基因1b型不同准种株HCV核心蛋白原核表达质粒pTrc-CKS构建及诱导表达  

Construction and expression of plasmids containing the truncated core protein of different quasispecies genotype 1b hepatitis C virus

作　　者：汪明珊[1] 颜学兵[1] 周林福[2] 侯晓丽[2] 李杨霞[2] 温晓芳[2] 姜云水[2] 陈智[2] 

机构地区：[1]徐州医学院附属医院感染病科,徐州市221002 [2]浙江大学医学院附属第一医院传染病研究所

出　　处：《中华实验和临床感染病杂志（电子版）》2009年第2期5-8,共4页Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition)

基　　金：江苏省自然科学基金(BK2007031);江苏省高校自然科学基金(08KJB320017)

摘　　要：目的为研究与不同准种HCV核心蛋白(Core)相互作用的细胞蛋白质组,构建并表达基因1b型的不同准种Core的原核表达质粒pTrc-CKS。方法用聚合酶链反应(PCR)扩增并获得等长度片段的基因1b型的不同准种株丙型肝炎病毒(HCV)Core基因,氨基酸(aa)长度分别为癌中心株(T):1～172 aa、癌旁株(NT):1～172 aa及C191(HCV-J6):1～172 aa。扩增产物用BamHⅠ及EcoRⅠ双酶切后插入到原核表达质粒pTrc-CKS中。阳性克隆转化到大肠埃希菌Top 10F’中,异丙基-β-D-S-代半乳糖苷(IPTG)诱导表达获得融合蛋白CKS-Core-His,经Ni2+10F’柱纯化并经Westernblot验证。结果PCR及双酶切验证表明3个基因为1b型的不同准种Core基因成功构建到原核质粒pTrc-CKS中,体外诱导并获得了相应的融合蛋白。结论构建不同准种Core基因的原核表达质粒获得成功,体外诱导表达并纯化获得了CKS-Core-His融合蛋白,为后续研究与Core相互作用的细胞蛋白质组奠定了基础。Objective To study the proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein （ HCV Core）, plasmids containing the truncated core protein of different quasispecies of genotype lb hepatitis C virus were constructed and induced to express. Methods The gene sequences of different quasispecies genotype lb Core were amplified from plasmids containing core sequences derives from tumor （T） and non-tumor （NT） tissues from one patient infected with HCV. Amimo acid （aa） lengths of HCV T were 1-172 aa, NT of 1-172 aa and HCV C191 （ HCV J-6） of 1-172 aa. PCR products were cleaved with restriction enzymes Bam H I and Eco R I and cloned into pTrc-CKS. Positive clones were transfected into E. coli Top 10F＇ and CKS-Core-His fusion proteins were expressed with isopropylthio-β-D- galactoside （IPTG） induction, then purified and verified by Western blot. Results Three different HCV quasispecies were correctly inserted into pTrc-CKS vector ：respectively and the fusion proteins were expressed well in vitro. Conclusions Successful construction of different CKS-Core-His expression plasmids lays a basis for future study of the proteomics interaction with the Core.

关 键 词：丙型肝炎病毒 核心蛋白 pTrc-CKS 质粒构建 

分 类 号：R512.6[医药卫生—内科学]