报告基因标记的HCV全基因组复制细胞模型的建立  

Establishment of reporter gene labeled HCV full-length genome replication cell model

作　　者：高博[1] 贾帅争[1] 彭剑淳[1] 王怡[1] 樊炜[1] 李银太[1] 詹林盛[1] 许金波[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《中华微生物学和免疫学杂志》2011年第6期523-527,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金，北京市自然科学基金

摘　　要：目的建立稳定的报告基因标记的HCV全基因组复制细胞模型，对细胞内病毒蛋白和核酸的表达水平进行定量。方法把neo抗性基因插入到Luc-JCl中，构建Luc-JC。将体外转录的Luc-JCRNA转染Huh7细胞，经筛选后获得的G418抗性克隆，通过荧光素酶活性检测、Westernblot和HCVNS3／4A对eYFP—MAVS的切割试验进行鉴定。用IFN-α处理筛选到的细胞，观察细胞中荧光素酶活性、HCV相关蛋白和RNA的变化，对复制细胞在抗病毒药物评价方面的应用进行初步验证。结果G418加压筛选得到了多个含HCV全基因组的细胞克隆。在细胞克隆中，荧光素酶活性检测观察到萤火虫荧光素酶；Westernblot检测到HCVNS3和NS5A蛋白的表达；由于NS3／4A的切割，eYFP—MAVS的定位由线粒体转移到细胞质。IFN-α处理阳性克隆细胞，荧光素酶活性、HCV蛋白表达和RNA水平明显降低。结论成功建立稳定的报告基因标记的HCV全基因组复制细胞模型，报告基因能用来指示细胞内HCV蛋白和RNA的表达水平，为进一步开展HCV致病机制研究和治疗药物筛选奠定了基础。Objective To establish a stable HCV full-length genome replication cell model which is labeled with reporter gene and easyly to quantify intracellular HCV proteins and RNA level . Methods neo gene was inserted into Luc-JC1 to make Luc-JC construct. Luc-JC RNA was obtained by in vitro tran- scription and then delivered into Huh7 cells by transfection. Gdl8-resistant clones of Huh7 cells were obtained by selection. Clones of HCV full-length genome replication cell were confirmed by luciferase activity assay, Western blot and cleaveage of eYFP-MAVS by HCV NS3/4A protease. Then, HCV replication cell colonies were treated by different dose IFN-α in order to observe the change of luciferase activity, HCV pro- tein and RNA level. Results At 3-,4 weeks post-transfection, visible colonies were selected and stained by crystal violet. Luciferase activity and HCV NS3, NS5A protein were detected by luciferase activity assay and Western blot, respectively. Subcellular localization of eYFP-MAVS transferred from mitochondria to cyto- plasms by cleavage of NS3/4A protease in cell colonies. Luciferase activity, HCV protein and RNA dimin- ished obviously after IFN-ct treatment. Conclusion A stable HCV full-length genome replication cell model labeled by reporter gene was successfully established and reporter activity can be used to indicate level of HCV proteins and RNA in cells. This cell model is a useful tool for the study on HCV pathogenesis and the screening of antiviral drugs.

关 键 词：丙型肝炎病毒 萤火虫荧光素酶 细胞模型 复制 

分 类 号：R512.63[医药卫生—内科学]