丙型肝炎病毒非结构蛋白NS5A上调硫氧还蛋白还原酶1基因表达的研究  

The study of up-regulating effect of hepatitis C virus NS5A protein on the transcription activity of thioredoxin reductase 1 gene promoter of hepatitis B virus X protein

作　　者：纪冬[1] 成军[1] 郭江[1] 杨瑗[1] 董菁[1] 王建军[1] 刘妍[1] 

机构地区：[1]解放军第302医院传染病研究所基因治疗研究中心,北京100039

出　　处：《胃肠病学和肝病学杂志》2005年第4期341-345,共5页Chinese Journal of Gastroenterology and Hepatology

基　　金：国家自然科学基金攻关项目;No.C03011402;No.C30070689;军队"九;五"科技攻关项目;No.98D063;军队回国留学人员启动基金项目;No.98H038;军队"十;五"科技攻关青年基金项目;No.01Q138;军队"十;五"科技攻关项目;No.01MB135

摘　　要：目的探讨丙型肝炎病毒(HCV)非结构蛋白NS5A对硫氧还蛋白还原酶1(TXNRD1)启动子转录的激活作用。方法以我室构建的丙型肝炎病毒非结构蛋白NS5A反式调节基因的cDNA文库抑制性消减杂交(SSH)筛选结果及基因表达谱芯片结果为基础,利用生物信息学技术确定TXNRD1的启动子区域(TXNRD1p),聚合酶链反应(PCR)扩增TXNRD1p,克隆至真核报告载体pCAT3_Basic中,构建pCAT3_TXNRD1p报告载体;以该质粒转染肝癌细胞系HepG2,用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性;并与pcDNA3.1(-)_NS5A共转染HepG2细胞系,用ELISA法检测CAT的表达活性。结果pCAT3_TXNRD1p和pcDNA3.1(-)_NS5A瞬时转染的HepG2细胞的CAT表达活性是pCAT3_Basic空载体的9.6倍,pCAT3_TXNRD1p的2.1倍。本实验进一步验证了我室利用SSH技术及基因表达谱技术发现的HCVNS5A蛋白反式激活TXNRDl基因转录作用的结果。结论我室克隆的TXNRD1启动子有顺式激活下游基因的活性;HCV的NS5A蛋白具有对TXNRD1基因启动子的反式激活作用。Objective To investigate transactivating activity of HCV NS5A protein on thioredoxin reductase 1 （TXNRD1 ） gene promoter. Methods The TXNRDI promoter sequence was identified in GenBank by bioinformatics and amplified from HepG2 genome by polymerase chain reaction （PCR）. The amplified product was cloned into pCAT3-Basic reporter vector,named pCAT3-TXNRDIp. The HepG2 cell line were transfected by pCAT3-TXNRD1p, and co-tranfected by pCAT3-TXNRD1p and pcDNA3.1（ - ）-NS5A plasmids, respectively. The choloraphenical acetyltransferase （CAT） activity was detected by enzyme linked immunosorbent assay （ELISA）. Results pCAT3-TXNRD1p had higher activity of CAT than pCAT3-Basicdid.The expression of CAT in co-transfection of pCAT3-TXNRD1p and pcDNA3.1 （ - ） - NS5A was 2.2 times as higher as that of pCAT3-TXNRD1p plasmid. Conclusion The TXNRD1 gene promoter identified in this study has transcription activity and HCV NS5A protein could transactivate the expression of TXNRD1 gene.

关 键 词：丙型肝炎病毒 非结构蛋白 NS5A 硫氧还蛋白还原酶1 基因表达 

分 类 号：R373.21[医药卫生—病原生物学]