A recombinant attenuated strain of African swine fever virus gene deletion and its preparation method and application

Abstract

The present invention discloses a recombinant attenuated strain with a gene deletion of African swine fever virus and a preparation method and application thereof, which belongs to the technical field of veterinary biological products. The purpose of the present invention is to provide a strategy for constructing an attenuated strain of African swine fever. The present invention provides a recombinant attenuated strain with a gene deletion of African swine fever virus, wherein the recombinant attenuated strain is a recombinant attenuated strain that uses genetic engineering means to jointly delete the functions of the H240R gene and the MGF505‑7R gene encoding protein in the African swine fever virus strain Pig / CN / HLJ / 2018, thereby obtaining a highly safe attenuated African swine fever candidate vaccine strain. The attenuated candidate vaccine strain completely weakens the pigs after immunizing the pigs, and can provide 100% immune protection against the strong strain of Pig / CN / HLJ / 2018, and can be used as a safe and effective vaccine for preventing and controlling the African swine fever epidemic in China, which has great social value.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and specifically relates to a recombinant attenuated strain of African swine fever virus gene deletion, and a preparation method and application thereof. Background Art

[0002] African swine fever (ASF) is an acute, severe, and highly contagious disease caused by African swine fever virus (ASFV), characterized by fever and systemic bleeding in pigs. The mortality rate of strong strains is close to 100%. Domestic pigs, wild boars, and soft ticks at all stages are natural hosts of African swine fever. It can be transmitted directly between domestic pigs and wild boars, through tick bites, and across regions and countries through swill, feed, and meat products contaminated with the virus. Currently, there are no commercial vaccines or specific drugs. Once an African swine fever epidemic occurs, it can only be controlled by killing, but this method not only causes significant economic losses, but also fails to meet the needs of large-scale pig farming in my country. Therefore, it is urgent to develop an effective and safe vaccine.

[0003] At present, the development of African swine fever vaccine mainly adopts three methods: 1. Directly inactivate the original African swine fever virus to obtain an inactivated vaccine; 2. Screen out the antigen protein that induces immune response of the virus, and then prepare subunit vaccine; 3. Use gene deletion to knock out the virulence gene to obtain a recombinant virus vaccine. The first method is the most commonly used method for preparing virus vaccines. Due to the characteristics of African swine fever virus infection such as antibody-dependent enhancement (ADE) and immunosuppression, the inactivated vaccine cannot provide effective immune protection for pigs; the limiting factor of the second method is that the African swine fever virus genome is huge, the results are complex, and most of the functions are unknown. The antigens that induce immune response are not clear at present, and the known antigens cannot induce effective immune protection. The vaccine with the most promising breakthrough at present is the gene knockout attenuated vaccine. By knocking out virulence-related genes such as immunosuppression, a vaccine candidate strain that is both immunogenic and non-pathogenic to pigs is obtained, which provides complete protection after immunizing pigs. For example, Chinese patent CN110093324A provides a seven-gene deleted weak strain of African swine fever virus, which is based on the seven-gene deleted weak strain of African swine fever virus Pig / CN / HLJ / 2018 strain, and lacks the functional proteins of the following genes: CD2v gene encoding products and six multigene family genes (multigene family 360 genes 12L, 13L, 14L and multigene family 505 genes 1R, 2R, 3R) encoding products. After 28 days of immunization of the inoculated piglets, the ASFV original virus was used for the challenge test and 100% immune protection was obtained; for another example, Chinese patent CN114107228A discloses a twelve-gene deleted attenuated African swine fever vaccine. In ASFV CN / GS The 2018 strain jointly lacked G-ACD-001900, MGF110-9L, G-ACD-0021 and nine multigene family genes (multigene family 360 genes 9L, 10L, 11L, 12L, 13L, 14L and multigene family 505 genes 1R, 2R, 3R). After inoculation of piglets, the parental strain was used for the challenge test and 100% immune protection was obtained.

[0004] Research on vaccines that knock out the virulence genes of African swine fever virus requires consideration of the immune response and protection of pigs, as well as safety performance. In existing technologies, the following factors also need to be considered: (1) Whether the virulence gene is knocked out and whether the pigs carry or excrete the virus is an important indicator of the safety of gene-deleted vaccines; (2) African swine fever virus encodes more than 160 proteins and has many virulence genes. Which virulence gene deletion has the best immune protection and safety is a long-term and arduous task; (3) Different strains have different effects when the same gene is deleted, and the low virus titer caused by multiple gene deletions will also reduce the immunogenicity or protective effect of the attenuated strains.

[0005] Therefore, in the preparation of African swine fever attenuated vaccines, identification of gene functions and their effects on pathogenicity and immune response provide a basis for the selection of knockout target genes and the construction of gene knockout. The screening of new target genes is crucial and determines the safety of vaccine candidate strains and the effectiveness of immunity. In the study of the MGF505-7R gene, the African swine fever virus Pig / CN / HLJ / 2018 strain was knocked out of the MGF505-7R gene, and the immunization dose was 10 5 HAD 50 After infecting pigs, the mortality rate is significantly reduced, but some virulence still exists, and the pig survival rate is about 60% (Li et al.pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1βandtype I IFN production.PLOS PATHOGENS.2021); the MGF505-7R gene of the African swine fever virus CN / GS / 2018 strain is knocked out, and a lower immune dose (10HAD 50 ) all survived after infecting pigs, but still had obvious clinical symptoms, such as fever and virus carriage (Li et al. African swine fever virus protein MGF-505-7R promotes virulence and pathogenesis by inhibiting JAK1-and JAK2-mediated signaling. Journal of Biological Chemistry. 2021).

[0006] The African swine fever virus Pig / CN / HLJ / 2018 strain has its H240R gene knocked out (ASFV-ΔH240R), and the protein encoded by the H240R gene has lost its function. Infecting pig alveolar macrophages induces a higher level of interferon response and high expression of antiviral genes. 5 HAD 50 ASFV-ΔH240R infects pigs, and the body temperature rises on the 6th day after infection, and the pigs are infected in the blood after immunization. Now there is an urgent need for a fully attenuated and immunogenic vaccine virus strain. Summary of the invention

[0007] The purpose of the present invention is to provide a strategy for constructing an attenuated African swine fever strain and to provide a vaccine virus strain that is completely attenuated and has good immunogenicity.

[0008] The present invention provides a recombinant attenuated strain with a deleted African swine fever virus gene. The recombinant attenuated strain is obtained by taking the African swine fever virus strain as a starting strain and mutating the H240R gene and the MGF505-7R gene of the African swine fever virus.

[0009] It is further defined that the African swine fever virus strain is genotype II African swine fever virus.

[0010] It is further defined that the African swine fever virus pig is the Pig / HLJ / 2018 strain.

[0011] It is further defined that the nucleotide sequence of the H240R gene of the African swine fever virus is shown as SEQ ID NO.1; the nucleotide sequence of the MGF505-7R gene of the African swine fever virus is shown as SEQ ID NO.2.

[0012] It is further defined that the amino acid sequence of the H240R gene of the African swine fever virus is shown as SEQ ID NO.3; the amino acid sequence of the MGF505-7R gene of the African swine fever virus is shown as SEQ ID NO.4.

[0013] The present invention provides a method for constructing the above-mentioned recombinant attenuated strain, characterized in that the method is to mutate the H240R gene and the MGF505-7R gene in the African swine fever virus by genetic engineering means.

[0014] It is further defined that the specific steps of the method are as follows:

[0015] Step 1, constructing a recombinant virus lacking the H240R gene: the H240R gene as shown in SEQ ID NO.2 is connected to the p72-EGFP plasmid to obtain the p72-EGFP-H240R vector, the sgRNA of the MGF505-7R gene is connected to the pX330 plasmid to obtain the pX330-sgH240R vector, and then the p72-EGFP-H240R vector and the pX330-sgH240R vector are co-transfected into porcine alveolar macrophages, and then the ASFV / CN / HLJ / 18 virus is infected in the transfected porcine alveolar macrophages obtained above to obtain the recombinant virus lacking the H240R gene;

[0016] Step 2: The MGF505-7R gene as shown in SEQ ID NO.1 was connected to the p72-mCherry plasmid to obtain p72-mCherry-MGF505-7R, and the sgRNA targeting the H240R gene was combined with the pX330 plasmid to obtain the pX330-sgMGF505-7R vector, and then the p72-mCherry-MGF505-7R and pX330-sgMGF505-7R vectors were co-transfected into porcine alveolar macrophages, and then the recombinant virus lacking the H240R gene obtained in step 1 was infected into the transfected porcine alveolar macrophages obtained above to obtain the recombinant virus lacking the H240R gene and the MGF505-7R gene.

[0017] It is further defined that the p72-EGFP vector is obtained by connecting the SEQ ID NO.7 sequence with the pBluescript II KS(+) vector; and the p72-mCherry vector is obtained by connecting the SEQ ID NO.8 sequence with the pBluescript II KS(+) vector.

[0018] The present invention provides a recombinant attenuated strain with a deleted African swine fever virus gene. The recombinant attenuated strain is based on an African swine fever virus strain as a starting strain, and the recombinant attenuated strain of African swine fever virus is prepared by jointly losing the functions of proteins encoded by the H240R gene and the MGF505-7R gene in the African swine fever virus.

[0019] It is further defined that the above-mentioned recombinant attenuated strain is used as the basic strain, and the CD2V gene and the MGF family gene are mutated to obtain a virus with multiple gene deletions.

[0020] The present invention provides a vaccine for African swine fever, which contains the above-mentioned recombinant attenuated strain.

[0021] It is further defined that the vaccine is a monovalent vaccine, a bivalent vaccine or a multivalent vaccine.

[0022] The present invention provides the use of the above-mentioned vaccine in the preparation of a medicament for treating or preventing African swine fever.

[0023] The present invention provides the use of the above-mentioned vaccine in the preparation of health products for assisting in the treatment or prevention of African swine fever.

[0024] Beneficial effect: The present invention obtains a safe attenuated African swine fever virus strain by jointly losing the function of the proteins encoded by the H240R gene and the MGF505-7R gene; immunization with the attenuated African swine fever virus strain (10 5 HAD 50) pigs did not get sick, and the nucleic acid and antibody of the cohabiting pigs were negative, indicating that there was no horizontal transmission; the challenge experiment showed that the attenuated African swine fever virus strain could provide 100% immune protection against the Pig / CN / HLJ / 2018 strong strain, and could be used as a safe and effective vaccine for the prevention and control of African swine fever in China, which has great social value. In addition, the protein sequences encoded by the H240R gene and the MGF505-7R gene in different genotypes of African swine fever viruses are highly homologous ( Figure 1 and Figure 2 ), therefore, the H240R gene and the MGF505-7R gene can be knocked out in different genotypes of African swine fever viruses, and an attenuated African swine fever virus vaccine can be successfully constructed. However, the present invention is not limited to the Pig / CN / HLJ / 2018 isolate. The protein sequences encoded by the H240R gene and the MGF505-7R gene in different genotypes of African swine fever viruses were compared, and it was found that the proteins encoded by the H240R gene and the MGF505-7R gene were highly homologous ( Figure 1 and Figure 2 ), therefore, the proteins encoded by the H240R gene and the MGF505-7R gene in different genotypes of African swine fever viruses have similar functions, and the recombinant virus with the H240R gene and the MGF505-7R gene knocked out can also successfully construct a candidate attenuated African swine fever virus vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the sequence comparison result of the protein encoded by the MGF505-7R gene in different genotypes of African swine fever viruses;

[0026] Figure 2 This is the sequence comparison result of the protein encoded by the H240R gene in different genotypes of African swine fever viruses;

[0027] Figure 3 Schematic diagram of the construction of ASFV MGF505-7R gene deletion vector (expressing red fluorescence);

[0028] Figure 4 Schematic diagram for the construction of ASFV H240R gene deletion vector (expressing green fluorescence);

[0029] Figure 5 To express red and green dual-color fluorescent protein for ASFV-ΔH240R-Δ7R;

[0030] Figure 6It is the result of ASFV-ΔH240R-Δ7R identification, wherein, Figure a is the result of H240R-JD-OF / R primer pair detecting recombinant virus ASFV-ΔH240R-Δ7R, and Figure b is the result of 7R-JD-OF / R primer pair detecting recombinant virus ASFV-ΔH240R-Δ7R;

[0031] Figure 7 For the identification of ASFV-ΔH240R, wherein Figure (a) is the result of observing the recombinant virus ASFV-ΔH240R under a fluorescence microscope, and Figure (b) is the result of detecting ASFV-ΔH240R with the H240R-JD-OF / R primer;

[0032] Figure 8 This is a graph showing the changes in body temperature of pigs after ASFV-ΔH240R immunization and control strain infection; the horizontal axis is the number of days after infection, and the vertical axis is the body temperature;

[0033] Fig. 9 The survival rate of pigs after ASFV-ΔH240R immunization and control strain infection; the horizontal axis is the number of days after infection, and the vertical axis is the survival rate;

[0034] Fig.10 The results of ASFV-ΔH240R immunization and control strain infection in pig blood; the horizontal axis is the number of days after infection, and the vertical axis is the number of viral genome copies (Log 10 );

[0035] Fig.11 This is a graph showing changes in body temperature of pigs after ASFV-ΔH240R-Δ7R immunization and infection with the control strain; the horizontal axis is the number of days after infection, and the vertical axis is the body temperature;

[0036] Fig.12 The survival rate of pigs after ASFV-ΔH240R-Δ7R immunization and control strain infection; the horizontal axis is the number of days after infection, and the vertical axis is the survival rate;

[0037] Fig.13 The results of ASFV-ΔH240R-Δ7R immunization and control strain infection in pig blood; the horizontal axis is the number of days after infection, and the vertical axis is the number of viral genome copies (Log 10 );

[0038] Fig.14 The results of P30 antibodies after ASFV-ΔH240R-Δ7R immunization and control strain infection; the horizontal axis is the number of days after infection, and the vertical axis is the ASFV p30 antibody level (P / N);

[0039] Fig.15This is a graph showing the changes in body temperature of pigs immunized with gene-deficient viruses and their cohabiting pigs after virus infection; the horizontal axis is the number of days after virus infection, and the vertical axis is body temperature;

[0040] Fig.16 The survival rate of pigs immunized with gene-deficient virus and their cohabiting pigs after virus infection; the horizontal axis is the number of days after virus infection, and the vertical axis is the survival rate;

[0041] Fig.17 The results of pig blood virus infection after the pigs immunized with the gene-deficient virus and their cohabiting pigs were challenged with the virus; the horizontal axis is the number of days after the virus attack, and the vertical axis is the number of viral genome copies (Log 10 );

[0042] Fig.18 The results of IFN-β transcription in the lungs of pigs immunized with gene-deficient viruses and their cohabiting pigs after virus challenge; the horizontal axis is the group, and the vertical axis is the relative expression of IFN-β mRNA; DETAILED DESCRIPTION

[0043] The Chinese epidemic strain of African swine fever virus (ASFV / CN / HLJ / 18 strain), also known as the Chinese epidemic strain of African swine fever virus Pig / CN / HLJ / 2018 (Wen Lili. Histopathological study of artificially infected SPF pigs with African swine fever virus Pig / HLJ / 2018 strain [D]. Beijing: Chinese Academy of Agricultural Sciences, 2021.) was isolated and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (its full-length sequence can be found in GenBank: MK333180.1).

[0044] Porcine alveolar macrophages (PAMs) were obtained from 30-50 day old SPF pigs and cultured in 1640 medium containing 10% FBS. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from SPF pig anticoagulated blood using a PBMC isolation kit (purchased from TBD, China).

[0045] Construction of green fluorescent protein (EGFP) gene screening expression cassette: Referring to the design strategy in the literature (Li et al. pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production. PLOS PATHOGENS. 2021), the p72 promoter and green fluorescent protein (EGFP) were fused by gene synthesis. The gene sequence is shown in SEQ ID NO.7. The synthesized sequence was connected to the cloning vector pBluescript II KS(+) through HindIII and BamHI. The constructed vector was named p72-EGFP.

[0046] Construction of red fluorescent protein (mCherry) gene screening expression cassette: Referring to the design strategy in the reference (Li et al. pMGF505-7Rdetermines pathogenicity of African swine fever virus infection byinhibiting IL-1βand type I IFN production. PLOS PATHOGENS.2021), the p72 promoter and red fluorescent protein (mCherry) were fused by gene synthesis. The gene sequence is shown in SEQ ID NO.8. The synthesized sequence was connected to the cloning vector pBluescript II KS(+) through HindIII and BamHI, and the constructed vector was named p72-mCherry.

[0047] p72-EGFP vector is SEQ ID NO.7

[0048] AAGCTTTTGTTATTATCAAGATCCTTCGCATAAACCGCCATATTTAATAAAAACAATAAATTATTTTTATAACATTATATAGCCACCATGgattacaaggatgacgacgataagACGCGTGCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGgaattcATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAgaattc

[0049] The p72-mCherry vector is obtained by inserting SEQ ID NO.8

[0050] AAGCTTTTGTTATTATCAAGATCCTTCGCATAAACCGCCATATTTAATAAAAACAATAAATTATTTTTATAACATTATATAGCCACCATGgattacaaggatgacgacgataagACGCGTGCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGgaattcatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagTAAgaattcTTTTTTTTTTGGATCC;

[0051] Example 1. Construction of a recombinant vector for an attenuated vaccine strain

[0052] The nucleotide sequence of the H240R gene is shown in SEQ ID NO.1:

[0053] Atggctgcaaacattattgcaacaagagccgtgccaaagatggccagcaaaaaagagcatcaatactgtctgctagactcccaggaaaagcgtcatgggcattatcccttttcatttgaattaaagccttatgggcaaacaggcgcaaatatcataggagtacagggctcacttacccatgttatcaaaatgacagtatttccatttatgattccttttcctttacaaaaaactcatatagatgattttattggtggacgcatttatttattttttaaggaactggacatgcaagcagtttctgatgtaaatggaatgcaataccacttcgagttcaaggttgttcctgtaagccccaaccaagtagagcttcttcctgtgaataataaatataaatttacatatgctataccggtagtgcaataccttaccccaatcttttatgatctttcgggaccgctagatttcccattagatactctttcggtccatgtggatatcctctccaatcatatacagcttcctatccaaaaccataacctaacaacgggtgatcgtgtttttatttctggatataaacacctgcaaacgattgaattatgtaaaaataacaagatttttatcaaaaatataccgccgctttcatccgaaaaaataaaactatatatactaaaaaatcgaatcagaattccgctatactttaaatctttaaaaacgtctaagtaa;

[0054] The amino acid sequence of the H240R gene is shown in SEQ ID NO.3:

[0055] MAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSK;

[0056] The nucleotide sequence of the MGF505-7R gene is shown in SEQ ID NO.2:

[0057]

[0058] The amino acid sequence of the MGF505-7R gene is shown in SEQ ID NO.4:

[0059] ;

[0060] according to Figure 3 The schematic diagram of the construction of the ASFV MGF505-7R gene deletion vector (expressing red fluorescence) shown in the figure is used to construct the ASFV MGF505-7R gene deletion vector;

[0061] 1. The approximately 1000 bp of the genome to the left of the MGF505-7R gene ORF (NCBI accession number: MK333180.1) (position 40763-42346 relative to the full-length sequence) was used as the left homologous arm sequence (approximately 1000 bp to the left of the 5'-ttaatttgggtagggaaa-3' sequence of the ASFV genome), and the approximately 1000 bp of the genome to the right of the MGF505-7R gene ORF was used as the right homologous arm sequence (approximately 1000 bp to the right of the 5'-atttctgaatcagtaagcaa-3' sequence of the ASFV genome). The left and right homologous arms were respectively inserted into the XhoI and BamHI sites of the p72-mCherry vector using a homologous recombination cloning kit. The successfully constructed homologous arm transfer vector was named p72-mCherry-MGF505-7R. The plasmid p72-mCherry-MGF505-7R was digested with KpnI, linearized, and then recovered for later use.

[0062] according to Figure 4 The schematic diagram of the construction of the ASFV H240R gene deletion vector (expressing green fluorescence) shown is used to construct the ASFV H240R gene deletion vector;

[0063] 2. The genome of about 1000 bp on the left of the H240R gene ORF (NCBI accession number: MK333180.1) (position 155375-156100 relative to the full-length sequence) was used as the left homology arm sequence (about 1000 bp on the left of the 5'-ttaaaccattgaattattta-3' sequence of the ASFV genome), and the genome of about 1000 bp on the right of the H240R gene ORF was used as the right homology arm sequence (about 1000 bp on the right of the 5'-taacatttttatagtctact-3' sequence of the ASFV genome). The left and right homology arms were respectively inserted into the XhoI and BamHI sites of the p72-EGFP vector using a homologous recombination cloning kit. The successfully constructed homology arm transfer vector was named p72-EGFP-H240R. The plasmid p72-EGFP-H240R was cut with KpnI, linearized, and then recovered for use.

[0064] 3. The sgRNA targeting the MGF505-7R gene (target sequence: 5'-GTATAACCCCAGCAAATGCA-3', shown in SEQ ID NO.5) and the sgRNA targeting the H240R gene (target sequence: 5'-GATTGGAGAGGATATCCACA-3', shown in SEQ ID NO.6) were inserted into the pX330 vector, respectively. The successfully constructed vectors were named pX330-sgMGF505-7R and pX330-sgH240R.

[0065] Example 2. Construction and identification of recombinant virus ASFV-ΔH240R-Δ7R

[0066] 1. The linearized p72-EGFP-H240R vector and the pX330-sgH240R vector obtained in Example 1 were co-transfected into PAMs at a ratio of 1:1 using a transfection reagent (X-treme GENE HP). After 12 hours, porcine alveolar macrophages (PAMs) were infected with ASFV / CN / HLJ / 18 at a dose of 1 MOI. Recombinant viruses with green fluorescence were observed after 24-36 hours. When 80% of the cells showed lesions, they were placed in a -20°C refrigerator and repeatedly frozen and thawed 3 times to collect the virus solution.

[0067] The collected virus liquid was inoculated onto new PAMs, and the virus liquid was discarded after 2 hours. The surface was covered with 1640 culture medium containing 1% low-melting point agarose, and plaques containing green fluorescence were selected after 48 hours. The plaques were picked repeatedly 5-8 times until a pure recombinant virus lacking the H240R gene (ASFV-ΔH240R) was obtained.

[0068] 2. The linearized p72-mCherry-MGF505-7R and pX330-sgMGF505-7R obtained in Example 1 were co-transfected into PAMs at a ratio of 1:1 using a transfection reagent (X-treme GENE HP). After 12 hours, PAMs were infected with ASFV-ΔH240R at a dose of 1 MOI. Recombinant viruses with red fluorescence were observed after 24-36 hours. When 80% of the cells showed lesions, they were placed in a -20°C refrigerator and repeatedly frozen and thawed 3 times to collect the virus solution.

[0069] The collected virus liquid was inoculated onto new PAMs, and the virus liquid was discarded after 2 hours. The surface was covered with 1640 culture medium containing 1% low-melting point agarose, and plaques containing red fluorescence were selected after 48 hours. The plaques were picked repeatedly 5-8 times until a pure recombinant virus lacking the H240R gene and the MGF505-7R gene (ASFV-ΔH240R-Δ7R) was obtained.

[0070] The results of fluorescence microscopy observation were as follows Figure 5 As shown, the results showed that the purified gene-deficient recombinant virus ASFV-ΔH240R-Δ7R simultaneously expressed red and green dual-color fluorescence when infecting PAMs, indicating that the recombinant virus was successfully constructed.

[0071] Virus purity detection: H240R-JD-OF / R primer pair (H240R-JD-OF: Gcaaacattattgcaacaag (SEQ ID NO.9) and H240R-JD-OR: Cagcctatttcggaactagg (SEQ ID NO.10)); 7R-JD-OF / R primer pair (7R-JD-OF: Agagtagccatgtattaac (SEQ ID NO.11) and 7R-JD-OR: Gaatatgctgtattaag (SEQ ID NO.12) were selected for purity detection. The genomic DNA of the recombinant virus and the parental virus DNA were extracted using a virus genome extraction kit (purchased from Qiagen), and PCR amplification was performed using this as a template. The results are shown in FIG. Figure 6 As shown, Figure 6 As shown in a, through the primer detection at both ends of the H240R gene, the amplified fragment of the recombinant virus ASFV-ΔH240R-Δ7R virus is 1048bp, showing a single band, which is consistent with the expected size, while the amplified fragment of the parental strain (ASFV-WT) is 840bp;

[0072] The primers 7R-JD-OF / R at both ends of the MGF505-7R gene were used to identify Figure 6 As shown in b, the amplified fragment of the recombinant virus ASFV-ΔH240R-Δ7R virus is 1122bp, showing a single band, which is consistent with the expected size, while the amplified fragment of the parent strain (ASFV-WT) is 1770bp; the amplified ASFV-WT fragment is 1770bp, and the sequencing results show that the H240R gene and MGF505-7R gene in the recombinant virus ASFV-ΔH240R-Δ7R are completely missing, and the corresponding positions are replaced by the reporter gene expression cassette. The results show that the recombinant virus lacking the H240R and MGF505-7R genes was successfully constructed without parental virus contamination.

[0073] The results are as follows Figure 7 As shown, Figure 7 As shown in (a), the recombinant virus ASFV-ΔH240R expressed green dual-color fluorescence when infecting PAMs under a fluorescence microscope; primers at both ends of the H240R gene (H240R-JD-OF / R) were used for detection, as shown in FIG. Figure 7The amplified fragment of the recombinant virus ASFV-ΔH240R shown in (b) is 1048bp, showing a single band, which is consistent with the expected size, while the amplified fragment of the parent strain (ASFV-WT) is 840bp. The sequencing results show that the H240R gene in the recombinant virus ASFV-ΔH240R is completely missing, and the corresponding position is replaced by the reporter gene expression cassette. The results show that the recombinant virus ASFV-ΔH240R with the missing H240R gene was successfully constructed without parental virus contamination.

[0074] Example 3. Preparation of attenuated vaccines

[0075] 1. Use the recombinant virus ASFV-ΔH240R-Δ7R as a vaccine.

[0076] 2. Using the recombinant virus ASFV-ΔH240R-Δ7R as the basic strain, mutate the CD2V gene and the MGF family gene attenuated gene to construct a virus with one or more gene deletions as a vaccine.

[0077] The following experiments are used to verify the experimental results:

[0078] 1. Titration of Virus Titer

[0079] The titration of African swine fever virus is expressed by 50% haemadsorption (HAD50), and the operation is performed according to the reference (Zhao D, Liu R, Zhang X, Li F, Wang J, Zhang J, Liu X, Wang L, Zhang J, WuX, Guan Y, Chen W, Wang X, He X, Bu Z. 2019. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microbes Infect 8: 438-447.), and appropriate adjustments are made: PBMC is inoculated in a 96-well cell culture plate, the sample to be tested is diluted 10 times, and 0.02 ml is inoculated in each well. Viral infection can be determined by the rosette formed by the aggregation of red blood cells around the infected cells. After 7 days of observation, the half haemadsorption (HAD50) is calculated according to the Reed and Muench method. The recombinant virus titer is qualified and the pathogenicity is evaluated. For subsequent pathogenicity evaluation, the titer was determined.

[0080] 2. Immunity test of gene-deleted strain ASFV-ΔH240R

[0081] The virulence evaluation experiment was conducted in the Biosafety Level 4 Laboratory Animal Room Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. 7-week-old Large White and Landrace inbred SPF pigs (purchased from the Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were used. The experimental animals were divided into 3 groups, including 5 pigs in the gene-deficient strain ASFV-ΔH240R group, 4 pigs in the ASFV CN / HLJ / 18 group, and 4 control pigs. The control group was pigs that had not been treated in any way. The immunization dose of the ASFV CN / HLJ / 18 group was 10 3 HAD 50 The immunization dose of ASFV-ΔH240R group was 10 5 HAD 50 .

[0082] After immunization, the animals' mental state and feeding conditions were continuously observed, their body temperature was monitored, and oral and anal swabs, anticoagulated blood, and separated serum were collected. Reference was made to the literature (Zhao D, Liu R, Zhang X, Li F, Wang J, Zhang J, Liu X, Wang L, Zhang J, Wu X, Guan Y, Chen W, Wang X, He X, Bu Z. 2019. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microbes Infect 8: 438-447.), and the ASFV content in the blood was determined by quantitative PCR. The survival rate, body temperature changes, and ASFV content in the blood of each group of animals were counted.

[0083] The results of body temperature changes are as follows Figure 8 As shown, the body temperature of pigs injected with the gene-deficient strain ASFV-ΔH240R increased on the 6th day after injection, and returned to normal levels after the 10th day; while the body temperature of the ASFV CN / HLJ / 18 group increased on the 3rd day after injection, and all died on the 10th day.

[0084] The survival rate results are as follows Fig. 9 As shown, one pig injected with the gene-deficient strain ASFV-ΔH240R died on the 17th day after injection, and the rest all survived, with a survival rate of 80%; while the ASFV CN / HLJ / 18 group began to die on the 9th day after injection, and all died on the 10th day.

[0085] The results of ASFV genome content detection in blood are as follows Fig.10 As shown, about 10 6The number of viral DNA copies was detected until the 28th day, while in the ASFV CN / HLJ / 18 group, about 10 9 The number of copies of viral DNA increases until all of them die.

[0086] The above experimental results show that the gene-deficient strain ASFV-ΔH240R is significantly weakened, but still retains some virulence.

[0087] 3. Immunity test of gene-deleted strain ASFV-ΔH240R-Δ7R

[0088] The virulence evaluation experiment was conducted in the Biosafety Level 4 Laboratory Animal Room Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. 7-week-old Large White and Landrace inbred SPF pigs (purchased from the Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were used. The experimental animals were divided into 4 groups, and the immunization route was intramuscular injection, including 5 pigs in the gene-deficient strain ASFV-ΔH240R-Δ7R group, 4 pigs in the gene-deficient strain ASFV-ΔH240R-Δ7R cohabitation group, 4 pigs in the ASFV CN / HLJ / 18 group, and 4 control pigs. The control pigs were pigs that had not undergone any treatment. The immunization dose of the ASFV CN / HLJ / 18 group was 10 3 HAD 50 The immunization dose of ASFV-ΔH240R-Δ7R group was 10 5 HAD 50 ASFV-ΔH240R-Δ7R cohabiting pigs refer to pigs that live with the ASFV-ΔH240R-Δ7R group but do not receive any treatment.

[0089] After immunization, the animals' mental state and feeding conditions were continuously observed, their body temperature was monitored, and oral and anal swabs, anticoagulated blood, and separated serum were collected. Reference was made to the literature (Zhao D, Liu R, Zhang X, Li F, Wang J, Zhang J, Liu X, Wang L, Zhang J, Wu X, Guan Y, Chen W, Wang X, He X, Bu Z. 2019. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microbes Infect 8: 438-447.), and the ASFV content in the blood was determined by quantitative PCR. The survival rate, body temperature changes, and ASFV content in the blood of each group of animals were counted.

[0090] The results of body temperature changes are as follows Fig.11As shown, the body temperatures of pigs injected with the gene-deficient strain ASFV-ΔH240R-Δ7R were normal after injection, without any fever; the body temperatures of pigs living together were also normal, without any fever; while the body temperatures of the ASFV CN / HLJ / 18 group increased on the third day after injection, and all died on the 10th day.

[0091] The survival rate results are as follows Fig.12 As shown, pigs injected with the gene-deleted strain ASFV-ΔH240R-Δ7R were observed for 28 days after injection. All pigs had no clinical symptoms and were in a healthy state with a survival rate of 100%. The cohabiting pigs also showed healthy signs with a survival rate of 100%. However, the ASFV CN / HLJ / 18 group began to die on the 9th day after injection and all died on the 10th day.

[0092] The results of ASFV genome content detection in blood are as follows Fig.13 As shown in Figure 2, extremely low viral genome copy numbers of less than 10 were detected in pigs injected with the gene-deficient strain ASFV-ΔH240R-Δ7R on day 7 after injection. 2 The number of viral DNA detected in the cohabiting pigs was less than 10 until the 28th day; while the number of genome copies detected in the ASFV CN / HLJ / 18 group was about 10 on the 4th day after injection. 9 The number of copies of viral DNA increases until all of them die.

[0093] ASFV p30 antibody test results in blood Fig.14 As shown, pigs injected with the gene-deleted strain ASFV-ΔH240R-Δ7R had elevated P30 antibodies on the 7th day after injection, and the antibodies reached a high level on the 11th day until the 28th day; no P30 antibodies were detected in cohabiting pigs, indicating that the cohabiting pigs were not infected with the virus.

[0094] The above experimental results show that when the H240R gene and the MGF505-7R gene were simultaneously knocked out of the parental strain ASFV CN / HLJ / 18, the function of the proteins encoded by them was lost. The virulence of the gene-deleted attenuated African swine fever virus strain ASFV-ΔH240R-Δ7R was completely weakened, no detoxification occurred, and the safety was relatively good.

[0095] 4. Animal challenge experiment

[0096] After 28 days of immunization, the ASFV-ΔH240R-Δ7R immunized group and the cohabiting pigs were challenged with ASFV virulent virus at a dose of 10 2.5 HAD 50The route of challenge was intramuscular injection. After challenge, the animals' mental state and feeding were continuously observed, their body temperature was monitored, oral and anal swabs, anticoagulant blood and serum were collected. The ASFV content in the blood was determined by quantitative PCR. The survival rate, body temperature changes and ASFV content in the blood of each group of animals were counted. The immunization dose of the ASFV-ΔH240R-Δ7R group was 10 5 HAD 50 ASFV-ΔH240R-Δ7R cohabiting pigs refer to pigs that live with the ASFV-ΔH240R-Δ7R group but do not receive any treatment.

[0097] The results of body temperature changes are as follows Fig.15 As shown, the ASFV-ΔH240R-Δ7R immunized group was challenged with ASFV strong virus. After the attack, the body temperature of one pig increased on the 12th day after the attack, and began to decrease on the 19th day, while the body temperatures of the other four pigs were at normal levels; the body temperature of the cohabiting pigs increased significantly on the 4th day after the attack and maintained a high temperature until all died on the 11th day.

[0098] The survival rate results are as follows Fig.16 As shown, the ASFV-ΔH240R-Δ7R immunized group was attacked with ASFV strong virus. After 21 days of observation, all pigs survived with a survival rate of 100%; the body temperature of the cohabiting pigs was also healthy, with a survival rate of 100%; however, the cohabiting pigs began to die on the 10th day after the attack, and all died on the 12th day, with a survival rate of 0.

[0099] The results of ASFV genome content detection in blood are as follows Fig.17 As shown in the figure, the ASFV-ΔH240R-Δ7R immunization group was challenged with ASFV virulent virus, and extremely low viral genome copy numbers were detected from the first to the seventh day after the challenge, less than 10 2 The number of viral DNA began to rise on the 11th day, and the number of viral gene copies detected in pigs remained at a low level, about 10 2 Up to 10 3 About 10 6 The number of copies of viral DNA increases until all of them die.

[0100] Interferon test results in the lungs Fig.18 As shown, the ASFV-ΔH240R-Δ7R immunized group was challenged with ASFV virulent virus, and higher levels of IFN-β transcription were detected on the 21st day after the challenge, which was significantly different from that of the cohabiting pigs; low levels of IFN-β transcription were detected in the lungs of the cohabiting pigs after the challenge.

[0101] The above experimental results show that after the H240R gene and the MGF505-7R gene are knocked out of the parent strain ASFV CN / HLJ / 18 at the same time, the protein function encoded by them is lost, and the virulence of the gene-deleted attenuated African swine fever virus strain ASFV-ΔH240R-Δ7R is completely weakened, and after the parent strain ASFV CN / HLJ / 18 is attacked by a lethal dose, the pig body temperature is normal and the pig survival rate is 100%. Therefore, the gene-deleted attenuated African swine fever virus strain ASFV-ΔH240R-Δ7R described in the present invention has a complete immune protection effect on the parent strain ASFV CN / HLJ / 18.

Claims

1. A recombinant attenuated strain of African swine fever virus gene deletion, characterized in that: The recombinant attenuated strain is based on the African swine fever virus strain as the starting strain, and the recombinant attenuated strain of African swine fever virus is prepared by jointly losing the functions of the proteins encoded by the H240R gene and the MGF505-7R gene in the African swine fever virus; the African swine fever virus strain is a genotype II African swine fever virus.

2. The recombinant attenuated strain according to claim 1, characterized in that The African swine fever virus strain is Pig / HLJ / 2018 strain.

3. The recombinant attenuated strain according to claim 1, characterized in that The amino acid sequence of the H240R gene of the African swine fever virus is shown in SEQ ID NO.3; the amino acid sequence of the MGF505-7R gene of the African swine fever virus is shown in SEQ ID NO.4; the nucleotide sequence of the H240R gene of the African swine fever virus is shown in SEQ ID NO.1; the nucleotide sequence of the MGF505-7R gene of the African swine fever virus is shown in SEQ ID NO.

2.

4. A method for preparing attenuated African swine fever virus, characterized in that: The method described is to knock out the H240R gene and the MGF505-7R gene in the African swine fever virus by genetic engineering means; the African swine fever virus strain is a genotype II African swine fever virus.

5. A vaccine for African swine fever, characterized in that: The vaccine contains the recombinant attenuated strain according to any one of claims 1-3.

6. The vaccine according to claim 5, characterized in that The vaccine is a bivalent vaccine or a multivalent vaccine.

7. Use of the recombinant attenuated strain according to any one of claims 1 to 3 or the method according to claim 4 in the preparation of a vaccine for preventing African swine fever.

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