SARS冠状病毒S蛋白部分序列1的克隆与表达

目的　构建SARS冠状病毒S蛋白部分序列 1(S1)的原核重组表达质粒 ,并分析其在大肠杆菌中的表达状况。方法　采用逆转录 -多聚酶链反应 (RT -PCR)技术从SARS冠状病毒RNA中扩增出编码S1蛋白的基因片段 ,并克隆至pMD18-T载体上 ;菌落PCR鉴定阳性克隆并测序列分析 ;将阳性克隆的插入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 ;将阳性菌株经IPTG诱导 ,SDS -PAGE和免疫印迹分析截短型S蛋白的表达。结果　RTPCR扩增出S1蛋白的特异片段 ,其阳性克隆的序列存在 2处碱基突变第 36位G变为A ;第 333位由C变为T) ,其中第 36位为有义突变 ,第 333位为同义突变 ;阳性克隆中截短型S1蛋白的基因片段被亚克隆到表达载体pGEX - 4T - 2而构建成重组表达质粒 ,并在JM 10 9中表达了S1蛋白的融合蛋白 ,表达的蛋白能与GST免疫血清和SARS病人血清反应。结论　成功构建了SARS冠状病毒S1蛋白的重组表达质粒 ,该质粒在大肠杆菌中表达了截短型S蛋白的融合蛋白。

Cloning and expression of SARS Coronavirus spike protein fragment 1

Aim To construct an expression plasmind encoding SARS Coronavirus spike protein fragment 1 and analyze its expression in E.coli JM109.Methods The gene fragment encoding spike protein fragment 1 was amplified by RT-PCR from SARS Coronavirus RNA,and was cloned into pMD18-T vector,positive clone was identified by clony PCR and sequencing with ABI PRISM TM 377XL DNA Sequencer;The gene fragement encoding spike protein fragment 1 in positive clone was digested with BamHⅠ and SalⅠ and was subcloned into pGEX-4T-2 which was also digested with BamHⅠ and SalⅠ;the recombinant plasmid was transformed into E.coli JM109 and the positive recombinant plasmid was induced with IPTG to express target protein and was characterized by SDS-PAGE and Western-blot.Results The gene fragment encoding spike protein fragement 1 was amplified by RT-PCR from SARS Coronavirus RNA;there were two mutatios in the insert of TA clone,one mutation in position 36 was non-synonymous,another mutation in position 333 was synonymous;the insert of S1 gene in positive clone was subcloned into pGEX-4T-2 correctly;recombinant fusion protein was expressed in the positive recombinant clone when induced with IPTG and the fused protein reacted with goat polyclonal antibodies to Schistosoma japonicum 26kDa GST and sera of SARS patients.Conclusion The recombinant plasmid expressing truncated spike protein has been successfully constructed ant the recomlbinant protein was expressed in E.coli JM109.