SARS冠状病毒受体结合域在大肠杆菌中的融合表达和鉴定  

Fusion Expression and Identification of Receptor Binding Domain of the SARS-CoV Spike Protein in the Escherichia coli

作　　者：高俊[1] 肖洪广[1] 林勇平[1] 

机构地区：[1]广州医学院第一附属医院检验科,广州510120

出　　处：《热带医学杂志》2007年第1期11-14,共4页Journal of Tropical Medicine

基　　金：广东省自然科学基金面上项目(No.06022094);广州市科技局应用基础项目(No.2004J1-C0211);广州市教委科研项目(No.1040;No.1047)。

摘　　要：目的为了研究SARS冠状病毒感染与免疫和跨种属传播机制,从来源于人和果子狸的SARS冠状病毒Spike蛋白基因中,克隆病毒受体结合域(receptor binding domain,RBD)基因片段,在大肠杆菌中进行表达。方法用PCR方法扩增RBD基因,先经T-A连接,转化大肠杆菌DH5α,挑选阳性克隆测序鉴定,双酶切后进一步亚克隆至质粒pGEX-6p-3,构建原核表达重组质粒,通过IPTG诱导在大肠杆菌BL21中表达,并用SDS-PAGE和Western-blotting方法检测表达情况。结果扩增了RBD的编码基因并成功构建了其原核表达载体pGEX-6p-3-hsRBD和pGEX-6p-3-csRBD,RBD能够在大肠杆菌中获得良好的表达,表达的融合蛋白相对分子质量约为47000。结论本工作利用基因工程技术表达了两物种来源的SARS冠状病毒的RBD,两者具有高度的同源性,因此我们可通过配基受体结合实验,为进一步研究SARS冠状病毒感染与免疫及跨种属传播机制奠定了基础。Objective To expressed receptor binding domain （RBD） of the SARS-CoV Spike protein in Escherichia coli. Methods The receptor binding domain region of the SARS-CoV Spike protein gene was amplified by PCR.PCR products were ligated to T-vector and transformed into Escherichia coli DH Set. Positive clones were sequenced. RBD fragments were subeloned into plasmid pGEX-6p-3 and expressed in Escherichia coli BL21. The recombinant protein was detected and identified by the SDS-PAGE and Western-blottlng. Results The RBD encode sequence was successfully amplified and its Prokaryotic Expression Vector was constructed. The RBD was expressed as fusion protein in Escherichia coli, with a molecular weight of 47 kD. Conclusion The recombinant receptor binding domain of the SARS-CoV Spike protein was successfully expressed as a fusion protein in E. coll. This recombinant protein can be used for the further study on the molecular mechanism of SARS-CoV infection, immunity and interspecies tranamission.

关 键 词：SARS-COV 受体结合域 融合蛋白表达 

分 类 号：R373[医药卫生—病原生物学]