The Prokaryotic And Eukaryotic Expression Of ASFV Protein P54, Development Of Indirct ELISA Method And Generation Of P54Monoclonal Antibody

Objective: PET prokaryotic expression system and pFastBac/NT-TOPOeukaryotic expression system were used to obtain African swine fever virus proteinp54. Further more, indirect ELISA to detect antibody of African swine fever in serumwas established. At the same time, p54monoclonal antibody was prepared.Methods: Gene of p54was cloned into pET-52b(+)3C/LIC vector, and thentransform into BL21(ED3) to get recombinant protein p54. The recombinantBL21(ED3) was broken by ultrasonication, and then centrifuged. The supernantantafter centrifuge was analysed by SDS PAGE and Western blot. The expressioncondition was optimized; the recommbinan protein p54was purified by using6-Histag. The indirect ELISA to detect surm antibody of ASF was developed by usingpurified recombinant p54as coating antigen.Gene of p54was cloned into pFastBac-NT-TOPO vector, then transform intoE.coil DH5α. After selecting, the recombinant pFastBac-NT-TOPO vector wasobtained, the recombinant baculovirus plasmid was identified by PCR and thentransformed into E.coil DH10Bac. After that, recombinantbaculovirus was preparedby lipsome mediate method.72h later, the recombinantbaculovirus infected sf9cellwas collected and broken by ultrasonication, and then centrifuged. The supernant wasused for SDS PAGE and Western blot analysis. The indirect ELISA was establishedby using the supernant as coating antigen.Protein p54expressed by prokaryotic expression system was used forimmunization, protein p54expressed by eukaryotic expression system was used forscreening. The hybridoma was prepared with traditional methods to generatemonoclonal antibody.Result: Protein p54was expressed by prokaryotic and eukaryotic expressionsystem respectly. The recommbinan protein p54can react with ASF standard positiveserum through Western blot analysis, the results showed that the recombinant proteinhas good immunoreactivity. The indirect ELISA was developed by using prokaryotic/ eukaryotic expression system expressed protein p54respectly, the ASF standardpositive serum can be detected after diluting1:1280and1:320dilution by two ELISArespectly, inter and intra CV is less than10%and15%. The coincidence rate betweencommercial ELISA kit and two indirect ELISA is90.1%and84.4%. Specificity of thetwo methods are100%. The sensitivity of the two method is88.2%and80.3%respectly.Conclusion: In this study, two indirect ELISAs detecting ASF antibody in serumwere established. The two methods are sensitive, specific, stable and easy to operate.Meanwhile, the p54monoclonal antibody was generated. The achievement of thisstudy will provide a technical preparation for detection of ASF.