丙型肝炎病毒(HCV)截短型E2的原核表达和蛋白纯化  

Prokaryotic Expression and Purification of Truncated Hepatitis C Virus Envelope Glycoprotein E2

作　　者：姚敏[1] 方海亮[1] 尹文[1] 雷迎峰[1] 杨敬[1] 孙梦宁[1] 田江红[1] 吕欣[1] 

机构地区：[1]第四军医大学基础部微生物学教研室,西安710032

出　　处：《科学技术与工程》2009年第9期2296-2298,2303,共4页Science Technology and Engineering

基　　金：国家自然科学基金(30600021)资助

摘　　要：对丙型肝炎病毒(HCV)截短型包膜糖蛋白E2-661基因进行原核表达和纯化。利用PCR法扩增出661bp的截短型HCVE2区基因片段,并将测序正确的E2-661基因克隆入原核表达载体pET28a中,转化大肠杆菌BL21(DE3),经IPTG诱导表达后,通过SDS-PAGE和Western-blot对表达产物进行分析和鉴定。结果表明目的蛋白分子量约为35kDa,主要以包涵体形式大量存在。Western-blot结果显示目的蛋白与抗His标签单抗及HCV阳性血清均具有良好的反应原性。并通过镍离子亲和层析方法获得纯化的重组蛋白。以上结果为HCVE2功能的进一步研究奠定了基础。To construct a recombinant prokaryotic plasmid of truncated HCV E2 （E2-661） and observe its expression in E. coli, HCV E2-661 gene fragment was amplified by PCR. After verified by sequencing. It was inserted into the prokaryotic expression vector plasmid pET28a. The recombinant plasmid pET28a-E2-661 was then transformed into E. coil BL21 （DE3） to express the fusion protein induced with IPTG. The expression product was detected by SDS-PAGE and western-blot. The fusion protein mainly aggregated into the inclusion body. The results of western-blot showed that the expressed protein showed good reactivity to anti-His tag antibody and HCV positive serum. The protein E2-661 fused with His tag was purified by Ni-NTA resin column. The expression and purification of HCV E2-661 protein will be useful for further research on the function of envelope glycoprotein E2.

关 键 词：丙型肝炎病毒 包膜糖蛋白E2 原核表达 

分 类 号：R512.63[医药卫生—内科学]