组蛋白变异体macroH2A1真核表达载体构建及其细胞内定位的研究  

The construction of eukaryotic expression vector and localization of core histone macro-H2A.1 in the cells

作　　者：张菁[1] 罗海华[1] 王娟[1] 卢康荣[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东广州510515

出　　处：《热带医学杂志》2012年第5期506-509,F0002,共5页Journal of Tropical Medicine

基　　金：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(81030055);广东省自然科学基金(10251051501000003;S2011010003917)

摘　　要：目的构建带有血凝素(HA)标签的小鼠组蛋白变异体macroH2A1(mH2A1)的真核表达载体,并观察其在人胚肾293T细胞中的表达定位情况。方法提取内毒素休克的BALB/c小鼠肝脏组织的总RNA,通过逆转录-聚合酶链反应获得内毒素休克小鼠肝脏组织的cDNA,以cDNA为模板使用PCR方法扩增得到mH2A1编码序列,并将其酶切后连接至带有HA标记的载体pcDNA3-HA上;对阳性克隆进行酶切、PCR和测序鉴定。随后将重组质粒瞬时转染293T细胞,利用荧光显微镜观察。结果 PCR、双酶切和测序鉴定表明pcDNA3-mH2A1-HA真核表达质粒构建正确;经转染实验发现,该质粒能够在293T细胞中表达,表达产物主要定位在细胞核中。结论 mH2A1真核表达载体pcDNA3-mH2A1-HA的成功构建及明确其在哺乳动物细胞中的具体核定位,为进一步研究mH2A1作用细胞的信号通路提供了一个重要的工具。Objective To construct eukaryotic expression vector for HA-tagged mouse core histone macro-H2A.1（mH2A1）,and to detect the expression and localization of mH2A1 in 293T cells.Methods Total RNA from the hepatic tissue of BALB/c mice subjected to injection with 25 mg/kg LPS was extrated,and the cDNA was amplified by reverse transcription-polymerase chain reaction（RT-PCR）.The coding sequence of mH2A1 was amplified by PCR with cDNA as the template and subcloned into the hemagglutinin（HA）-tagged mammalian cell expression vector pcDNA3-HA.The positive clones were identified with enzyme digestion,PCR and sequencing.The recombinant plasmid pcDNA3-mH2A1-HA was transfected into 293T cells,which were observed under fluorescence microscope.Results The recombinant plasmid pcDNA3-mH2A1-HA was verified by polymerase chain reaction（PCR）,enzyme digestion and sequence analysis.With fluorescence microscopy,it was found that the mH2A1-HA fusion protein was highly expressed in 293T cells with a nuclear distribution.Conclusion The mH2A1-HA expression vector has been successfully constructed,with an clear nuclear localization in mammalian cells,which provides an important tool for the study on the signaling pathway induced by mH2A1.

关 键 词：组蛋白变异体macroH2A1 血凝素类 细胞内定位 载体构建 

分 类 号：Q512.7[生物学—生物化学]