Septin 8红色荧光蛋白融合载体的构建及表达  

Construction and Expression of Setpin 8-red Fluorescent Fusion Protein Vector

作　　者：王丹[1] 梅柱中[1] 刘爱华[1] 明小燕[1] 王达安[1] 王旭[1] 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

出　　处：《热带医学杂志》2010年第2期120-122,136,F0002,共5页Journal of Tropical Medicine

基　　金：长江学者和创新团队发展计划项目(No.IRT0731);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重点项目(No.U0632004);国家自然科学基金(No.30670828;30572151)

摘　　要：目的构建在哺乳动物细胞中表达的septin 8红色荧光蛋白融合表达载体(pmCherry-C2/septin 8),并观察其在细胞内的表达和定位情况。方法采用PCR的方法从人脑胶质细胞瘤cDNA文库中扩增获得septin 8基因编码区序列,将其克隆至红色荧光蛋白载体pmCherry-C2上,重组质粒经PCR、酶切以及序列分析正确无误后转染NIH3T3细胞,并利用Western blotting和细胞免疫化学技术分析重组蛋白在细胞内的表达和定位。结果重组pmCherry-C2/septin 8融合蛋白在NIH3T3细胞中得到高量表达且主要分布于细胞浆。结论成功构建了pmCherry-C2/septin 8融合表达载体,该载体能在哺乳动物细胞NIH3T3中表达,为进一步研究septin 8细胞内生物学功能打下了良好的基础。Objective To construct eukaryotic cell expression plasmid of pmCherry-C2 fusion protein and analysis its expression and intracellular localization in NIH3T3 cell. Methods Septin 8 coding region was amplified by PCR from human glioma cells cDNA library and subcloned into pmCherry-C2 vector. After comfirmed by PCR, enzyme digestion and DNA sequencing,the construct was then transfected into NIH3T3 cells and analyzed by fluorescence microscopy. Results pmCherry-C2/septin 8 fusion protein was highly expressed in NIH3T3 cell. Fluorescence microscope analysis revealed that the fusion protein is mainly distributed in the cytoplasm. Conclusion The pmCherry-C2/septin 8 fusion protein plasmid has been successfully constructed and expressed in NIH3T3 cell, which laid the foundation for the functional analysis of septin 8 in the future.

关 键 词：septln 8 红色荧光蛋白 载体构建 基因表达 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]