Expression,purification and antigen analysis of SARS-associated coronavirus nucleocapsid protein in Pichia pastoris system / 第三军医大学学报

ABSTRACT

Objective To study the expression of SARS-associated coronavirus nucleocapsid(N) gene in Pichia pastoris and to obtain recombinant N protein with good biological activity.Methods The gene encoding the full N protein of SARS-CoV was amplified by PCR and cloned into Pichia pastoris expression vector pPICZA.The recombinant expression plasmid pPICZA/N was transformed into X-33,KM71H and GS115. The positive insert transformants were screened,cultured and induced by methanol.The recombinant protein was further purified with Ni affinity chromatography.Antigen activity was detected with anti-N monoclonal antibody,polyclonal antibody and sera from SRAS patients.Results SDS-PAGE and immunological analysis demonstrated that only Pichia pastoris transformants KM71H/pPICZA/N and X-33/pPICZA/N expressed Mr 70 000 fusion protein with special antigenicity.Conclusion SARS-CoV N protein expression in Pichia pastoris has been achieved,establishing the basis for further study of biological and immunological function of N antigen.