African swine fever virus single-component subunit vaccine compositions.

[0117]

[0118] Table 10 Immunogenicity Test Results of African Swine Fever Virus Single-Component Subunit Vaccine Compositions 6

[0119]

[0120] Table 11 Immunogenicity Test Results of African Swine Fever Virus Single-Component Subunit Vaccine Compositions 7

[0121]

[0122] Table 12 Immunogenicity Test Results of African Swine Fever Virus Single-Component Subunit Vaccine Compositions 8

[0123]

[0124] Table 13 Immunogenicity Test Results of African Swine Fever Virus Single-Component Subunit Vaccine Compositions 9

[0125]

[0126] The results showed that all single-component subunit African swine fever virus vaccine compositions induced varying degrees of immune responses, and antibody tests were positive in all cases. This indicates that the African swine fever virus protein antigens used in the African swine fever virus subunit vaccines of the present invention are all immunogenic, and the multi-component subunit African swine fever virus vaccines prepared in this manner have better immunizing effects and higher antibody levels.

[0127] Comparing the OD values ​​detected in Tables 2 and 5-13, the African swine fever virus subunit vaccine composition of the present invention, when the total amount of antigen used (i.e., the total content of each antigen protein in the African swine fever virus subunit vaccine composition of the present invention in an immunization dose of 2 ml) is only 176 μg (vaccine 1) or 352 μg (vaccine 2), has a lower detected OD value. 400nmThe values ​​were 2.016 and 2.448, respectively. The antigen content (i.e., the antigen protein content in a 2ml immunization dose of the African swine fever virus single-component subunit vaccine composition) detected in Tables 5-13 was 352 μg, which was higher or comparable to the total antigen content of vaccine 1 or vaccine 2. Its detected OD... 400nm The values ​​are all much smaller than the antibody OD in serum after immunization with vaccine 1 or vaccine 2. 400nm The value indicates that the various antigenic components in the African swine fever virus subunit vaccine composition of the present invention have a synergistic effect, which can produce a stronger immune response and a higher antibody titer.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the technical solution of the present invention. Any simple modifications, equivalent changes and alterations 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 shall still fall within the scope of the technical solution of the present invention. SEQUENCE LISTING <110> Pulike Biotechnology Co., Ltd. <120> African swine fever virus subunit vaccine composition, its preparation method and application <160> 10 <170> PatentIn version 3.3 <210> 1 <211> 975 <212> DNA <213> African swine fever virus <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 tctggaaaa ctcttgcgct 960 aaaaaaggtg gttaa 975 <210> 2 <211> 465 <212> DNA <213> African swine fever virus <400> 2 gacgaagaaa aaaccccgct ggaccgtcgt atcgaagctc agcgtctgga ccgtaaacac 60 atcctgatgg aattcctgaa caaatctacc caggcttaca acgacttcct ggaaaacgtt 120 aaaaaaatcg gtatcaaact gttaaagaa atcgctctga ccccgaacat cacccgtctg 180 cgtgacgctc tgtctcgtat caacgacatg ggtaccatcg ctctggacct gtctctgatc 240 ggttctaca ccaacgctgc tgctcgtgaa gaacgtgaaa ccttcctgac ccagctgacc 300 ctggttaaaa acgttctgga agaaatctct aaaccgacc cgaacttcaa aaacctgtac 360 gactcttgct ctcgtctgct gcagatcatc gacttctaca ccgacatcgt tcagaaaaaa 420 tacggtggtg aagaagactg cgaatgcacc cgtgttggtg gttaa 465 <210> 3 <211> 390 <212> DNA <213> African swine fever virus <400> 3 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 390 <210> 4 <211> 291 <212> DNA <213> African swine fever virus <400> 4 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> 5 <211> 315 <212> DNA <213> African swine fever virus <400> 5 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> 6 <211> 399 <212> DNA <213> African swine fever virus <400> 6 tcttctcgta aaaaaaaagc tgctgctatc gaagaagaag acatccagtt catcaacccg 60 taccaggacc agcagtgggt tgaagttacc ccgcagccgg gtacctctaa accggctggt 120 gctaccaccg cttctgttgg taaaccggtt accggtcgtc cggctaccaa ccgtccggct 180 accaacaaac cggttaccga caacccggtt accgaccgtc tggttatggc taccggtggt 240 ccggctgctg ctccggctgc tgcttctgct ccggctcacc cggctgaacc gtacaccacc 300 gttaccaccc agaacaccgc ttctcagacc atgtctgcta tcgaaaacct gcgtcagcgt 360 aacacctaca cccacaaaga cctggaaaac tctctgtaa 399 <210> 7 <211> 594 <212> DNA <213> African swine fever virus <400> 7 atgagatcct ccaagaagat caacaacaag aagaacatgt tcaacatcaa gatgactatc 60 tccactctgc tgatcgctct gatcatcctg ctgatcatta tcctggtggt gttcctgtac 120 tacaagaagc agcagccacc aaagaaggtg tgcaaggtgg acaaggactg cggttccggt 180 gagcactgcg tgagaggttc ctgctcctcc ctgtcctgcc tggacgctgt gaagatggac 240 aagagaaaca tcaagatcga ctccaagatc tcctcctgcg agttcactcc aaacttctac 300 agattcactg acactgctgc tgacgagcag caagagttcg gtaagactag acacccaatc 360 aagatcactc catccccatc cgagtcccac tccccacaag aggtgtgcga gaagtactgc 420 tcctggggta ctgacgactg cactggttgg gagtacgtgg gtgacgagaa ggagggtact 480 tgctacgtgt acaacaaccc acaccaccct gtgctgaagt acggtaagga ccacatcatc 540 gctctgccaa gaaaccacaa gcacgctcac caccatcacc accaccacca ctaa 594 <210> 8 <211> 1965 <212> DNA <213> African swine fever virus <400> 8 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> 9 <211> 630 <212> DNA <213]] African swine fever virus <400> 9 atggacttca tcctgaacat cagcatgaag atggaagtga tcttcaagac cgacctgcgt 60 agcagcagcc aggtggtgtt ccacgctggt agcctgtaca actggttcag cgtggagatc 120 atcaacagcg gtcgcatcgt gaccaccgct atcaagaccc tgctgagcac cgtgaagtac 180 gacatcgtga agtccgctcg tatctacgcc ggccagggtt acaccgagca ccaggcccag 240 gaagaatgga acatgatcct gcacgtgctg ttcgaagaag aaaccgagag ctccgcttcc 300 tccgaaaaca tccacgagaa gaacgacaac gaaaccaacg agtgtaccag ctccttcgaa 360 accctgtcg aacaggagcc ttcctccgag gtgcctaagg acagcaagct gtacatgctg 420 gcccagaaga ccgtgcagca catcgaacag tacggcaagg ctcctgactt caacaaggtt 480 atccgcgctc acaacttcat ccagaccatc tacggtaccc ctctgaagga agaggaaaag 540 gaagtggtgc gtctgatggt tatcaagctg ctgaagaaga tcagctttct cctgacctac 600 atccaccacc accatcacca ccaccactaa 630 <210> 10 <211> 5369 <212> DNA <213> BL21(DE3)(E.coli BL21(DE3)) <400> 10 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgct tcttcccttc 120 cttctcgcc acgtcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt tttcgccct ttgacgttgg agtccacgtt 300 cttaatagt ggactcttgt tccaaactgg aacaacactc 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 attackacaac aggaatcgaa tgcaccggc gcaggacc tgccagcgca tcacaat 1020 ttcacctga atcaggatat tcttctaata cctggaatgc tgttcccg 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 subunit vaccine composition, wherein, The protein antigens of the African swine fever virus subunit vaccine composition consist of African swine fever virus p34, p14, C129R, DP96R, A104R, p54, p22, p72, and p30 proteins, and the African swine fever virus subunit vaccine composition also includes a pharmaceutically acceptable carrier. The African swine fever virus (ASFV) p34 protein is encoded by SEQ ID NO.1 or its degenerate sequence; the ASFV p14 protein is encoded by SEQ ID NO.2 or its degenerate sequence; the ASFV C129R protein is encoded by SEQ ID NO.3 or its degenerate sequence; the ASFV DP96R protein is encoded by SEQ ID NO.4 or its degenerate sequence; the ASFV A104R protein is encoded by SEQ ID NO.5 or its degenerate sequence; the ASFV p54 protein is encoded by SEQ ID NO.6 or its degenerate sequence; the ASFV p22 protein is encoded by SEQ ID NO.7 or its degenerate sequence; the ASFV P72 protein is encoded by SEQ ID NO.8 or its degenerate sequence; and the ASFV p30 protein is encoded by SEQ ID NO.9 or its degenerate sequence.

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

3. The African swine fever virus subunit vaccine composition according to claim 1, wherein, The African swine fever virus subunit vaccine composition is available in the form of a solution injection or a suspension injection; the African swine fever virus p34 protein content is ≥8 μg / ml; the African swine fever virus p14 protein content is ≥8 μg / ml; the African swine fever virus C129R protein content is ≥8 μg / ml; the African swine fever virus DP96R protein content is ≥8 μg / ml; the African swine fever virus A104R protein content is ≥8 μg / ml; the African swine fever virus p54 protein content is ≥12 μg / ml; the African swine fever virus p22 protein content is ≥12 μg / ml; the African swine fever virus P72 protein content is ≥12 μg / ml; and the African swine fever virus p30 protein content is ≥12 μg / ml.

4. The African swine fever virus subunit vaccine composition according to claim 1, wherein, The African swine fever virus (ASFV) p34 protein content is 8-24 μg / ml; the African swine fever virus (ASFV) p14 protein content is 8-24 μg / ml; the African swine fever virus (ASFV) C129R protein content is 8-24 μg / ml; the African swine fever virus (ASFV) DP96R protein content is 8-24 μg / ml; the African swine fever virus (ASFV) A104R protein content is 8-24 μg / ml; the African swine fever virus (ASFV) p54 protein content is 12-36 μg / ml; the African swine fever virus (ASFV) p22 protein content is 12-36 μg / ml; the African swine fever virus (ASFV) P72 protein content is 12-36 μg / ml; and the African swine fever virus (ASFV) p30 protein content is 12-36 μg / ml.

5. The African swine fever virus subunit vaccine composition according to claim 1, wherein, The African swine fever virus (ASFV) p34 protein content is 8-16 μg / ml; the African swine fever virus (ASFV) p14 protein content is 8-16 μg / ml; the African swine fever virus (ASFV) C129R protein content is 8-16 μg / ml; the African swine fever virus (ASFV) DP96R protein content is 8-16 μg / ml; the African swine fever virus (ASFV) A104R protein content is 8-16 μg / ml; the African swine fever virus (ASFV) p54 protein content is 12-24 μg / ml; the African swine fever virus (ASFV) p22 protein content is 12-24 μg / ml; the African swine fever virus (ASFV) P72 protein content is 12-24 μg / ml; and the African swine fever virus (ASFV) p30 protein content is 12-24 μg / ml.

6. The African swine fever virus subunit vaccine composition according to claim 1, wherein, The African swine fever virus (ASFV) p34 protein content is 8 μg / ml; the African swine fever virus (ASFV) p14 protein content is 8 μg / ml; the African swine fever virus (ASFV) C129R protein content is 8 μg / ml; the African swine fever virus (ASFV) DP96R protein content is 8 μg / ml; the African swine fever virus (ASFV) A104R protein content is 8 μg / ml; the African swine fever virus (ASFV) p54 protein content is 12 μg / ml; the African swine fever virus (ASFV) p22 protein content is 12 μg / ml; the African swine fever virus (ASFV) P72 protein content is 12 μg / ml; and the African swine fever virus (ASFV) p30 protein content is 12 μg / ml.

7. The African swine fever virus subunit vaccine composition according to claim 1, wherein, The African swine fever virus (ASFV) p34 protein content is 16 μg / ml; the African swine fever virus (ASFV) p14 protein content is 16 μg / ml; the African swine fever virus (ASFV) C129R protein content is 16 μg / ml; the African swine fever virus (ASFV) DP96R protein content is 16 μg / ml; the African swine fever virus (ASFV) A104R protein content is 16 μg / ml; the African swine fever virus (ASFV) p54 protein content is 24 μg / ml; the African swine fever virus (ASFV) p22 protein content is 24 μg / ml; the African swine fever virus (ASFV) P72 protein content is 24 μg / ml; and the African swine fever virus (ASFV) p30 protein content is 24 μg / ml.

8. The African swine fever virus subunit vaccine composition according to claim 1, wherein, The pharmaceutically acceptable carriers include adjuvants, which 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 one or more 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 ISA206, and gel adjuvant. The adjuvant content is 5%-60% V / V; Alternatively, the pharmaceutically acceptable carrier may include a lyophilization protectant selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

9. The African swine fever virus subunit vaccine composition according to claim 8, wherein, The saponins are Quil A, QS-21, or GPI-0100.

10. The African swine fever virus subunit vaccine composition according to claim 8, wherein, The adjuvant content is 30%-60% V / V.

11. The African swine fever virus subunit vaccine composition according to claim 10, wherein, The adjuvant content is 50% V / V.

12. A method for preparing the African swine fever virus subunit vaccine composition according to claim 1, the method comprising: (1) The African swine fever virus protein gene was artificially synthesized and linked to a cloning vector, i.e., a protein cloning vector, by genetic engineering means; (2) The constructed protein cloning vector and expression vector are digested with enzymes to construct an expression vector containing the protein gene, i.e., the protein expression vector; (3) The expression vector of the protein is introduced into the recipient bacteria for induced expression, and the expressed protein is identified to obtain African swine fever virus protein; (4) Mix the obtained African swine fever virus protein with a pharmaceutically acceptable carrier to obtain an African swine fever virus subunit vaccine composition.

13. The use of the African swine fever virus subunit vaccine composition according to any one of claims 1 to 11, wherein, The application refers to its use in the preparation of drugs for the prevention of African swine fever virus infection.

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