Prokaryotic Expression Of E0 And E2 Genes Of Classical Swine Fever Virus And Establishment Of Indirect ELISA For Detection Antibody

Classical swine fever(CSF)is an acute,intense,highly contagious disease caused by classical swine fever virus(CSFV).Since its first report in the United States in the 19 th century,CSFV has caused a serious impact on the healthy development of the world pig industry.As a Class A disease prescribed by the World Health Organization(OIE),CSFV are mainly controlled by injection of attenuated vaccines.The traditional swine fever attenuated vaccine has good immune efficacy and provides better protection for the pig herd.However,it is impossible to distinguish between infected antibodies and vaccine antibodies after using this type of vaccine.The E2 subunit vaccine is the earliest CSF marker vaccine.Immunization of the E2 subunit vaccine not only protects against CSFV wild-type strains,but also easily distinguishes between immunized pigs and infected pigs by detecting antibodies against E0 protein.Among the structural proteins encoded by CSFV,both E0 and E2 glycoproteins can induce the production of neutralizing antibodies,and are also the main antigens for establishing CSFV serological assays.Therefore,on the basis of immune E2 subunit vaccine,the detection method constructed by the E0 and E2 genes of CSFV can be used to analyze the immune effect of vaccine and preliminarily distinguish vaccine antibody from infection antibody,which provides a reference for the further development of CSFV detection kit.This experiment conducted a series of studies:(1)Specific primers were designed and synthesized according to the E0 and E2 genes sequences of CSFV registered in GeneBank,and referring to the characteristics of pET32 a vector and protein of E0 and E2,and the 5,end of the primers of the upstream and downstream were inserted into EcoRI and XhoI respectively.The E0 and E2 genes were amplified by RT-PCR,and the gene fragments were 699 bp and 558 bp,respectively.The plasmids identified by cloning and sequencing were named pMD19T-E0,pMD19T-E2.The pET32 a linearized vector was ligated with the E0 and E2 genes excised from the cloning plasmid to construct the recombinant expression plasmid pET32a-E0 and pET32a-E2,and then the recombinant plasmid was transferred into the competent cell BL21(DE3)and induced to express and detected by SDS-PAGE.A large amount of protein expressed in the expected size is detected,and the protein is mainly present in the inclusion body.The pET32a-E0 and pET32a-E2 proteins were purified by nickel column affinity chromatography.Western-blotting showed that the pET32a-E0 and pET32a-E2 proteins had good reactogenicity.(2)An indirect ELISA method was constructed using the purified pET32a-E0 recombinant protein as an antigen.The optimized reaction conditions showed that the optimal antigen coating concentration of pET32a-E0 recombinant protein was 0.6?g /mL,and coating at 37°C for 2 h and incubated at 4°C overnight;the best sealing condition was that fetal bovine serum(5%)diluted with PBST buffer was incubated at 37? for 1 h;the optimal dilutions of serum and enzyme-labeled antibodies were 1:100 and 1:20000,respectively,both of them were reacted at 37°C for 1 h;TMB substrate was reacted in the dark room for 10 min.The method based on pET32a-E0 protein established repeatability test(both intra-batch and inter-batch)showed a coefficient of variation of less than 10%,and there was no antigen cross-reactivity with PRRSV,PCV,PRV,PEDV,TGEV positive serum,with high repetition and specificity.(3)The indirect ELISA method was established using the purified pET32a-E2 recombinant protein as the coating antigen.The optimization of the reaction conditions and the selection results showed that the suitable concentration of pET32a-E2 recombinant protein was 1.6 ?g / mL and the temperature was constant at 37°C for 2 h.After being placed at 4°C overnight,tne best blocking condition was incubation with the skim milk powder(5 %)dissolved in PBST buffer,and the reaction was constant at 37°C for 1 h;the optimal dilution ratios of serum and enzyme secondary antibody were 1:80 and 1: 20000,both were reacted at 37°C for 1 h;TMB substrate color solution was best reacted in the dark at room temperature for 15 min.The established method's repeatability test(intra-batch and interbatch)showed that the coefficient of variation was less than 10%,and there was no antigenic cross-reaction with PRRSV,PCV,PRV,PEDV and TGEV positive serum,showing high repeatability and specificity.It provides a relatively practical ELISA diagnostic method for the detection of CSFV antibodies.