SARS冠状病毒N蛋白的表达及其诱导的免疫应答  

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

作　　者：袁仕善[1] 吴少庭[2] 秦莉[3] 张仁利[2] 高世同[2] 黄达娜[2] 余新炳[4] 

机构地区：[1]中山大学基础医学博士后流动站深圳市疾病预防控制中心科研基地,深圳518020 [2]深圳市疾病预防控制中心 [3]华中科技大学同济医学院 [4]中山大学

出　　处：《中国人兽共患病杂志》2005年第10期855-859,共5页Chinese Journal of Zoonoses

摘　　要：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。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.

关 键 词：SARS 冠状病毒 N蛋白 纯化 免疫应答 

分 类 号：R373.9[医药卫生—病原生物学]