丙型肝炎病毒全长基因嵌合萤光素酶重组质粒转染细胞系的建立与初步应用

Development of a cell culture system with consistently expression of whole hepatitis C virus gene and Renilla luciferase and its application

目的构建能产生较高效价重组病毒的HCV细胞感染模型，为HCV致病机制的研究和抗病毒药物的筛选提供一个有效的体外细胞培养系统。方法利用PCR技术在HCVNS5AC端插人海肾萤光素酶（Renilla Luciferase，Rluc）报告基因，并引入能提高HCV效价的突变，酶切鉴定重组基因序列构建成功后，转染入肝癌细胞系Huh7．5，用细胞免疫荧光、免疫印迹法和萤光素酶活性分析检测病毒复制和感染水平。用IFN“鉴定该系统用于抗丙肝药物筛选的可行性。结果在重组病毒JFH1—2440-RlucRNA转染的细胞中可检测到Rluc的活性，而阴性对照JFH1-GND和野生株JFH1-wtRNA转染的细胞未检测到Rluc的活性，经连续传代，JFH12440-RlucRNA转染细胞所产生的病毒效价可达到1．5×10^4FFU／mL，Rluc的活性随着IFNa浓度的增加而逐渐减低，呈现明显的剂量依赖特性。结论构建的重组HCV-JFH1-Rluc报告基因系统具有经济、快速、敏感等优点，为抗HCV药物的筛选提供了有利平台。

Objective To develop a cell culture system with consistent expression of whole hepatitis C virus （HCV） gene and Renilla luciferase gene and to facilitate the study on HCV pathogenesis and the screening of new antiviral drugs. Methods Renilla luciferase （RLuc） reporter gene and a mutation that could yield higher virus gene expression were introduced into the C terminus of non-structural protein 5A （NSSA） of the JFH1 viral genuine by using recombinant PCR. The viral RNA was transfected into Huh7.5 cells. Naive Huh7.5 cells were infected by the supernatant from the viral RNA transfected cells. HCV replication and infection were determined by virus titration, Renilla luciferase assay, immunofluorescence assay and western blotting. IFN a was used to evaluate the feasibility of this system for anti-HCV new drug screening. Results The viral RNA replicated efficiently in transfected cells. These cells could produce high titer of HCV-Rluc reporter virus and the virus titer reached to 1.5 X 10＇ FFU/ml at day 15 of posttransfection. The activity of Renilla luciferase was inhibited by IFN-a in a dose dependent manner in HuhT. 5 cells infected by HCV-Rluc reporter virus. Conclusion The recombinant HCV-JFH1 Rluc reporter gene system is sensitive and efficient. It can be a useful tool for high throughput screening of anti HCV drugs.