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。 Vero-E6 cells were transfected with a vector (pHCV) containing the HCV whole genome and bacteriophage T7 promoter and terminator sequences followed by infection with a recombinant poxvirus (vTF7-3) highly expressing T7 RNA polymerase through vTF7-3 Auxiliary role in Vero-E6 cells efficiently proliferate HCV virion, and establish a new HCV in vitro cell culture system. The results of RT-PCR and real-time PCR assay of HCV titers in transfected cell lysates showed that the copies of HCV gene in pHCV transfected cells reached 107-108 / mL with HCV positive strand RNA synthesis. Western blotting showed that the culture system HCV protein in the structure, non-structural protein expression; pHCV transfected cells were observed by transmission electron microscopy, showing a clear HCV virion diameter 40-50nm. The initial establishment of this new system laid the foundation for the study of HCV replication mechanism, preparation of HCV vaccine and development of antiviral drugs.