不含POU结构域的八聚核苷酸结合蛋白(NONO)真核表达载体的构建及其细胞内定位  

Construction of eukaryotic expression vector NONO expression product and its intracellular localization in cells

作　　者：吴翠玲[1] 张秀娟[1] 王娟[1] 刘爱华[2] 姜勇[1] 

机构地区：[1]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,广州510515 [2]南方医科大学南方医院呼吸科,广州510515

出　　处：《解放军医学杂志》2011年第4期365-367,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金重点项目(81030055);国家自然科学基金委员会-广东省人民政府自然科学联合基金(U0632004);长江学者和创新团队发展计划(IRT0731);“973”计划项目(2010CB529704);高等学校博士学科点专项科研基金(20069981001);广州市科技计划项目(2007J1-C0301)

摘　　要：目的构建小鼠的不包含POU结构域的八聚核苷酸结合蛋白(NONO)真核表达载体,并观察其在NIH3T3细胞中的表达和定位。方法提取BALB/c小鼠肝脏组织总RNA,通过RT-PCR扩增得到NONO编码序列,将该序列克隆至带有血凝素(HA)标记的载体pcDNA3-HA上,构建质粒pcDNA3-NONO-HA。通过PCR、双酶切鉴定构建正确后以脂质体为媒介转染NIH3T3细胞,采用荧光显微镜观察该融合蛋白在细胞内的表达及定位情况。结果重组质粒经PCR、酶切和测序鉴定证明构建正确,且能在NIH3T3细胞中得到大量表达。荧光显微镜观察发现NONO-HA融合蛋白在静息状态下主要分布于胞质中。结论成功构建带HA标签的NONO真核表达载体,该载体能在哺乳动物细胞中有效表达NONO-HA融合蛋白。Objective To construct an eukaryotic expression vector NONO（containing nucleotide octamer-binding protein without POU domain） of mouse,and detect its expression and intracellular localization in NIH3T3 cells,so as to obtain a tool to assist the study of intracellular biological functions of NONO.Methods The total RNA was extracted from the liver of BALB/c mice,the corresponding coding sequences of mouse NONO（GenBank accession No.53237024） were amplified by RT-PCR and then cloned into hemagglutinin（HA）-tagged vector of pcDNA3-HA to form a new recombinant plasmid named pcDNA3-NONO-HA.The recombinant plasmid was verified by polymerase chain reaction（PCR） and double digestion by restricted endonuclease,followed by sequencing.The recombinant plasmid was then transfected into NIH3T3 cells with the liposome transfection reagent Polyfect as a medium.Twenty-four hours later,immunofluorescence was performed.After detection of fusion protein NONO-HA by specific antibody of HA tag and the Alexa Fluor 488 coupled secondary antibody,the expression and localization of the fusion protein were observed by fluorescence microscopy.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the recombinant plasmid pcDNA3-NONO-HA was correctly constructed.After transfection of the recombinant plasmid,the fusion protein was found to highly express in NIH3T3 cells and distribute mainly in the cytoplasm.Conclusion The eukaryotic expression vector for HA-NONO fusion protein is successfully constructed and effectively expressed in mammalian cells.The constructed vector may serve as an assistant tool in the study of intracellular biological functions of NONO.

关 键 词：血凝素类 基因表达 重组融合蛋白质类 

分 类 号：Q78[生物学—分子生物学]