人LIF基因与增强型绿色荧光蛋白融合表达载体的构建及转染

目的：构建携带人LIF基因与增强型绿色荧光蛋白（EGFP）的融合表达载体，并转染人胚肺成纤维细胞，为研究LIF在维持人胚胎生殖细胞自我更新中的作用机制奠定基础．方法：以重组质粒pcDNA3．1／lif为模板，PCR扩增LIF基因，将扩增片断双酶切后连接到质粒pEGFP-N1中，形成重组表达载体pEGFP—N1／lif．并用脂质体法转染人胚肺成纤维细胞，荧光显微镜下观察，RT—PCR及Western杂交检测融合蛋白的表达．结果：成功构建融合表达载体pEGFP—N1／lif，在人胚肺成纤维细胞实现表达，检测到融合蛋白EGFP—LIF．结论重组pEGFP—N1／lif载体构建成功，可用于标记LIF蛋白、为进一步研究LIF在维持人胚胎生殖细胞自我更新中的作用机制奠定基础．

Construction and Expression of Eukaryotic Expression Vector Bearing Fusion Gene of Human LIF Gene and EGFP

Objective To construct a recombinant eukaryotic vector bearing the fusion gene of human LIF gene and EGFP(enhanced green fluorescent protein),and to transfect it into human embryonic lung fibroblasts(hELF).Methods The LIF gene was amplified by PCR with the recombinant plasmid pcDNA3.1/ lif as the template and the fragment was combined with plasmid pEGFPN1.The recomninant expression vector,pEGFP-Nl/lif,was transfected to hELF using Lipofectamin.The expressed fusion protein was examined by fluorescence microscopy,RT-PCR and Western-blot.Results The eukaryotic expression vector pEGFP-N1/lif was successfully constructed.The fusion protein EGFP-LIF was detected in hELF.Conclusion The successful construction and expression of the eukaryotic expression vector pEGFP-N1/lif has been achieved.