SARS冠状病毒N蛋白的表达及其诱导的免疫应答

目的表达和纯化SARS冠状病毒的N蛋白,分析其诱导的免疫应答。方法从重组克隆pMD18-N中切取N基因的插入片段,亚克隆至原核表达质粒pET-23a(+)中,转化大肠杆菌BL21,PCR和酶切鉴定转化菌落的插入序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE分析重组蛋白的表达;大量诱导表达N蛋白,金属螯合层析予以纯化,将纯化蛋白免疫小鼠,观察其诱导的免疫应答。结果N基因的特异片段被亚克隆到原核表达质粒pET-23a(+),在大肠杆菌表达了SARS冠状病毒的N蛋白;表达的蛋白经金属螯合层析获得纯化;纯化蛋白能被重组N蛋白的GST融合蛋白的免疫血清识别,并诱导小鼠产生了高滴度的抗体和Th1/Th2型的免疫应答。结论成功构建了N蛋白的原核重组表达质粒,纯化的蛋白具有良好的抗原性。

Expression of the recombinant nucleocapsid protein of SARS coronavirus and its induction of immune responses

To express and purify the recombinant nucleocapsid protein(N-protein) of SARS coronavirus and to analyze its induction of immune responses induced by this protein,the right gene fragment encoding the nucleocapsid protein in the recombinant clone pMD18-N was digested with restrictive endonucleases and was subcloned into prokaryotic expression vector pET-2a(+).The recombinant plasmid pET-N was then transformed into E.coli BL21,and the recombinant clone was characterized by PCR,and digested with restriction endonucleases;meanwhile the positive clone was induced with IPTG to express the target protein,this protein was characterized by SDS-PAGE;the recombinant protein was purified from E.coli cell lysate by metal-chelating chromatography,and the immunological activities of this protein were analyzed by immunoblotting and the immune responses induced in mice.It was demonstrated that the pET-N was constructed through subcloning the right insert of N-protein into pET-23a(+),and the recombinant protein could be expressed in clone containing pET-N when induced with IPTG and the expressed protein was purified with metal-chelating chromatograpjhy.The purified protein reacted with sera of mice immunized GST-N and could induce a moderate degree of immune responses in mice.It is concluded that the prokaryotic expression plasmid pET-N has been successfully constructed,and the purified recombinant protein shows good antigenicity.