Expression Of African Swine Fever Virus EP153R Gene And Construction Of Gene Deletion Virus

African swine fever(ASF)is an acute,hot,highly contagious animal infectious disease.Its viral genome is about 170-193 kb in length,contains 151?167 open reading frames(ORFs),and can encode 150-200 kinds of proteins.C-type lectin protein is a non-structural protein encoded by the African swine fever virus EP153 R gene.It participates in the erythrocyte adsorption process after the African swine fever virus infects cells,and can inhibit the expression of MHC-I and inhibit the apoptosis.Objective: To prepare the African swine fever virus C-type lectin protein antisera and ASFV-SY18?EP153R gene deletion recombinant virus to establish the African swine fever serology detection method and the C-type lectin protein biological function Research lays the foundation.Methods:(1)In this experiment,the p Shuttle-CTLA4-FLAG-EP153-Myc-EP402-HA plasmid was used as a template to PCR amplify the EP153 R gene,and then the target gene and the prokaryotic expression vector p ET-32 a were digested and ligated to construct a prokaryotic recombinant plasmid designated as p ET-32a-EP153 R.Recombinant plasmids identified by digestion and sequencing were transformed into E.coli BL21(DE3)competent cells,and IPTG was used to induce the expression of host bacteria to purify the recombinant protein of African swine fever virus C-type lectin.The purified protein was used to immunize BALB/c mice to prepare mouse antibody specific to C-type lectin protein,and the reactivity and specificity of the antibody were identified by Western blot and indirect immunofluorescence technology etc.(2)The homology arms of the left and right arms of the EP153 R gene from the genomic DNA of the ASFV-SY18 strain were amplifed by PCR,and then were subcloned into the p MD18-T vector.Using p MD18-T-p72-EGFP as template,the left arm of the EP153 R gene homology arm,the P72-EGFP gene expression box and the right arm of the EP153 R gene homology arm were sequentially cloned into the p UC19 vector to construct the p UC19-EP153RLR-EGFP homologous recombination vector.Based on the ASFV-SY18 strain EP153 R gene sequence,sg RNA was designed to construct p X335-?NLS-sg RNA recombinant plasmid.Co-transfection into the primary porcine alveolar macrophages(PAM)with p UC19-EP153RLR-EGFP homologous recombination vector and p X335-?NLS-sg RNA recombinant plasmid,and then ASFV-SY18 strain virus was inoculated for 12 hours until green fluorescentce appears with CPE.After that,it was purified by limiting dilution method to obtain SY18?EP153R recombination.Result:(1)The results of sequencing showed that the prokaryotic expression recombinant plasmid p ET-32a-EP153 R was successfully constructed.SDS-PAGE and Western blot results showed that the prokaryotic expression of recombinant protein C-type lectin was successfully induced by IPTG,and its size was about 38 k Da.It was mainly expressed in the form of inclusion bodies and could react with His-tagged monoclonal antibody.(2)The purified C-type lectin protein was used to immunize BALB/c mice,and the mouse anti-sera against C-type lectin protein was successfully prepared.By Western blot and indirect immunofluorescence,the results showed that the antibodies can specifically recognize C-type lectin protein when infection.(3)Successfully p UC19-EP153RLR-EGFP homologous recombination vector and p X335-?NLS-sg RNA recombinant plasmid were constructed.After purification,ASFV SY18 ?EP153R recombinant virus was obtained using homologous recombination and CRISPR / cas9 technology.Conclusion: The African swine fever virus C-type lectin protein antibodies and SY18?EP153R gene deletion recombinant virus were successfully prepared.