| African Swine Fever（ASF）is a highly contagious and acute infectious disease caused by African Swine Fever Virus（ASFV）infecting pigs.It is currently unable to be controlled by vaccines and drugs.Since the outbreak of ASF in 2018,ASF has caused serious economic losses to China’s pig breeding industry.ASF monitoring and culling of positive pigs are the effective means to control the ASF epidemic.Rapid and accurate detection is the basis of ASF epidemic prevention and control,but currently there is a lack of effective and rapid detection of ASF detection method.ASFV P72 protein is the main structural protein with good reactivity and immunogenicity.In this context,this study studied ASFV P72 protein and established an indirect ELISA method for ASFV P72 antibody detection on this basis.Based on the genes of ASFV China/2018/Anhuix CGQ strain,the surface antigen sites of P72 Protein were predicted.The N-terminal amino acid sequence with good antigenicity was selected as the study object,and Enhanced Green Fluorescent Protein（eGFP）was used as the reporter gene.The specific primers were designed.The p72 and eGFP genes were obtained by PCR amplification,then the p72 and eGFP genes were linked by overlapping PCR and cloned into pMD-18T vector.The p72-eGFP gene was subcloned into the eukaryotic expression vector pIRESpuro2 using the recombinant plasmid pMD18T-p72-eGFP as a template.The results of double enzyme digestion,PCR and sequence analysis showed that the eukaryotic expression vector pIRESpuro2-p72-eGFP was successfully constructed,and the p72-eGFP gene fragment was 1 727 bp.The lowest concentration of purinomycin on CHO-k1 cells was obtained by adding different concentrations of purinomycin into F12K medium for continuous culture of CHO-k1 cells.The eukaryotic expression plasmid of pirespuro2-p72-eGFP was transfected into CHO-k1 cells,and obvious green fluorescence was observed 24 h later,which proved that the eukaryotic expression plasmid of pirespuro2-p72-eGFP was successfully expressed in CHO-k1 cells.The CHO-k1-p72-eGFP monoclonal cell line was successfully obtained by continuous culture for 28 days under the effect of purinomycin.The purified P72 recombinant protein was identified by Coomasses bright blue staining.The results showed that there were obvious bands at about 60 ku,which was close to the size of the target band.Western Blot was used to identify and purify the P72 protein,and the results showed that the P72 recombinant protein could produce specific reaction with ASFV antibody positive serum,and had good reactivity.Using the purified recombinant protein as antigen to established ASFV P72 indirect ELISA detection method and the optimization of its various working conditions,the results showed that the best package is P72 protein concentration(including 0.3244 g/m L,serum best dilution proportion of 1:100,the best sealing fluid 1%BSA solution,enzyme mark antibody to 1:50000 best dilution degree,best time for 40 min,TMB color liquid optimal duration of 15min.The established ELISA method was used to conduct the intra-batch and inter-batch repeatability tests on 5 ASFV antibody positive serum samples and 5 negative serum samples.The coefficient of variation of OD450nmin the intra-batch repeatability test was4.19%,2.56%,4.82%,8.99%,6.39%,3.89%,8.04%,4.36%,8.52%,2.58%.The coefficients of variation of OD450nmbetween batches were 4.00%,7.51%,5.79%,3.96%,9.40%,5.73%,4.43%,7.30%,4.14%and 3.52%,and the coefficients of variation of OD450nmin both the intra batch and the interbatch repeated tests were all less than 10%,which proved that the established ELISA method had good repeatability.The established ASFV P72 antibody indirect ELISA method was used to detect the positive sera of swine antibodies to common virus diseases.The results of ELISA were all negative,which proved that the method had good specificity.Forty-four clinical serum samples were tested and compared using the established ELISA method and the Spanish INGENASA ASFV-blocking ELISA antibody test kit.The test results of INGENASA kit showed 28positive results,4 suspected results and 12 negative results.There were 28 positive results,2 suspected results and 14 negative results by the indirect ELISA method for ASFV P72antibody detection.The results showed that the concordant rate of the test method established in this study was 94.45%compared with the test kit of INGENASA.Based on the study of ASFV P72 protein,an indirect ELISA method for dectecting ASFV P72 antibody was established in this study.The ELISA method showed no significant difference in reproducibility,good specificity and high sensitivity,providing a technical reserve for the development of commercial ELISA kits. |
