基于非洲猪瘟病毒D250R蛋白间接ELISA抗体检测方法的建立OA北大核心

D250R protein of African swine fever virus(ASFV),a non-structural protein with decap-ping enzyme activity,is expressed during early infection.In this study,the recombinant prokaryotic expression plasmid pET-32a-D250R was constructed,and the recombinant protein His-D250R was success-fully obtained through IPTG induction and nickel affinity purification.Both His-D250R and the recom-binant protein Flag-D250R expressed in eukaryotic cells can be recognized by ASFV standard positive serum,indicating that D250R protein is a potential target for serum detection.Additionally,mouse polyclonal antibody was prepared using the purified His-D250R protein as the immunogen.The antibody exhibited an ELISA titer as high as 1∶409 600,and can react with both Flag-D250R and His-D250R protein in Western-blot,demonstrating the good antigenicity of the recombinant protein.Using His-D250R pro-tein as coating antigen,an indirect ELISA method for antibody detection was established through opti-mization of conditions such as coating,blocking,antibody incubation,and substrate reaction condi-tions.The method exhibited a cut-off value of 0.359,and the positive serum results of PRV,PRRSV,PCV2,CSFV were all negative.Sensitivity of this method reached 1∶3 200,and coefficients of variation for intra-and inter-batch repeatability tests were less than 10％.The concordance rate with commercial kits was 98.8％.These results indicated that the indirect ELISA method based on D250R protein had good specificity,sensitivity,and repeatibility,and possesses potential clinical application value,and could provide important support for the development of diagnostic methods for African swine fever.