Developmentof Indirect ELISA Antibody Test Kit For African Swine Fever Virus

African Swine Fever （ASF） is an acute infectious swine disease caused by ASF virus （AFSV）. Being infected by strong AFSV stains, swine’s fatality rate can reach 100%. Moreover, there is no vaccine that can effectively prevent ASF. By now ASF has not been found in China. However, with the increasing prosperity of international trade, it is possible that ASF appears in China. Therefore, it is very necessary to work out corresponding laboratory diagnosis methods. In our research, molecular biologic methods were used to express ASFV’s protein pK205, laying the foundation for working out quick, sensitive, and specific serological diagnostic methods. The research has produced the following results:1) ASFV K205R gene was amplifiedAccording to published K205R gene sequence design primers, chemically-synthesized plasmids that contain K205R gene were used as a template to amplify K205 gene and were inserted into vector pMD-18T for sequencing. The result of sequencing was identical to published ASFV-K205R （GenBank ID:NC₀01659.1）.2) ASFV K205R gene’s cloning and expression in Escherichia coliBgⅢ & NotI were used to carry out double enzyme cutting for recombinant plasmid pMD18T-K205R, which contains K205R gene, and vector pET-32a. Prokaryotic expression plasmid, named pET32a-K205R, was generated after reclamation, purification, and connection procedures. Recombinant plasmid pET32a-K205R was transferred into Escherichia coli BL21 （DE3） to express protein pK205R after IPTG induction. It was indicated via SDS-PAGE that pET32a-K205R was expressed efficiently in Escherichia coli in a dissolvable form and expression product’s molecular weight was 44.0KD approximately. Subsequently, rabbit immune serum was used for western blotting. Test results showed that antiserum could react specifically with protein pK205R. This proved that protein pK205R was of antigenicity and it could be used as an antigen to test specific ASF antibody. According to the 6×His label attached to the recombinant protein, HisTrap FF chromatographic column was used to purify pK205R. Purer recombinant proteins were obtained.3) ASFV indirect ELISA antibody test methods were worked outWith purified protein pK205R as a coating antigen, optimal operating conditions were identified through the optimization of optimal antigen coating concentration, optimal serum dilution concentration, antigen coating condition, blocking solution sort, optimal enzyme-labeled secondary antibody dilution, optimal serum acting time, optimal enzyme-labeled secondary antibody acting time, and substrate coloration time. Results showed that optimal antigen coating concentration was 6.5μg/mL and optimal serum dilution concentration 1:320; antigen should be coated at 37℃ for 1 hour and then coated at 4℃for overnight; optimal blocking solution was 1%BSA and optimal enzyme-labeled secondary antibody dilution concentration 1:4000; both serum and enzyme-labeled secondary antibody’s optimal acting time was 60min; and optimal substrate coloration time was 15min.4) AFSV indirect ELISA antibody test kit assembling and quality evaluationASFV antibody test kit was developed by using the indirect ELISA antibody test methods worked out. We also made a study of its specificity, sensibility, reproducibility, and shelf life. Results showed that the kit reacted positively with a standard positive serum but it did not react crossly with the positive serum of six diseases, including HC, PR, PRRS, Hps, E.coli and SB. When a standard positive serum was diluted as per 1:5120, the result was lower than the critical value and the ELISA method worked out was very sensitive. Both within-batch and batch-to-batch repeated experiments’ coefficient of variation was less than 10%. After the kit was kept at 4℃ for four months, test results were basically identical.