Expression Of Major Antigen Proteins Of Classical Swine Fever Virus In P.Pastoris And E.coli

Classical swine fever virus(CSFV) is the causative agents of classical swine fever which is a highly contagional disease of pig. Proteins E2 and Erns are the major antigenic proteins of CSFV and also the main target for developing subunit marker vaccine of CSF. To explore the possibility to produce CSF antigenic protein in vitro by genetic engineering methods, the genomic fragment Ems/E1/E2 of lapinized Chinese strain (C-strain) was amplified by RT-PCR and nPCR, and then cloned into eukaryotic expression vector pPIC9K, recombinant plasmid pPSWE was constructed. After linearized by restriction enzyme(RE) SalI, the recombination plasmid were transformed into P.Pastoris SMD1168 competent cells by electroporation, recobinant with multiple inserts were screen by different concentration of G418, and then induced with 0.5% methanol. The induced culture was centrifuged and its supernate was concentrated by aminosulfate precipitation, the dialysed sample was analyzed by SDS-PAGE and Western blotting. The results indicated the interest protein got expression, but the expression level was low. In order to improve the expression level of antigenic protein, then the E2 gene fragment was cloned into prokaryotic expression vector pGEX4T-1. The resulted expression plasmid, referred as pGST-E2 was used to transform E.coli strain BL21( Star)pLysS and eventual E2 expression in the form of inclusion body at high level was gained after induction with IPIG. To increase the ratio of soluble protein in expression products, the recombinant bacteria was induced with 0.1mmol/L IPTG at 16℃. The results of SDS-PAGE showed that, 40% expression product exist in soluble form, after purified by affinity chromatography, its purity is up to 85%.