南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (05): 545-548.

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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

郭爱华； 刘志锋； 孙学刚； 李海玉； 邓鹏； 姜勇；   

  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； 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； 广东广州510515；
  • 出版日期:2006-05-20 发布日期:2006-05-20
  • 基金资助:
    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)~~

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

GUO Ai-hua,LIU Zhi-feng,SUN Xue-gang,LI Hai-yu,DENG Peng,JIANG Yong Department of Pathophysiology/Key Laboratory of Functional Proteomics of Guangdong Province,Southern Medical University,Guangzhou 510515,China   

  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； 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； 广东广州510515；
  • Online:2006-05-20 Published:2006-05-20

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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。 

Abstract: 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-PAGE.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 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. 

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