增强型绿色荧光蛋白标记的重组丙型肝炎病毒体外细胞培养系统的建立

Development of a cell culture system based on recombinant hepatitis C virus expressing enhanced green fluorescent protein

目的构建一个易检测且灵敏度高的丙型肝炎病毒（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体系。方法利用重组PCR技术在HCV的非结构蛋白NSSA的c端引入优势突变点V2440L，然后在其RsrU酶切位点插入增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）报告基因，基因测序及酶切鉴定重组基因序列构建成功后，体外转录获得RNA，然后转染入肝癌细胞系Huh7．5，用Westernblot检测EGFP与NSSA融合蛋白，用细胞免疫荧光（IFA）检测病毒复制水平和EGFP表达情况。用RT—PCR检测转染细胞培养液不同时间点的HCVRNA水平。用IFN-d鉴定该系统用于抗丙型肝炎药物筛选的可行性。结果在重组病毒JFHl-2440．EGFPRNA转染的细胞内可检测到EGFP的表达，EGFP表达水平与病毒复制水平一致。转染细胞的培养上清液能感染新的Huh7．5细胞，IFA结果显示转染后第9天培养上清液的病毒滴度为104FFU／ml，提示JFHl-2440-EGFPRNA转染的细胞能释放有传染性的HCV病毒颗粒。RT．PCR检测结果显示转染细胞上清液中的HCVRNA在转染72h后可达到3．06x10^5拷贝／ml，第9天可达到7．96^10。拷贝／ml。EGFP的表达呈干扰素浓度依赖性。结论构建的重组病毒HCVJFHI-2440．EGFP体外细胞培养系统具有经济、快速、敏感等优点，为HCV致病机制的研究和抗病毒药物的筛选提供了一个有效的工具。

Objective To develop a time saving and sensitive cell culture system based on hepatitis C virus chimera expressing enhanced green fluorescent protein（EGFP） and to facilitate the study on HCV pathogenesis and screening of anti-HCV drugs. Methods Enhanced green fluorescent protein reporter gene and a mutation V2440L that can yield higher virus titers were introduced into the C-terminus of non-structural protein 5A （NSSA） of the JFH1 viral genome by using recombinant PCR. The viral RNA was transfected into Huh7. 5 cells. Viral RNA in supematant of HCV RNA-transfected cells was determined after transfeetion by RT-PCR. HCV replication and infection were determined by immunofluorescence assay. IFN-awas used to evaluate the feasibility of this system for anti-HCV drugs screening. Results The viral RNA replicated efficiently in transfected cells. These cells can produce HCV-EGFP reporter virus. Viral RNA levels in supernatant were 3.06x10^5 copies/ml and 7. 96x10^6 copies/ml at 72 h and 9 d after transfection,respectively. The virus titer reached to 10^4 FFU/ml 9 d after transfection. The expression of EGFP was inhibited by IFN-a in a dose dependent manner in Huh7.5 cells infected by HCV-EGFP reporter virus. Conclusion The recombinant HCV JFH1-EGFP reporter gene system is a time saving, cost effective and sensitive method for studying viral replication cycles and screening of anti-HCV drugs.