Establishment And Application Of Indirect ELISA Detection Methods Against African Swine Fever Virus Antibodies Based On CHO Cells System

African swine fever(ASF)is a highly contagious disease that has devastated the global pig industry,and there is no safe and effective vaccine or drug available to prevent and control this disease.The virulent strain of ASF virus(ASFV)caused high mortality.ASFV is a large DNA virus with complex and variable genome.It is urgent to develop an efficient,sensitive and specific detection technology and a safe and effective vaccine.In this study,ASFV pK205R,p17 and p12 were successfully expressed using CHO suspension cells,and the antigenicity as diagnostic antigens was analyzed.An indirect ELISA method was developed against ASFV pK205R protein;a monoclonal antibody against pK205R(2H11)was prepared using the purified pK205R as the immunogen and the linear B-cell epitope recognized by 2H11 was identified.On the other hand,a recombinant PRRSV expressing ASFV pK205R was rescued by reverse genetic manipulation and evaluated for immune antibody levels.The main contents of the study were as follows:To verify the potential of ASFV pK205R,p17 and p12 as diagnostic antigens,eukaryotic expression plasmids pCDNA3.1-pK205R-strep,pCDNA3.1-p 17-strep and pCDNA3.1-p12-strep were constructed using homologous recombination by ligating them to the pCDNA3.1 vector based on the reported gene sequences of K205R,D117L and 061R of ASFV SY18(GenBank ID:MH766894.1).The eukaryotic expression plasmids were transfected into CHO cells and the pK205R,p17 and p12 were purified and the purified proteins were used as antigens to immunize BALB/c mice.The polyclonal antibodies were prepared and the specificity was verified by Western blotting(WB)and indirect immunofluorescence assay(IFA).In order to construct CHO cell line that can stably and efficiently express ASFV pK205R and achieve large-scale production,the K205R gene fragment fused with the Twin strep tag was homologously recombined into the pCDNA3.4 vector,and a CHO cell line 205-3 that can secretly express ASFV pK205R protein was constructed by G418 screening and limited dilution methods.The K205R gene was amplified by RT-PCR from 20 consecutive generations of cell samples,indicating that the gene was successfully integrated into the cell genome;the viability of recombinant protein-expressing cell line was verified by CCK-8 and cell counting.The recombinant protein was purified by strep beads and the results showed that the protein could be purified in amounts of 0.3-0.43 mg/mL.The purified pK205R was immunized in BALB/c mice,and the spleen cells of the mice immunized four times were fused with SP2/0 hybridoma cells,and two monoclonal strains of anti-ASFV pK205R protein were obtained by three times subcloning.The 2H11 monoclonal antibody,which is more stable in antibody secretion and has a higher potency,was subtyped and its recognized B-cell epitope was identified.The results showed that the heavy chain of the 2H11 monoclonal antibody is IgG1 and the light chain is the kappa chain.The epitope was highly conserved among the different strains by bioinformatics analysis.In order to establish a sensitive ELISA for the early diagnosis of ASFV,the pK205R recombinant protein was purified from the stable cell line.The recombinant pK205R was used as an antigen to coat the enzyme standard plate and the reaction conditions were optimized to establish an indirect ELISA method for both the detection of wild virus and the recombinant PRRSV expressing ASFV pK205R.The indirect ELISA method can be used to monitor ASFV clinical infection and evaluate the level of antibodies of the recombinant PRRSV.This method was found to be specific,sensitive and stable,with a 98.1%compliance rate with the commercial kit.ASFV pK205R is immunogenic and has potential for vaccine development.In this study,a full-length infectious clone of PRRSV containing the K205R gene was obtained by inserting the K205R gene between ORF1b and ORF2 of the PRRSV vHuN4-F112 genome using reverse genetic manipulation,and a recombinant virus vA-ASFV-K205R strain was obtained by viral rescue,with similar growth characteristics to those of the parental virus.The recombinant virus was immunized in PRRSV-free and ASFV-free piglets and antibody levels were monitored by the commercial kit and the indirect ELISA developed in this study.The genetically engineered live vector vaccine candidate induced high levels of PRRSV-specific antibodies and also produced specific antibodies against pK205R.