双荧光素酶标记丙型肝炎病毒亚基因组复制子细胞模型的建立

Establishment of a hepatitis C virus subgenomic replicons cell model labeled with dual luciferase reporter

目的构建双荧光素酶标记丙型肝炎病毒(HCV)亚基因组复制子细胞模型,为HCV研究提供有效工具。方法通过单克隆筛选及慢病毒感染构建双荧光素酶标记的HCV亚基因组复制子细胞模型(HCV Fluc-UbiGluc)。利用荧光素酶检测、Western印迹检测鉴定细胞模型,利用IFN-α对HCV抗病毒效果评价验证细胞模型的有效性。结果筛选所得HCV亚基因组细胞克隆检测到荧光素酶活性,Western印迹检测到HCV NS5A蛋白表达。NS3/4A对黄色荧光蛋白(YFP)标记的MAVS(YFP-MAVS)切割作用使亚细胞定位发生转移。IFN-α处理后荧光素酶活性、HCV蛋白表达水平明显降低。结论该模型采用萤火虫荧光素酶报告基因指示HCV亚基因组在细胞内的复制水平,Gaussia荧光素酶指示细胞的生长数量,能准确反映候选药物对HCV复制水平的影响,并有效排除药物的细胞毒性干扰,具有操作简单、快速等优点,在抗病毒药物高通量筛选、HCV致病机制等研究方面具有一定的价值。

Objective To establish an HCV subgenomic replicons cell model that is labeled with reporter gene and to provide a useful tool for HCV research.Methods An HCV subgenomic replicons cell model [HCV-firefly luciferase（Fluc）-Ubi-Gaussia luciferase（Gluc） ]containing dual luciferase reporters was established by monoclonal selection and lentivirus infections.The reporter and HCV protein expression were confirmed by luciferase activity assay and Western blotting,respectively.The efficacy of the cell model was validated by IFN-α which was shown to inhibit HCV replication.Results Luciferase activity and HCV NS5A protein were detected by luciferase activity assay and Western blotting,respectively.Subcellular localization of yellow fluorescence protein（YFP）-MAVS was transferred from mitochondria to nucleis by cleavage of NS3 /4A protease in HCV replicons cells.HCV proteins and luciferase activity were diminished obviously after IFN-α treatment.Conclusion In this cell model,the HCV RNA and host cells are labeled with firefly luciferase and Gaussia luciferase,respectively.The mold can indicate the level of HCV replication and eliminate the cytotoxicity antivirus drugs induced effectively.It will be a valuable tool for studying HCV pathogenesis and screening antiviral drugs.