表达丙肝病毒核心蛋白基因的人肝细胞模型的建立  

Establishment of human hepatocyte model expressing HCV-C gene

作　　者：单于[1] 陈系古[1] 黄冰[1] 胡安斌[2] 郭中敏[1] 赵宁[1] 

机构地区：[1]中山大学实验动物中心,广州510080 [2]华中科技大学同济医学院附属协和医院普外科

出　　处：《中华实验外科杂志》2003年第11期1043-1045,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目 (1 0 30 55)

摘　　要：目的　构建表达丙型肝炎病毒核心蛋白 (HCV C)基因的人肝细胞模型。方法　用聚合酶链反应 (PCR)方法从 pHCV MA质粒中钩出HCV C基因 ,酶切纯化后克隆于质粒 pTRE中 ,建立重组质粒 pTRE C。pTRE C经酶切、纯化后目的片段与质粒PcDNA3连接 ,通过限制性内切酶酶切分析及PCR鉴定筛选出正确重组质粒PcDNA3 tRe C ,由脂质体介导转染Chang liver人肝细胞 ,建立表达HCV C基因的人肝细胞模型 ,PCR方法鉴定Chang liver人肝细胞内HCV C基因 ,反转录聚合酶链反应 (RT PCR)方法检测HCV C基因在Chang liver人肝细胞内的表达 ,抗生物素蛋白 生物素 过氧化物酶复合体法 (ABC法 )方法分析HCV核心蛋白在人肝细胞内的表达及定位。结果　构建了表达HCV C基因的重组真核表达载体 ,并在Chang liver人肝细胞中表达出HCV C蛋白。结论　成功地建立了表达HCV C基因的人肝细胞模型 ,该模型为进一步研究HCV C的生物学特性及HCV感染所致肝硬化、肝癌的致病机制提供基础材料。Objective To construct the human hepatocyte model expressing HCV-C gene.Methods The HCV-C gene was amplified by PCR from the plasmid pHCV-MA,then it was cut with restriction enzymes and cloned into the plasmid pTRE to form recombinant plasmid pTRE-C.The plasmid pTRE-C was cut with restriction enzymes,and the aim fragment was ligated to the plasmid PcDNA3.The correct recombinant plasmid PcDNA3-tRe-C was screened out with analysis of restriction enzymes and PCR,then transferred into cultured human hepatocytes of Chang-liver by lipofectin,and established the cell model which expressed HCV-C gene.The HCV-C gene in Chang-liver hepatocytes was identified by PCR.The expression of HCV-C gene in Chang-liver hepatocytes was detected by RT-PCR.The expression and location of HCV core protein were identified by immunocytochemistry.Results The eukaryote expressing recombinant plasmid vector was constructed.Furthermore,the recombinant plasmid expressed the expected HCV-C protein in the human hepatocytes.Conclusion The human hepatocyte model expressing HCV-C gene was successfully established.This model was very useful for the future research of HCV-C biological characters and of mechanism of hepatocirrhosis and cancinogenesis resulting from HCV infection.

关 键 词：人肝细胞模型 丙型肝炎病毒核心蛋白基因 聚合酶链反应 丙型肝炎病毒感染 C蛋白 

分 类 号：R-331[医药卫生]