基础医学与临床 ›› 2009, Vol. 29 ›› Issue (6): 570-574.

• 研究论文 • 上一篇    下一篇

支持HCV 1b亚基因组复制的体外细胞模型的建立

陈慧毅 卢捷 李瑞 辛中帅 阴彬 龚燕华 彭小忠   

  1. 中国医学科学院基础医学研究所 北京协和医学院基础学院 医学分子生物学国家重点实验室 中国科学院上海巴斯德所 中国科学院上海巴斯德所 中国医学科学院基础医学研究所 北京协和医学院基础学院 医学分子生物学国家重点实验室 中国医学科学院基础医学研究所 北京协和医学院基础学院 医学分子生物学国家重点实验室 中国医学科学院 基础医学研究所 北京协和医学院 基础学院 医学分子生物学国家重点实验室
  • 收稿日期:2009-04-15 修回日期:2009-04-20 出版日期:2009-06-25 发布日期:2009-06-25
  • 通讯作者: 彭小忠

Establishment of a cell model supporting HCV1b subgenomic replication

Hui-yi CHEN, Jie LU, Rui LI, Zhong-shuai XIN, Bin YIN, Yan-hua GONG, Xiao-zhong PENG   

  1. Institute of Basic Medical Sciences, CAMS Institut Pasteur of Shanghai, CAS National Laboratory of Medicine Molecular Biology, IBMS, CAMS,School of Basic Medicine PUMC National Lab of Medical Molecular Biology, Institute of Basic Medical Science& School of Basic Medicine, CAMS & PUMC
  • Received:2009-04-15 Revised:2009-04-20 Online:2009-06-25 Published:2009-06-25
  • Contact: Xiao-zhong PENG

摘要: 目的 建立一个稳定支持中国人群HCV 1b亚基因组高效复制的体外细胞模型。方法 构建中国人群HCV 1b亚型亚基因组复制子质粒,通过体外转录的方法获得亚基因组复制子RNA,采用电穿孔技术把RNA转染到Huh-7.5.1细胞中,经过G418筛选含有HCV 1b亚基因组的细胞集落。RT-PCR检测细胞集落中的HCV亚基因组以及Western blot检测细胞集落中的HCV蛋白的表达。用干扰素(IFN)处理含有HCV亚基因组的细胞,观察细胞中HCV RNA的变化。结果 G418药物筛选得到了支持HCV1b亚基因组复制的细胞集落。RT-PCR 检测有HCV RNA NS4B的表达,Western blot检测有HCV的非结构蛋白NS5B的表达。IFN处理含有HCV复制子的细胞,HCV RNA水平明显降低。结论 成功建立了支持中国人群HCV 1b亚基因组高效复制的体外细胞模型,为进一步研究HCV致病机制、治疗药物筛选及疫苗方面的研究打下了基础。

Abstract: Objective To establish a cell model for supporting HCV 1b subgenomic replicon of Chinese population replication in vitro. Methods Plasmid containing HCV 1b subgenomic replicon of Chinese population was constructed. HCV subgenomic replicon RNA was obtained by in vitro transcription, and then delivered into Huh-7.5.1 cell by electroporation. After screening the cells in the medium containing G418, we detected the HCV RNA in the obtained cell colonies by RT-PCR and the HCV NS5B protein by western blot. The cell colonies which contain HCV RNA were treated by IFN in order to observe the diminished level of HCV RNA. Results In 4-6 weeks after transfection, visible colonies were stained by crystal violet. The HCV subgenomic replicon RNA NS4B was detected by RT-PCR and the HCV NS5B protein was detected by western blot. After treatment by IFN, HCV RNA diminished obviously. Conclusion We have successfully established a cell model for supporting HCV 1b subgenomic replicon of Chinese population replication in vitro. This cell model is a useful tool for the study on HCV pathogenesis, the screening of antiviral drugs and the development of vaccines.