《一种基于HIV-1 TAT蛋白质转导结构域细胞内转导系统的成功改建》.doc

一种基于HIV-1 TAT蛋白质转导结构域细胞内转导系统的成功改建 作者：郭爱华；刘志锋；孙学刚；李海玉；邓鹏；姜勇　　(南方医科大学病理生理学教研室/广东省功能蛋白质组学重点实验室，广东 广州 510515) 摘要：目的??探索本室构建的pET14b-His-TAT-Flag重组载体表达的融合蛋白在细胞中转导效率低的原因，建立高效的细胞内转导系统。方法??通过PCR方法去除原有载体中的Flag序列，在改建正确的pET14b-His-TAT重组载体基础上插入EGFP编码序列，将经酶切、DNA测序鉴定正确的pET14b-His-TAT-EGFP质粒转化E. coli BL21(DE3)，经IPTG诱导表达、SDS鉴定正确，再经蛋白透析、过滤处理。将His-TAT-EGFP融合蛋白加入ECV304细胞中，用荧光显微镜观察。结果??重组质粒pET14b-His-TAT融合表达载体和pET14b-His-TAT-EGFP重组载体经酶切、DNA测序鉴定证实构建成功。表达纯化出了高纯度His-TAT-EGFP融合蛋白并具有较高细胞内转导活性。结论 成功改建pET14b-His-TAT-Flag重组载体，正确构建pET14b-His-TAT-EGFP载体，His-TAT-EGFP融合蛋白高效表达，并具有明确的细胞内转导活性。 关键词：HIV-1反式激活因子；蛋白质转导结构域；融合蛋白；细胞内转导 中图分类号：Q291??文献标识码：A??文章编号：1673-4254(2006)05-0545-04 Reconstruction of an intracellular transduction system based on HIV-1 TAT protein transduction domainGUO Ai-hua； LIU Zhi-feng； SUN Xue-gang； LI Hai-yu； DENG Peng； JIANG YongDePartment of Pathophysiology/Key Laboratory of Functional Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, ChinaAbstract: Objective To explore the reasons for the low intracellular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system. Methods The Flag tag of pET14b-His- Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein (EGFP) coding sequence was inserted into the new pET14b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET14b-His-TAT-EGFP vector, which was then transformed into E. coli BL21(DE3). After IPTG induction, the recombinant protein of His-TAT-EGFP was isolated and analyzed with SDS. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency. Results pET14b-His-TAT vector and pET14b-His-TAT- EGFP vector were successfully constructed, which was identified with enzyme digestion and DNA sequencing. His-TAT-EGFP fusion protein was expressed and purified suc