转甲状腺素蛋白真核表达载体的构建及其细胞内定位  

Construction and intracellular localization of eukaryotic expression vector of transthyretin

作　　者：胡水旺[1] 孙明晶[1] 夏高晓[1] 陈丽[1] 赵明哲[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：《解放军医学杂志》2009年第6期722-724,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金项目(30670828、30572151);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(U0632004);广州市科技计划项目(2007J1-C0301);长江学者和创新团队发展计划项目(IRT0731)

摘　　要：目的构建转甲状腺素蛋白(TTR)的真核表达载体,观察其在NIH3T3细胞中的表达及定位。方法提取BALB/c小鼠肝脏组织的总RNA,通过RT-PCR扩增得到TTR编码序列,将该编码序列克隆到带有血凝素(HA)标记的载体pcDNA3-HA上,构建质粒pcDNA3-TTR-HA。重组质粒通过PCR、酶切测序等证明构建正确后经脂质体转染NIH3T3细胞,固定并染色后通过荧光显微镜观察该融合蛋白的表达及定位。结果重组质粒经鉴定证明构建正确。转染实验发现,该质粒能够在NIH3T3细胞中表达,表达产物主要分布在细胞质中。结论成功构建带HA标签的TTR真核表达载体,该载体能在哺乳动物细胞中有效表达并正确定位,为深入研究TTR在细胞内的相关生物学功能奠定了基础。Objective To construct an eukaryofic expression vector of Transthyretin （TTR） and detect its expression and localization in NIH 3T3 cells. Methods The total RNA was extracted from the liver tissue of BALB/c mice, and the corresponding coding se quences of mouse TTR （GenBank accession No. NM-013697） were amplified by RT-PCR and then cloned into the hemagglutinin （HA）- tagged vector pcDNA3-HA to construet a new recombinant plasmid named pcDNA3-TTR-HA. The recombinant plasmid was verified by PCR, double digested with restriction endonuclease, and followed by sequencing, Subsequently, the correct construct was then transfected into NIH 3T3 cells with liposome transfection reagent Polyfect. After fixing the cells with formaldehyde and staining the fusion protein with specific antibody of HA tag, the expression and localization of the fusion protein were observed with fluorescence microscope. Results All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant plasmid pcDNA3-TTR-HA was correctly constructed. After transfection into NIH 3T3 cells with the recombinant plasmid, it was found that the fusion protein was highly expressed in NIH 3T3 cells and distributed mainly in the cytoplasm as shown by fluorescence microscopy. Conclusions The eukaryotic expression vector of TTR-HA fusion protein has been successfully constructed and effectively expressed in mammalian cells with a correct localization, and it can serve as a convenient and useful tool for the further study of the related biological functions of TTR in eukaryotic cells.

关 键 词：前白蛋白 克隆 分子 基因表达 

分 类 号：Q51[生物学—生物化学] Q78