一种基于HIV-1TAT蛋白质转导结构域细胞内转导系统的成功改建  

Reconstruction of an intracellular transduction system based on HIV-1 TAT protein transduction domain

作　　者：郭爱华[1] 刘志锋[1] 孙学刚[1] 李海玉[1] 邓鹏[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室/广东省功能蛋白质组学重点实验室,广东广州510515

出　　处：《南方医科大学学报》2006年第5期545-548,共4页Journal of Southern Medical University

基　　金：国家高技术研究发展(863)计划课题(2001AA234061);广东省科技计划项目(A1090202);广东省自然科学基金重点项目(NO.13058);广州市科技计划项目(2001-Z-035-01-1)~~

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.coliBL21(DE3),经IPTG诱导表达、SDS-PAGE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。Objective To explore the reasons for the low intraceUular transduction efficiency of a previously constructed His-TAT-Flag recombinant protein and establish a more efficient transduction system. Methods The Flag tag ofpET14b-His-Tat-Flag vector was deleted with PCR mutant kit, and enhanced green fluorescent protein （EGFP） coding sequence was inserted into the new pETI4b-His-TAT recombinant vector. Enzyme digestion and DNA sequencing were performed for identification of pET I 4b-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-PAGE. Purified His-TAT-EGFP recombinant protein was added to ECV304 cells and the fluorescence was observed to evaluate the transduction efficiency. Results pETI4b-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 successfully and showed cellular transduction activity. Conclusion The prokaryotic expression vector has been successfully constructed by modifying pET14b-His-TAT-Flag, and the expressed and purified recombinant protein of His-TAT-EGFP possesses high efficiency of intracellular transduction activity.

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 

分 类 号：R341[医药卫生—基础医学]