HCV2a(JFH1)NS5A蛋白在原核和真核细胞中的表达、鉴定及分析  

Expression, Identification and Characterization of HCV2a(JFH1) NS5A Protein in E.coli and HEK 293T Cells

作　　者：王永智[1] 任浩[1] 丁惠[1] 赵平[1] 薛利军[1] 潘卫[1] 戚中田[1] 

机构地区：[1]第二军医大学微生物学教研室全军医学微生物学重点实验室,上海200433

出　　处：《热带医学杂志》2007年第11期1039-1043,F0004,共6页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30600529)。

摘　　要：目的在原核与真核细胞中表达丙型肝炎病毒(hepatitis C virus,HCV)2a型JFH1(japanese fulminanthepatitis 1)株NS5A蛋白,分析JFH1NS5A蛋白与其它基因型NS5A蛋白间的差异,为进一步研究NS5A蛋白在HCV病毒复制中的作用奠定基础。方法PCR特异扩增JFH1NS5A基因,克隆入pET-32a和pEGFP-N1载体中,构建JFH1NS5A的原核和真核重组表达质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。将pET-JFH1NS5A转化大肠杆菌BL21(DE3),pEGFP-JFH1NS5A转染HEK293T细胞,在原核与真核系统中进行表达,并以SDS-PAGE、荧光显微术和Westernblot方法检测。利用PubMedBLAST软件对JFH1与HCV1a、1b、2b和2c型NS5A蛋白间的同源性进行了分析。结果酶切鉴定及测序结果表明成功构建了重组质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。SDS-PAGE电泳检测到融合蛋白在原核细胞中的表达,荧光显微镜观察和Westernblot检测到了GFP-JFH1NS5A融合蛋白在HEK293T细胞中表达。BLAST分析显示,JFH1和H77、HC-J4、HCV-J8和BEBE1病毒株NS5A蛋白的同源性分别为57%、60%、68%和74%。结论JFH1NS5A蛋白在原核与真核细胞中表达成功,为研究HCVNS5A在JFH1株病毒高效复制中的作用提供了材料。Objective To express HCV genotype 2a JFH1 NS5A protein both in prokaryotic and eukaryotic cells, analyze the amino acid sequence homology between the NS5A proteins of different genotypes,and produce recombinant JFH1 NS5A protein for the study of the role of JFH1 NS5A in the replication of HCV. Methods The full coding sequence of JFH1 NSSA was amplified by PCR and cloned into pET32a and pEGFP-N1 vector to construct recombinant expression plasmids pET-JFH1 NS5A and pEGFP-JFH1 NS5A,respectively. pET-JFH1 NS5A was transformed into BL21 （DE3） and induced by IPTG. pEGFP-JFH1 NS5A was transfected into HEK 293T cells using LipofectamineTM 2000. The expression of target protein was identified and verified by SDS-PAGE, fluorescence microscopy and immunoblotting. The amino acid sequence homology between JFHI NS5A with NS5A of the other genotypes was analyzed by BLAST software. Results The recombinant plasmids were constructed successfully and identified by restriction endonuclease analysis and DNA sequencing. SDS-PAGE analysis of the recombinant fusion protein produced in E.coli showed a band between 60 to 116 kDa. Green fluorescence signal was observed in the 293T cells expressing the GFP-JFH1 NS5A fusion protein under fluorescence microscopy. The expression was also verified by Western blot. The BLAST results revealed that the JFH1 NS5A protein showed 57%, 60%, 68% and 74% homology with NS5A proteins from HCVla, HCVlb, HCV2b and HCV2c, respectively. Conclusion JFH1 NS5A protein was expressed successfully in both prokaryotic and eukaryotic cells, which can also provide material for studying the role of NS5A in the replication of HCV.

关 键 词：丙型肝炎病毒 JFH1株 NS5A蛋白 表达 鉴定 

分 类 号：R373.21[医药卫生—病原生物学] Q939.93[医药卫生—基础医学]