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。
To 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 study the 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.
出处
《现代农业科技》
2011年第8期23-24,28,共3页
Modern Agricultural Science and Technology
基金
湖南省自然科学基金项目(09JJ6048)
湖南省教育厅科学研究项目(08C457)
