【摘要】 目的建立HCV体外复制细胞模型。方法用含有生长HCV基因的p90/HCVFL-longpU质粒,转化Sure细胞,36℃摇菌18h,提取质粒,以线性化DNA为模板,用T7RNA体外转录系统作体外转录,转录产物RNA作RT-PCR、普通琼脂糖凝胶电泳鉴定。采用lipofectin包裹HCVRNA转染生长良好的HepG2细胞。转染48h后收集细胞,TRIzol提取RNA,分别用正链及负链引物作RT-PCR,确定转染细胞中有无HCV的复制中间体及病毒核酸。作免疫组化确定有无病毒NS3、NS5抗原。结果体外可获得高浓度全长HCV-RNA,但转染未获成功。结论全长HCV-RNA转染细胞模型的建立尚需进一步研究。更多还原

【Abstract】 Objective To Establish a model of Hepatitis C virus vitro Replication in cell culture. Methods We took p90/HCVFL-longp U plasmid containing HCV gene to convert Sure cell, jolted the bacterium for 18 hours at 36 degrees, extracted plasmid, used linearization DNA as follow board, and used T7RNA in vitro transcription system to get transcript-RNA as PT-PCR and accredited through plain agarose gel electrophoresis. We used lipofectin to package HepG2 cell which grew well and was transfected by HCV RNA. After 48 hours, we collected cells, used Trzzol to extract RNA and used plus strand and minor strand primer as RT-PCR. Then to make sure whether there were replicative intermediate and viral nucleic acid in transfected cells. At last ,we did immunohistochemistrical experiment to ascertain whether there was antigen to virus NS3,NS5. Results Hypsi-concentration and full-length HCV-RNA was acquired out-of-body, but the transfection of HepG2 was not successful. Conclusions Establishment of Hepatitis C virus replication in cell cultures needs to be further researched.更多还原