His-TAT-mCherry融合蛋白的跨膜转导及其在细胞内的定位  

Membrane translocation and location in cells of red fluorescent protein fusion vector incorporated HIV-TAT protein

作　　者：陈茜[1] 宋方丽[2] 刘亚伟[2] 杨勤[1] 姜勇[2] 

机构地区：[1]贵阳医学院病理生理教研室,贵阳 550000 [2]南方医科大学病理生理教研室

出　　处：《解放军医学杂志》2009年第2期147-150,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30670829);国家高技术研究发展计划(863计划)课题(2001AA234061);广东省科技计划项目(A1090202);广州市科技计划项目(2002Z3-C70812007J1-C0301)

摘　　要：目的构建在原核中表达的人类免疫缺陷病毒转录活化因子(HIV-TAT)红色荧光蛋白(mCherry)融合表达载体,荧光显微镜观察HIV-TAT的跨膜转导及其在细胞内的定位,为进一步研究HIV-TAT的跨膜转导机制及其定位提供重要工具。方法采用基因重组技术构建含HIV-TAT以及红色荧光蛋白的质粒pET14b-His-TAT-mCherry,对阳性克隆进行PCR、酶切和测序鉴定,将该质粒转化至E.coli BL21(DE3)感受态细胞,使其在体外表达,并进行纯化、除菌。将纯化的His-TAT-mCherry融合蛋白与Hela细胞共同孵育,荧光显微镜下观察。结果PCR、双酶切和DNA测序证明所构建质粒正确;表达纯化出了高纯度的His-TAT-mCher-ry融合蛋白。荧光显微镜下见Hela细胞中红色荧光蛋白主要分布在胞质中,细胞膜上也有一定分布。结论成功构建了pET14b-His-TAT-mCherry原核表达质粒,纯化了高纯度的His-TAT-mCherry融合蛋白,该蛋白在哺乳动物细胞中有跨膜转导活性,为研究HIV-TAT的跨膜转导机制提供了一个重要的工具。Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein （mCherry） in mammalian cells, and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells, in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV- TAT. Methods With the designed primer coding mCherry sequence, the mCherry gene was amplified by PCR with the vector pmCherry C2 as template, and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT mCherry. The constructed vector was then transformed into E.coil BL21 （DE3）, which had been identified by PCR and double digested with restriction endonuclease, followed by sequencing. After IPTG induction, the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE. Purified His-TAT-mCherry recombinant protein was added to Hela ceils and the fluorescence was observed to evaluate the transduction efficiency. Results The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed. His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully, and the fusion protein showed cellular transduction activity. It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm, and also the membrane to some extent. Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCberry sequence. Effective expression and purification of this fusion protein is achieved. It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition. Thus, it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.

关 键 词：基因产物 tat 红色荧光蛋白 跨膜转导 

分 类 号：R318[医药卫生—生物医学工程]