泛素结合酶UFC1基因的荧光表达载体构建.docVIP

泛素结合酶UFC1基因的荧光表达载体构建 　　摘要利用pEGFP-C3作为载体，构建与绿色荧光蛋白基因相连的泛素结合酶UFC1（ubiquitin-fold modifier conjugating enzyme 1）序列的真核表达质粒，观察其在真核细胞中的表达和定位，为进一步研究UFC1基因功能打下了基础。采用RT-PCR方法从宫颈癌Hela细胞中扩增UFC1基因全长cDNA，双酶切后克隆到真核表达载体pEGFP-C3中，构建并鉴定pEGFP-C3-UFC1质粒。经脂质体介导转染Hela细胞后，在荧光显微镜下观察目的蛋白在真核细胞内的表达情况。双酶切鉴定和测序分析均证实，已成功构建pEGFP-C3-UFC1重组载体。重组载体转染Hela细胞，观察到绿色荧光蛋白表达，还可见绿色荧光呈点状分布于细胞质中；而对照pEGFP-C3质粒转染Hela细胞，绿色荧光蛋白则呈弥散状分布。成功克隆了UFC1基因，构建了pEGFP-C3-UFC1重组质粒。 　　关键词UFC1基因；克隆；真核表达载体 　　中图分类号Q78文献标识码A文章编号 1007-5739（2011）08-0023-02 　　 　　ConstructionofFluorescenceExpressionVectorofUbiquitin-foldModifierConjugatingEnzyme（UFC1）Gene 　　HE Ai-lan 1PENG Dong-hai 1WU Xiao-ling 2LIAO Yan-yang 2 * 　　（1 Hunan Institute of Humanities，Loudi Hunan 417000； 2 Hunan Agricultural University） 　　AbstractTo construct eukaryotic expression plasmid of ubiquitin-fold modifier conjugating enzyme（UFC1） gene linked with GFP gene with pEGFP-C3 vector and observe its expression and location in eukaryotic cells，thus it can provide a powerful tool to further studythe function of UFC1 gene.The full-length cDNA of UFC1 gene was amplified from human cervical carcinoma Hela cell by RT-PCR；UFC1 gene was cloned into eukaryotic expression vector pEGFP-C3 after double-digestion；To construct and identify the recombinant plasmid pEGFP-C3-UFC1. Then the plasmid was transfected into Hela cells by liposome，and its expression was detected by observing the expression of enhanced green fluorescent protein（EGFP）through fluorescent microscope. Recombinant pEGFP-C3-UFC1 vector was confirmed by double-digestion and sequencing. The green fluorescence was observed in transfected Hela cells. It was mostly located in the cytoplasm and distributed unequally with pEGFP-C3-UFC1 vector transfected；but it was located in cell whole and distributed equally with control vector pEGFP-C3 transfected. UFC1 gene was successfully cloned and recombinant pEGFP-C3-UFC1 vector was successfully constructed. 　　Key wordsUFC1 gene；clone；eukaryotic expression vector