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

GUO Jia; YAO Xiang-jie; ZHENG Cong-yi; FANG Cheng-xiang; QU San-fu; LI Xin-qiang; LI Wei-yun

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August 2004

GUO Jia, YAO Xiang-jie, ZHENG Cong-yi, FANG Cheng-xiang, QU San-fu, LI Xin-qiang and LI Wei-yun. Study of Cell Culture System Transfected with Full-length cDNA Clone of Hepatitis C Virus*[J]. Virologica Sinica, 2004, 19(4): 320-324.

Citation: GUO Jia, YAO Xiang-jie, ZHENG Cong-yi, FANG Cheng-xiang, QU San-fu, LI Xin-qiang, LI Wei-yun. Study of Cell Culture System Transfected with Full-length cDNA Clone of Hepatitis C Virus* .VIROLOGICA SINICA, 2004, 19(4) : 320-324.

丙肝病毒全基因组克隆转染细胞体系的初步研究*

姚相杰 ¹ ,
郑从义 ¹ ,
方呈祥 ^1*,, ,
屈三甫 ¹ ,
李信墙 ¹ ,
李卫云 ,

1.
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

通讯作者： 方呈祥, ;

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T7 RNA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。
- HCV全基因组
- , 克隆
- , 转染细胞体系
- , 重组痘病毒

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

1.
1. College of Life Scinces, Wuhan University, Wuhan 430072, China
2.
Chinawave Biotechnology Co. Ltd, Guangzhou 510010, China

Corresponding author: ZHENG Cong-yi,

Abstract

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.
- Full genome of HCV
- , Clone
- , Transfected cell culture system
- , Recombinant vaccinia virus

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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

Corresponding author: ZHENG Cong-yi,

1. 1. College of Life Scinces, Wuhan University, Wuhan 430072, China
2. Chinawave Biotechnology Co. Ltd, Guangzhou 510010, China

Abstract: 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.