Cloning and expression of nucleocapsid protein of SARS coronavirus

Objective To clone and express SARS coronavirus(SARS-CoV) nucleocapsid protein and analyze its immunological activity. Methods The gene encoding SARS-CoV nucleocapsid protein was amplified by RT-PCR from SARS-CoV RNA,and was cloned into pMD18-T vector. Positive clones were identified by PCR and sequencing with ABI PRISM TM 377XL DNA sequencer;The insert digested with BamHⅠ and SalⅠ was subcloned into pGEX-4T-2 which was also digested with BamHⅠ and SalⅠ;The recombinant plasmids were transformed into E.coli JM109 and were identified by clone PCR and double enzyme digestion;The recombinant clone was induced with IPTG to express target protein and characterized by SDS-PAGE;Expressed recombinant protein was purified by affinity chromatography;The immunological activity of this protein was analyzed by reacting with the sera from SARS patients and inducing antibodies in mice. Results The gene fragment encoding nucleocapsid protein was amplified by RT-PCR from SARS-CoV RNA;The insert of positive clone was subcloned into pGEX-4T-2 correctly;Recombinant protein was expressed in the positive recombinant clone when induced with IPTG and was purified by affinity chromatography from E.coil JM109 lysate;The purified protein reacted with the sera of SARS patients and induced antibodies with a high titer in mice. Conclusion The recombinant plasmid expressing N protein was successfully constructed and the purified recombinant protein showed good immunological activity.