Prokaryotic Expression Of CSFV E0 Gene And Establishment Of Indirect ELISA For Detection Of Antibody

Classical swine fever(CSF)is one of the epidemic disease in OIE reported. CSF ofen takes place frequently in our country. The break out of CSF brougt great economy loss and far-reaching community impact .So it is important to defend and control to our country . Injecting inactived vaccine is a mainly means to defend CSF in many countries.Through prokaryotic expression of E0 gene of classical swine fever virus (CSFV) from wild strain FJ237,we have established indirect ELISA for detection of antibody ,which provide much information for a diagnosis kit.Using a pair of primers designed according to the nucleotide sequence from wild strain FJ237,the genes of E0 were amplified. The special primer pair containing XhoⅠand EcoRⅠ.The fragment was amplifed with the primer pair by PCR ,a 687 bp fragment was gained.Recombinant plasmid and pET32a vector was digested with XhoⅠand EcoRⅠrespectively. The target gene of E0 was subcloned into pET32 a vector. Positive clone with interest gene was indentificated by restriction analysis,PCR and DNA sequencing. Then the recombinant plasmid was transformed into Escherichia coli(E. coli )BL21(DE3) for E0 expression. The interest gene was induced to express in E.coli with different concentration IPTG. The bacteria containing pET32a-E0 was examined by SDS-PAGE and western-blot. Result showed that the pET32a-E0 protein can expresse in E.coli and melecular weight of the protein was 50 ku. The condition of expression was optimized ,in 37℃E. coli BL21(DE3) for E0 expression. It was up to OD600 0.6 ,the IPTG concentration is 0.1 mmol/L,cultured 3h in 30℃continued,the amount of expression is higher,the amount of protein is about 25% of the total bacteria protein. Western-blot shows that the protein can be recognized by the positive serum of CSFV . This study showed that the E0 gene expressed highly in E.coli and the E0 protein has character of antigen.The protein was expressed massly.Soluble test showed that the protein exsits in suspension. The fusion protein was purified by affinity chromatography .The indirected ELISA for detection anti-CSFV antibody was established with the purified His-E0. The optimal reaction conditions were determined. It showed that the concentration of coated antigen was 58ug /mL and the dilution of serum samples was 1:60. The blocking condition of CSFV for ELISA was 10g/L BSA ,incubated at 37℃for 2h.The working concentration of HRP-labed goat anti-swine IgG was 1:2000,incubated at 37℃for 1h.The substrate for ELISA was incubated at RT for 15 min before reading the OD450. The development of the CSFV indirect ELISA afforded a simple and applied way for monitoring antibody of CSF which provide much information for a diagnosis kit.