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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 construct dual luciferase-labeled hepatitis C virus (HCV) subgenomic replicon cell model and provide an effective tool for HCV research. Methods Dual luciferase-labeled HCV subgenomic replicon cell model (HCV Fluc-UbiGluc) was constructed by monoclonal screening and lentivirus infection. The cell model was identified by luciferase assay and Western blotting, and the effectiveness of HCV anti-viral effect was verified by IFN-α. Results The sub-genomic HCV subgenomic cells were screened for luciferase activity and HCV NS5A protein was detected by Western blotting. NS3 / 4A yellow fluorescent protein (YFP) labeled MAVS (YFP-MAVS) cleavage subcellular localization of the transfer. After treatment with IFN-α, luciferase activity and HCV protein expression were significantly decreased. Conclusion This model uses firefly luciferase reporter gene to indicate the level of HCV subgenomic replication in cells. Gaussia luciferase indicates the number of cells that can accurately reflect the effect of candidate drugs on the level of HCV replication, and effectively exclude the cytotoxicity of the drug Interference, has the advantages of simple operation and fastness, and has certain value in the research of high-throughput screening of antiviral drugs and the pathogenesis of HCV.