Pin1基因真核表达载体的构建及在宫颈癌HeLa细胞中的表达  

Construction of pEGFP-Pin1 Recombinant Plasmid and Its Expression in Cervical Cancer Cell Line

作　　者：李红雨[1] 朱涛[1] 周金华[1] 徐茜[1] 胡春霞[1] 邓东锐[1] 王世宣[1] 卢运萍[1] 马丁[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科

出　　处：《华中科技大学学报（医学版）》2007年第5期633-636,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家重点基础研究发展计划(973)基金(No2002CB513107);国家自然科学基金(No30271358)资助项目

摘　　要：目的构建一种含人肽基脯氨酰顺反异构酶Pin1的质粒,以绿色荧光蛋白作为报告基因,观察其在宫颈癌HeLa细胞中的表达。方法从人宫颈癌HeLa细胞中,以RT-PCR方法扩增出人Pin1基因全长cDNA序列(CDS),插入增强绿色荧光蛋白表达质粒pEGFP-C1,脂质体法转染HeLa细胞,荧光显微镜下观测绿色荧光蛋白的表达,免疫印迹法检测GFP-Pin1蛋白的表达。结果RT-PCR扩增出人Pin1基因;构建pEGFP-C1-Pin1重组质粒,体外成功转染入宫颈癌HeLa细胞,在荧光显微镜下可见强绿色荧光蛋白的表达,免疫印迹法证实存在GFP-Pin1蛋白高表达;经G418筛选获得单细胞克隆。结论重组质粒pEGFP-Pin1体外转染HeLa细胞后,目的基因能够在细胞内有效表达,检测方法简便可靠,为利用转染该目的基因的细胞进行下一步研究提供了实验基础。Objective To construct a vector containing human peptidyl-prolyl cis/trans isomerase Pin1 with green fluorescence protein as the reporter gene,and then to observe its expression in HeLa cells.Methods The CDS of Pin1 was amplified from HeLa cells by RT-PCR,then ligated with pEGFP-C1 to form recombinant plasmid pEGFP-Pin1 and transfected into HeLa cells.GFP-tagged Pin1 subcellular localization was analyzed by fluorescent microscopy,and the GFP-Pin1 was detected by immunoblotting.Results The recombinant plasmid pEGFP-Pin1 was constructed and transfected into HeLa cells successfully.Pin1 localized almost exclusively in the cell nucleus speckles and concentrated at discrete structures.The GFP-Pin1 was strongly expressed in HeLa cells.The stable transfected clone was selected by G418.Conclusion Recombinant plasmid pEGFP-Pin1 was effectively expressed after being transfected into HeLa cells,which provided the lab support for further study on Pin1.

关 键 词：肽基脯氨酰顺反异构酶Pin1 质粒构建 基因转染 

分 类 号：R737.33[医药卫生—肿瘤]