Prokaryotic Expression,Reactogenicity Analysis And ELISA Method Of Various Targeting Proteins Of African Swine Fever Virus

African swine fever(ASF)is an acute,severe and highly contagious animal disease caused by African swine fever virus(ASFV)infection of swine.Since the first case was found in Africa in 1909,the epidemic has spread widely in dozens of countries in Europe and America,and then spread to Russia and other countries.In August 2018,the first case of African swine fever was found in Shenyang,Liaoning Province,my country.Since then,the epidemic has spread across the country,causing huge economic losses to my country’s aquaculture industry.In this experiment,the published African swine fever virus gene nucleic acid sequence was used as the basis,and the four target gene fragments obtained were cloned into the p Clod Ⅰ prokaryotic expression vector by PCR amplification with primers,and four recombinant plasmids were obtained: K145 R,A240L,CP204 L,K205R.The 4correctly identified recombinant plasmids and the 20 correctly identified positive recombinant plasmids stored in the laboratory were transformed into the host strain E.coli BL21(DE3),induced at 15°C for 24 hours,and analyzed by SDS-PAGE.The expression of target protein was analyzed by protein electrophoresis.All recombinant proteins were expressed in two forms: soluble expression and inclusion body expression.The soluble protein was taken from the supernatant after sonication,filtered and sterilized,and purified using a purification kit.The denaturing agent of m M urea or 8m M guanidine hydrochloride was dissolved,and the supernatant and precipitate collected after high-speed centrifugation were collected and verified by SDS-PAGE protein electrophoresis analysis.The experimental results showed that the actual size of each recombinant protein in the supernatant was consistent with the theoretical value,and the size was about 10-70 kd.After data inquiry,these proteins include ASFV structural proteins,enzymes related to viral replication,proteins related to immunity and inflammation,proteins related to viral replication and virion maturation,and some proteins whose functions are not yet known.The 24 ASFV recombinant proteins reacted with the positive sera of pigs at different times after the immunization challenge,and Western blotting analysis was used to identify the time and intensity of the antibodies produced by these proteins in vivo after the host was infected with ASFV.The results showed that the 24 recombinant proteins showed good results.reactogenicity.The purified recombinant protein K205 R was selected as the coating antigen,the positive serum after challenge of immunized pigs was used as the primary antibody,and the rabbit anti-pig antibody labeled with horseradish peroxidase was used as the secondary antibody.Indirect ELISA method for ASFV antibody detection.Compared with commercial kits,the positive coincidence rate was 96.6%.This experiment will provide technical support for African swine fever monitoring,epidemic prevention and control,and purification in my country.