SARS冠状病毒S蛋白部分序列2的克隆与表达

Cloning and expression of SARS Coronavirus spike protein fragment 2

目的 构建SARS冠状病毒棘突蛋白部分序列 2 (S2 )的原核表达质粒 ,分析其在大肠杆菌中的表达状况。方法 采用逆转录聚合酶链式反应技术从SARS冠状病毒基因组中扩增出编码S蛋白的第 2 170到 2 814位碱基的基因片段 ,克隆至 pMD18-T载体 ,PCR筛选阳性克隆并测序。用限制性内切酶消化后 ,目的片段亚克隆至表达载体pGEX - 4T - 2 ,转化大肠杆菌JM10 9,PCR和双酶切鉴定转化菌落。将阳性菌落经IPTG诱导 ,SDS -PAGE和免疫印迹分析靶蛋白的表达。结果 RT -PCR扩增出S2特异片段 ,经测序与GenBank比对存在 1处碱基突变。S2基因片段亚克隆至表达载体 pGEX - 4T - 2构建成重组表达质粒 ,并在JM 10 9中表达了S2融合蛋白。结论 成功构建了SARS冠状病毒S2的重组表达质粒 ,并在大肠杆菌中表达了S2融合蛋白。

Aim To construct an expressional plasmid encoding partial sequence of SARS Coronavirus spike protein from 2170 bp to 2814bp,which we called fragment-2 (S2) here and analyze its expression in E coli JM109.Methods The sequence encoding S2 was amplified by RT-PCR fom SARS Coronavirus genome RNA,then was cloned to pMD18-T vector After identified by PCR and sequencing with ABI PRISM TM 377XL DNA Sequencer,the positive clone encoding S2 was digested with BamHⅠand SalⅠ,then S2 fragment was subcloned into pGEX-4T-2 which was digested with the same restricted endonucl eareases.The recombinant plasmid pGEX-S2 was transformed into E.Coli JM109 and induced with IPTG to express target protein and was characterized by SDS-PAGE and Western-blot.Results S2 fragment was amplified by RT-PCR and subcloned into pGEX-4T-2 properly.Recombinant fusion protein was examined in the positive clone when induced with IPTG and the fused protein could react wigoat polyclonal antibody to Schistosoma japonicum 26kDa GST.Conclusion The recombinant plasmid pGEX-S2 has been successfully constructed and the recombinant protein is expressed in E.coli JM109.