Composition of African swine fever virus (ASFV) protein and vaccine prepared from composition

Abstract

The invention discloses an ASFV (African swine fever virus) protein composition and a vaccine prepared from the ASFV protein composition. The invention provides an African swine fever virus protein combination. The African swine fever virus protein combination comprises at least three combinations of four proteins, the four proteins are an EP84R protein or a truncated body thereof, a D129L protein or a truncated body thereof, an E146L protein or a truncated body thereof, and a K421R protein or a truncated body thereof. After the recombinant virus or subprotein vaccine or mRNA vaccine constructed by using the African swine fever viruses EP84R, D129L, E146L and K421R is used for immunizing pigs, the immunized pigs can play a role in resisting ASFV infection, the immunized pigs can completely resist attack of ASFV virulent viruses without attack, susceptible pigs can be protected from natural infection or artificial attack of ASFV, and the recombinant virus or subprotein vaccine or mRNA vaccine is used for preventing African swine fever.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products and relates to a combination of African swine fever virus (ASFV) proteins and a vaccine prepared thereby. Background Art

[0002] African swine fever (ASF) is a contagious, febrile, hemorrhagic, and highly lethal infectious disease of pigs caused by African swine fever virus (ASFV). The mortality rate of acute infection is as high as 100%. It is currently a major threat to the pig farming industry in my country and even the world, and has caused significant losses in epidemic areas. Due to unsatisfactory safety or efficacy, neither the inactivated vaccine nor the live vaccine prepared by the African swine fever virus has been approved for marketing. The prevention and control of African swine fever has always mainly adopted the methods of culling and carcass removal, and strict biosafety measures have been taken for prevention.

[0003] The genome of African swine fever virus is 170-193kb in length, contains 160-234 ORFs, and is estimated to encode about 165 proteins. So far, a large number of genes related to virus virulence, immunosuppression, and cell apoptosis have been discovered, including genes from multigene families. Studies have shown that the virulence factors of African swine fever virus include 9GL, UK, I177L, I226R, A137R, L60L, etc. (Lewis et al., 2000)(Zsak et al., 1998)(O'Donnell et al., 2015)(Borca et al., 2020), immunosuppressive factors include MGF100, MGF110, MGF300, MGF360 and MGF505, etc. (Afonso et al., 2004)(Li et al., 2021)(Reis et al., 2016), and blood adsorption factors such as CD2v, EP153R, etc. (Rodriguez et al., 1993). In recent years, attenuated vaccine strains with deletion of virulence genes such as ASFV MGF360, MGF505, CD2v, UK, A238L, and natural attenuated strains isolated from domestic or wild boar herds have been shown to have certain immune protection effects in pigs, but some strains have been shown to produce chronic lesions in field trials, such as skin ulcers, fever, joint swelling, stiff pigs, sow abortion, and clinical abnormal symptoms during fattening (King et al., 2011; Leitao et al., 2001; Revilla et al., 1992, personal communication). Artificial gene-deficient virus strains such as MGF / CD2v, I177L, I226R, A137R, UK and other gene-deficient strains have good immune effects. However, these gene-deficient live vaccines have successively shown obvious adaptive mutations after being used in target animals, resulting in a decrease in the immune level of vaccinated pigs and non-immune pigs and the occurrence of multiple diseases. Therefore, gene-deficient live vaccines have obvious biosafety risks. Recombinant vaccines and subunit vaccines constructed using some ASFV genes such as p30, p54, p72, etc. are reported to have certain immune effects, but no better vaccines have been applied and promoted.

[0004] Therefore, discovering effective target proteins and exploring new, safer and more effective vaccines have always been important and realistic issues facing the prevention and control of African swine fever in the pig industry. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a new African swine fever vaccine.

[0006] In order to solve the above technical problems, in the first aspect, the present invention provides an African swine fever virus protein combination, which includes at least 3 combinations of the following 4 proteins; the 4 proteins are EP84R protein or its truncation, D129L protein or its truncation, E146L protein or its truncation and K421R protein or its truncation.

[0007] In an embodiment of the present invention, the EP84R protein truncation (fEP84L) is the EP84L protein amino acid sequence from position 16 to position 70;

[0008] The D129L protein truncation (fD129LR) is the amino acid sequence of the D129L protein at positions 30-90;

[0009] The E146L protein truncation (fE146L) is the amino acid sequence of the E146L protein at positions 25-105;

[0010] The K421R protein truncation (fK421R) is the 58th to 300th position of the K421R protein amino acid sequence.

[0011] In the African swine fever virus protein combination described above, the African swine fever virus protein combination also includes other protein combinations;

[0012] The other protein combination consists of at least one of B646L protein, E183L protein, CP204L protein and CP2457L protein.

[0013] The above protein combination can be a combination of individual proteins or a fusion protein obtained by the combined expression of multiple proteins.

[0014] In an embodiment of the present invention, the above protein combination is any one of the following:

[0015] 1)EP84R, D129L, K421R;

[0016] 2)EP84R, E146L, K421R;

[0017] 3)D129L, E146L, K421R;

[0018] 4)EP84R, D129L, E146L, K421R;

[0019] 5)EP84R, D129L, E146L, K421R, B646L

[0020] 6)EP84R, D129L, E146L, K421R, E183L, B646L

[0021] 7)EP84R, D129L, E146L, K421R, CP204L, E183L, B646L

[0022] 8)EP84R, D129L, E146L, K421R, CP2457L, CP204L, E183L, B646L;

[0023] 9) fEP84, fD129LR, fE146L, fK421R;

[0024] 10) EP84R, D129L, E146L;

[0025] The above proteins or their truncations are all derived from African swine fever virus; the truncations have the same functions as the full-length proteins.

[0026] The above-mentioned EP84R protein, D129L protein, E146L protein, K421R protein, B646L protein, E183L protein, CP204L protein and CP2457L are the proteins shown in GenBank accession number: MH766894.3 (submitted on July 12, 2023), and can also be proteins obtained by adding tags to the ends of the amino acid sequences of each protein.

[0027] The above protein can be a product expressed by a recombinant vector virus, or a product expressed by a recombinant vector or cell such as Escherichia coli, yeast, animal cells, or plant cells.

[0028] In a second aspect, the present invention provides an African swine fever virus nucleic acid combination, which includes at least three combinations of the following four nucleic acids; the four nucleic acids are nucleic acids encoding EP84R protein or its truncation, nucleic acids encoding D129L protein or its truncation, nucleic acids encoding E146L protein or its truncation and nucleic acids encoding K421R protein or its truncation.

[0029] In the African swine fever virus nucleic acid combination described above, the nucleic acid molecule is DNA or mRNA.

[0030] The above-mentioned nucleic acid molecule can be any of the following: the natural sequence of the ASFV gene, the same PCR amplification sequence, the artificially synthesized same sequence or degenerate sequence, and a sequence that is optimized and truncated on this basis but still encodes the protein product of each gene.

[0031] The above-mentioned protein encoding nucleic acids can be combined in a single form, or multiple protein encoding nucleic acids can be connected via a linker to obtain a fusion gene.

[0032] In a third aspect, the present invention provides a nucleic acid delivery vector or a nucleic acid delivery vector set containing any three or four of the following four nucleic acids;

[0033] The four nucleic acids are nucleic acids encoding EP84R protein or a truncation thereof, nucleic acids encoding D129L protein or a truncation thereof, nucleic acids encoding E146L protein or a truncation thereof, and nucleic acids encoding K421R protein or a truncation thereof.

[0034] In the above, the nucleic acid delivery vector may be a lipid nanoparticle or other nucleic acid delivery vector.

[0035] The lipid nanoparticles can be a combination of 4 mRNA vaccines, such as the combination shown in Table 19 in the embodiment. The titer of each group of mRNA vaccines is 10 8.0 TCID 50 / ml mixture, each mRNA vaccine is a lipid nanoparticle containing the African swine fever virus nucleic acid combination described in the second aspect.

[0036] In the above-mentioned nucleic acid delivery vector or nucleic acid delivery vector group, the nucleic acid is mRNA.

[0037] Each of the above mRNAs is obtained by transcribing the nucleic acid encoding each protein or its truncated form.

[0038] In an embodiment of the present invention, the preparation is specifically as follows: the 5'UTR, the nucleic acid encoding the protein or its truncation, the 3'UTR and polyA are cloned in series in the order of sequence into the fragment between the kpn I and xba I sites of the transcription plasmid pGEM-3zf(+) containing the T7 promoter to obtain a recombinant transcription plasmid; and then transcribed after Hind III digestion to obtain mRNA.

[0039] In a fourth aspect, the present invention provides a recombinant virus combination, which includes a recombinant virus or a recombinant virus group expressing any three or four of the following four nucleic acids;

[0040] The recombinant virus is a recombinant virus obtained by packaging a recombinant vector expressing any three or four of the following four nucleic acids;

[0041] The recombinant virus group is a recombinant virus group composed of recombinant viruses respectively packaged by recombinant vectors expressing single or multiple nucleic acids;

[0042] The four nucleic acids are nucleic acids encoding EP84R protein or a truncation thereof, nucleic acids encoding D129L protein or a truncation thereof, nucleic acids encoding E146L protein or a truncation thereof, and nucleic acids encoding K421R protein or a truncation thereof.

[0043] Each gene is distributed alone or in multiples on each recombinant virus; that is, each recombinant virus can express a single gene or a coupled gene composed of multiple genes.

[0044] The above-mentioned recombinant virus combination can be a combination of vector viruses of the same type or a combination of vector viruses of different types.

[0045] In the recombinant virus combination described above, the recombinant vector is derived from human adenovirus type 5, poxvirus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine adenovirus, porcine Japanese encephalitis virus, classical swine fever virus, retrovirus, paramyxovirus or lactic acid bacteria, but is not limited to these; each vector can produce a viral vector system and a bacterial vector system that can produce transient infection, stable infection or continuous expression of exogenous genes in pigs but does not cause any abnormal clinical symptoms.

[0046] In the embodiments of the present invention, examples are as follows:

[0047] The recombinant adenovirus is a recombinant adenovirus vector expressing the full length or part of the target gene and the backbone plasmid pacAd59.2-100 that is transfected into cells to package the recombinant adenovirus. The recombinant adenovirus group can be a combination of 1, 3 or 4 recombinant adenoviruses, and the amount of each virus used is greater than or equal to 10 8.0 TCID 50 ; Combinations as shown in Table 4, Table 5, Table 8 or Table 11 in the Examples. The recombinant adenoviral vector can express one or more target genes in full or in part.

[0048] The recombinant blue ear virus is obtained by transfecting cells with a recombinant blue ear virus vector expressing a target gene and packaging the recombinant blue ear virus. The recombinant blue ear virus group can be a combination of 4 recombinant blue ear viruses, and the amount of each virus used is greater than or equal to 10 8.0 TCID 50 ; A combination as shown in Table 14 in the Examples.

[0049] The recombinant pseudorabies virus is a recombinant pseudorabies virus vector expressing a target gene that is transfected into cells to package the recombinant pseudorabies virus. The recombinant pseudorabies virus group can be a combination of 4 recombinant pseudorabies viruses, with each virus used in an amount greater than or equal to 10 8.0 TCID 50 ; A combination as shown in Table 17 in the Examples.

[0050] The recombinant virus group can also be a combination of 4 recombinant blue ear viruses and 4 recombinant pseudorabies viruses. The recombinant virus group is allocated for 2 injections, first the recombinant PRRSV combination immunization and then the recombinant pseudorabies virus combination immunization. The titer of each recombinant PRRSV is 10 5.5 TCID 50 / ml were mixed in equal proportions, and the titer of each recombinant pseudorabies virus was 10 6.5 TCID 50 / ml are mixed in equal proportions, as shown in the combination in Table 18 in the embodiment;

[0051] In a fifth aspect, the present invention provides a biomaterial, which is any of the following:

[0052] 1) A recombinant vector group or recombinant vector expressing the African swine fever virus protein combination described in the first aspect;

[0053] 2) A recombinant microorganism group or recombinant microorganism expressing the African swine fever virus protein combination described in the first aspect;

[0054] 3) A recombinant cell group or recombinant cell expressing the African swine fever virus protein combination described in the first aspect.

[0055] The above-mentioned recombinant vector is a recombinant vector obtained by inserting each nucleic acid or multiple nucleic acids into an expression vector;

[0056] The above-mentioned recombinant vector group is a recombinant vector group composed of the above-mentioned individual recombinant vectors;

[0057] Each of the above nucleic acids can be a full-length protein-encoding nucleic acid or a protein-truncated nucleic acid fragment.

[0058] Each nucleic acid is distributed on each recombinant vector individually or in multiple forms; that is, each recombinant vector can express a single nucleic acid or multiple nucleic acid-coupled genes. Protein-encoding nucleic acids and protein-truncation-encoding nucleic acid fragments can be constructed into a single recombinant vector or cell that can express protein in pigs, or two or more protein-encoding nucleic acids or protein-truncation-encoding nucleic acid fragments can be cloned into the same recombinant vector or cell separately or together in a fusion form.

[0059] The above-mentioned expression vectors include, but are not limited to: human adenovirus type 5, poxvirus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine adenovirus, porcine Japanese encephalitis virus, classical swine fever virus, rabies virus, retrovirus, paramyxovirus or other viral vector systems or bacterial vector systems that can produce transient infection, stable infection or continuous expression of foreign genes in mammals but do not cause any abnormal clinical symptoms.

[0060] In the embodiments of the present invention, the recombinant vector is exemplified by a recombinant adenovirus vector, a recombinant porcine reproductive and respiratory syndrome virus vector (recombinant porcine reproductive and respiratory syndrome virus vector) or a recombinant porcine pseudorabies virus vector.

[0061] In a sixth aspect, the present invention provides the African swine fever virus protein combination described in the first aspect, the African swine fever virus nucleic acid combination described in the second aspect, the nucleic acid delivery vector or nucleic acid delivery vector group described in the third aspect, the recombinant virus combination described in the fourth aspect, or the biomaterial described in the fifth aspect in any of the following applications:

[0062] 1) Preparation of products for treating diseases caused by African swine fever virus infection;

[0063] 2) Preparation of products for inducing immune response to African swine fever virus antigens;

[0064] 3) Preparation of products for preventing diseases caused by African swine fever virus infection;

[0065] 4) Prepare anti-African swine fever virus products.

[0066] The product is a vaccine, medicine, combination, health product, functional food, food for special medical use or other biological product. The above vaccine also includes adjuvant or immunopotentiator or immunomodulator or other vaccine.

[0067] The above-mentioned adjuvant can be a salt adjuvant such as aluminum gel, different polysaccharide adjuvants, biological protein adjuvants, nucleic acid adjuvants or nanomaterial adjuvants, etc.; in an embodiment of the present invention, further, the adjuvant is a polysaccharide adjuvant, specifically Poria cocos polysaccharide or Schisandra chinensis polysaccharide.

[0068] The above vaccines are subunit vaccines, nucleic acid vaccines or recombinant virus vaccines.

[0069] In a seventh aspect, the present invention provides an African swine fever product, including any one of the following:

[0070] 1) The African swine fever virus protein combination described in the first aspect;

[0071] 2) The African swine fever virus nucleic acid combination of the second aspect;

[0072] 3) the nucleic acid delivery vector or nucleic acid delivery vector group described in the third aspect;

[0073] 4) The recombinant virus combination described in the fourth aspect;

[0074] 5) The biological material described in the fifth aspect.

[0075] The recombinant virus combination in the above-mentioned product or vaccine can be any three or more recombinant virus combinations, which are mixed together in a certain ratio and used directly or in batches.

[0076] The present invention also provides a method for preventing African swine fever virus, which is to immunize animals with the above product to achieve immunization. In an embodiment of the present invention, the recombinant viruses in the product are mixed in a certain proportion before immunization.

[0077] The present invention provides a combination of recombinant viruses of ASFV genes and a vaccine prepared therefrom, namely, utilizing genes such as EP84R protein, D129L protein, E146L protein and K421R protein of ASFV as target genes, respectively constructing recombinant viruses or recombinant bacteria of different viral or bacterial vectors, and combining the recombinant viruses or recombinant bacteria of these different gene, and the cultured virus liquid or bacterial culture liquid is mixed in equal proportions or different proportions and used directly, or is mixed with an adjuvant or an immunopotentiator and used, and the immunized susceptible animals can show good immune protection against African swine fever virus, can protect susceptible pigs from natural infection or artificial attack by ASFV strong toxin, and can be used for the prevention of African swine fever.

[0078] The vaccine of the present invention can be used to directly immunize the target animal - pigs, or can be mixed with adjuvants and immunopotentiators to immunize the target animal, both of which can provide good immune protection and protect susceptible pigs from natural infection or artificial attack by ASFV, and can be used for the prevention of African swine fever.

[0079] The beneficial effects of the present invention are as follows: After immunizing pigs, the recombinant virus or subprotein vaccine or mRNA vaccine constructed using African swine fever virus EP84R, D129L, E146L, and K421R can enable the immunized pigs to resist infection with ASFV. The immunized pigs can completely resist the attack of ASFV virulent virus without becoming ill, and can protect susceptible pigs from natural infection or artificial attack of ASFV, and are used for the prevention of African swine fever. DETAILED DESCRIPTION

[0080] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0081] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0082] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0083] The cells, strains and vectors used in the following examples are as follows:

[0084] 1. HEK293AD cells (purchased from Invitrogen, USA) are stored in the laboratory of this unit and need to be cultured in DMEM medium (a product of Gibco, catalog number C119955bbBT) or full suspension medium containing 1%-10% calf serum. BHK-21 cells (purchased from China Veterinary Drug Inspection Institute) are stored in the laboratory of this unit and need to be cultured in DMEM medium or full suspension medium containing 1%-10% calf serum. Marc-145 cells (purchased from China Veterinary Drug Inspection Institute) are stored in the laboratory of this unit and need to be cultured in DMEM medium containing 1%-10% calf serum. Vero cells (purchased from China Veterinary Drug Inspection Institute) are stored in the laboratory of this unit and need to be cultured in DMEM medium containing 1%-10% calf serum.

[0085] 2. The expression plasmid vector pacAd5 CMVK-NpA (purchased from Invitrogen, USA) and the backbone plasmid pacAd5 9.2-100 (purchased from Invitrogen, USA) of the foreign gene were amplified and extracted in E. coli. The expression plasmid of the foreign gene in the stable cell line was pcDNA3.1 plasmid (purchased from Invitrogen, USA).

[0086] 3. The in vitro transcription vector pGEM-3ZF(+) was purchased from Promega. The 5'UTR, 3'UTR and polyA structure were biosynthetically subcloned into pGEM-3ZF(+). The constructed recombinant plasmid was propagated in E. coli and the plasmid was extracted.

[0087] 4. PRRSV-A1 strain (the full-length gene was synthesized by Jilin Kumei Biotechnology Co., Ltd., 59-15453 bp of sequence 1) is a rescued attenuated vaccine strain, and the infectious genome is cloned in a plasmid.

[0088] 5. The pseudorabies virus strain is the vaccine candidate strain JL14-△gI / gE / TK. The specific construction method is: the virulent JL strain isolated and identified in our laboratory was weakened into the vaccine candidate strain JL14-△gI / gE / TK by homologous recombination deletion of gI and gE genes (recorded in the following literature: Zhou Xintao. Construction and characteristics study of pseudorabies virus gene-deficient strain JL14-△gI / gE / TK [D]. Jilin Agricultural University, 2018).

[0089] 6. African swine fever virus SY-18 strain (recorded in the following literature: Zhou X, Li N, Luo Y, Liu Y, Miao F, Chen T, Zhang S, Cao P, Li X, Tian K, Qiu HJ, Hu R (2018) Emergence of African Swine Fever in China, 2018. Transboundary and emerging diseases 65 (6): 1482-1484. doi: 10.1111 / tbed.12989), isolated by the Epidemiology Research Laboratory of the Military Veterinary Research Institute in 2018. During isolation, 0.1 ml of sterile diseased material (blood) from infected pigs was inoculated in a 25CM 2 The primary porcine alveolar macrophages were cultured in a culture flask at 37°C for 4-6 days, and then the culture medium was collected after freezing and thawing. The culture medium was used for continuous passage 4 times, and then the culture medium was collected after freezing and thawing. An appropriate amount was taken for serial dilution, and the virus titer was measured on a 96-well cell culture plate. The half-cultured cell infection dose (TCID) of the virus was calculated according to the Reed-Muench method. 50 ). The virus titer was 10 6.0 TCID 50 The above cultures are reserved for future use. The GenBank accession number of the genome sequence of this virus is: MH766894.3 (submitted on July 12, 2023). The strain used in this study is the fourth-generation propagation virus using PAM cells and is stored in aliquots at -80°C.

[0090] African swine fever virus target genes EP84R, D129L, E146L, K421R, these genes and amino acid sequences can be found in GenBank accession number: MH766894.3 (submitted on July 12, 2023).

[0091] Example 1. Construction and immune effect of recombinant adenovirus expressing African swine fever virus EP84R, D129L, E146L, K421R genes

[0092] 1. Construction of recombinant adenovirus expressing African swine fever virus EP84R, D129L, E146L, K421R genes

[0093] The construction of recombinant adenovirus expressing EP84R, D129L, E146L, and K421R genes is as follows (the construction and use strategies of other replication-deficient and replication-competent vector DNA viruses are similar):

[0094] 1. Construction of homologous recombination plasmid

[0095] The genome of African swine fever virus SY-18 strain was extracted by conventional methods, and primers of ASFV EP84R, D129L, E146L, and K421R genes with homologous arms of expression plasmid vector pacAd5CMVK-NpA were designed, as shown in Table 1. Using the genome of SY-18 strain as a template, the designed primers were used to amplify by PCR method to obtain homologous recombinant gene fragments of ASFV EP84R, D129L, E146L, and K421R genes.

[0096] Table 1 shows the primer sequences for EP84R, D129L, E146L, and K421R gene amplification

[0097]

[0098] The following recombinant plasmids are obtained by inserting each homologous recombination gene fragment into the EcoRI site of plasmid pacAd5 CMVK-NpA (human adenovirus type 5 expression vector). The details are as follows:

[0099] The homologous recombination gene fragments of each gene amplified above were connected with the pacAd5 CMVK-NpA plasmid linearized by EcoRI in a certain ratio, and then transformed into Escherichia coli competent cells for homologous recombination to obtain recombinant plasmids pAdCMV-EP84R, pAdCMV-D129L, pAdCMV-E146L, and pAdCMV-K421R, respectively.

[0100] The recombinant plasmid pAdCMV-EP84R is a plasmid obtained by inserting the EP84R gene into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the EP84R gene;

[0101] The recombinant plasmid pAdCMV-D129L is a plasmid obtained by inserting the D129L gene into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the D129L gene;

[0102] The recombinant plasmid pAdCMV-E146L is a plasmid obtained by inserting the E146L gene into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the E146L gene;

[0103] The recombinant plasmid pAdCMV-K421R is a plasmid obtained by inserting the K421R gene into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the K421R gene.

[0104] 2. Rescue and identification of recombinant adenovirus

[0105] The recombinant plasmids pAdCMV-EP84R, pAdCMV-D129L, pAdCMV-E146L, pAdCMV-K421R and the backbone plasmid pacAd5 9.2-100 were linearized by PacI digestion to obtain four kinds of recombinant plasmids after digestion and the backbone plasmid pacAd5 9.2-100 after digestion.

[0106] The above-mentioned recombinant plasmids after enzyme digestion were mixed with the enzyme-cut backbone plasmid pacAd59.2-100 (4 μg) in a certain ratio (2 μg), and then mixed with 200 μl of transfection reagent. 2000) after mixing, add 2ml of cell culture medium, add evenly to the cell monolayer, and transfect 25CM 2 HEK293AD cells grown into a monolayer were cultured for 3 days, and the transfected cells were passaged. After growing into a monolayer, the second transfection was performed according to the above steps.

[0107] Transfect three times according to the above steps. When a large number of cell rounding and other pathological changes are observed, freeze-thaw the cells, collect and save the freeze-thawed fluid, and record them as P0 recombinant adenovirus EP84R, P0 recombinant adenovirus D129L, P0 recombinant adenovirus E146L, and P0 recombinant adenovirus K421R, respectively. Different gene fragments are transferred as different recombinant viruses. The P0 recombinant adenovirus is inoculated into a 25cm 2 HEK293AD cells (cell number: 2×10 6 ), and freeze-thawed 2 days after the disease, and the freeze-thawed liquid was collected and preserved, which was the P1 generation recombinant adenovirus; the above steps were repeated until the P3 generation recombinant adenovirus EP84R, D129L, E146L, K421R were collected.

[0108] 3. Detection

[0109] The titer of each P3 recombinant adenovirus was determined by the following method: the P3 recombinant adenovirus was diluted 10-fold in series, a total of 12 dilutions were made, 8 replicate wells were inoculated for each dilution, and the cells were inoculated on HEK293AD cells in a 96-well plate with an inoculation volume of 0.1 ml / well. After inoculation, the cell lesions were observed under a light microscope at 37°C for 5-6 days, and the TCID of the virus was calculated according to the Reed-Muench method. 50 .

[0110] The results are shown in Table 2.

[0111] Table 2 shows the titer determination results of each P3 generation recombinant adenovirus

[0112] Recombinant adenovirus <![CDATA[Titer TCID 50 / mL]]> rAdv-EP84R <![CDATA[10 8.50 ]]> rAdv-D129L <![CDATA[10 8.250 ]]> rAdv-E146L <![CDATA[10 8.875 ]]> rAdv-K421R <![CDATA[10 8.625 ]]>

[0113] The nucleic acid of each P3 generation recombinant adenovirus was extracted, and the recombinant adenovirus was identified using identification primers (upstream primer: CGCAAATGGGCGGTAGGCGTG, downstream primer: CACTGCATTCTAGTTGTGGTTT). The expected identification product size is shown in Table 1. The target size fragment amplified is the target recombinant adenovirus.

[0114] Results All the recombinant adenoviruses obtained were target adenoviruses.

[0115] The recombinant adenoviruses expressing the target genes were named as recombinant adenoviruses rAdv-EP84R, rAdv-D129L, rAdv-E146L, and rAdv-K421R, respectively.

[0116] II. Immune protection experiment of recombinant adenovirus expressing EP84R, D129L, E146L, and K421R genes

[0117] 1. Immunoprotection test of any combination of 2, 3, or 4 of EP84R, D129L, E146L, or K421R gene recombinant adenovirus (other replication-deficient and replication-competent vector DNA viruses and other combinations and usage strategies of these genes are similar)

[0118] 1) Immunity

[0119] The recombinant adenovirus obtained above was combined in the form of 2, 3, or 4 recombinant adenovirus combinations as shown in Table 4 (10 of each virus was used). 8.0 TCID 50 ) to obtain vaccines of different combinations with a total volume of 2 ml, and 100 micrograms of Poria polysaccharide were added to each vaccine.

[0120] Poria polysaccharide is prepared as follows: 10 kg of Poria tubers are crushed, 50 kg of distilled water is added and soaked overnight, heated to 80°C, maintained for 2 hours, and stirred continuously, and the supernatant is collected by centrifugation at 10000 rpm (centrifugal force is 12857 g g) for 15 minutes by a continuous centrifuge, and the water is evaporated to 1 / 10 of the supernatant volume by a rotary evaporator. Some of the supernatant is taken, 1 / 5 volume of petroleum ether is added, refluxed at 37°C for 60 minutes, anhydrous ethanol is added to the aqueous phase to a final concentration of 65% ethanol, and after standing for 1 hour, 8000 rpm (8228 g) is centrifuged for 30 minutes, and the precipitate is collected, and the precipitate is resuspended in a physiological saline (NaCl aqueous solution) with a mass volume ratio (g: ml) of 0.9%, so that the final concentration of the precipitate reaches 100 mg / ml, which is Poria polysaccharide.

[0121] Experimental group: Each combination of the above vaccines with added Poria polysaccharide (10 8.0 TCID 50 , and then add 100 micrograms of Poria polysaccharide to each portion (2 ml) after mixing. ) Five pigs were immunized by intramuscular injection. Immunization was performed twice, with an interval of 14 days, and the dose was the same each time.

[0122] Control immune group: A mixture of recombinant adenovirus expressing p30, p54, p72, pCD2v, pF317L, pp62, and pp220 and a mixture of recombinant adenovirus expressing D129L, EP84R, E146L, NP419L, and S273R genes were used as controls (10 cells / mL of each virus were used). 8.0 TCID 50 , and then add 100 micrograms of Poria polysaccharide to each portion (2 ml) after mixing. ), and 5 pigs were immunized by intramuscular injection. Immunization was performed twice, with an interval of 14 days, and the dose was the same each time.

[0123] The recombinant adenoviruses expressing the p30, p54, p72, pCD2v, pF317L, pp62, and pp220 genes were prepared according to the construction method of the recombinant adenovirus in 1 (the sequences of each gene can be found in GenBank accession number: MH766894.3, and the submission date is July 12, 2023), the template is the genome of the African swine fever virus SY 18 strain, and the primer sequences and amplified gene sizes are shown in Table 3.

[0124] Table 3 shows the primer sequences and product sizes for each gene amplification

[0125]

[0126]

[0127] The recombinant adenovirus was identified using the upstream primer: CGCAAATGGGCGGTAGGCGTG, and the downstream primer: CACTGCATTCTAGTTGTGGTTT. The expected size of the identified product is shown in Table 3. The target size fragment amplified is the target recombinant adenovirus.

[0128] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0129] Follow daily feeding.

[0130] 2) Anti-virus

[0131] On the 14th day after the second immunization (the first day of the second immunization was recorded as the 1st day), the above experimental groups, control immunization group and challenge control group were challenged with strong toxicity using African swine fever virus SY18 strain: 10 3.0TCID 50 / 2ml ASFV virulent SY18 strain. Observe the survival of the pigs.

[0132] The recombinant viruses of each combination and their immune challenge results are shown in Table 4. It can be seen that the recombinant adenovirus of each combination has an obvious challenge protection effect, which is different from the traditional reported gene selection.

[0133] Table 4 shows the results of immune challenge with different recombinant virus combinations and traditional target protein recombinant viruses

[0134]

[0135]

[0136] 2. Immunization and challenge experiments with recombinant adenovirus combinations expressing EP84R, D129L, E146L, K421R and other genes (5, 6, 7, 8)

[0137] 1) Immunity

[0138] The recombinant adenovirus obtained above was combined according to the combination shown in Table 5 (10 of each virus was used). 8.0 TCID 50 ) to obtain vaccines in different combinations with a total volume of 2 ml, and 100 micrograms of Poria polysaccharide were added to each vaccine.

[0139] Experimental group (Group 1): The recombinant adenovirus obtained above was added with each combination of Poria cocos polysaccharide vaccines (10 8.0 TCID 50 After mixing, 100 micrograms of Poria polysaccharide were added to each portion (2 ml) and injected intramuscularly into 5 pigs. Immunization was performed twice, with an interval of 14 days, and the dose was the same each time.

[0140] Experiment + other protein group (Groups 2-5): The recombinant adenovirus obtained above was mixed with the recombinant adenovirus expressing other proteins (10 8.0 TCID 50 After mixing, 100 micrograms of Poria polysaccharide were added to each portion (2 ml) and injected intramuscularly into 5 pigs. Immunization was performed twice, with an interval of 14 days, and the dose was the same each time.

[0141] The recombinant adenoviruses expressing other proteins are CP2457L, CP204L, E183L, and B646L (the preparation method is based on the construction method of the recombinant adenovirus in 1, and the sequence of each gene can be found in GenBank accession number: MH766894.3, and the submission date is July 12, 2023);

[0142] Other combination groups: Recombinant adenovirus expressing pCD2v, pF317L, pp62, pp220 and control group recombinant adenovirus CP2457L, CP204L, E183L, B646L were mixed (10 of each virus were used). 8.0 TCID 50 After mixing, 100 micrograms of Poria polysaccharide were added to each portion (2 ml) and injected intramuscularly into 5 pigs. Immunization was performed twice, with an interval of 14 days, and the dose was the same each time.

[0143] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0144] Follow daily feeding.

[0145] 2) Anti-virus

[0146] On the 14th day after the second immunization (the first day of the second immunization was recorded as the 1st day), the above experimental groups, control immunization group and challenge control group were challenged with strong toxicity using African swine fever virus SY18 strain: 10 3.0 TCID 50 / 2ml ASFV virulent SY18 strain. The patients were observed for 28 days, and the clinical manifestations and final outcomes were recorded.

[0147] The recombinant viruses of each combination and their immune challenge results are shown in Table 5. It can be seen that through observation within 28 days, all the non-immunized control pigs became ill and died, all the pigs immunized with the traditional vector vaccine combination died, and all 5 pigs in the other immunized groups were alive and well.

[0148] Table 5 shows the results of immunizing pigs with more than 4 gene combinations of different recombinant adenoviruses

[0149]

[0150] Example 2: Construction of recombinant adenovirus expressing two fusion genes of African swine fever virus and its immune effect

[0151] The present invention provides the construction and immune effect of ASFV two-gene fusion recombinant adenovirus (the construction and combined use strategies of any two or more fusion genes of other replication-deficient and replication-competent vector DNA viruses are similar), as follows:

[0152] 1. Construction of ASFV II gene fusion recombinant adenovirus

[0153] The EP84R gene sequence + linker sequence + D129L gene tandem sequence were connected by a synthetic method to obtain a two-gene fusion fragment (EP84R-D129L);

[0154] The E146L gene sequence + linker sequence + K421R gene tandem sequence were connected by a synthetic method to obtain a two-gene fusion fragment (E146L-K421R);

[0155] The linker sequence above is: gcaacaaacttctctctgctgaaacaagccggagatgtcgaagagaatcctggaccg.

[0156] According to the recombinant adenovirus construction method in Example 1, two-gene fusion recombinant adenoviruses were constructed respectively: first, each two-gene fusion fragment was amplified to obtain a homologous recombination fragment (using the two-gene fusion fragment as a template, the primers required for amplification and the product size are shown in Table 6), and then constructed into the EcoRI restriction site of the pacAd5 CMVK-NpA plasmid through homologous recombination to obtain a recombinant plasmid, and then the recombinant plasmid and the backbone plasmid pacAd5 9.2-100 were transfected into HEK293AD cells, and the freeze-thaw fluid was harvested until each P3 generation two-gene fusion recombinant adenovirus was obtained, which were named rAdv-EP84R-D129L and rAdv-E146L-K421R, respectively.

[0157] The nucleic acid of the P3 generation recombinant adenovirus expressing each target gene was extracted, and the recombinant adenovirus was identified using identification primers (upstream primer: CGCAAATGGGCGGTAGGCGTG, downstream primer: CACTGCATTCTAGTTGTGGTTT). The expected identification product size is shown in Table 6. The target size fragment amplified is the target recombinant adenovirus.

[0158] Table 6 shows the primer sequences and product sizes for the amplification of the two gene fusion fragments

[0159]

[0160] The titer of each recombinant virus was determined according to conventional methods. During the determination, each virus culture fluid was diluted 10 times in series, for a total of 12 dilutions, and 8 replicates were inoculated for each dilution. The cells were inoculated on HEK293AD cells in a 96-well plate with an inoculation volume of 0.1 ml / well. After inoculation, the cell lesions were observed under a light microscope at 37°C for 5-6 days, and the TCID of the virus was calculated according to the Reed-Muench method. 50 The results are shown in Table 7.

[0161] Table 7 shows the titer of fusion recombinant adenovirus

[0162] Recombinant adenovirus <![CDATA[Titer TCID 50 / mL]]> rAdv-EP84R-D129L <![CDATA[10 8.375 ]]> rAdv-E146L-K421R <![CDATA[10 8.625 ]]>

[0163] II. Immune protection test of recombinant adenovirus with two gene fusions

[0164] 1. Immunity

[0165] Experimental group: 10 samples of each of the two gene fusion recombinant adenoviruses prepared above (as shown in Table 7) were taken. 8.0 TCID 50 , added 100 micrograms of Poria polysaccharide respectively; injected into the neck muscles of pigs respectively, immunized 5 pigs in each group.

[0166] Control immune group: A mixture of three recombinant adenoviruses expressing p30, p54, and p72 was used as a control. Each virus was used at 10 8.0 TCID 50 After mixing, 100 micrograms of Poria polysaccharide were added, and 5 heads were immunized in each group.

[0167] The above two groups were immunized twice, with an interval of 14 days, and the dose of each immunization was the same.

[0168] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0169] Follow daily feeding.

[0170] 2. Attack the virus

[0171] On the 14th day after the second immunization (the first day of the second immunization was recorded as day 1), the above experimental groups (EP84R-D129L and E146L-K421R), control immunization groups (rdv-p30, rdv-p54, rdv-p72) and the challenge control group were challenged with African swine fever virus SY18 strain, and 10 3.0 TCID 50 / 2ml ASFV virulent SY18 strain. Observe the survival of the pigs.

[0172] The results are shown in Table 8. It can be seen that all the non-immunized control pigs became ill and died, all the pigs immunized with the traditional vector vaccine combination died, and more than 60% to 80% of the 5 pigs in the other immunized groups survived. This shows that the selected genes and fusion combinations have obvious protective effects against viruses, which is different from traditional gene selection.

[0173] Table 8 is a comparison of the immune effects of different two-gene fusion recombinant viruses and traditional target protein recombinant viruses

[0174]

[0175] Example 3: Construction of recombinant adenovirus expressing a partial coding fragment of African swine fever virus gene and its immune effect

[0176] The present invention provides the construction and immune test of recombinant adenovirus encoding partial gene fragments of EP84R9L, D129L, E146L, and K421R (the construction and use strategies of recombinant viruses encoding partial gene fragments of ASFV of other replication-deficient and replication-competent vector DNA viruses are similar), as follows:

[0177] 1. Construction of recombinant adenovirus encoding partial ASFV gene fragments

[0178] 1. Construction of homologous recombination plasmid

[0179] The recombinant plasmid pAdCMV-fEP84L is a plasmid obtained by inserting bases 10-150 of the EP84R gene (MH766894.3, submitted on July 12, 2023) into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the gene encoding the fEP84L protein (amino acid sequence of the EP84L protein, bases 16-70);

[0180] The recombinant plasmid pAdCMV-fD129LR is a plasmid obtained by inserting bases 88-211 of the D129L gene into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the fD129LR protein (D129L protein amino acid sequence bases 30-90) gene;

[0181] The recombinant plasmid pAdCMV-fE146L is a plasmid obtained by inserting bases 58-353 of the E146L gene into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the gene encoding the fE146L protein (amino acid sequence of the E146L protein, bases 25-105);

[0182] The recombinant plasmid pAdCMV-fK421R is a plasmid obtained by inserting the 241-930 bases of the K421R gene into the EcoRI site of the pacAd5 CMVK-NpA plasmid (human adenovirus type 5 expression vector), and the plasmid expresses the gene encoding the fK421R protein (58-300 bases of the amino acid sequence of the K421R protein). The amplification primer sequences are shown in Table 9.

[0183] Table 9 shows the primer sequences and product sizes for each part of the gene amplification

[0184]

[0185] 2. Rescue and identification of recombinant adenovirus

[0186] According to the method of Example 1, the recombinant plasmids pAdCMV-fEP84L, pAdCMV-fD129LR, pAdCMV-fE146L, pAdCMV-fK421R and the backbone plasmid pacAd5 9.2-100 were respectively linearized by PacI digestion to obtain four types of recombinant plasmids after digestion and the backbone plasmid pacAd5 9.2-100 after digestion.

[0187] The above-mentioned recombinant plasmids after enzyme digestion were mixed with the enzyme-cut backbone plasmid pacAd59.2-100 (4 μg) in a certain ratio, and then mixed with 12 μl of transfection reagent. 2000) after mixing, add 2 ml DMEM cell culture medium, add evenly to the cell monolayer, and transfect 25CM 2 HEK293AD cells grown into a monolayer were transfected multiple times according to the method of Example 1 to prepare P3 generation recombinant adenovirus.

[0188] 3. Detection

[0189] The titer of the P3 generation recombinant adenovirus was measured by the following method: refer to Example 1.

[0190] The results are shown in Table 10.

[0191] Table 10 shows the titer determination results of each recombinant adenovirus of generation P3

[0192] Recombinant adenovirus <![CDATA[Titer TCID 50 / mL]]> rAdv-fEP84R <![CDATA[10 8.50 ]]> rAdv-fD129L <![CDATA[10 8.375 ]]> rAdv-fE146L <![CDATA[10 8.625 ]]> rAdv-fK421R <![CDATA[10 8.50 ]]>

[0193] The nucleic acid of the P3 generation recombinant adenovirus was extracted and the identification primers (upstream primer: CGCAAATGGGCGGTAGGCGTG, downstream primer: CACTGCATTCTAGTTGTGGTTT) were used to identify the recombinant adenovirus. The expected identification product size is shown in Table 9. The target size fragment amplified is the target recombinant adenovirus. The recombinant adenoviruses obtained were all target adenoviruses.

[0194] Each rescued target adenovirus was named recombinant adenovirus rAdv-fEP84R, rAdv-fD129L, rAdv-fE146L, and rAdv-fK421R, respectively.

[0195] II. Immune protection experiment of recombinant adenovirus

[0196] The constructed gene recombinant vector viruses such as fEP84R, fD129L, fE146L, and fK421R were inoculated into HEK293AD cells, respectively, and the culture was expanded, the virus liquid was harvested, and the titer was determined; 8 TCID 50 Three or more combinations of the above recombinant vector virus solutions are mixed in equal proportions.

[0197] 1. Immunity

[0198] The rAdv-fEP84R, rAdv-fD129LR, rAdv-fE146L, and rAdv-fK421R recombinant adenoviruses were mixed in equal proportions, and the virus titer of each recombinant virus was 10 8 TCID 50 / ml to obtain a combined vaccine with a total volume of 2ml, and 100 micrograms of pachymaran were added to each vaccine.

[0199] fEP84R, fD129LR, fE146L, rfK421R recombinant adenovirus combination group: 5 pigs were immunized intramuscularly with the above combination vaccine supplemented with Poria cocos polysaccharide. The immunization was repeated twice, with an interval of 14 days, and the dose was the same each time.

[0200] p30, p54, p72, pCD2v, pF317L, pp62, pp220 virus combination group: The recombinant adenovirus mixture expressing p30, p54, p72, pCD2v, pF317L, pp62, pp220 was used as the control immunization group (10 8.0 TCID 50 After mixing, 100 micrograms of Poria polysaccharide were added to each portion (2 ml) and injected intramuscularly into 5 pigs. Immunization was performed twice, with an interval of 14 days, and the dose was the same each time.

[0201] The above two groups were immunized twice, with an interval of 14 days, and the dose of each immunization was the same.

[0202] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0203] Follow daily feeding.

[0204] 2. Attack the virus

[0205] On the 14th day after the second immunization (the first day of the second immunization was recorded as the 1st day), the above-mentioned immunization groups and the control group were challenged with African swine fever virus SY18 strain, and 10 3.0 TCID 50 / 2ml ASFV virulent SY18 strain. The patients were observed for 28 days, and the clinical manifestations and final outcomes were recorded.

[0206] The results are shown in Table 11. Through observation within 28 days, all the control pigs infected with the virus became ill and died, all the pigs immunized with the traditional vector vaccine combination (p30, p54, p72, pCD2v, pF317L, pp62, pp220 virus combination) died, and all 5 pigs in the immunization group with the recombinant virus of 4 gene fragments (fEP84, fD129LR, fE146L, fK421R recombinant adenovirus combination) were alive and well.

[0207] Table 11 shows the results of pig challenge with recombinant adenovirus combination with different gene fragments

[0208]

[0209] Example 4: Construction of recombinant blue ear virus (PRRSV) expressing African swine fever virus gene and its immune effect

[0210] 1. Construction of recombinant PRRSV expressing EP84R, D129L, E146L, and K421R genes

[0211] 1. Construction of virus rescue plasmid

[0212] Virus rescue plasmid pcDNA-HH-PRRSV-TRS: A plasmid obtained by inserting the HamRz-PRRSV (5'UTR-ORF1-PacⅠ-TRS-ORF2-ORF3-ORF4-ORF5-ORF6-ORF7-3'UTR)-HdvRz (sequence 1) fragment into the EcoRV restriction site of plasmid pcDNA3.1 (Invitrogen, catalog number: V790-20).

[0213] The nucleotide sequence of the HamRz-PRRSV (5'UTR-ORF1-PacⅠ-TRS-ORF2-ORF3-ORF4-ORF5-ORF6-ORF7-3'UTR)-HdvRz fragment is sequence 1, in which positions 1-58 are the hammerhead ribozyme encoding gene, positions 12048-12048 are the restriction site PacⅠ, positions 12049-12087 are the transcription regulatory sequence TRS, and positions 15454-15537 are the delta-hepatitis ribozyme encoding gene.

[0214] Using ASFV SY18 strain DNA as a template, primers were designed referring to the instructions of Takara's seamless cloning enzyme In-Fusion, and PCR amplified the corresponding fragments of EP84R, D129L, E146L, and K421R genes (primer sequences are shown in Table 12), and connected with the plasmid pcDNA-HH-PRRSV-TRS digested with PacⅠ to construct recombinant PRRSV rescue plasmids pcDNA-HH-PRRSV-EP84R, pcDNA-HH-PRRSV-D129L, pcDNA-HH-PRRSV-E146L, and pcDNA-HH-PRRSV-K421R.

[0215] The above recombinant plasmid is a plasmid obtained by inserting each gene fragment into the PacⅠ site of the plasmid pcDNA-HH-PRRSV-TRS.

[0216] Table 12 shows the primer sequences and product sizes for each gene amplification

[0217]

[0218] 2. Rescue and identification of recombinant viruses

[0219] Take the rescue and identification of EP84R gene recombinant PRRSV as an example.

[0220] The recombinant PRRSV rescue plasmid pcDNA3.1-HH-PRRSV-EP84R was transfected into BHK-21 cells, and frozen and thawed twice after 3 days. The supernatant was collected and transferred to Marc145 cells and observed for 4 days. If cell lesions were found, it proved that the recombinant virus was successfully rescued and marked as rPRRSV-EP84R.

[0221] The recombinant virus rPRRSV-EP84R was identified by RT-PCR using identification primers (upstream primer: 5'-TGCTGGAAAGTGATGTTGGAC-3', downstream primer: 5'-TGCTCAGGGTGAACGGTAGA-3') to obtain the target product.

[0222] In the same way, BHK cells were transfected with the corresponding rescue plasmids to rescue recombinant PRRSV viruses rPRRSV-D129L, rPRRSV-E146L, and rPRRSV-K421R expressing D129L, E146L, and K421R genes, respectively, and RT-PCR was performed using identification primers. The sequences of primers for each gene amplification and the product sizes are shown in Table 12.

[0223] 3. Titer detection

[0224] The titer of the African swine fever virus gene-recombinant PRRSV was determined. During the determination, the culture fluid of each recombinant PRRSV virus was diluted 10 times in series, for a total of 12 dilutions, and 8 replicates were inoculated at each dilution. The cells were inoculated on Marc-145 cells in a 96-well plate with an inoculation volume of 0.1 ml / well. After inoculation, the cell lesions were observed under a light microscope at 37°C for 4-5 days, and the TCID of the virus was calculated according to the Reed-Muench method. 50 The results are shown in Table 13 below.

[0225] Table 13 is the results of African swine fever virus gene-recombinant PRRSV titer determination

[0226] Recombinant virus <![CDATA[Titer TCID 50 / mL]]> rPRRSV-EP84R <![CDATA[10 7.250 ]]> rPRRSV-D129L <![CDATA[10 7.375 ]]> rPRRSV-E146L <![CDATA[10 7.125 ]]> rPRRSV-K421R <![CDATA[10 7.0 ]]>

[0227] II. Immunization and challenge experiments with recombinant PRRSV expressing EP84R, D129L, E146L, and K421R genes

[0228] 1. Immunity

[0229] The recombinant PRRSV viruses expressing EP84R, D129L, E146L, and K421R genes were mixed in 2, 3, or 4 combinations of recombinant viruses (Table 14). The virus titer of each recombinant virus in each combination was 10 5.7 TCID 50 / ml, each recombinant virus was mixed in equal proportions, 0.3ml of each virus, and 100 micrograms of Schisandra polysaccharide were added to each dose of vaccine.

[0230] Schisandra polysaccharide was prepared as follows: 10 kg of Schisandra leaves were crushed, 50 kg of distilled water was added and soaked overnight, heated to 80°C, maintained for 2 hours, and stirred continuously, and the supernatant was collected by centrifugation at 10000 rpm (centrifugal force of 12857 g) for 15 minutes in a continuous centrifuge, and the water was evaporated to 1 / 10 of the supernatant volume by a rotary evaporator. Some of the supernatant was taken, 1 / 5 volume of petroleum ether was added, refluxed at 37°C for 60 minutes, anhydrous ethanol was added to the aqueous phase until the final concentration of ethanol was 70%, and after standing for 1 hour, 8000 rpm (8228 g) was centrifuged for 30 minutes, and the precipitate was collected, and the precipitate was resuspended in a physiological saline (NaCl aqueous solution) with a mass volume ratio (g: ml) of 0.9%, so that the final concentration of the precipitate reached 100 mg / ml, which was Schisandra polysaccharide.

[0231] Immunization experimental group: 10 recombinant PRRSV viruses were taken from each combination prepared above (Table 14 rPRRSV-EP84R, rPRRSV-D129L, rPRRSV-E146L, rPRRSV-K421R combination, rPRRSV-EP84R, rPRRSV-D129L combination, rPRRSV-EP84R, rPRRSV-D129L, rPRRSV-E146L combination) 5.7 TCID 50 / ml each recombinant virus was mixed in equal proportions, 0.3ml of each virus, and then 100 micrograms of Schisandra polysaccharide were added after mixing; the mixture was injected into the neck muscles of pigs, and 5 pigs were immunized in each group.

[0232] Traditional vector vaccine combination group: p30, p54, p72, pCD2v, pF317L, pp62, pp220 recombinant adenovirus mixture was used as a control, and 10 of each virus was used. 8.0 TCID 50 , and then add 100 micrograms of Schisandra polysaccharide after mixing, and 5 heads were immunized in each group.

[0233] The above two groups were immunized twice, with an interval of 14 days, and the dose of each immunization was the same.

[0234] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0235] Follow daily feeding.

[0236] 2. Attack the virus

[0237] On the 14th day after the second immunization (the first day of the second immunization was recorded as the 1st day), the above experimental groups, control immunization group and challenge control group were challenged with African swine fever virus SY18 strain, and 10 3.0 TCID 50 / 2mlASFV virulent SY18 strain. The patients were observed for 28 days, and the clinical manifestations and final outcomes were recorded.

[0238] The results are shown in Table 14. It can be seen that through observation within 28 days, all the non-immunized control pigs became ill and died, all the pigs immunized with the traditional vector vaccine combination died, and some or all of the 5 pigs immunized with the combination of recombinant viruses with 2, 3, and 4 genes survived.

[0239] Table 14 shows the results of the challenge of pigs immunized with recombinant porcine reproductive and respiratory syndrome virus with different gene combinations

[0240]

[0241] Example 5: Construction of recombinant pseudorabies virus expressing EP84R, D129L, E146L, K421R genes and immune protection test

[0242] 1. Construction of recombinant pseudorabies virus expressing EP84R, D129L, E146L, and K421R genes

[0243] 1. Homologous recombination plasmid

[0244] The homologous recombination plasmid pUC-ΔTK-EGFP was obtained by inserting the TKRight Arm-EGFP-TKLeft Arm fragment (sequence 2) between the XbaⅠ and SphⅠ restriction sites of the pUC19 plasmid (Takara, Code No. 3219).

[0245] The pUC-ΔTK-EGFP linearized fragment was obtained by PCR amplification using pUC-ΔTK-EGFP as a template and primers PUC-F and PUC-R.

[0246] PUC-F: ctaccggtcgccaccatggtgagcaaggg, PUC-R: gaccggtagcgctagcggatctgacggt;

[0247] Using ASFV SY18 strain DNA as a template, primers were designed according to the instructions of Takara's seamless cloning enzyme In-Fusion (Table 15). PCR amplified the corresponding fragments of EP84R, D129L, E146L, and K421R genes, and cloned them into the pUC-ΔTK-EGFP plasmid respectively to construct recombinant plasmids pUC-ΔTK-EGFP-EP84R, pUC-ΔTK-EGFP-D129L, pUC-ΔTK-EGFP-E146L, and pUC-ΔTK-EGFP-K421R.

[0248] The above recombinant plasmid is obtained by recombining the gene fragments obtained by the above PCR amplification under the action of In-Fusion enzyme and inserting them between the GCCACC (Kozak sequence) of the pUC-ΔTK-EGFP linearized fragment obtained by PCR and the EGFP gene.

[0249] Table 15 shows the recombinant PRV virus-specific primers and product sizes

[0250]

[0251] 2. Rescue, purification and identification of recombinant viruses

[0252] Take the rescue, purification and identification of EP84R gene recombinant pseudorabies virus as an example.

[0253] The recombinant plasmid pUC-ΔTK-EGFP-EP84R was transfected into BHK cells, and 5 hours later, the JL14-ΔgI / gE / TK virus lacking the gI and gE genes was infected at an MOI of 0.1; after 24 hours of culture, cells with green fluorescence were picked and added to fresh normal BHK cells to complete a round of purification; after 4 rounds of purification in this step, the fluorescent cells were collected, frozen and thawed three times, and 3 rounds of limiting dilution were performed on BHK cells, and the fluorescent cells in the last round of limiting dilution wells were collected and preserved; the identification primers (upstream primer: 5'-GGCTGACCGCCCAACGA-3', downstream primer: 5'-CCTTGCTCACCATCGGTCC-3') were used for purity identification; the purified recombinant pseudorabies virus strain was named PRV-EP84R (referred to as PRV-EP84R gene recombinant pseudorabies virus).

[0254] In the same way, the corresponding recombinant plasmids were used to transfect BHK cells, and the recombinant pseudorabies viruses PRV-D129L, PRV-E146L, and PRV-K421R were rescued and purified. After correct identification with identification primers, they were stored at -40°C.

[0255] The titer of the recombinant pseudorabies virus of the African swine fever virus gene was determined. During the determination, the culture fluid of each recombinant virus was serially diluted 10 times, for a total of 12 dilutions. Eight replicates were inoculated for each dilution and inoculated onto Vero cells in a 96-well plate. The inoculation volume was 0.1 ml / well. After inoculation, the cell lesions were observed under a light microscope at 37°C for 3-4 days, and the TCID of the virus was calculated according to the Reed-Muench method. 50 The results are shown in Table 16.

[0256] Table 16 shows the results of the African swine fever virus gene-recombinant PRV titer determination

[0257] Recombinant virus <![CDATA[Titer TCID 50 / mL]]> PRV-EP84R <![CDATA[10 7.50 ]]> PRV-D129L <![CDATA[10 7.625 ]]> PRV-E146L <![CDATA[10 7.875 ]]> PRV-K421R <![CDATA[10 7.625 ]]>

[0258] II. Immunization and challenge experiments with recombinant pseudorabies virus expressing ASFV EP84R, D129L, E146L, and K421R genes

[0259] 1. Group immunization

[0260] Experimental group (PRV-EP84R, PRV-D129L, PRV-E146L, PRV-K421R combination): The recombinant pseudorabies viruses PRV-EP84R, PRV-D129L, PRV-E146L, and PRV-K421R prepared above were combined according to the form of Table 17, and the virus titer of each recombinant virus was 106.5 TCID 50 / ml or above, each recombinant virus is mixed in equal proportion, 0.1ml of each virus, and 100 micrograms of 4% nano-aluminum gel adjuvant is added to each mixed vaccine.

[0261] The preparation steps of 4% nano-aluminum gel adjuvant are as follows:

[0262] Aluminum salt dissolution: dissolve an appropriate amount of aluminum salt (such as aluminum hydroxide) in deionized water to prepare an aluminum salt solution with a mass volume ratio (g:ml) of 5%.

[0263] Acid-base adjustment: Adjust the pH value of the aluminum salt solution (usually 6-8). Dilute hydrochloric acid or sodium hydroxide can be used to promote the formation of aluminum gel.

[0264] Colloid formation: Stirring (e.g., stirring, heating) at 10-70° C. until the aluminum ions in the aluminum salt solution form colloids to form nano-aluminum particles. This step can be continued for several hours until the colloid is fully formed.

[0265] Washing of nano-aluminum particles: Unreacted aluminum salts and impurities were removed by centrifugation at 800 r / min for 10 minutes to obtain high-purity aluminum colloid.

[0266] Concentration adjustment: According to the needs, adjust the concentration of aluminum gel so that its final mass volume percentage (g: ml) concentration reaches 4%. This can be achieved by diluting or concentrating the liquid.

[0267] Stabilization and preservation: Add stabilizers to prevent the aggregation of particles. Finally, store the prepared nano aluminum gel under appropriate conditions (such as low temperature) to ensure its stability.

[0268] Traditional vector vaccine combination group (p30, -p54, -p72, -pCD2v, -pF317L, -pp62, -pp220 combination): p30, p54, p72, pCD2v, pF317L, pp62, pp220 recombinant adenovirus mixture was used as a control, and 10 of each virus was used 8.0 TCID 50 After mixing, 100 micrograms of 4% nano-aluminum gel adjuvant was added, and 5 heads were immunized in each group.

[0269] The above two groups were immunized twice, with an interval of 14 days, and the dose of each immunization was the same.

[0270] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0271] Follow daily feeding.

[0272] 2. Attack the virus

[0273] On the 14th day after the second immunization (the first day of the second immunization was recorded as the 1st day), the above experimental groups, control immunization group and challenge control group were challenged with strong toxicity (10 g / kg / day per head) using African swine fever virus SY18 strain. 3.0 TCID 50 / 2ml ASFV virulent SY18 strain). The patients were observed for 28 days, and the clinical manifestations and final outcomes were recorded.

[0274] The results are shown in Table 17. Through 28 days of observation, all the non-immunized control pigs became ill and died, all the pigs immunized with the traditional vector vaccine combination became ill and died, and all the pigs immunized with the PRV-EP84R, PRV-D129L, PRV-E146L, and PRV-K421R gene recombinant pseudorabies virus combination survived.

[0275] Table 17 shows the results of ASFV D129L and other gene recombinant pseudorabies virus immunization pig virus challenge

[0276]

[0277]

[0278] Example 6: Immunization and challenge experiments of recombinant PRRSV and recombinant pseudorabies virus expressing ASFV EP84R, D129L, E146L, K421R genes

[0279] 1. Group immunization

[0280] Two groups of recombinant virus combinations were used: rPRRSV-EP84R, rPRRSV-D129L, rPRRSV-E146L, rPRRSV-K421R recombinant PRRSV virus and PRV-D129L, PRV-E146L, PRV-K421R, PRV-EP84R recombinant pseudorabies virus. The titer of each recombinant rPRRSV was 10 5.5 TCID 50 / ml, mixed in equal proportions, 0.1ml of each virus, used directly after mixing; the titer of each recombinant pseudorabies virus is 10 6.5 TCID 50 / ml, mixed in equal proportions, 0.1ml of each recombinant virus, and freeze-dried after mixing. The two groups of vaccine combinations were first immunized with (recombinant rPRRSV combination) and then (recombinant pseudorabies virus combination) by intramuscular injection into the neck muscle of pigs, with an interval of 14 days between the two groups of immunizations. A total of 5 pigs were immunized.

[0281] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0282] Follow daily feeding.

[0283] 2. Attack the virus

[0284] 14 days after the second immunization, the immunized pigs and the non-immunized control pigs were given 10 3.0 TCID 50 / 2mlASFV virulent SY18 strain, observed for 28 days, and recorded clinical manifestations and final outcomes.

[0285] The results are shown in Table 18. It can be seen that after 28 days of observation, all non-immunized control pigs became ill and died, and the dead pigs were tested positive for ASFV. All 5 pigs in the immunized group survived after the virus challenge.

[0286] Table 18 shows the results of the virus challenge of pigs immunized with four-gene recombinant rPRRSV and recombinant pseudorabies virus

[0287]

[0288] Example 7. Preparation and immunogenicity of mRNA vaccines expressing EP84R, D129L, E146L, and K421R

[0289] 1. Preparation of mRNA vaccines expressing EP84R, D129L, E146L, and K421R

[0290] 1. Construction and amplification of transcription plasmid

[0291] Change 5'UTR(GAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATG

[0292] ), DNA coding sequence (including stop codon) of EP84R, D129L, E146L or K421R gene, 3'UTR (AGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAGCTCGCTTTCTTGCTGTCCAATTT CTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTG CCTAATAAAAAACATTTATTTTCATTGCAGCTCGCTTTCTTGCTGTCCAATTT CTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTG CCTAATAAAAAACATTTATTTTCATTGC) and poly A

[0293] (AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA), were cloned in tandem in the order of chronological arrangement into the transcription plasmid pGEM-3zf(+) containing T7 promoter, transformed into Escherichia coli, and the plasmids were extracted by bacterial enrichment culture to obtain recombinant transcription plasmids pGEM-3zf(+)-EP84R, pGEM-3zf(+)-D129L, pGEM-3zf(+)-E146L, and pGEM-3zf(+)-K421R.

[0294] The recombinant transcription plasmid pGEM-3zf(+)-EP84R is a plasmid obtained by replacing the fragment between the kpn I and xba I sites of the plasmid pGEM-3zf(+) with a fragment consisting of 5'UTR, the DNA coding sequence of the EP84R gene (including the stop codon), 3'UTR and the polyA sequence.

[0295] The recombinant transcription plasmid pGEM-3zf(+)-D129L is obtained by replacing the fragment between the kpn I and xba I sites of the plasmid pGEM-3zf(+) with a fragment consisting of 5'UTR, the DNA coding sequence of the D129L gene (including the stop codon), 3'UTR and the polyA sequence.

[0296] The recombinant transcription plasmid pGEM-3zf(+)-E146L is a plasmid obtained by replacing the fragment between the kpn I and xba I sites of the plasmid pGEM-3zf(+) with a fragment consisting of the DNA coding sequence (including the stop codon) of the 5'UTRE146L gene, 3'UTR and polyA sequence.

[0297] The recombinant transcription plasmid pGEM-3zf(+)-K421R is a plasmid obtained by replacing the fragment between the kpn I and xba I sites of the plasmid pGEM-3zf(+) with a fragment consisting of 5'UTR, the DNA coding sequence of the K421R gene (including the stop codon), 3'UTR and the polyA sequence.

[0298] 2. Linearization of transcription plasmid and transcription of mRNA

[0299] The recombinant transcription plasmids pGEM-3zf(+)-EP84R, pGEM-3zf(+)-D129L, pGEM-3zf(+)-E146L, and pGEM-3zf(+)-K421R were digested with Hind III and the linearized fragments were recovered. The fragments were added into the in vitro transcription system (the in vitro transcription system used T7 High Yield RNA Transcription Kit (E131, Novoprotein Co., Ltd.), for specific operations, refer to the instructions of the kit, and N1-Me-Pseudo UTP was used instead of UTP of the T7 kit to reduce immunogenicity). The reaction was carried out at 37°C for 3 hours, and then DNase I was added and incubated at 37°C for 15 minutes to digest the transcribed DNA template, and the mRNA was purified by RNA purification kit.

[0300] 3. mRNA capping

[0301] The purified mRNA was capped using an RNA capping kit (T7 ARCA mRNA in vitro Synthesis kit, Catalog No.: MT0125, Beijing Biolab Technology Co., Ltd.) at 37°C for 0.5 h, and the capped mRNA was purified using an RNA purification kit or LiCl precipitation centrifugation method.

[0302] 4. DNA Removal

[0303] An appropriate amount of DNase I was added to the reaction system obtained in 3 above, and the mixture was incubated at 37° C. for 15 min to digest the DNA template, followed by purification to obtain mRNA.

[0304] 5. Purification of mRNA and preparation of vaccines

[0305] Purification method (purification by lithium chloride, other kits can also be used for purification): add 30uL of LithiumChcoride Precipitation Solution (7.5M LithiumChcoride, 50mM EDTA) and 30uL RNaseFree Water, mix well and place in -20℃ for at least 30min, centrifuge at 4℃, centrifuge at 1200rpm for 15min, remove the supernatant, and collect the precipitate. Wash with pre-cooled 70% ethanol 3 times, centrifuge at 4℃ for 2min each time (70% ethanol must be prepared and used immediately), open the lid and stand in the clean bench for 5-8min until the ethanol evaporates, re-dissolve in RNaseFreeWater and test the concentration, and store in a -80℃ refrigerator for use.

[0306] The purified mRNAs were diluted with citric acid buffer to 80-120 ng / μL as the aqueous phase, and liposomes were dissolved with anhydrous ethanol as the organic phase. The liposome components were distearoyl phosphatidylethanolamine (DSPE), cholesterol, DLIN-MC3-DMA and DMG-PEG2000, and the mass ratios of the components were 10:38.5:50:1.5. The final concentration of liposomes in the organic phase was 5-7 mg / ml. The organic phase and the aqueous phase were mixed in a nucleic acid nano-drug preparation instrument at a certain mass ratio (1:2-1:4), and the flow rate was 12 ml / min. The mixed liquid was the mRNA liquid encapsulated by lipid nanoparticles, and the anhydrous ethanol was removed by dialysis to obtain the mRNA vaccine encapsulated by lipid nanoparticles that can be used for animal immunization.

[0307] We have obtained mRNA vaccines that can express four types of proteins, namely EP84R-mRNA vaccine, D129L-mRNA vaccine, E146L-mRNA vaccine, and K421-mRNA vaccine.

[0308] The above four mRNA vaccines, EP84R-mRNA vaccine, D129L-mRNA vaccine, E146L-mRNA vaccine, and K421-mRNA vaccine, were mixed in equal proportions according to mass ratio to prepare a total of 300 bottles, with each bottle containing 5 doses (each protein dose in 1 dose is 30 micrograms), totaling 1,500 doses.

[0309] 2. Immunization of mRNA vaccines expressing EP84R, D129L, E146L, and K421R (only mRNA vaccines and immune test results of the 4-gene combination are provided, and the mRNA vaccines and immune tests of other combinations are similar)

[0310] 1. Group immunization

[0311] EP84R-mRNA, D129L-mRNA, E146L-mRNA, and K421-mRNA vaccines were injected intramuscularly into the neck muscles of pigs. A total of 5 pigs were immunized, with each pig immunized with one dose. The vaccines were administered twice, with an interval of 14 days.

[0312] At the same time, a combination of five traditional immune vector vaccines (p30, p54, p72, pCD2v, pF317L, pp62, and pp220) was set up: a mixture of recombinant adenovirus expressing p30, p54, p72, pCD2v, pF317L, pp62, and pp220 was used as a control (10 of each virus was used). 8.0 TCID 50 After mixing, add 100 micrograms of Poria polysaccharide to each portion (2 ml).

[0313] Virus-challenged control group: The control group was not injected with any virus, with 5 animals in each group.

[0314] Follow daily feeding.

[0315] 2. Attack the virus

[0316] 14 days after the second immunization, each pig in the immunized group and the control group was given 10 3.0 TCID 50 / 2ml ASFV virulent SY18 strain, observed for 28 days, and recorded clinical manifestations and final outcomes.

[0317] The results are shown in Table 19. Through 28 days of observation, all the non-immunized control pigs became ill and died, all the pigs immunized with the traditional vector vaccine combination became ill and died, and all the pigs immunized with the mRNA vaccine survived.

[0318] Table 19 shows the results of the challenge of pigs with mRNA vaccines of four proteins including ASFV D129L

[0319]

[0320]

[0321] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A combination of African swine fever virus proteins, comprising a combination of at least 3 of the following 4 proteins; The four proteins are EP84R protein or a truncation thereof, D129L protein or a truncation thereof, E146L protein or a truncation thereof and K421R protein or a truncation thereof.

2. The African swine fever virus protein combination according to claim 1, characterized in that: The African swine fever virus protein combination also includes other protein combinations; The other protein combination consists of at least one of B646L protein, E183L protein, CP204L protein and CP2457L protein.

3. African swine fever virus nucleic acid combination, which includes at least 3 combinations of the following 4 nucleic acids; The four nucleic acids are nucleic acids encoding EP84R protein or a truncation thereof, nucleic acids encoding D129L protein or a truncation thereof, nucleic acids encoding E146L protein or a truncation thereof and nucleic acids encoding K421R protein or a truncation thereof.

4. The African swine fever virus nucleic acid combination according to claim 3, characterized in that: The nucleic acid molecule is DNA or mRNA.

5. A nucleic acid delivery vector or a nucleic acid delivery vector set containing any three or four of the following four nucleic acids; The four nucleic acids are nucleic acids encoding EP84R protein or a truncation thereof, nucleic acids encoding D129L protein or a truncation thereof, nucleic acids encoding E146L protein or a truncation thereof, and nucleic acids encoding K421R protein or a truncation thereof.

6. A recombinant virus combination, comprising a recombinant virus or a recombinant virus group expressing any three or four of the following four nucleic acids; The four nucleic acids are nucleic acids encoding EP84R protein or a truncation thereof, nucleic acids encoding D129L protein or a truncation thereof, nucleic acids encoding E146L protein or a truncation thereof, and nucleic acids encoding K421R protein or a truncation thereof.

7. The recombinant virus combination according to claim 6, characterized in that: The recombinant vector is derived from human adenovirus type 5, poxvirus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine adenovirus, porcine Japanese encephalitis virus, classical swine fever virus, retrovirus, paramyxovirus or lactic acid bacteria.

8. Biological material, any of the following: 1) A recombinant vector group or recombinant vector expressing the African swine fever virus protein combination according to claim 1 or 2; 2) A recombinant microorganism or recombinant microorganism expressing the African swine fever virus protein combination according to claim 1 or 2; 3) A recombinant cell group or recombinant cell expressing the African swine fever virus protein combination according to claim 1 or 2.

9. The African swine fever virus protein combination of claim 1 or 2, the African swine fever virus nucleic acid combination of claim 3 or 4, the nucleic acid delivery vector or nucleic acid delivery vector group of claim 5, the recombinant virus combination of any one of claims 6-7, or the biomaterial of claim 8, in any of the following applications: 1) Preparation of products for treating diseases caused by African swine fever virus infection; 2) Preparation of products for inducing immune response to African swine fever virus antigens; 3) Preparation of products for preventing diseases caused by African swine fever virus infection; 4) Prepare anti-African swine fever virus products.

10. African swine fever products, including any of the following: 1) The African swine fever virus protein combination according to claim 1 or 2; 2) The African swine fever virus nucleic acid combination according to claim 3 or 4; 3) The nucleic acid delivery vector or nucleic acid delivery vector set according to claim 5; 4) The recombinant virus combination according to any one of claims 6-7; 5) The biomaterial according to claim 8.

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