丙肝病毒全基因组克隆转染细胞体系的初步研究  

Study of Cell Culture System Transfected with Full-length cDNA Clone of Hepatitis C Virus

作　　者：郭佳[1] 姚相杰[1] 郑从义[1] 方呈祥[1] 屈三甫[1] 李信墙 李卫云 

机构地区：[1]武汉大学生命科学学院,湖北武汉430072 [2]广州中海潮生物技术有限公司,广东广州510010

出　　处：《中国病毒学》2004年第4期320-324,共5页Virologica Sinica

基　　金：国家高技术研究发展计划(863)(2002AA216031)

摘　　要：采用一种含有HCV全基因组和噬菌体T7启动子和终止子序列的载体(pHCV)转染Vero-E6细胞,随后感染高效表达T7RNA聚合酶的重组痘病毒(vTF7-3),通过vTF7-3的辅助作用,使Vero-E6细胞高效增殖HCV病毒体,建立了一种新的HCV体外细胞培养体系。RT-PCR、荧光定量PCR检测转染细胞裂解液中HCV滴度的结果显示:pHCV转染细胞内HCV基因拷贝达107-108/mL,同时有HCV正链RNA合成;免疫印迹显示该培养体系中有HCV结构蛋白、非结构蛋白的表达;pHCV转染细胞经透射电镜观察,可见清晰的HCV病毒体,直径在40-50nm。这一新体系的初步建立,为研究HCV的复制机制、制备HCV疫苗和研发抗病毒药物奠定了基础。A Vero-E6 culture system was developed to assay Hepatitis C virus (HCV) replication by plasmid (pHCV) transfection,which contains a T7 promoter at the 5’end, full-length cDNA of the HCV genome and a T7 terminator. To facilitate high-level transcription of HCV RNA, Vero-E6 cells were transfected with pHCV and then infected with recombinant vaccinia virus (vTF7-3) containing the T7 RNA polymerase gene. This transfection-based cell culture system produced high levels of HCV genome (107-108 copies/mL) detectable by real-time quantitative PCR and the production of HCV RNA transcripts was confir med by RT-PCR. Western blot analysis revealed that HCV structural and non-structural proteins were correctly processed. The transfected Vero-E6 cells assembled 40-50nm virus-like particles were analysed with transmission electron microscope. This model can be utilized for studying mechanisms of HCV replication, preparation of HCV vaccine and to test potential antiviral drugs.

关 键 词：丙型肝炎病毒 HCV 全基因组 克隆 转染细胞体系 重组痘病毒 

分 类 号：R346[医药卫生—基础医学]