Nine-component antigen African swine fever recombinant adenovirus vaccine

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a nine-component antigen African swine fever recombinant adenovirus vaccine. The present invention first provides a nine-component antigen African swine fever recombinant adenovirus combination consisting of African swine fever virus antigen P34 protein, P30 protein, P54 protein, A104R protein, X protein, E165R protein, C129R protein, P72 protein and Y protein, wherein the coding gene of at least one antigen or its antigenic epitope is inserted into an adenovirus backbone vector to construct a recombinant adenovirus expressing the antigen or its antigenic epitope; the nine-component antigen African swine fever recombinant adenovirus combination can produce a strong immune response; the prepared nine-component antigen African swine fever recombinant adenovirus vaccine has good immune protection against the parent African swine fever virulent strain, and has no biosafety risk; and the nine-component antigen African swine fever recombinant adenovirus vaccine combined with the nine-component subunit vaccine has better immune protection efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a nine-component antigen African swine fever recombinant adenovirus vaccine and a preparation method thereof. Background Art

[0002] African swine fever (ASF) is the "number one killer" that threatens the world's pig farming industry and has been prevalent for more than a hundred years. In August 2018, African swine fever was introduced into my country, severely damaging my country's pig farming industry and causing unprecedented losses. So far, no commercial ASF vaccine has been available, which has become a pain point and problem for the pig farming industry, and is also a major national need. Scientific research at home and abroad has shown that ASF inactivated vaccines, subunit vaccines, and live vector vaccines still have not solved the problem of immune efficacy. Although gene-deficient vaccines have solved the problem of immune efficacy, there are residual virulence and biosafety risks. The African swine fever virus (ASFV) has a large genome and encodes more than 150 viral proteins, most of which have unclear functions. Little is known about the antigens that induce and stimulate the immune system, which has become a key issue in creating an African swine fever vaccine with ideal immune efficacy.

[0003] Adenovirus vectors have high gene transfer efficiency, convenient production and purification, and good safety. They are the most commonly used vectors in gene therapy clinical trials; human adenovirus type 5 (HAdV-5) is an ideal vaccine vector and is widely used in recombinant vaccine research. There are currently a variety of commercialized replication-deficient vector systems based on HAdV-5. Compared with wild-type adenovirus, the mainstream Ad5 adenovirus vector lacks the early gene E1 and E3 regions, allowing the virus vector to accommodate exogenous genes of 8Kb and below. The exogenous gene here usually refers to the exogenous target gene that needs to be studied. Recombinant adenovirus vector vaccines are vaccines that use genetic engineering technology to introduce genes encoding pathogen protective antigens into adenovirus vectors and express them. This vaccine brings antigen genes into human cells for immune effects. Since adenovirus itself has weak virulence, it can be used to bring viral antigen proteins into the body to stimulate the body's immune system to produce an immune response. Recombinant adenovirus vectors have been widely used in the development of a variety of viral vaccines, such as hepatitis B / C vaccines and Ebola vaccines.

[0004] Chinese patent CN115814071A discloses a subunit vaccine composed of African swine fever virus p34, p14, C129R, DP96R, A104R, p54, p17, p22, P72, and p30 proteins; Chinese patent CN115702928A discloses a subunit vaccine composed of African swine fever virus p34, p14, C129R, DP96R, A104R, p54, p22, P72, and p30 proteins. Although the above studies have disclosed subunit vaccines composed of different antigens, none of them systematically evaluated the immune protection efficacy of the vaccine. Further research by the applicant's team also found that some of the antigenic components involved in the above-mentioned subunit vaccine have an ADE effect, which may seriously affect the protective efficacy of the subunit vaccine against strong strains of African swine fever. The ADE effect refers to the fact that after the virus-specific antibodies bind to the virus, the antibodies bound to the virus can bind to certain cells expressing FcR on their surface through the Fc segment, causing the virus to enter these cells, thereby enhancing the virus's infectivity.

[0005] Based on the above problems, the applicant first discovered a nine-component antigen African swine fever subunit vaccine composed of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, and the subunit vaccine has an ideal protective efficacy against the virulent parent strain of African swine fever; on this basis, the applicant replaced at least one of the self-component antigens in the subunit vaccine with adenovirus in the present invention to obtain a nine-component antigen African swine fever recombinant adenovirus vaccine; the recombinant adenovirus vaccine has a certain protective efficacy against the virulent parent strain of African swine fever; at the same time, the applicant found that the nine-component antigen African swine fever recombinant adenovirus vaccine described in the present invention combined with the above-mentioned nine-component antigen African swine fever subunit vaccine achieved better immune protection efficacy. Summary of the invention

[0006] In response to the above technical problems, the present application has discovered a nine-component antigen African swine fever recombinant adenovirus vaccine after a large number of experimental screening and evaluation studies. The nine-component antigen African swine fever recombinant adenovirus vaccine can not only produce a better immune response and have good safety, but also provide ideal protective efficacy when challenged with the parent African swine fever virulent strain, specifically including the following contents:

[0007] In a first aspect, the present invention provides a nine-component antigen African swine fever recombinant adenovirus combination, characterized in that the nine-component antigen African swine fever recombinant adenovirus combination consists of African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, wherein a coding gene of at least one antigen or its antigenic epitope is inserted into an adenovirus backbone vector to construct a recombinant adenovirus expressing the antigen or its antigenic epitope; the X protein is DP96R protein or a fusion protein of DP96R protein and P12 protein; the Y protein is P22 protein, or P17 protein, or a combination of P22 protein and P17 protein, or a fusion protein of P22 protein and P17 protein, or a fusion protein of P22 protein fragment and P17 protein fragment.

[0008] Preferably, the African swine fever virus is type II African swine fever virus.

[0009] Preferably, the type II African swine fever virus is ASFV CN / GS 2018.

[0010] Preferably, the amino acid sequence of the African swine fever virus P34 protein is shown in SEQ ID NO.1; the amino acid sequence of the African swine fever virus P30 protein is shown in SEQ ID NO.3; the amino acid sequence of the African swine fever virus P54 protein is shown in SEQ ID NO.5 or 7; the amino acid sequence of the African swine fever virus A104R protein is shown in SEQ ID NO.9; the amino acid sequence of the African swine fever virus E165R protein is shown in SEQ ID NO.11; the amino acid sequence of the African swine fever virus DP96R protein is shown in SEQ ID NO.13, and the amino acid sequence of the fusion protein of the DP96R protein and the P12 protein is shown in SEQ ID NO.15; the amino acid sequence of the African swine fever virus C129R protein is shown in SEQ ID NO.17; the amino acid sequence of the African swine fever virus P72 protein is shown in SEQ ID NO.19; the amino acid sequence of the African swine fever virus P17 protein is shown in SEQ ID NO.21; the amino acid sequence of the African swine fever virus P22 protein is shown in SEQ ID NO. NO.23; the amino acid sequence of the fusion protein of the African swine fever virus P17 protein fragment and the P22 protein fragment is shown in SEQ ID NO.25; the amino acid sequence of the fusion protein of the African swine fever virus P17 protein and the P22 protein is shown in SEQ ID NO.27.

[0011] Preferably, the nine-component antigen African swine fever recombinant adenovirus combination is obtained by separately or simultaneously inserting genes encoding African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein into an adenovirus backbone vector to obtain the nine-component antigen African swine fever recombinant adenovirus combination.

[0012] Preferably, the nine-component antigen African swine fever recombinant adenovirus combination is constructed by inserting genes encoding African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein into an adenovirus backbone vector to obtain a nine-component antigen African swine fever recombinant adenovirus combination that expresses P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, respectively.

[0013] Preferably, the adenovirus backbone vector includes a replication-defective adenovirus backbone vector and a replication-competent adenovirus backbone vector.

[0014] Preferably, the adenovirus is selected from human adenovirus type 5 (Ad5), human adenovirus type 26 (Ad26) and chimpanzee adenovirus (ChAds).

[0015] Preferably, the adenovirus backbone vector is a replication-deficient adenovirus backbone vector, and the recombinant adenovirus vector is derived from the AdMax adenovirus system.

[0016] Preferably, the method for constructing the recombinant adenovirus comprises the following steps:

[0017] (1) codon optimization was performed on the gene sequences of African swine fever virus antigen-toxin P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, HA or flag tag was introduced at the 3' end of the gene sequence for fusion expression, the synthesized fragment was cloned into the transition vector pCDNA3.1(+), and the plasmid was extracted;

[0018] (2) using restriction endonucleases Nhe I / BamH I to digest the transition plasmid and the replication-defective adenovirus backbone vector described in step (1), respectively, and connecting and screening to obtain positive plasmids;

[0019] (3) transfecting the positive plasmid described in step (2) into HEK-293A cells to package adenovirus;

[0020] (4) Obtain recombinant adenovirus by subculturing.

[0021] Preferably, the adenovirus backbone vector is a replicative adenovirus backbone vector, and the replicative adenovirus backbone vector comprises a backbone plasmid, an intermediate plasmid and a shuttle plasmid.

[0022] Preferably, the method for constructing the recombinant adenovirus comprises the following steps:

[0023] (1) codon optimization was performed on the gene sequences of African swine fever virus antigen P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, HA or flag tag was introduced at the 3' end of the gene sequence for fusion expression, the synthesized fragment was cloned into the transition vector pcDNA3.1(+), and the plasmid was extracted;

[0024] (2) using restriction endonucleases Kpn I / Bgl II to digest the transition plasmid described in step (1) and the shuttle vector plasmid of the replicative adenovirus backbone vector, respectively, and then connecting and screening to obtain positive plasmids;

[0025] (3) transfecting HEK-293 cells with the positive plasmid described in step (2) and the backbone plasmid and intermediate plasmid of the replicative adenovirus backbone vector to package adenovirus;

[0026] (4) Obtain recombinant adenovirus by subculturing.

[0027] Preferably, the concentration ratio of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein recombinant adenovirus in the nine-component antigen African swine fever recombinant adenovirus combination is: 1-6: 1-6: 1-6: 1-6: 1-6: 1-6: 1-6: 1-6: 1-6.

[0028] Preferably, the concentration ratio of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein recombinant adenovirus in the nine-component antigen African swine fever recombinant adenovirus combination is: 1-3:1:1-3:1:1:1-3:1:1:1.

[0029] Preferably, the concentration ratio of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein recombinant adenovirus in the nine-component antigen African swine fever recombinant adenovirus combination is: 1-2:1:1-2:1:1:1-2:1:1:1.

[0030] Preferably, the concentration ratio of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein recombinant adenovirus in the nine-component antigen African swine fever recombinant adenovirus combination is: 1:1:1-2:1:1:1-2:1:1:1.

[0031] Preferably, the titers of the recombinant adenoviruses of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the nine-component antigen African swine fever recombinant adenovirus combination are all greater than or equal to 10 9 ifu / ml.

[0032] Preferably, the titers of the recombinant adenoviruses of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the nine-component antigen African swine fever recombinant adenovirus combination are all greater than or equal to 10 10 ifu / ml.

[0033] Preferably, the titers of the recombinant adenoviruses of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the nine-component antigen African swine fever recombinant adenovirus combination are all greater than or equal to 10 11 ifu / ml.

[0034] In a second aspect, the present invention provides the use of the nine-component antigen African swine fever recombinant adenovirus combination described in the first aspect above in the preparation of a drug for preventing or treating African swine fever virus infection.

[0035] In a third aspect, the present invention provides the use of the nine-component antigen African swine fever recombinant adenovirus combination described in the first aspect above in the preparation of biological products for preventing African swine fever virus infection.

[0036] In a fourth aspect, the present invention provides a nine-component antigen African swine fever recombinant adenovirus vaccine, which is composed of the nine-component antigen African swine fever recombinant adenovirus combination described in the first aspect and a pharmaceutically acceptable adjuvant.

[0037] Preferably, the adjuvant includes one or more of chemical immune adjuvants, microbial immune adjuvants, plant immune adjuvants, and biochemical immune adjuvants.

[0038] In a fifth aspect, the present invention provides a nine-component antigen African swine fever recombinant adenovirus vaccine combination, wherein the nine-component African swine fever combination vaccine combination comprises the nine-component antigen African swine fever recombinant adenovirus vaccine described in the fourth aspect and a subunit vaccine made from the African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein described in the first aspect.

[0039] Preferably, the subunit vaccine consists of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in a concentration ratio of 1-6:1-6:1-6:1-6:1:1-6:1-6:1-6:1.

[0040] Preferably, the subunit vaccine consists of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, X protein, C129R protein and Y protein in a concentration ratio of 1-3:1-3:1-3:1-3:1-3:1-3:1-3:1-3.

[0041] Preferably, the subunit vaccine consists of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in a concentration ratio of 1-2:1:1-2:1:1-2:1:1.

[0042] Preferably, the subunit vaccine consists of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in a concentration ratio of 1:1:1-2:1:1:1-2:1:1:1.

[0043] Preferably, the subunit vaccine consists of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in a concentration ratio of 2:1:2:1:1:2:1:1:1.

[0044] Preferably, the concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the subunit vaccine are all greater than or equal to 50 μg / ml.

[0045] Preferably, the concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the subunit vaccine are all greater than or equal to 90 μg / ml.

[0046] Preferably, the concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the subunit vaccine are all greater than or equal to 150 μg / ml.

[0047] Preferably, the concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the subunit vaccine are all 150-2400 μg / ml.

[0048] Preferably, the concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the subunit vaccine are 400 μg / ml, 200 μg / ml, 400 μg / ml, 200 μg / ml, 200 μg / ml, 400 μg / ml, 200 μg / ml, 200 μg / ml, and 200 μg / ml, respectively.

[0049] The beneficial effects of the present invention are as follows: (1) The present invention provides a nine-component antigen African swine fever recombinant adenovirus combination, wherein the nine-component antigen African swine fever recombinant adenovirus combination consists of African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, wherein at least one antigen is a recombinant adenovirus expressing an antigen; the X protein is DP96R protein or a fusion protein of DP96R protein and P12 protein; the Y protein is P22 protein, or P17 protein, or a combination of P22 protein and P17 protein, or a fusion protein of P22 protein and P17 protein, or a fusion protein of P22 protein fragment and P17 protein fragment; the nine-component antigen African swine fever recombinant adenovirus combination can produce Good immune response; (2) The nine-component combination vaccine prepared by adding a vaccine adjuvant to the nine-component antigen African swine fever recombinant adenovirus combination of the present invention can provide good protection efficiency when challenged with the parent African swine fever virulent strain; (3) The nine-component antigen African swine fever recombinant adenovirus vaccine described in the present application has good safety; (4) The preparation process of the nine-component combination vaccine described in the present application is simple, suitable for large-scale industrial production, and has great social value and economic value; (5) The nine-component antigen African swine fever recombinant adenovirus vaccine of the present invention is combined with a subunit vaccine made of African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein to further enhance the immune protection efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Construction map of recombinant adenovirus shuttle vector plasmid;

[0051] Figure 2 Recombinant adenovirus packaging results;

[0052] Figure 3 Recombinant adenovirus passage results;

[0053] Figure 4 Identification of protein expression in cells infected with recombinant adenovirus, including H1: P34 protein; H2: P30 protein; H3: P54 protein; H4: A104R protein; H5: E165R protein; H6: X protein; H7: C129R protein; H8: P72 protein; H9: Y protein;

[0054] Figure 5 Recombinant adenovirus titer test results;

[0055] Figure 6 Results of the test of the titer of the recombinant adenovirus-immunized rabbit serum;

[0056] Figure 7 Recombinant adenovirus vaccine immune cytokine results. DETAILED DESCRIPTION

[0057] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further described below in conjunction with specific implementation methods. However, the protection scope of the present invention is not limited to the following embodiments.

[0058] The experiments described in the following examples were performed with biosafety permits and African swine fever laboratory activity permits:

[0059] In accordance with the relevant requirements of biosafety level 3 laboratories (BSL-3) and African swine fever-related biosafety, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences has obtained the Ministry of Agriculture's permission to conduct research on highly pathogenic ASFV pathogens and animals through the Biosafety Committee of the Lanzhou Veterinary Research Institute, the Laboratory Animal Ethics Committee, the Biosafety Committee of the Chinese Academy of Agricultural Sciences, the Laboratory Animal Ethics Committee of the Lanzhou Veterinary Research Institute, and the Biosafety Committee of the Lanzhou Veterinary Research Institute. The research has been filed with the Ministry of Agriculture and Rural Affairs and meets the national biosafety level requirements.

[0060] The experimental cells, viruses and plasmids described in the following examples are from:

[0061] Adenovirus AdMax expression system and HEK-293A cell line were purchased from Shanghai Hanheng Biotechnology Co., Ltd.; replication-competent adenovirus vectors were preserved by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Primary porcine alveolar macrophages (PAM) were prepared and preserved by our laboratory (separated from porcine lung washing).

[0062] LB liquid medium (dry powder) and LB solid medium (dry powder) were purchased from Beijing Solebow Technology Co., Ltd.; kanamycin, IPTG, and X-Gal were purchased from Shanghai Solebow Technology Co., Ltd.; Gel Extraction Kit (100) and PlasmidMini Kit I (100) were purchased from OMEGA, USA; conventional molecular and chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. PureLinK HiPure Plasmid Maxiprep plasmid extraction kits were purchased from Invitrogen; DNAMarker was purchased from TaKaRa, and Adeno-X Rapid Titer Kit adenovirus titer determination kit was purchased from Clontech.

[0063] Type II African swine fever virus strain ASFV CN / GS 2018 was from the National African Swine Fever Regional Laboratory (Lanzhou), belonging to genotype II, with a virus titer of 1×10 5 HAD 50 / mL, which is the 4th generation seed virus after PAM cell expansion. It was deposited in the China Center for Type Culture Collection on December 21, 2020, with the deposit number CCTCC NO: V202096; deposit address: Wuhan University, Wuhan, China; Tel: 027-68752319.

[0064] The recombinant adenovirus shuttle vector plasmids expressing African swine fever virus proteins are cloned by classical molecular gene cloning methods. According to the viral protein gene sequence encoded by the ASFV CN / GS / 2018 isolate, different viral protein coding region fragments are designed and synthesized to construct shuttle expression vectors. Each gene expression sequence is designed through codon optimization (including elimination of rare codons, adjustment of GC content, etc.), and the optimized gene sequence is sent to the company for synthesis to construct a recombinant adenovirus shuttle vector.

[0065] Experimental rabbits: Healthy New Zealand male rabbits weighing about 2.0 kg were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0066] Pigs: healthy pigs, all purchased from farms without ASFV prevalence, PCV2 antigen test was negative, and ASFV, PRRSV antigen and antibody tests were all negative.

[0067] definition:

[0068] The term "antigen" refers to a substance that can induce an immune response in the body, that is, a substance that can be specifically recognized and bound by the 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 corresponding products in vivo and in vitro.

[0069] The terms "vaccine", "vaccine composition" and "subunit vaccine" refer to a pharmaceutical composition expressed by a viral vector or containing African swine fever virus protein antigens, which can induce, stimulate or enhance the immune response of pigs against African swine fever, a biological preparation that can provide a protective response in animals, wherein the vaccine has been delivered and cannot cause severe disease.

[0070] Furthermore, the “vaccine”, “vaccine composition”, and “subunit vaccine” comprise one or more adjuvants, excipients, carriers, and diluents.

[0071] Furthermore, the adjuvant includes one or more of chemical immune adjuvants, microbial immune adjuvants, plant immune adjuvants, and biochemical immune adjuvants.

[0072] Further, the chemical immune adjuvants include aluminum hydroxide, Freund's adjuvant, mineral oil, Span, etc.; the microbial immune adjuvants include mycobacteria, lipopolysaccharide, muramyl dipeptide, murine peptide, fat-soluble wax D, short bacillus; the plant immune adjuvants include polysaccharides extracted from plants or large fungi, such as tuckahoe polysaccharide, safflower polysaccharide, Chinese herbal medicine, etc.; the biochemical immune adjuvants include thymosin, transfer factor, interleukin, etc. Preferred adjuvants can be nano-adjuvants, biological adjuvants, interleukins, interferons, etc.

[0073] Furthermore, the "vaccine", "vaccine composition" and "subunit vaccine" can also be used to prepare combined vaccines, such as combined with other vaccines for pigs;

[0074] Further, the administration of the "vaccine", "vaccine composition", "subunit vaccine" can be conveniently administered, such as intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal routes. The "vaccine", "vaccine composition", "subunit vaccine" can be administered after a prime-boost regimen; for example, after the first vaccination, the subject can receive a second booster administration after a period of time (e.g., about 7, 14, 21 or 28 days). Typically, the dose of the booster administration is the same as or lower than the dose used for the primary immunization. In addition, a third booster immunization may also be performed, such as 2-3 months, 6 months or one year after immunization.

[0075] The term "prevention" when referring to African swine fever virus infection means inhibiting the replication of African swine fever virus, inhibiting the spread of African swine fever virus or preventing African swine fever virus from taking root in its host, and alleviating the symptoms of diseases or conditions caused by African swine fever virus infection.

[0076] Example 1 Preparation of nine-component antigen recombinant adenovirus

[0077] The expression and identification of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein were carried out with reference to the content of patent document CN202310374606.0.

[0078] According to the P34, P30, P54, A104R, E165R, X (DP96R protein or fusion protein of DP96R protein and P12 protein), C129R, P72 and Y genes of the ASFV CN / GS / 2018 isolate, the sequence was optimized and designed to construct the shuttle vector plasmid for recombinant adenovirus expression. The P34, P30, P54, A104R, E165R, X, C129R, P72 and Y gene sequences were codon optimized (including elimination of rare codons, adjustment of GC content, etc.), HA or flag tags were introduced at the 3' end of the gene for fusion expression, and the synthesized fragments were cloned in the transition vector pCDNA3.1(+), the plasmids were extracted, the transition plasmid and the shuttle vector plasmid were digested with restriction endonucleases Nhe I / BamH I, respectively, the target DNA fragment was recovered and purified after nucleic acid electrophoresis, and the target gene fragment and the vector fragment were ligated using T4 ligase from NEB (vector such as Figure 1). The amino acid sequence of the P34 protein is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2; the amino acid sequence of the P30 protein is shown in SEQ ID NO.3, and the coding gene sequence is shown in SEQ ID NO.4; the amino acid sequence of the P54 protein is shown in SEQ ID NO.7, and the coding gene sequence is shown in SEQ ID NO.8 (in order to facilitate purification and expression, the present application uses a truncated protein of the P54 protein (removing the transmembrane region), the amino acid sequence is shown in SEQ ID NO.5, and the coding gene sequence is shown in SEQ ID NO.6); the amino acid sequence of the A104R protein is shown in SEQ ID NO.9, and the coding gene sequence is shown in SEQ ID NO.10; the amino acid sequence of the E165R protein is shown in SEQ ID NO.11, and the coding gene sequence is shown in SEQ ID NO.12; the X protein is the DP96R protein, the amino acid sequence is shown in SEQ ID NO.13, and the coding gene sequence is shown in SEQ ID NO.14 (in the present application, when the DP96R protein is expressed, the P12 protein (DP96R / P12) is fused, the amino acid sequence is shown in SEQ ID NO.15, and the coding gene sequence is shown in SEQ ID NO.16); the amino acid sequence of the C129R protein is shown in SEQ ID NO.17, and the coding gene sequence is shown in SEQ ID NO.18; the amino acid sequence of the P72 gene is shown in SEQ ID NO.19, and the coding gene sequence is shown in SEQ ID NO.20; the Y protein is the P22 protein (the amino acid sequence is shown in SEQ ID NO.23, and the gene sequence is shown in SEQ ID NO.24), or the P17 protein (shown in SEQ ID NO.21, and the gene sequence is shown in SEQ ID NO.22), or a combination of the P22 protein and the P17 protein, or a fusion protein of a P22 protein fragment and a P17 protein fragment (a fusion protein of the extracellular region of the P22 protein and the extracellular region of the P17 protein, the amino acid sequence is shown in SEQ ID NO.25, and the gene sequence is shown in SEQ ID NO.26). IDNO.26), or a fusion protein of P22 protein and P17 protein (amino acid sequence is shown in SEQ ID NO.27, gene sequence is shown in SEQ ID NO.28); this embodiment takes the fusion protein of the extracellular region of P22 protein and the extracellular region of P17 protein as an example.

[0079] The connection system is as follows: the molar ratio of the gel recovery product vector fragment to the target gene fragment product is 1:3, T4Ligase 1μL, T4 Ligase Buffer 2μL, water to 20μL, incubate at 16℃ for 12h. The connection product is transformed into Trans10 Escherichia coli competent cells as follows: take out the ampicillin-resistant LB medium plate, put the competent cells on ice to melt, add 10μL of the connection product, gently pipette and mix evenly, and place on ice for 30min; place the centrifuge tube in a 42℃ water bath, heat shock for 90s, and place on ice for 2min; use ampicillin resistance to screen transformants. The positive plasmids obtained by gene sequencing were named pH1#, pH2#, pH3#, pH4#, pH5#, pH6#, pH7#, pH8#, and pH9#.

[0080] 2. Packaging and Passaging of Recombinant Adenovirus

[0081] HEK-293A cells are used to package adenovirus. Cells should be grown in monolayers under optimal growth conditions of 37°C and 5% CO2. Adenovirus shuttle vectors and backbone plasmids containing target genes must be extracted in large quantities, with a concentration greater than 1μg / μL and an A260 / 280 ratio in the range of 1.7 to 1.8 before they can be used for virus packaging.

[0082] Prepare virus packaging cells: Prepare cells one day before transfection. Inoculate the HEK-293A cells to be transfected into a 6 cm culture dish and culture them at 37°C, 5% CO2 for 24 hours. On the day of transfection, the cells should have a confluence of 50-70%.

[0083] Transfection: Transfect each culture dish with a diameter of 6 cm with the prepared viral plasmid. The composition of the transfection complex is as follows: add 400 μL of transfection buffer, 2 μg of pHBAd shuttle plasmid, 4 μg of pBHGlox (delta) E1,3Cre plasmid, and 12 μL of JetPrime to a 1.5 mL EP tube. Mix the above reagents thoroughly and incubate at room temperature for 10 minutes. Add the transfection complex to the prepared cells and continue to culture at 37°C and 5% CO2. Observe the cells for CPE every day. Figure 2 shown.

[0084] Virus passage: Add the virus in the culture medium to fresh HEK-293A cell culture medium to amplify the virus in small quantities. When the cells show plaques again, collect the cells and supernatant, freeze and thaw repeatedly three times to collect the virus, and use this virus as the P1 generation virus. Infect HEK-293A cells with the P1 generation adenovirus, and continue to infect for three generations. Amplify the adenovirus in large quantities at the P4 generation (such as Figure 3After plaques are formed, the virus is collected and the virus titer is determined.

[0085] 3. Adenovirus Expression Verification

[0086] 1) Cell preparation: Pipette 0.5 mL / well of cell solution into a 24-well plate and place in a 37°C, 5% CO2 incubator for continued culture. When the HEK-293A cells in the 24-well plate reach a confluence of 80%, infection is performed;

[0087] 2) Take 50 μL of recombinant adenovirus supernatant to infect cells and harvest the cells after 24 hours;

[0088] 3) Western blot detection of the tagged protein and the specific band of the target protein, such as Figure 4 As shown, it is consistent with the expected molecular weight. It can be seen that the recombinant adenovirus that has been successfully packaged can infect cells and express target proteins, with sizes of approximately 37kDa, 45kDa, 24kDa, 31kDa, 36kDa, 35kDa, 34kDa, 72kDa and 44kDa, respectively. According to Western blot analysis, the proteins can react with African swine fever virus positive serum. Among them, H1 is P34 protein, H2 is P30 protein, H3 is P54 protein, H4 is A104R protein, H5 is E165R protein, H6 is X protein, H7 is C129R protein, H8 is P72 protein, and H9 is Y protein.

[0089] 4. Titer determination of recombinant adenovirus

[0090] Dilute 10 -2 ~10 -7 HEK-293A cells were inoculated with recombinant adenovirus at a concentration of 100 times, and the titer of the recombinant adenovirus was determined using the Adeno-X Rapid TiterKit adenovirus titer determination kit. The field of view with 5-50 positive cells was selected for counting and the titer of the recombinant adenovirus was calculated. The result was 10 10 ~10 12 ifu / ml range (results as Figure 5 shown).

[0091] Although the embodiments of the present application specifically limit the method for preparing the recombinant adenovirus of the nine-component protein, it is not limited to the above method. Those skilled in the art can construct the recombinant adenovirus of the nine-component protein described in the present application by conventional methods. The expression vector is not limited to the AdMax system described above. Those skilled in the art can also select conventional plasmids according to the selected expression system, including expression plasmids known in the art.

[0092] When expressing the above nine-component protein, the present application may also optimize the sequence of the above antigen protein, including truncation, mutation, introduction of fusion tags, etc., to improve the expression efficiency of the target protein;

[0093] The truncation strategy is to remove the transmembrane region of the target protein, remove the localization signal, remove the special domain, etc.;

[0094] The fusion tag is selected from any one or a combination of the following: HA, Flag, Myc, V5, His, GST, etc.

[0095] On the basis that the nine-component antigen African swine fever recombinant adenovirus vaccine constructed using the AdMax system as described in this application has good immune protection efficacy, other African swine fever recombinant adenovirus vaccines containing the nine-component antigens described in this application obtained by using other adenovirus vectors known to those skilled in the art also have the same immune protection efficacy.

[0096] Example 2 Immunogenicity test of nine-component antigen African swine fever recombinant adenovirus

[0097] Twenty-seven healthy male rabbits weighing about 2.0 kg were selected and allowed to adapt to the environment for two days. Three rabbits were immunized with each of the recombinant adenoviruses expressing nine antigenic proteins (H1# to H9#, representing P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, respectively) by subcutaneous injection at multiple points on the back. The immunization dose was 10 10 ifu / head. Reinforce immunization twice with equal amounts of recombinant virus on the 15th and 30th days after the first immunization, respectively. The immunization site and method are the same as the first immunization. 14 days after the third immunization, a small amount of blood is collected through the ear vein, and the serum is separated. The antibody titer is determined by the indirect ELISA method. The maximum dilution of the antibody when the S / N value is ≥2.1 is used as the antibody titer. If the serum titer is not less than 1:64000, blood is collected and serum is separated according to the conventional method and stored below -20°C. If the titer of the immune serum is low, it can be reinforced once, and the antibody titer can be tested again after 14 days. If the serum titer meets the requirements, the serum can be prepared according to the above method. Otherwise, it is discarded and re-prepared.

[0098] The nine-component antigen African swine fever recombinant adenovirus was diluted to 1 μg / ml with coating buffer, and then added to the ELISA plate at 100 μL / well, and coated at 2-8℃ for 16 hours. The plate was washed twice with washing working solution and the liquid in the well was shaken off, patted dry, and the blocking solution was added at 150 μL / well, blocked at 37℃ for 2 hours, the liquid in the well was shaken off, patted dry, and the ELISA plate was dried in a dry and ventilated environment. It was vacuum packed with desiccant in an aluminum foil bag and stored at 4℃ for later use.

[0099] To determine the antibody titer of each protein immunized rabbit polyclonal antiserum, the prepared rabbit antiserum was diluted with PBS at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000, and the rabbit antiserum titer was detected by indirect ELISA. At the same time, non-immunized rabbit antiserum was used as a negative control, and the specific judgment standard was: S / N ≥ 2.1 (S represents the sample OD 450nm Value, N represents the negative control OD 450nm The maximum dilution multiple of rabbit antibody at the value (0.05) is the rabbit antibody titer.

[0100] 14 days after the third immunization, blood was collected from the rabbit's ear vein, serum was separated, and ELISA titer test was performed. The results showed that the titer of the nine-component antigen African swine fever recombinant adenovirus immune antibody was greater than 1:64000 (such as Figure 6 shown).

[0101] Example 3 Immune protection test of nine-component antigen African swine fever recombinant adenovirus vaccine combined with subunit vaccine

[0102] In the early stage of this study, the components of the nine-component antigen African swine fever subunit vaccine were replaced one by one with a combination vaccine with a proportion of recombinant adenovirus components, and then different antigen combinations were fully mixed and emulsified with an equal amount of ISA 201 adjuvant (Seppic, France) to prepare a nine-component antigen African swine fever subunit-recombinant adenovirus combination vaccine for animal immunization evaluation. The early results showed that on the basis of the strong immune protection efficacy of the nine-component antigen African swine fever subunit vaccine, after replacing at least one antigen protein with a recombinant adenovirus expressing the antigen protein, the obtained recombinant adenovirus vaccine also had different degrees of protection. On this basis, this example selected three vaccine combinations for immune protection efficacy evaluation. The details are as follows:

[0103] 1. Configuration of the nine-component antigen African swine fever vaccine

[0104] Vaccine 1 (nine-component antigen African swine fever subunit vaccine combined with nine-component recombinant adenovirus vaccine), including:

[0105] Preparation of nine-component antigen African swine fever subunit vaccine: the concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein are 400μg / ml, 200μg / ml, 400μg / ml, 200μg / ml, 200μg / ml, 400μg / ml, 200μg / ml, 200μg / ml, 200μg / ml, respectively; mix and emulsify with an equal amount of ISA 201 adjuvant (Seppic, France) to prepare a nine-component antigen African swine fever subunit vaccine.

[0106] Preparation of nine-component recombinant adenovirus vaccine: 2 ml of P34 recombinant adenovirus (10 10 ifu / mL), P30 recombinant adenovirus 2ml (10 10 ifu / mL), P54 recombinant adenovirus 2ml (10 10 ifu / mL), A104R recombinant adenovirus 2ml (10 10 ifu / mL), E165R recombinant adenovirus 2ml (10 10 ifu / mL), X recombinant adenovirus 2ml (10 10 ifu / mL), C129R recombinant adenovirus 2ml (10 10 ifu / mL), P72 recombinant adenovirus 2ml (10 10 ifu / mL), Y recombinant adenovirus 2ml (10 10 ifu / mL); and an equal amount of ISA 201 adjuvant (Seppic, France) were thoroughly mixed and emulsified to prepare a nine-component antigen African swine fever recombinant adenovirus vaccine.

[0107] Vaccine 2 (9-component recombinant adenovirus vaccine 1): P34 recombinant adenovirus 2ml (10 10 ifu / mL), P30 recombinant adenovirus 2ml (10 10 ifu / mL), P54 recombinant adenovirus 2ml (10 10 ifu / mL), A104R recombinant adenovirus 2ml (10 10 ifu / mL), E165R recombinant adenovirus 2ml (10 10 ifu / mL), X recombinant adenovirus 2ml (10 10 ifu / mL), C129R recombinant adenovirus 2ml (10 10 ifu / mL), P72 recombinant adenovirus 2ml (10 10 ifu / mL), Y recombinant adenovirus 2ml (10 10 ifu / mL); adjuvant 50%;

[0108] Vaccine 3 (9-component recombinant adenovirus vaccine 2): P34 recombinant adenovirus 2ml (10 9 ifu / mL), P30 recombinant adenovirus 2ml (10 9 ifu / mL), P54 recombinant adenovirus 2ml (10 9 ifu / mL), A104R recombinant adenovirus 2ml (10 9 ifu / mL), E165R recombinant adenovirus 2ml (10 9 ifu / mL), X recombinant adenovirus 2ml (10 9ifu / mL), C129R recombinant adenovirus 2ml (10 9 ifu / mL), P72 recombinant adenovirus 2ml (10 10 ifu / mL), Y recombinant adenovirus 2ml (10 9 ifu / mL); adjuvant 50%.

[0109] 2. Immunoassay

[0110] Detection kits: African swine fever virus fluorescence PCR detection kit, African swine fever virus blocking ELISA antibody detection kit, porcine circovirus type II direct amplification fluorescence quantitative PCR antigen detection kit, provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; RealPCR PRRSV-2RNA detection premix, porcine reproductive and respiratory syndrome virus antibody X3 detection kit, purchased from IDEXX.

[0111] The test virus ASFV CN / GS / 2018 strain, numbered: ASFV CN / GS / 2018 / 1901, ASFV CN / GS / 2018 / 2001, was provided by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0112] Experimental animals: 60-75 days old experimental pigs were all purchased from pig farms where ASFV, PRRSV and PCV-2 were not prevalent. The ASFV and PRRSV antigen and antibody tests were negative, and the PCV-2 antigen test was negative.

[0113] 2.1 Animal immunization

[0114] The nine-component antigen African swine fever recombinant adenovirus vaccines 1-3 prepared above were used to immunize 60-75 day old test pigs, with 5-10 pigs immunized with each vaccine, by intramuscular injection behind the ear, 2-3 ml / head (different antigen composition and content). 21 days after the first immunization, the pigs were injected intramuscularly behind the ear of the other side, 2-3 ml / head (different antigen composition and content) for booster immunization. At the same time, 5 non-immunized controls were set up.

[0115] 2.2 Evaluation of virus attack

[0116] 14 days after booster vaccination, ASFV CN / GS / 2018 strain was used for challenge evaluation, with a challenge dose of 1.25HAD 50 / 3ml / head (equivalent to 5MLD). Observe continuously for 21 days after the infection, measure the rectal temperature daily, and record the clinical symptoms. All the control pigs became ill, and 5 / 5 died during the test period. The infection model was successfully constructed; the rectal temperature of the immunized pigs should not exceed 40.5℃; if the rectal temperature is ≥40.5℃, the retention should not exceed 2 days; no typical clinical symptoms caused by African swine fever virus infection (such as depression, loss of appetite or loss of appetite, vomiting, cyanosis of the ears or the whole body, bleeding from natural orifices) appeared; no death caused by African swine fever virus infection occurred during the test period. If the above 3 items are met, it is judged as protection. The vaccine protection rate is calculated based on the number of protected immunized pigs.

[0117] 2.3 Sample collection

[0118] On the 5th, 10th, 15th and 21st days after infection, 5 ml of blood was collected from each pig (4 ml for serum separation and 1 ml for preparation of anticoagulant blood (EDTA-K2)), and anal swab samples were also collected and stored at -70°C or below.

[0119] 3. Results

[0120] 3.1 Immune efficacy of nine-component antigen African swine fever recombinant adenovirus vaccine

[0121] The nine-component antigen African swine fever recombinant adenovirus vaccine described in this application (Vaccine 2, high dose; Vaccine 3, low dose) can induce the immunized animals to produce higher levels of cytokines IFN-γ, IL-10 and IL-2 (the results are shown in Figure 7 The results of the immune protection efficacy of the above three groups of vaccines are shown in Table 1, indicating that the nine-component antigen African swine fever recombinant adenovirus vaccine described in the present application has a certain protective efficacy against the parental strong strain challenge; and the nine-component antigen African swine fever recombinant adenovirus vaccine and the nine-component antigen African swine fever subunit vaccine combined immunization have better protective efficacy against the parental strong strain challenge.

[0122] Table 1 The protection ratio of different vaccines against virus

[0123] Vaccine No. Attack and protection ratio Vaccine 1 5 / 5 Vaccine 2 4 / 5 Vaccine 3 3 / 5 Comparison 0 / 5

[0124] 3.2 Body temperature monitoring and clinical symptom observation of experimental pigs after virus challenge

[0125] The immune-protected test pigs had normal body temperature or body temperature ≥40.5℃ for no more than 2 days during the test period, grew healthily, and did not show typical clinical symptoms caused by African swine fever virus infection (depression, loss of appetite or loss of appetite, vomiting, cyanosis of the ears or skin, bleeding from natural orifices); the unprotected pigs began to have fever from the third day after the challenge, with body temperature reaching above 41℃, and showed typical clinical symptoms of African swine fever, and all died from the 7th to the 16th day after the challenge. The pigs in the control group began to have fever from the third day after the challenge, with body temperature reaching above 41℃, and all died from the 8th to the 15th day after the challenge.

[0126] 3.2 Results of nucleic acid testing of experimental pigs after virus challenge

[0127] No ASFV CN / GS / 2018 was detected in the immune-protected test pigs during the test period.

[0128] It should be noted that when the concentration of each component in the nine-component subunit vaccine described in the present application is greater than or equal to 50 μg / ml, the titer of each component in the recombinant adenovirus nine-component vaccine is greater than or equal to 1×10 9 ifu / mL, it has ideal protection efficiency.

[0129] The above results show that: (1) the present invention provides a nine-component antigen African swine fever recombinant adenovirus combination, the nine-component antigen African swine fever recombinant adenovirus combination is composed of African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, wherein at least one antigen is a recombinant adenovirus expressing an antigen; the X protein is DP96R protein or a fusion protein of DP96R protein and P12 protein; the Y protein is P22 protein, or P17 protein, or a combination of P22 protein and P17 protein, or a fusion protein of P22 protein and P17 protein, or a fusion protein of P22 protein fragment and P17 protein fragment, and the nine-component antigen African swine fever recombinant adenovirus The combination can produce a good immune response; (2) the nine-component combination vaccine prepared by adding a vaccine adjuvant to the nine-component antigen African swine fever recombinant adenovirus combination of the present invention can provide good protection efficiency when challenged with the parental African swine fever virulent strain; (3) the nine-component antigen African swine fever recombinant adenovirus vaccine described in the present application has good safety; (4) the preparation process of the nine-component combination vaccine described in the present application is simple, suitable for large-scale industrial production, and has great social and economic value; (5) the nine-component antigen African swine fever recombinant adenovirus vaccine of the present invention is combined with a subunit vaccine made of African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, X protein, C129R protein and Y protein, which further enhances the immune protection efficacy.

Claims

1. A nine-component antigen African swine fever recombinant adenovirus combination, characterized in that: The nine-component antigen African swine fever recombinant adenovirus combination is constructed by inserting the coding genes encoding the African swine fever virus antigen P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein into an adenovirus backbone vector respectively or simultaneously to obtain the nine-component antigen African swine fever recombinant adenovirus combination; the concentration ratio of the recombinant adenovirus of P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the nine-component antigen African swine fever recombinant adenovirus combination is: 1-6: 1-6: 1-6: 1-6: 1-6: 1-6: 1-6: 1-6; the titer of the recombinant adenovirus is greater than or equal to 10 9 IFU / ml; the X protein is a fusion protein of DP96R protein and P12 protein; the Y protein is a fusion protein of P22 protein fragment and P17 protein fragment; the amino acid sequence of the P34 protein is shown in SEQ ID NO.1; the amino acid sequence of the P30 protein is shown in SEQ ID NO.3; the amino acid sequence of the P54 protein is shown in SEQ ID NO.5 or 7; the amino acid sequence of the A104R protein is shown in SEQ ID NO.9; the amino acid sequence of the E165R protein is shown in SEQ ID NO.11; the amino acid sequence of the fusion protein of the DP96R protein and P12 protein is shown in SEQ ID NO.15; the amino acid sequence of the C129R protein is shown in SEQ ID NO.17; the amino acid sequence of the P72 protein is shown in SEQ ID NO.19; the amino acid sequence of the fusion protein of the P22 protein fragment and the P17 protein fragment is shown in SEQ ID NO.

25.

2. The nine-component antigen African swine fever recombinant adenovirus combination according to claim 1, characterized in that: The African swine fever virus is type II African swine fever virus.

3. The nine-component antigen African swine fever recombinant adenovirus combination as claimed in claim 2, characterized in that: The type II African swine fever virus is ASFV CN / GS2018.

4. The nine-component antigen African swine fever recombinant adenovirus combination according to claim 1, characterized in that: The nine-component antigen African swine fever recombinant adenovirus combination is constructed by inserting genes encoding African swine fever virus antigens P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein into an adenovirus backbone vector to obtain a nine-component antigen African swine fever recombinant adenovirus combination that expresses P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, respectively.

5. The nine-component antigen African swine fever recombinant adenovirus combination as claimed in claim 4, characterized in that: The adenovirus backbone vector includes a replication-deficient adenovirus backbone vector and a replication-competent adenovirus backbone vector.

6. The nine-component antigen African swine fever recombinant adenovirus combination according to claim 5, characterized in that: The adenovirus backbone vector is a replication-deficient adenovirus backbone vector, and the replication-deficient adenovirus backbone vector is derived from the AdMax adenovirus system.

7. The nine-component antigen African swine fever recombinant adenovirus combination according to claim 6, characterized in that: The method for constructing the recombinant adenovirus comprises the following steps: (1) codon optimization was performed on the gene sequences of African swine fever virus antigen-toxin P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, HA or flag tag was introduced at the 3' end of the gene sequence for fusion expression, the synthesized fragment was cloned into the transition vector pCDNA3.1(+), and the transition plasmid was extracted; (2) using restriction endonucleases Nhe I / BamH I to digest the transition plasmid and the replication-defective adenovirus backbone vector described in step (1), respectively, and connecting and screening to obtain positive plasmids; (3) transfecting the positive plasmid described in step (2) into HEK-293A cells to package adenovirus; (4) Obtain recombinant adenovirus by subculturing.

8. The nine-component antigen African swine fever recombinant adenovirus combination as claimed in claim 5, characterized in that: The adenovirus backbone vector is a replicative adenovirus backbone vector, and the replicative adenovirus backbone vector includes a backbone plasmid, an intermediate plasmid and a shuttle plasmid; the method for constructing the recombinant adenovirus includes the following steps: (1) codon optimization was performed on the gene sequences of African swine fever virus antigen P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein, HA or flag tag was introduced at the 3' end of the gene sequence for fusion expression, the synthesized fragment was cloned into the transition vector pcDNA3.1(+), and the transition plasmid was extracted; (2) using restriction endonucleases Kpn I / Bgl II to digest the transition plasmid described in step (1) and the shuttle vector plasmid of the replicative adenovirus backbone vector, respectively, and then connecting and screening to obtain positive plasmids; (3) transfecting HEK-293 cells with the positive plasmid described in step (2) and the backbone plasmid and intermediate plasmid of the replicative adenovirus backbone vector to package adenovirus; (4) Obtain recombinant adenovirus by subculturing.

9. Use of the nine-component antigen African swine fever recombinant adenovirus combination as described in any one of claims 1 to 8 in the preparation of a drug for preventing or treating African swine fever virus infection.

10. A nine-component antigen African swine fever recombinant adenovirus vaccine, characterized in that: The nine-component antigen African swine fever recombinant adenovirus vaccine consists of the nine-component antigen African swine fever recombinant adenovirus combination described in any one of claims 1-8 and a pharmaceutically acceptable adjuvant.

11. The nine-component antigen African swine fever recombinant adenovirus vaccine according to claim 10, characterized in that: The adjuvant includes: one or more of chemical immune adjuvants, microbial immune adjuvants, plant immune adjuvants, and biochemical immune adjuvants.

12. A nine-component antigen African swine fever recombinant adenovirus vaccine combination, characterized in that: The nine-component antigen African swine fever recombinant adenovirus vaccine combination comprises the nine-component antigen African swine fever recombinant adenovirus vaccine according to claim 11 and a subunit vaccine made of the African swine fever virus antigen P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein according to claim 1; the subunit vaccine comprises a concentration ratio of 1-6:1-6:1-6:1-6:1:1 -6:1-6:1:1-6 of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein; the concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the subunit vaccine are all greater than or equal to 50 μg / ml.

13. The nine-component antigen African swine fever recombinant adenovirus vaccine combination according to claim 12, characterized in that: The concentrations of African swine fever virus P34 protein, P30 protein, P54 protein, A104R protein, E165R protein, X protein, C129R protein, P72 protein and Y protein in the subunit vaccine are 400 μg / ml, 200 μg / ml, 400 μg / ml, 200 μg / ml, 200 μg / ml, 400 μg / ml, 200 μg / ml, 200 μg / ml and 200 μg / ml, respectively.

14. Use of the nine-component antigen African swine fever recombinant adenovirus combination as described in any one of claims 1 to 8 in the preparation of a vaccine for preventing African swine fever virus infection.

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