摘要
目的探讨跨膜递送短肽——TAT蛋白转导结构域(简称TAT)介导的与其融合的活性蛋白在活体的跨膜递送作用。方法以融合蛋白GST-TAT-GFP,GST-GFP-TAT和GST-GFP为研究模型蛋白,不经过蛋白质的变性处理、直接通过向小鼠腹腔注射和皮肤涂抹这两种含TAT的融合蛋白及作为对照的融合蛋白GST-GFP,一定时间作用后取体内器官和皮肤做冷冻切片,荧光显微镜检测这些融合蛋白的跨膜递送情况;并对分别融合在C端或者N端的TAT介导GFP在活体动物体内和皮肤的跨膜递送作用进行对比。结果腹腔注射实验结果表明,TAT可以介导不经过蛋白质的变性处理的融合蛋白GST-TAT-GFP和GST-GFP-TAT跨膜递送进入到小鼠的心脏、肝、肾、脾和肺,甚至脑组织;其中GST-GFP-TAT跨膜递送效率比GST-TAT-GFP更高。结构模拟分析提示GST-GFP-TAT与GST-TAT-GFP中的TAT的暴露情况不同可能是造成两种蛋白跨膜递送活性差异的重要因素。皮肤实验的结果则表明TAT不仅介导融合蛋白GST-TAT-GFP和GST-GFP-TAT进入小鼠表皮,而且使其进入小鼠皮肤的真皮层。结论 TAT可以跨膜递送不经过变性处理的融合蛋白进入小鼠皮肤和体内,递送效率可能与TAT的暴露程度相关;这些结果为在蛋白质疗法方面应用TAT提供了进一步的理论依据。
Objective To investigate the transmembrane delivery in vivo of bioactive protein fused with TAT protein transduction domain（abbreviated as TAT）,the well Known peptide with transmembrane delivery activity.Methods Fusion proteins GST-TAT-GFP and GST-GFP-TAT were used as model protein,and GST-GFP as control one in this study.They were i.p.injected into mice or topically applied onto the mouse skin.Certain time later,frozen sections of the organ tissues or skin were analyzed by fluorescence microscopy.The transmembrane delivery efficiency of GST-TAT-GFP and GST-GFP-TAT was examined comparatively.Results The data revealed that substantial fluorescence intensity could be detected in different organ tissues of mice i.p.injected with GST-TAT-GFP and GST-GFP-TAT without unfolding treatment,suggesting that TAT efficiently delivered bioactive protein into the heart,liver,kidney,spleen,lung and even brain of the mice.GST-GFP-TAT showed a higher delivery efficiency than GST-TAT-GFP.Analysis of structure simulation suggested that the difference might be resulted from the different levels of exposedness of TAT among fusion protein.Thefusion proteins topically applied onto skin was proved to be delivered into not only the epidermis but also the derma.Conclusion TAT efficiently transducts fusion protein into organs and skin of mice even without denaturing treatment.These results provide further support for the application of TAT in the field of protein therapies.
出处
《中国实验动物学报》
CAS
CSCD
2010年第6期463-466,I0003,I0004,共6页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金(No.30800285)
福建省卫教联合攻关项目(WKJ2008-2-47)
福建省高等学校新世纪优秀人才支持计划项目(SW2006-15)资助
关键词
TAT蛋白转导结构域
融合蛋白
跨膜递送
皮肤
器官
TAT protein transduction domain
Transmembrane delivery
Fusion protein
Skin
Organ
Mice
作者简介
刘树滔（1970-），男，副教授，博士，研究方向为生物技术。
