支持HCV 1b亚基因组复制的细胞模型的建立

Establishment of a cell model supporting HCV 1b subgenomic replication

目的建立一个稳定支持中国人群HCV1b亚基因组高效复制的体外细胞模型。方法构建中国人群HCV1b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HCV1b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HCVR NANS4B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HCV1b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。

Objective To establish a cell model for supporting HCV lb subgenomic replicon from Chinese population. Methods Plasmid containing HCV 1 b subgenomic replicon was constructed. HCV snbgenomic replicon RNA was obtained by 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 NSSB 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 a period of 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 1 b 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.