反式激活蛋白转导结构域介导绿色荧光蛋白在小鼠体内的跨膜转运

TAT protein transduction domain mediates transmembrane delivery of EGFP into tissue cells of mice in vivo

目的探讨HIV-1反式激活蛋白(TAT)的蛋白转导结构域(PTD)介导绿色荧光蛋白(EGFP)在小鼠体内的跨膜转运作用。方法采用PCR及克隆技术构成表达载体pET28a-TAT-EGFP,在大肠杆菌中表达TAT-EGFP融合蛋白,将纯化后的融合蛋白注入小鼠尾静脉,取脑、心肌、肝、脾和肾等器官冰冻切片,荧光显微镜下观察融合蛋白在组织中的分布。结果表达和纯化了分子量为28KD的TAT-EGFP融合蛋白,在小鼠的肝、心肌、脑、肾和脾等组织切片荧光检测呈阳性。结论TAT可介导EGFP在广泛组织内的跨膜转导,这一研究为外源活性大分子物质进入组织细胞提供了一个新的途径。

[Objective] To explore the transmembrane delivery effect of EGFP into tissue cells of the mice mediated by protein transduetion domain （PTD） of HIV-1 trans aefivitor transcription （TAT）. [Methods] An express vector pET28a-TAT-EGFP was eontrueted By PCR and gene clone. The TAT-EGFP fusion protein was expressed in E. eoli and purified with affinity chromatography. The purified fusion protein was injected into vena eaudalis of mice and then examined tissue section of brain, cardic muscle, liver, spleen, kidney by fluorescence microscope. [Results] TAT-EGFP fusion protein with molecular weight of 28 KD was successfully expressed positively. The fusion protein was detected in tissue cells from the liver, cardiac muscle, kindey, brain and spleen. [Conclusion] TAT can mediate fusion protein with larger molecular weight to enter into tissue cells, it provide a novel approach for the treatment with exogenous and bioactive proteins.