HCVIRES介导萤火虫荧光素酶翻译的双顺反子载体的构建与在小鼠体内的表达

将HCVIRES插入双报告基因海肾荧光素酶 (Rluc)基因和萤火虫荧光素酶 (Fluc)基因之间 ,建立了“依赖帽子的扫描机制”翻译表达Rluc ,HCVIRES调控Fluc翻译的双顺反子表达载体pCI Rluc HCVIRES Fluc ,通过酶切反应及转染HepG2细胞鉴定双荧光素酶瞬间表达活性等试验 ,证实获得了表达双荧光素酶的双顺反子载体 .并应用水压转染法将双顺反子表达质粒导入小鼠体内 ,在小鼠肝脏检测到高水平表达的Rluc和Fluc .该研究成功构建一种HCVIRES介导萤火虫荧光素酶基因表达的双顺反子载体 ,并在HepG2细胞及小鼠体内进行了瞬时表达 ,为进一步建立稳定评价靶向HCVIRES药物作用的细胞及小动物模型研究奠定了基础

Construction and Expression of the Bicistronic Reporter Plasmid of Firefly Luciferase Controlled by HCV IRES in Mice

The bicistronic reporter constructs containing Renilla and firefly luciferase reporter genes was developed. The Renilla luciferase(Rluc)gene was expressed by a cap dependent mechanism,and the firefly luciferase(Fluc)was controlled by HCV internal ribosomal entry site(IRES). Expression plasmid (pCI Rluc HCV IRES Fluc) was confirmed by endonuclease digestion as well as luciferase transient expression in HepG2 cell. The bicistronic reporter was transfected into mice by hydrodynamics based transfection. The liver of mice showed the high level of Renilla and firefly luciferase activity.These results suggested that the bicistronic reporter plasmid of firefly luciferase controlled by HCV IRES was successfully constructed,and the dual luciferase could be transiently expressed in mice and cultured cell model systems. The results were the basis for further establishing small animal and cultured cell model system which stably express firefly luciferase controlled by HCV IRES.