过氧化物酶蛋白1真核表达载体构建及在Hela细胞的表达与定位  

Construction of Eukaryotic Expression Vector of Peroxiredoxin Ⅰ and its Expression and Localization in Hela Cells

作　　者：刘震西[1,2] 康瑞霞[1,2] 刘芸[2] 胡水旺[2] 樊启黄[2] 李玉花[1] 姜勇[2] 

机构地区：[1]东北林业大学发育生物学实验室,黑龙江哈尔滨150040 [2]南方医科大学病理生理学教研室广东省蛋白质组学重点实验室,广东广州510515

出　　处：《现代生物医学进展》2012年第14期2615-2618,共4页Progress in Modern Biomedicine

基　　金：国家重大基础理论研究发展(973)计划项目(2010CB529704);教育部长江学者和创新团队发展计划项目(IRT0731);国家自然科学基金重点项目(81030055);广东省自然基金项目(10251051501000003)

摘　　要：目的:构建过氧化物酶蛋白1(PRDX 1)的真核表达载体,观察其在Hela细胞内表达及定位。方法:提取BALB/c小鼠肝脏组织总RNA,通过RT-PCR方法扩增得到小鼠PRDX 1编码序列,酶切后克隆至pcDNA3-myc载体。重组质粒通过PCR、酶切、测序证明构建正确后经脂质体转染Hela细胞,然后利用Western blot和荧光显微镜技术观察该融合蛋白在细胞内表达及定位。结果:经鉴定证明重组质粒构建正确;Western blot实验显示,该质粒能够在Hela细胞中特异表达;免疫荧光试验显示,蛋白产物分布在胞浆和胞核,证明该蛋白在细胞内高表达。结论:成功构建带有myc标签的PRDX 1真核表达载体,该质粒能够在哺乳细胞中特异表达并且外源性PRDX 1蛋白分布在Hela细胞胞浆胞核内,为深入研究PRDX 1蛋白在细胞内相关生物学研究奠定了基础。Objective： To construct an eukaryotic vector of Peroxiredoxin 1（PRDX 1） and detect its expression and localization in Hela cells.Methods： The total RNA was extracted from the liver tissues of BALB/c mice,and the corresponding coding sequences of mouse PRDX 1 were amplified by RT-PCR,then they were cloned into the pcDNA3-myc vector to construct a new recombinant plasmid pcDNA3-PRDX 1-myc.The recombinant plasmid was verified by PCR,double digested by restriction endonuclease,and followed by sequencing.Subsequently,the correct construct was transfected into Hela cells with liposome transfection reagent Polyfect.The expression and localization of the fusion protein were detected by Western blot and fluorescence microscope.Results： The recombinant plasmid pcDNA3-PRDX 1-myc was correctly constructed.After transfection the recombinant plamid into Hela cells,the fusion protein was detected in Hela cells by Western blot and the fusion protein was founded in whole cells as shown by fluorescence microscopy.Conclusion： The eukaryotic expression vector for Prdx 1-myc fusion protein has been successfully constructed and effectively expressed in mammalian cells,and it can serve as an important tool for the further study of the correlated biological functions of PRDX 1 in eukaryotic cells.

关 键 词：过氧化物酶1 分子克隆 蛋白表达定位 

分 类 号：Q75[生物学—分子生物学] Q78