重组质粒pEGFP—C1-STAT6的构建及在HeLa细胞中表达

Construction of pEGFP-C1-STAT6 Fusion Gene in Eukaryotic Expression Vector and its Expression in Hela Cells

目的构建以增强型绿色荧光蛋白（enhanced green fluorescence protein，EGFP）为报告基因的重组质粒pEGFP—C1-STAT6．观察其在HeLa细胞中的表达。方法以重组质粒pCLNEO—STAT6为模板，PCR法扩增出目的基因，将扩增片段双酶切后连接到质粒pEGFP—C1上，构建重组质粒pEGFP—C1-STAT6，脂质体法转染HeLa细胞，western印迹检测融合蛋白的表达，并在荧光显微镜下观察融合蛋白的定位和分布。结果成功构建质粒pEGFP—C1—STAT6，并在HeLa细胞中实现表达；Western印迹检测到融合蛋白EGFP—STAT6；在荧光显微镜下观察到绿色的融合蛋白表达和定位。结论重组质粒pEGFP—C1-STAT6构建成功，可用于标记STAT6蛋白，为进一步研究STAT6在信号转导中的作用机制奠定基础。

Objective To construct pEGFP-C1-STAT6 fusion gene in eukaryotic expression vector and detect its expression in Hela cells. Methods The STAT6 gene was amplified by PCR from the template plasmid pCLNEO-STAT6 and the PCR fragment was combined with pEGFP-C1. The recombinant expression vector pEGFP-C1-STAT6 was transfected into HeLa cells. The expression of fusion protein was examined by fluorescent microscopy and Western blot. Results The ex- pression vector pEGFP-C1-STAT6 was successfully constructed. The fusion protein GFP-STAT6 was detected in HeLa cells by Western blot. In addition, the localization of this fusion protein was detected by fluorescent microscopy. Conclusion The recombinant construct of pEGFP-C1-STAT6 may be used for further study on the role of STAT6 in signaling transduction.