肌动蛋白和Tau蛋白荧光蛋白融合载体的构建、表达及其细胞内定位  

Construction,expression and intracellular localization of fusion vectors carrying actin,Tau and fluorescent protein

作　　者：明小燕[1] 龚小卫[1] 王丹[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,广东省广州市510515

出　　处：《中国组织工程研究与临床康复》2009年第11期2033-2036,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:肌动蛋白和Tau蛋白分别是微丝和微管的重要组成成分,肌动蛋白和Tau蛋白的分布能否作为微丝和微管的观察指标。目的:构建肌动蛋白和Tau蛋白的红色和绿色荧光蛋白融合表达载体并转染真核细胞,观察其在细胞内的表达和定位情况。设计、时间及地点:单一样本观察实验,于2008-03/08在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共同完成。材料:克隆在pcDNA3载体上的肌动蛋白和Tau蛋白真核表达载体pcDNA3-actin和pcDNA3-Tau,以及红色荧光蛋白表达载体pmCherry-C2和绿色荧光蛋白表达载体pEGFP-C2、大肠杆菌株DH5α和小鼠NIH3T3细胞系由南方医科大学病理生理学教研室保存。方法:将克隆在pcDNA3上的肌动蛋白和Tau蛋白亚克隆到红色荧光蛋白载体pmCherry-C2和绿色荧光蛋白载体pEGFP-C2上,然后转染NIH3T3细胞,利用荧光显微镜观察重组载体的表达和细胞内定位。主要观察指标:NIH3T3细胞转染24h后,利用Leica荧光显微镜观察上述重组载体在细胞中的表达和定位情况。结果:重组质粒经酶切、PCR和测序鉴定正确无误,并在NIH3T3细胞中可获得高量表达。融合蛋白发出的红色和绿色荧光均表明,肌动蛋白在细胞质中呈短丝状弥散分布,Tau蛋白以细胞核为中心呈放射状排列。结论:成功构建了肌动蛋白和Tau蛋白的不同荧光蛋白融合表达载体,并在真核细胞中得到有效表达,这为研究细胞骨架在信号分子移位中的作用提供了一个重要的工具。BACKGROUND： The actin and Tau protein are important components of microfilament and microtubule, whether actin and Tau protein can be served as observation indexes of microfilament and microtubule？ OBJECTIVE： To construct eukaryotic cell expression vectors of actin and Tau fused with enhanced green fluorescent protein （EGFP） or red fluorescent protein （RFP）, further more, to study their expressions and intracellular localization in eukaryotic cells. DESIGN, TIME AND SETTING： A single sample experiment was conducted at the Department of Pathophysiology of Southern Medical University and Key Function Proteomics Laboratory of Guangdong Province from March to August 2008. MATERIALS： The eukaryotic vectors pcDNA3-actin, pcDNA3-Tau, pmCherry-C2 and pEGFP-C2, as well as DH5α and NIH3T3 cell lines were preserved at the Department of Pathophysiology of Southern Medical University. METHODS： The coding sequences of actin and Tau in pcDNA3 vectors were sub-cloned into pEGFP-C2 or pmCherry-C2 vectors, followed by transfected into NIH3T3 cells, the intracellular localization of their expression products were observed with fluorescence microscope. MAIN OUTCOME MEASURES： The expressions and localization of four recombinant plasmids were detected by Leica fluorescence microscope at 24 hours after transfected with NIH3T3. RESULTS： The four recombinant plasmids were verified by enzyme digestion, PCR and sequencing, and the fusion proteins were highly expressed in NIH3T3 cells. With fluorescence microscope observation, EGFP- or RFP-tagged actin distributed mainly in the cytoplasm as short, filamentous structures, while EGFP- or RFP-tagged Tau was radial around the nucleus. CONCLUSION： The fusion expression vectors of pEGFP-Tau, pEGFP-actin, pmCherry-Tau and pmCherry-actin proteins are successfully constructed and expressed efficiently in eukaryotic cells, which providing an important tool for studying the effect of cytoskeleton on signaling molecules translocation.

关 键 词：肌动蛋白 TAU蛋白 荧光蛋白 载体构建 细胞内定位 

分 类 号：R349.64[医药卫生—基础医学]