带荧光标记的新型丙型肝炎病毒感染性克隆的构建及其活性鉴定.docVIP

PAGE \* MERGEFORMAT PAGE \* MERGEFORMAT 1 PAGE \* MERGEFORMAT PAGE \* MERGEFORMAT 1 带荧光标记的新型丙型肝炎病毒感染性克隆的构建及其活性鉴定 陈晨 张永宏 【摘要】目的 构建携带有增强绿色荧光蛋白（EGFP）或带红色荧光蛋白（mCherry）的不同基因型丙型肝炎病毒（HCV）细胞感染性毒株,为HCV治疗提供新思路和药物作用靶点。方法 化学合成加入EGFP或mCherry基因片段，克隆入HCV全长基因组中，获得带加入EGFP/mCherry的病毒质粒，通过酶切鉴定、基因测序鉴定成功后，体外转录获得HCV的RNA,然后转染人肝癌细胞系Huh7.5,取其上清液去感染新的Huh7.5细胞。通过荧光显微镜检测EGFP/mCherry表达情况。收集带EGFP、mCherry的活病毒，测定病毒感染滴度。二次感染Huh7.5细胞，并对各组的二次感染受感染细胞数的比例进行分析。结果 成功构建带EGFP或带mCherry的不同基因型HCV细胞感染性毒株。收集病毒转染细胞的培养上清液具有感染活性，能感染新的Huh7.5细胞。进行二次感染结果显示同种基因型的EGFP或同种基因型的mCherry的共感染比不同基因型的病毒共感染更有利于病毒的产生。结论 构建带EGFP或mCherry报告基因的HCV毒株在肝癌细胞中可得到表达，为特异性抗HCV药物评价系统的建立提供实验依据。 【关键词】 丙型肝炎病毒;EGFP;mCherry;Huh7.5肝癌细胞;感染 Build a new clone of hepatitis C virus infection with fluorescent markers and activity identification Chen Chen,Zhang Yong-hong. *Department of Gastroentrology,Gui Zhou Medical University,Guizhou Guiyang 550001，China Corresponding author:Zhang Yonghong, Email:ZY163.com,cn 【Abstract】Objective To construct carrying enhanced green fluorescent protein (EGFP) or with a red fluorescent protein (mCherry) of different genotypes of hepatitis C virus (HCV) infection of cell strains, providing new ideas and drug targets for the treatment of HCV point. Method The chemical synthesis adding EGFP or mCherry gene fragment was cloned into the full-length HCV genomic clones, obtained with adding EGFP/mCherry viral plasmid by restriction enzyme digestion, after successful gene sequencing, in vitro transcribed RNA HCV obtained, and then transfected human hepatoma cell line Huh7.5, whichever Huh7.5 supernatant to infect new cells. By fluorescence microscopy to detect EGFP / mCherry expression. Collected with EGFP, mCherry live virus titer determination of viral infection. Huh7.5 cells secondary infection, and to analyze the percentage of each group of secondary infection of the infected cell. Results The band was successfully constructed with EGFP or mCherry cells infected with different genotypes of H