CN115717129A - Recombinant attenuated strain with African swine fever virus gene deletion and preparation method and application thereof - Google Patents
Recombinant attenuated strain with African swine fever virus gene deletion and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant attenuated strain with African swine fever virus gene deletion, a preparation method and application thereof, belongs to the technical field of veterinary biological products, and aims to provide a strategy for constructing the African swine fever attenuated strain. The invention provides a recombinant attenuated strain with deleted African swine fever virus genes, which jointly deletes the functions of H240R genes and MGF505-7R gene coding proteins in an African swine fever virus strain Pig/CN/HLJ/2018 by adopting a genetic engineering means, thereby obtaining an attenuated African swine fever candidate vaccine strain with high safety. The attenuated candidate vaccine strain is completely attenuated to pigs after immunizing pigs, can provide 100 percent immune protection for the virus attack of the Pig/CN/HLJ/2018 virulent strain, can be used as a safe and effective vaccine for preventing and controlling the epidemic situation of the Chinese African swine fever, and has great social value.
Description
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a recombinant attenuated strain with African swine fever virus gene deletion, and a preparation method and application thereof.
Background
African Swine Fenver (ASF) is an acute, virulent and highly contagious infectious disease which is caused by African Swine Fever Virus (ASFV) infection and is characterized by pig Fever and systemic organ bleeding, and the death rate of virulent strains is close to 100 percent. The domestic pig, the wild pig and the soft tick at each stage are natural hosts of African swine fever, can be directly transmitted between the domestic pig and the wild pig, can be transmitted by tick bite, and can be transmitted by swill, feed, meat products and the like which pollute viruses across regions and countries. At present, no commercial vaccine or specific medicine exists, once the epidemic situation of the African swine fever occurs, the epidemic situation can be controlled only by means of catching and killing, but the mode not only causes great economic loss, but also cannot meet the requirement of large-scale pig raising in China. Therefore, the development of effective and safe vaccines is urgent.
The current development of vaccines against african swine fever is mainly achieved by three ways: 1. directly inactivating the original African swine fever virus to obtain an inactivated vaccine; 2. screening out antigen protein of virus induced immune response to prepare subunit vaccine; 3. the recombinant virus vaccine is obtained after a virulence gene is knocked out by adopting a gene deletion means. The first method is the most common method for preparing virus vaccines, and because African swine fever virus infection has the characteristics of antibody-dependent enhancement (ADE), immunosuppression and the like, the inactivated vaccine cannot provide effective immunoprotection for pigs; the second method limiting factor is that the African swine fever virus has large genome, complex result and most of unknown functions, the antigen of the induced immune response is not clear at present, and the known antigen can not induce effective immune protection. The vaccine which is hopeful to break through at present is a gene knockout attenuated vaccine, and a vaccine candidate strain which has immunogenicity and has no pathogenic effect on pigs is obtained by knocking out virulence related genes such as immunosuppression and the like, so that a complete protective effect is provided after the pigs are immunized. For example, chinese patent CN110093324A provides an african swine fever virus seven-gene deletion low virulent strain, which is based on the african swine fever virus Pig/CN/HLJ/2018 strain, and which lacks functional proteins of the following genes: CD2v gene coding products and six polygene family genes (polygene family 360 genes 12L, 13L and 14L and polygene family 505 genes 1R, 2R and 3R) coding products, after piglets are immunized for 28 days, the ASFV original virus is used for carrying out challenge test to obtain 100 percent immune protection; for another example, chinese patent CN114107228A discloses a twelve-gene-deleted attenuated african swine fever vaccine, wherein 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) are jointly deleted in an ASFV CN/GS 2018 strain, and after a piglet is inoculated, a challenge test is performed with a parent strain to obtain 100% immune protection.
The research on the African swine fever virus virulence gene knockout vaccine needs to consider the immune response and protection to pigs and also considers the safety performance. The following factors also need to be considered in the prior art: (1) Whether the virulence genes are toxic or not after being knocked out is an important index for the safety of the gene deletion vaccine; (2) The African swine fever virus codes more than 160 proteins, has numerous virulence genes, and the deletion of which virulence genes has the best immunoprotection and safety, which is a long-term and arduous work; (3) Different strains have different effects caused by the deletion of the same gene, and the virus titer caused by the deletion of multiple genes is low, so that the risk of causing attenuated strains to reduce the immunogenicity or the protective effect and the like can be caused.
Therefore, in the preparation process of the attenuated African swine fever vaccine, the gene function and the influence of the gene function on pathogenicity and immune response are identified, a basis is provided for the selection of a knockout target gene and the construction of gene knockout, the screening of a new target gene is very important, and the safety and the immune effectiveness of a vaccine candidate strain are determined. In the study of MGF505-7R gene, the MGF505-7R gene is knocked out by African swine fever virus strain Pig/CN/HLJ/2018, and the immunizing dose is 10 5 HAD 50 The mortality rate after infecting pigs is obviously reduced, but partial toxicity still exists, and the survival rate of the pigs is about 60% (Li et al, pMGF505-7R definitions pathogenesis of African swine fever infection by inhibiting IL-1 beta and type I IFN production. PLOS PATHOGENS.2021); MGF505-7R gene is knocked out by African swine fever virus strain CN/GS/2018, and lower immunization dose (10 HAD) 50 ) All pigs survived after infection, but significant clinical symptoms such as increased body temperature, toxicity, etc. still remained (Li et. African swine virus protein MGF-505-7R proteins virus and pathogenesis by inhibition JAK1-and JAK2-mediated signaling. Journal of Biological chemistry. 2021).
H240R gene (ASFV-delta H240R) is knocked out by African swine fever virus Pig/CN/HLJ/2018 strain, the function of the protein coded by the H240R gene is lost, and porcine alveolar macrophage is infectedLeading to higher interferon response and high expression of antiviral genes. 10 is prepared by animal experiments 5 HAD 50 The ASFV-delta H240R infects pigs, the body temperature rises on the 6 th day after infection, and the pig blood has a toxic phenomenon after immunization. There is a need for a fully attenuated and well immunogenic vaccine virus strain.
Disclosure of Invention
The invention aims to provide a strategy for constructing an attenuated strain of African swine fever and provide a vaccine virus strain which is completely attenuated and has good immunogenicity.
The invention provides a recombinant attenuated strain with deletion of an African swine fever virus gene, which is obtained by mutating H240R of the African swine fever virus and MGF505-7R gene of the African swine fever virus by taking the African swine fever virus strain as an initial strain.
Further defined, the african swine fever virus strain is a genotype II african swine fever virus.
Further defined, the African swine fever virus Pig is the Pig/HLJ/2018 strain.
Further limited, 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.
Further limited, 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.
The invention provides a method for constructing the recombinant attenuated strain, which is characterized in that the method is used for mutating an H240R gene and an MGF505-7R gene in African swine fever virus by a genetic engineering means.
Further defined, the method comprises the following specific steps:
step 2: the MGF505-7R gene is shown as SEQ ID NO.1, and is connected with a p72-mCherry plasmid to obtain p72-mCherry-MGF505-7R, sgRNA of a targeted H240R gene and a pX330 plasmid obtain a pX330-sgMGF505-7R vector, then the p72-mCherry-MGF505-7R and the pX330-sgMGF505-7R vector are co-transfected into porcine alveolar macrophages, and the recombinant virus which is obtained in the step 1 and lacks the H240R gene is infected into the transfected porcine alveolar macrophages to obtain the recombinant virus which lacks the H240R gene and the MGF505-7R gene.
Further defined, the p72-EGFP vector was obtained by ligating the sequence of SEQ ID NO.7 to the pBluescript II KS (+) vector; the p72-mCherry vector was obtained by ligating the sequence of SEQ ID NO.8 with the pBluescript II KS (+) vector.
The invention provides a recombinant attenuated strain with deletion of an African swine fever virus gene, which is prepared by taking an African swine fever virus strain as an initial strain and jointly losing the functions of an H240R gene and an MGF505-7R gene coding protein in the African swine fever virus.
Further limiting, the recombinant attenuated strain is taken as a basic strain, and the CD2V gene and the MGF family gene are mutated to obtain the virus with multiple gene deletions.
The invention provides a vaccine for African swine fever, which contains the recombinant attenuated strain.
Further defined, the vaccine is a monovalent vaccine, a bivalent vaccine, or a multivalent vaccine.
The invention provides application of the vaccine in preparing a medicament for treating or preventing African swine fever.
The invention provides application of the vaccine in preparing a health-care product for adjuvant therapy or prevention of African swine fever.
Has the advantages that: the invention obtains an attenuated African swine fever with good safety by jointly losing the functions of the encoded proteins of the H240R gene and the MGF505-7R geneA viral strain; immunizing the attenuated African swine fever virus strain (intramuscular injection of 10 per pig) 5 HAD 50 ) The pig does not have diseases, and the nucleic acid and the antibody of the pig living in the same house are negative, which indicates that the horizontal transmission phenomenon does not exist; the virus attack experiment shows that the attenuated African swine fever virus strain can provide 100% immune protection for the virus attack of the Pig/CN/HLJ/2018 virulent strain, can be used as a safe and effective vaccine for preventing and controlling African swine fever, and has great social value, and because protein sequences coded by the H240R gene and the MGF505-7R gene in the African swine fever viruses with different genotypes have high homology (figure 1 and figure 2), the H240R gene and the MGF505-7R gene can be knocked out from the African swine fever viruses with different genotypes, and the attenuated African swine fever virus vaccine can also be successfully constructed. However, the present invention is not limited to the Pig/CN/HLJ/2018 isolate. Comparing the protein sequences coded by the H240R gene and the MGF505-7R gene in the African swine fever viruses with different genotypes, the protein coded by the H240R gene and the MGF505-7R gene is highly homologous (figure 1 and figure 2), so that the protein coded by the H240R gene and the MGF505-7R gene in the African swine fever viruses with different genotypes has similar functions, and the African swine fever virus attenuated candidate vaccine can be successfully constructed by knocking out the recombinant virus of the H240R gene and the MGF505-7R gene.
Drawings
FIG. 1 shows the sequence alignment of MGF505-7R gene encoded protein in African swine fever viruses of different genotypes;
FIG. 2 shows the comparison of the sequences of the proteins encoded by the H240R gene in African swine fever viruses of different genotypes;
FIG. 3 is a schematic diagram showing the construction of an ASFV MGF505-7R gene deletion vector (express red fluorescence);
FIG. 4 is a schematic diagram showing the construction of an ASFV H240R gene deletion vector (express green fluorescence);
FIG. 5 shows ASFV- Δ H240R- Δ 7R expressing red-green two-color fluorescent protein;
FIG. 6 shows the result of ASFV- Δ H240R- Δ 7R assay, in which, panel a shows the result of detecting recombinant virus ASFV- Δ H240R- Δ 7R by H240R-JD-O-F/R primer pair, and panel b shows the result of detecting recombinant virus ASFV- Δ H240R- Δ 7R by 7R-JD-O-F/R primer pair;
FIG. 7 shows the identification of ASFV-. DELTA.H 240R, in which (a) is a result of observation of recombinant virus ASFV-. DELTA.H 240R under a fluorescent microscope and (b) is a result of detection of ASFV-. DELTA.H 240R by the H240R-JD-O-F/R primer;
FIG. 8 is a graph of the change in body temperature of swine after infection with ASFV- Δ H240R and control strains; wherein the abscissa is the number of days after infection and the ordinate is body temperature;
FIG. 9 shows the survival rate of pigs infected with ASFV- Δ H240R immune and control strains; wherein the abscissa is the number of days after infection and the ordinate is the survival rate;
FIG. 10 shows the result of the virus carried in the pig blood after infection of ASFV- Δ H240R immune and control strains; wherein the abscissa is the number of days after infection and the ordinate is the viral genome copy number (Log) 10 )；
FIG. 11 is a graph of the change in pig body temperature following infection with ASFV- Δ H240R- Δ 7R immune and control strains; wherein the abscissa is the number of days after infection and the ordinate is body temperature;
FIG. 12 shows survival rates of swine after infection with ASFV- Δ H240R- Δ 7R and control strains; wherein the abscissa is the number of days after infection and the ordinate is the survival rate;
FIG. 13 shows the result of the pig blood after infection of ASFV- Δ H240R- Δ 7R and control strains; wherein the abscissa is the number of days after infection and the ordinate is the viral genome copy number (Log) 10 )；
FIG. 14 shows the results of P30 antibody after infection of ASFV- Δ H240R- Δ 7R immune and control strains; wherein the abscissa is the number of days post infection and the ordinate is the ASFV P30 antibody level (P/N);
FIG. 15 is a graph showing the change in body temperature of pigs after challenge with the gene-deficient virus immunized pig and its congeneric pig; wherein the abscissa is days after toxin attack, and the ordinate is body temperature;
FIG. 16 shows the survival rate of pigs challenged with the gene-deficient virus immunized pig and pigs of the same habitation; wherein the abscissa is days after challenge, and the ordinate is survival rate;
FIG. 17 shows the result of the virus carried in the blood of pigs challenged with the gene-deleted virus immunized pig and pigs living together with the immunized pig; wherein the abscissa is the number of days after challenge and the ordinate is the viral genome copy number (Log) 10 )；
FIG. 18 shows the lung IFN- β transcription results after challenge of the genetically deleted virus immunized pigs and their co-resident pigs; wherein the abscissa is the group and the ordinate is the relative expression level of IFN- β mRNA;
Detailed Description
The African swine fever virus Chinese epidemic strain (ASFV/CN/HLJ/18 strain), also known as African swine fever virus Chinese epidemic strain Pig/CN/HLJ/2018 (Wenleli African swine fever virus Pig/HLJ/2018 strain artificially infected SPF Pig histopathological study [ D ]. Beijing: chinese academy of agricultural sciences, 2021.) was isolated and stored by Harbin veterinary research institute of Chinese academy of agricultural sciences (full-length sequence thereof is referred to GenBank: MK 333180.1).
Porcine Alveolar Macrophages (PAMs) are derived from 30-50 day old SPF pigs cultured in 1640 medium containing 10% FBS. Porcine Peripheral Blood Mononuclear Cells (PBMCs) were isolated from SPF porcine anticoagulated blood using PBMC isolation kit (purchased from TBD, china).
Construction of green fluorescent protein (EGFP) gene screening expression cassette: the design strategy in the reference (Li et al. PMGF505-7 Rs Pathology of origin of human swing virus introduction by inhibition IL-1. Beta. And type I IFN production. PLOS PATHOGENS. 2021) was to fuse the p72 promoter with the green fluorescent protein (EGFP) by means of gene synthesis, the gene sequence is shown in SEQ ID NO.7, the synthesized sequence was ligated into the cloning vector pBluescript II KS (+) via HindIII and BamHI, and the constructed vector was named p72-EGFP.
Construction of a red fluorescent protein (mCherry) gene screening expression cassette: the vector constructed according to the design strategy in the reference (Li et al. PMGF505-7 Rs cloning strategy of African swing feeder infection by inhibiting IL-1. Beta. And type I IFN production. PLOS PATHOGENS. 2021) by fusing the p72 promoter and the red fluorescent protein (mCherry) by means of gene synthesis, whose sequence is shown in SEQ ID No.8, by ligating the synthesized sequence into the cloning vector pBluescript II KS (+) by means of HindIII and BamHI, was named p72-mCherry.
The p72-EGFP vector is the gene sequence shown in SEQ ID NO.7
AAGCTTTTGTTATTATCAAGATCCTTCGCATAAACCGCCATATTTAATAAAAACAATAAATTATTTTTATAACATTATATAGCCACCATGgattacaaggatgacgacgataagACGCGTGCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGgaattcATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAgaattc
The p72-mCherry vector is the same as SEQ ID NO.8
AAGCTTTTGTTATTATCAAGATCCTTCGCATAAACCGCCATATTTAATAAAAACAATAAATTATTTTTATAACATTATATAGCCACCATGgattacaaggatgacgacgataagACGCGTGCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGgaattcatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagTAAgaattcTTTTTTTTTTGGATCC；
Example 1 construction of recombinant vectors for attenuated vaccine strains
The nucleotide sequence of the H240R gene is shown as SEQ ID NO. 1:
Atggctgcaaacattattgcaacaagagccgtgccaaagatggccagcaaaaaagagcatcaatactgtctgctagactcccaggaaaagcgtcatgggcattatcccttttcatttgaattaaagccttatgggcaaacaggcgcaaatatcataggagtacagggctcacttacccatgttatcaaaatgacagtatttccatttatgattccttttcctttacaaaaaactcatatagatgattttattggtggacgcatttatttattttttaaggaactggacatgcaagcagtttctgatgtaaatggaatgcaataccacttcgagttcaaggttgttcctgtaagccccaaccaagtagagcttcttcctgtgaataataaatataaatttacatatgctataccggtagtgcaataccttaccccaatcttttatgatctttcgggaccgctagatttcccattagatactctttcggtccatgtggatatcctctccaatcatatacagcttcctatccaaaaccataacctaacaacgggtgatcgtgtttttatttctggatataaacacctgcaaacgattgaattatgtaaaaataacaagatttttatcaaaaatataccgccgctttcatccgaaaaaataaaactatatatactaaaaaatcgaatcagaattccgctatactttaaatctttaaaaacgtctaagtaa；
the amino acid sequence of the H240R gene is shown as SEQ ID NO. 3:
MAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSK；
the nucleotide sequence of MGF505-7R gene is shown in SEQ ID NO. 2:
atgttctcccttcaggacctctgtcggaagaacaccttcttccttccaagtgattttagcaagcataccctgcatttgctggggttatactggaaggggcatggatctatccaaaggataaagaatgatggtgtgcttatagagcatgatcttactctttccatcaatgaagccttaattcttgcaggagaagagggaaacaatgaagtagtaaagctcttgttactatgggaaggaaatcttcattatgccatcataggagctttgaggactgagaactataacctagtatgtgagtaccatagtcaaattcaggactggcatgttctcctccctttgattcaagatccagaaacattcgaaaaatgtcatgatttaagccttgaatgtgatctttcatgccttctccaacatgctgtaaaatataacatgctttcgattcttgttaaatataaagaggatctactaaatgtactatttaggcaacaaattcaaggactatttattttagcatgtgaaaatcggaagcttgagattcttacgtggatgggtcaaaatctgccaattcctgatcctgagcctatttttagcattgctgttgtcacaaaagatttagaaatgttttccttagggtacaagattgtttttgaatacatggaaaaccaaggacttcatttaacccaggtagttcgtatggttatgctaaatcatcactttggcatggtaataaataaaggacttttaccctttgtgctggaaattttaaattatggtgggaatgtaaatagagccttatcttatgctgtcacacaaaataaaagaaagattttagaccatgttgttcgccaaaagaatataccccataaaaccattgaaagaatgttgcatctggctgtaaaaaagcatgctcccaggaaaactctgaacttgttactatcttacataaattacaaggtgaaaaatgttaaaaagttgttagaacatgtagtgaaatacaactctactcttgtgataagactcttgttagaaaaaaagaaaaacctgctggatgctactttgacaagatatgtcaaagattctacatactttcaggtgaaagaatttatgcaagacttctccatcagcccagaaaaattcattaaaatagctgtgcgggaaaagagaaatgtgttgatcaagggtatttctgaagatatttgggaaaatcccgcggaaagaatcaggaatcttaagcagatagtgtgtaccataaaatatgaaagtggaagacaattcctgataaatatcattcacaccatttaccagagttattctttgaaacctgaagaaattcttaaattggcaacattttatgtcaaacacaatgcaaccacccattttaaagatctctgcaaatatctttggctgaacagaagaacagaaagtaagaaactgtttttagagtgcttggaaattgctgataagaaggagtttcctgatattaaaagtattgtgagtgaatacattaactatttgtttactgcaggagctattaccaaggaagaaatcatgcaagcctatgctttggagtatgccatgtattaa；
the amino acid sequence of the MGF505-7R gene is shown in SEQ ID NO. 4:
MFSLQDLCRKNTFFLPSDFSKHTLHLLGLYWKGHGSIQRIKNDGVLIEHDLTLSINEALILAGEEGNNEVVKLLLLWEGNLHYAIIGALRTENYNLVCEYHSQIQDWHVLLPLIQDPETFEKCHDLSLECDLSCLLQHAVKYNMLSILVKYKEDLLNVLFRQQIQGLFILACENRKLEILTWMGQNLPIPDPEPIFSIAVVTKDLEMFSLGYKIVFEYMENQGLHLTQVVRMVMLNHHFGMVINKGLLPFVLEILNYGGNVNRALSYAVTQNKRKILDHVVRQKNIPHKTIERMLHLAVKKHAPRKTLNLLLSYINYKVKNVKKLLEHVVKYNSTLVIRLLLEKKKNLLDATLTRYVKDSTYFQVKEFMQDFSISPEKFIKIAVREKRNVLIKGISEDIWENPAERIRNLKQIVCTIKYESGRQFLINIIHTIYQSYSLKPEEILKLATFYVKHNATTHFKDLCKYLWLNRRTESKKLFLECLEIADKKEFPDIKSIVSEYINYLFTAGAITKEEIMQAYALEYAMY；
constructing ASFV MGF505-7R gene deletion vector according to the construction scheme of ASFV MGF505-7R gene deletion vector (express red fluorescence) shown in FIG. 3;
1. the MGF505-7R gene ORF (NCBI accession No. MK 333180.1) (at positions 40763-42346 relative to the full-length sequence position) was flanked by about 1000bp of the left genome as the left homology arm sequence (about 1000bp to the left of the 5' ttaattgggtaggaaa-. The plasmid p72-mCherry-MGF505-7R is digested by KpnI, linearized and recovered for later use.
Constructing an ASFV H240R gene deletion vector according to the construction schematic diagram of the ASFV H240R gene deletion vector (express green fluorescence) shown in FIG. 4;
2. a genome of about 1000bp to the left of the H240R gene ORF (NCBI accession No. MK 333180.1) (at positions 155375-156100 relative to the full-length sequence position) was used as a left homology arm sequence (about 1000bp to the left of the 5 'and ttaacattgatattta-3' sequence of the ASFV genome), a genome of about 1000bp to the right of the H240R gene ORF was used as a right homology arm sequence (about 1000bp to the right of the 5 'and taacatttatattatactactact-3' sequence of the ASFV genome), and the left and right homology arms were inserted into XhoI and BamHI sites of the p72-EGFP vector using a homologous recombination cloning kit, respectively, and the successfully constructed homology arm transfer vector was named as p72-EGFP-H240R. The plasmid p72-EGFP-H240R is digested by KpnI, linearized and recovered for later use.
3. The sgRNA targeting MGF505-7R gene (target sequence: 5 'GTATAACCCCCAGCAAATGCA-3', SEQ ID NO. 5) and the sgRNA targeting H240R gene (target sequence: 5 'GATTGGAGGATATCCACA-3', SEQ ID NO. 6) were inserted into pX330 vector, respectively, and the vectors successfully constructed were named as pX330-sgMGF505-7R and pX330-sgH240R.
Example 2 construction and identification of recombinant Virus ASFV- Δ H240R- Δ 7R
1. The linearized p72-EGFP-H240R vector and the pX330-sgH240R vector obtained in example 1 are co-transfected with a transfection reagent (X-treme GENE HP) according to a proportion of 1.
Inoculating the collected virus liquid to new PAMs, discarding the virus liquid after 2H, covering 1640 culture solution containing 1% low-melting-point agarose on the surface, selecting plaques containing green fluorescence after 48H, and repeatedly picking the plaques for 5-8 times until pure recombinant virus (ASFV-delta H240R) lacking H240R genes is obtained.
2. The linearized p72-mCherry-MGF505-7R and the pX330-sgMGF505-7R obtained in example 1 are co-transfected with a transfection reagent (X-treme GENE HP) at the ratio of 1.
Inoculating the collected virus liquid to new PAMs, discarding the virus liquid after 2H, covering 1640 culture solution containing 1% low-melting-point agarose on the surface, selecting plaques containing red fluorescence after 48H, and repeatedly picking the plaques for 5-8 times until pure recombinant viruses (ASFV-delta H240R-delta 7R) lacking H240R genes and MGF505-7R genes are obtained.
The observation result under the fluorescence microscope is shown in figure 5, and the result shows that the purified gene deletion recombinant virus ASFV-delta H240R-delta 7R expresses red-green double-color fluorescence simultaneously when infecting PAMs, which indicates that the recombinant virus is successfully constructed.
And (3) virus purity detection: selecting a H240R-JD-O-F/R primer pair (H240R-JD-O-F: gcaaaatttgcaacaag (SEQ ID NO. 9)) and H240R-JD-O-R: cagcctatttcggaacagg (SEQ ID NO. 10)); the purity of 7R-JD-O-F/R primer pair (7R-JD-O-F: agagtagccatgtattaac (SEQ ID NO. 11) and 7R-JD-O-R: gaatgctgttataag (SEQ ID NO. 12) was tested, the genomic DNA of the recombinant virus and the parental viral DNA were extracted using a viral genome extraction kit (purchased from Qiagen Co.), and PCR amplification was performed using this as a template, and the results are shown in FIG. 6, in which the amplified fragment of the recombinant virus ASFV- Δ H240R- Δ 7R virus was 1048bp, exhibiting a single band, and was identical to the expected size, and the amplified fragment of the parental strain (ASFV-WT) was 840bp, as shown by a in A in FIG. 6;
identifying primers 7R-JD-O-F/R by using MGF505-7R gene both ends, as shown in b in FIG. 6, the virus amplification fragment of recombinant virus ASFV- Δ H240R- Δ 7R is 1122bp, presents a single band, is consistent with the expected size, and the amplification fragment of parent strain (ASFV-WT) is 1770bp; the amplified ASFV-WT fragment is 1770bp, and the sequencing result shows that H240R gene and MGF505-7R gene in the recombinant virus ASFV-delta H240R-delta 7R are completely deleted, and the corresponding position is replaced by a reporter gene expression cassette. The result shows that the recombinant virus lacking the H240R and MGF505-7R genes is successfully constructed without parental virus pollution.
As shown in fig. 7, the recombinant virus ASFV- Δ H240R expressed green bicolor fluorescence upon infection with PAMs, as shown in fig. 7 (a), under a fluorescence microscope; through primer detection of two ends (H240R-JD-O-F/R) of the H240R gene, as shown in (b) in FIG. 7, the virus amplification fragment of the recombinant virus ASFV-delta H240R is 1048bp, presents a single band and is consistent with the expected size, while the amplification fragment of the parent strain (ASFV-WT) is 840bp, and the sequencing result shows that the H240R gene in the recombinant virus ASFV-delta H240R is completely deleted and the corresponding position is replaced by a reporter gene expression cassette. The result shows that the H240R gene deletion recombinant virus ASFV-delta H240R is successfully constructed without parental virus pollution.
Example 3 preparation of attenuated vaccines
1. The recombinant virus ASFV-delta H240R-delta 7R is used as vaccine.
2. One or more gene-deleted viruses are constructed by taking recombinant virus ASFV-delta H240R-delta 7R as a basic strain, mutating CD2V genes and MGF family gene attenuated genes and are used as vaccines.
The experimental effect was verified using the following experiment:
1. titration of viral Titers
Titration of African swine fever viruses was performed using half the number of hemadsorptions (50% haemaddisplacement, HAD50) as described in the references (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 vision in pegs of the first African swine virus infected in China. Emerg Microbes infection 8 438-447.) with appropriate adjustments: PBMC were inoculated into a 96-well cell culture plate, the sample to be tested was diluted 10-fold, and 0.02ml was inoculated into each well, and virus infection was judged based on rosette formed by aggregation of erythrocytes around infected cells, observed for 7 days, and half the amount of adsorbed blood cells (HAD 50) was calculated according to the Reed and Muench method. And determining the titer of the recombinant virus to be qualified, and evaluating the pathogenicity. For later pathogenicity evaluation, titers were determined.
2. Gene deletion strain ASFV-delta H240R immunoassay
Toxicity evaluation experiments are carried out in the animal room center of the biological safety four-level laboratory of Harbin veterinary institute of Chinese agricultural science, 7-week-old Dabai and Changbai inbred SPF pigs (purchased from the animal center of Harbin veterinary institute of Chinese agricultural science) are adopted, experimental animals are divided into 3 groups, wherein the gene deletion strain ASFV-delta H240R comprises 5 heads, the ASFV CN/HLJ/18 comprises 4 heads, and the control pigs comprise 4 heads. The control group is pigs without any treatment, and the immune dose of the ASFV CN/HLJ/18 group is 10 3 HAD 50 The immunizing dose of ASFV- Δ H240R group was 10 5 HAD 50 。
Continuously observing the mental state and the ingestion condition of the animals after immunization, monitoring the body temperature of the animals, collecting the mouth and anus swab, anticoagulation and separating serum. Reference is 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 vision in pigs of the first air swing virus isolated in china. Emery microorganisms infection 8. And (4) counting the survival rate, the body temperature change and the ASFV content in blood of each group of animals.
The result of body temperature change is shown in fig. 8, the body temperature of the pig injected with the gene deletion strain ASFV- Δ H240R is increased on the 6 th day after injection, and the body temperature returns to the normal level after the 10 th day; whereas the ASFV CN/HLJ/18 group had elevated body temperature on day 3 post-injection until all died on day 10.
The survival rate results are shown in fig. 9, one pig of the pigs injected with the gene deletion strain ASFV-delta H240R died in the 17 th day after injection, and the rest pigs all survived with the survival rate of 80%; whereas the ASFV CN/HLJ/18 group began to die on day 9 post-injection and all died on day 10.
The results of measuring the content of ASFV genome in blood are shown in FIG. 10, and about 10 of pigs injected with the gene-deleted strain ASFV- Δ H240R were detected on the 4 th day after injection 6 Copy number of viral DNA until day 28; whereas ASFV CN/HLJ/18 group was detected on day 4 post-injectionAbout 10 9 Copies of viral DNA until total death.
The experimental result shows that the gene deletion strain ASFV-delta H240R is obviously weakened, but still keeps partial toxicity.
3. Gene deletion strain ASFV-delta H240R-delta 7R immunoassay
Toxicity evaluation experiments are carried out in the animal room center of the biological safety four-level laboratory of Harbin veterinary institute of Chinese agricultural science, 7-week-old large white and long white inbred SPF pigs (purchased from the animal center of Harbin veterinary institute of Chinese agricultural science) are adopted, the experimental animals are divided into 4 groups, and the immunization route is intramuscular injection, wherein the gene deletion strain ASFV-delta H240R-delta 7R group comprises 5 pigs, the gene deletion strain ASFV-delta H240R-delta 7R group comprises 4 pigs, the gene deletion strain ASFV/HLJ/18 group comprises 4 pigs and a control pig comprises 4 pigs. Control pigs were pigs without any treatment and the immunization dose in the ASFV CN/HLJ/18 group was 10 3 HAD 50 The immunizing dose of the ASFV- Δ H240R- Δ 7R group was 10 5 HAD 50 The ASFV- Δ H240R- Δ 7R-inhabiting pig refers to a pig which stays with the ASFV- Δ H240R- Δ 7R group but has not undergone any treatment.
Continuously observing the mental state and the ingestion condition of the animals after immunization, monitoring the body temperature of the animals, collecting the mouth and anus swab, anticoagulation and separating serum. Reference is 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 vision in pigs of the first air swing virus isolated in china. Emery microorganisms infection 8. And (4) counting the survival rate, the body temperature change and the ASFV content in blood of each group of animals.
The body temperature change result is shown in figure 11, the body temperature of the pigs injected with the gene deletion strain ASFV-delta H240R-delta 7R is normal level after injection, and the body temperature does not rise; the temperature of the pig in the same house is also normal level, and the body temperature is not increased; whereas the ASFV CN/HLJ/18 group had elevated body temperature on day 3 post-injection until all died on day 10.
The survival rate result is shown in figure 12, when the pigs injected with the gene deletion strain ASFV-delta H240R-delta 7R are observed for 28 days after injection, all the pigs have no clinical symptoms and are in a healthy state, and the survival rate is 100 percent; the physical signs of the pigs living together are in a healthy state, and the survival rate is 100 percent; whereas the ASFV CN/HLJ/18 group began to die on day 9 post-injection and all died on day 10.
The result of detecting the content of ASFV genome in blood is shown in FIG. 13, and the pig injected with the gene deletion strain ASFV- Δ H240R- Δ 7R detects the extremely low copy number of virus genome on the 7 th day after injection, which is less than 10 2 Up to day 28; the genome copy number of the pigs detected by the same residence is less than 10 until the 28 th day; whereas ASFV CN/HLJ/18 group detected about 10 on day 4 post-injection 9 Copies of viral DNA until total death.
The detection result of the ASFV P30 antibody in the blood is shown in FIG. 14, the P30 antibody of the pig injected with the gene deletion strain ASFV- Δ H240R- Δ 7R is increased on the 7 th day after the injection, and the antibody reaches a higher level on the 11 th day until the 28 th day; no P30 antibody was detected in the pigs of the same age, indicating that the pigs were not infected with the virus.
The experimental result shows that the H240R gene and the MGF505-7R gene are simultaneously knocked out by the parent strain ASFV CN/HLJ/18, and the obtained gene deletion attenuated African swine fever virus strain ASFV-delta H240R-delta 7R has completely weakened virulence after the function of the encoded protein is lost, does not generate the phenomenon of toxin expulsion, and has better safety.
4. Animal toxicity attacking experiment
After 28 days of immunization, ASFV-delta H240R-delta 7R immunization groups and the pigs living in the same house are subjected to ASFV virulent challenge with the challenge dose of 10 2.5 HAD 50 The toxic counteracting route is intramuscular injection. After the challenge, the mental state and the ingestion condition of the animals are continuously observed, the body temperature of the animals is monitored, and the anus and anus swab, the anticoagulation blood and the separated serum are collected. And the content of ASFV in blood is determined by a quantitative PCR method. And (4) counting the survival rate, the body temperature change and the ASFV content in blood of each group of animals. The immunization dose of the ASFV- Δ H240R- Δ 7R group was 10 5 HAD 50 The ASFV- Δ H240R- Δ 7R-inhabiting pig refers to a pig which stays with the ASFV- Δ H240R- Δ 7R group but has not undergone any treatment.
The body temperature change results are shown in fig. 15, the ASFV-delta H240R-delta 7R immune group performs ASFV virulent attack, the body temperature of 1 pig after the attack is increased on the 12 th day after the attack, the body temperature starts to decrease on the 19 th day, and the body temperatures of the other 4 pigs are all normal levels; the temperature of pigs living together is obviously increased in the 4 th day after the challenge, and the high temperature phenomenon is maintained until all pigs die in the 11 th day.
The survival rate results are shown in fig. 16, when the immune group of ASFV-delta H240R-delta 7R is attacked by ASFV virulent virus, all pigs survive after 21 days of virus attack, and the survival rate is 100%; the temperature of the pig in the same house is in a healthy state, and the survival rate is 100 percent; and the pigs in the same residence die after challenge on the 10 th day and all die on the 12 th day, and the survival rate is 0.
The results of the detection of the content of the ASFV genome in the blood are shown in FIG. 17, the ASFV- Δ H240R- Δ 7R immune group performs ASFV virulent attack, and the extremely low copy number of the virus genome, which is less than 10, is detected from the 1 st to the 7 th days after the virulent attack 2 The viral DNA of (1); the copy number began to rise at day 11, and pigs were detected to maintain a lower level of viral gene copy number, about 10 2 To 10 3 In the middle of; the pigs living together detected about 10 days 4 after challenge 6 Copy number of viral DNA until all die.
The detection result of interferon in the lung is shown in figure 18, the ASFV-delta H240R-delta 7R immune group carries out ASFV virulent attack, detects the high-level IFN-beta transcription on the 21 st day after the virulent attack, and has obvious difference compared with the pig with the same residence; syndromic pigs detected low levels of IFN- β transcription in the lungs after challenge.
The experimental result shows that H240R gene and MGF505-7R gene are knocked out simultaneously by parent strain ASFV CN/HLJ/18, the obtained gene deletion attenuated African swine fever virus strain has completely weakened virulence of ASFV-delta H240R-delta 7R after the function of the encoded protein is lost, and the pig temperature is normal and the survival rate of the pig is 100% after the parent strain ASFV CN/HLJ/18 lethal dose attacks. Therefore, the gene deletion attenuated African swine fever virus strain ASFV-delta H240R-delta 7R has complete immune protection effect on the parent strain ASFV CN/HLJ/18.
Claims (10)
1. The recombinant attenuated strain with the African swine fever virus gene deletion is characterized in that the recombinant attenuated strain takes the African swine fever virus strain as an initial strain, and the African swine fever virus recombinant attenuated strain is prepared by jointly losing the functions of H240R gene and MGF505-7R gene coding protein in the African swine fever virus.
2. The recombinant attenuated strain of claim 1, wherein the African swine fever virus strain is genotype II African swine fever virus.
3. The recombinant attenuated strain of claim 2, wherein the african swine fever virus strain is the Pig/HLJ/2018 strain.
4. The recombinant attenuated strain of claim 1, wherein 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 MGF505-7R gene of 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 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.
5. A method for preparing attenuated African swine fever virus, which is characterized in that the method mutates H240R gene and MGF505-7R gene in the African swine fever virus by genetic engineering means.
6. The recombinant attenuated strain of claim 5, wherein the genetic engineering means comprises gene deletion techniques, gene mutation techniques, gene insertion techniques and gene editing.
7. A vaccine against African swine fever comprising the recombinant attenuated strain of any one of claims 1-4.
8. The vaccine according to claim 7, wherein the recombinant attenuated strain of any one of claims 1 to 4 is used as a base strain, and the CD2V gene and the MGF family gene are mutated to obtain a virus with multiple gene deletions.
9. The vaccine according to claim 7 or 8, wherein the vaccine is a bivalent or multivalent vaccine.
10. Use of the recombinant attenuated strain of any one of claims 1-4, the method of claim 5 or 6 for the manufacture of vaccines and medicaments for the prevention or treatment of African swine fever.
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| CN116492455A (en) * | 2023-03-31 | 2023-07-28 | 中国人民解放军军事科学院军事医学研究院 | African swine fever virus K421R gene and replication-defective African swine fever vaccine prepared by using same |
| CN116590243A (en) * | 2023-04-07 | 2023-08-15 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Genetically engineered virus strain of African swine fever virus and preparation and application thereof |
| CN117701509A (en) * | 2023-11-20 | 2024-03-15 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | African swine fever virus passage attenuated strain and vaccine based on strain |
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| CN112063634A (en) * | 2020-05-01 | 2020-12-11 | 中国农业科学院兰州兽医研究所 | Attenuated African swine fever virus strain with gene deletion and application thereof |
| CN113215109A (en) * | 2020-07-10 | 2021-08-06 | 中国农业科学院兰州兽医研究所 | Construction of African swine fever polygene combined deletion attenuated strain and application of attenuated strain as vaccine |
| CN115094088A (en) * | 2022-04-19 | 2022-09-23 | 华南农业大学 | pSFV-flag X-CMV replicon plasmid, preparation method and cocktail mixed vaccine |
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| CN112063634A (en) * | 2020-05-01 | 2020-12-11 | 中国农业科学院兰州兽医研究所 | Attenuated African swine fever virus strain with gene deletion and application thereof |
| CN113025629A (en) * | 2020-05-01 | 2021-06-25 | 中国农业科学院兰州兽医研究所 | Attenuated African swine fever virus strain with gene deletion and application thereof |
| CN113215109A (en) * | 2020-07-10 | 2021-08-06 | 中国农业科学院兰州兽医研究所 | Construction of African swine fever polygene combined deletion attenuated strain and application of attenuated strain as vaccine |
| CN115094088A (en) * | 2022-04-19 | 2022-09-23 | 华南农业大学 | pSFV-flag X-CMV replicon plasmid, preparation method and cocktail mixed vaccine |
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| PINGPING ZHOU等: "Deletion of the H240R Gene of African Swine Fever Virus Decreases Infectious Progeny Virus Production Due to Aberrant Virion Morphogenesis and Enhances Inflammatory Cytokine Expression in Porcine Macrophages", 《J VIROL.》, vol. 96, no. 3, 9 February 2022 (2022-02-09), pages 5 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116492455A (en) * | 2023-03-31 | 2023-07-28 | 中国人民解放军军事科学院军事医学研究院 | African swine fever virus K421R gene and replication-defective African swine fever vaccine prepared by using same |
| CN116492455B (en) * | 2023-03-31 | 2024-04-23 | 中国人民解放军军事科学院军事医学研究院 | African swine fever virus K421R gene and replication-defective African swine fever vaccine prepared by using same |
| CN116590243A (en) * | 2023-04-07 | 2023-08-15 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Genetically engineered virus strain of African swine fever virus and preparation and application thereof |
| CN116590243B (en) * | 2023-04-07 | 2024-01-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Genetically engineered virus strain of African swine fever virus and preparation and application thereof |
| CN117701509A (en) * | 2023-11-20 | 2024-03-15 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | African swine fever virus passage attenuated strain and vaccine based on strain |
| CN117701509B (en) * | 2023-11-20 | 2024-05-28 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | African swine fever virus passage attenuated strain and vaccine based on strain |
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