HCV包膜蛋白E2的克隆、表达与检测  

Cloning,expression and detection of Hepatitis C virus envelope protein E2

作　　者：何素梅[1] 周见远[1] 肖建华[1] 聂东宋 

机构地区：[1]南华大学病原生物学研究所,衡阳421001 [2]湖南景达制药有限公司,岳阳414000

出　　处：《微生物学免疫学进展》2011年第1期20-23,共4页Progress In Microbiology and Immunology

摘　　要：构建丙型肝炎HCV包膜蛋白糖蛋白的E2基因原核表达载体,获得大量重组HCVE2蛋白,进行E2蛋白的抗原性及潜在保护作用研究。通过RT-PCR从HCVRNA阳性血清标本中扩增出975bp(383～708)E2基因片段,PCR产物经EcoR I和Sall I双酶切后连接到经同样酶切的PET-41a原核表达载体上,转化到大肠杆菌BL21(DE3)菌株,经Amp筛选,得到阳性重组质粒PET41a-HCVE2菌株,并以IPTG诱导蛋白表达,SDS-PAGE鉴定,表达产物经固定化金属配体亲和层析纯化,用ELLSA方法检测生物学活性。结果表明,构建的HCVE2包膜蛋白基因片段原核表达质粒所表达产物主要以包涵体形式存在,表达的融合蛋白与HCV阳性血清具有较好的反应原性。以HCVE2融合蛋白检测患者阳性血清具有良好的抗原性,有望能提高HCV抗体检测试剂盒的检出率。To study the antigenicity and the potential protection of recombinant HCV E2 protein and obtain large amount oftarget product in prokaryotic expression systerms.The HCV E2 gene was amplified by RT-PCR and the product was diges-ted by EcoR I and Sall I.The fragment was inserted into an Escherichia coli expression vector PET41a,and then used totransform E.coli BL21(DE3).The target protein was expressed in BL21(DE3) and induced by IPTG.The expressed pro-tein was indentified by SDS-PAGE and then purified by immobilized metal ion affinity chromatography(IMAC),and thebiological activity of product was detected by ELLSA.The results showed that the prokaryotic expression plasmid of HCVE2 is constructed successfully.The expression product is present in the from of inclusion bodies.The expression productsshow the good reactivity to the positive HCV serum.This result indicate that recombinant protein has high antigenicity,itmay be a useful agent for detecting the anti-HCV antibody.

关 键 词：丙型肝炎病毒 包膜蛋白 原核表达 载体构建 抗原性 

分 类 号：R373.21[医药卫生—病原生物学]