Prokaryotic Expression Of African Swine Fever P30 Protein And Establishment And Application Of Its Indirect ELISA Antibody Detection Method

African swine fever(ASF)is a kind of acute infectious disease caused by African swine fever virus(ASFV)infection.In the late summer and early autumn of 2018,the disease spread to my country for the first time,and it quickly broke out across the country.At present,the development of vaccines is still a big problem.Therefore,effective,rapid and sensitive clinical diagnostic methods are extremely important in the prevention and control of African swine fever.P30 protein is a structural protein expressed in the early stage of African swine fever virus,which is suitable for prompt diagnosis.For the early detection,early diagnosis and early elimination of African swine fever,this study established an indirect ELISA antibody detection method for African swine fever by constructing the expression vector p ET-28a-P30,using the prokaryotic expression system to express the African swine fever P30 recombinant protein.Carrying out the following test.1.Prokaryotic expression and purification of P30 protein of African swine fever virusAccording to the CP204 L gene sequence of the ASFV China/2018/Anhui strain published by Gen Bank,the target fragment was artificially synthesized and seamlessly cloned into the expression vector p ET-28a(+).After being converted to the BL21 strain,the African swine fever was induced by IPTG P30 protein.2.Establishment of ASFV indirect ELISA antibody detection methodThe purified recombinant protein P30 was used as the coating antigen,and the ELISA reaction conditions were optimized by a single variable method to determine the optimal coating concentration,serum dilution factor,blocking solution,enzyme-labeled secondary antibody dilution factor,serum incubation time,and two Anti-reaction time and substrate color time.3.Quality evaluation and application of ASFV indirect ELISA antibody detection methodThe established indirect ELISA detection method will be checked for specificity,sensitivity,repeatability,compliance rate and shelf life,and the established indirect ELISA detection method for African swine fever virus will be used for clinical application.After testing,the research finally achieved the following results:1.The target protein P30 was successfully obtained,which was partially soluble under the expression conditions of this study.After purification,SDS-PAGE showed that a single target band with a size of about 23.1 k Da was obtained,and the purity was high.2.The optimal coating concentration of antigen is 0.5 μg/m L,serum dilution ratio is1:40,blocking solution is 5% skimmed milk powder,working concentration of enzymelabeled secondary antibody is 1:8000,serum incubation time and secondary antibody reaction time Both are 30 minutes and the substrate color time is 10 minutes.The critical OD630 value of this method is found to be 0.238.3.The detection method does not cross-react with a variety of disease-positive sera,indicating good specificity;when the ASF-positive serum sample is diluted to 1:10 240,the result is still positive,indicating good sensitivity;inter-batch reproducibility and batch-tobatch reproducibility The coefficient of variation of internal reproducibility test results is below 10%;when the kit is stored at 4 ℃,there is a high consistency for at least 3 months;compared with commercial kits,the total coincidence rate reaches 100%;application The method tested 170 serum samples,and the results showed that 43 of them were positive and127 were negative.In summary,this study successfully utilized the expressed recombinant P30 protein to establish an ASFV indirect ELISA antibody detection method,which has good specificity,sensitivity and compliance,and achieved the expected results.