Cloning and expression of E,M and N protein of SARS-CoV in Escherichia coli and their antigenicities

【Objective】To obtain recombinant proteins of envelop gene (E),membrane gene(M) and nucleocapsid (N) gene from SARS-CoV,and to evaluate their antigenicities. 【Method】E,M and N genes obtained by PCR were cloned into prokaryotic expression vector pGEX-4T-1,and then transferred to E. coli BL21(DE3). The conditions in which the recombinant proteins were expressed were compared. The recombinant N protein was analyzed its antigenicity by western blotting.【Results】The recombinant N protein was highly expressed in E. coli and was observed by SDS-PAGE. It exists in the form of soluble protein. Its molecular weight was 71.97 kilodalton. Recombinant N protein could react with sera from mice immunized with pVAX1-N gene vaccine by western blotting. The recombi-nant E and M proteins were expressed at a very low level and was observed by western blotting analysis using mouse-anti-GST.tag as antibody. Their molecular weights were 35 and 46 kilodalton,respectively.【Conclusion】 Recombinant N protein could be highly expressed in E. coli BL21 (DE3) in the form of soluble protein. It had good immune reactivity and antigenic specificity,and might be a potential antigen for the serologic diagnosis.