穿膜肽TAT的表达与活性的初步研究

目的：构建原核表达载体,获得纯化的融合蛋白TAT-GFP,以便于考查TAT的细胞定位及穿膜功能。方法：将TAT基因与绿色荧光蛋白（GFP）基因串联于原核表达载体pET28a中,通过IPTG诱导融合表达。分离纯化的融合蛋白与BHK-21细胞共孵育一定时间后,激光共聚焦显微镜观察其在细胞内的定位;同时以尾静脉注射方式进行小鼠给药实验,一定时间后麻醉,将主要器官组织取出、固定、冰冻切片,荧光显微镜下观察蛋白的分布情况。结果：DNA测序证明成功构建融合蛋白表达载体pET28a-tat-gfp。经诱导表达、纯化可获得纯度为85%以上的融合蛋白TAT-GFP。细胞实验表明,融合蛋白可迅速透过细胞膜并广泛分布于BHK-21细胞内,其中尤以细胞核内最多,而对照组GFP蛋白则不能穿透细胞膜入胞;动物实验中,给药2h后即可观察到TAT携带GFP到达小鼠的各主要器官和组织,甚至穿透血脑屏障分布于脑组织部分。结论：融合表达的穿膜肽TAT具有一定的携带生物分子（GFP）穿膜功能,本研究为深入了解TAT的性质及其未来应用奠定了一定的基础。

Cloning,expression and activity anaylsis of cell-penetrating peptide TAT

Objective：To clone and express tat gene in Escherichia coli in order to study the property and location of cell-penetrating peptide TAT. Methods：Green fluorescence protein （GFP）was introduced as a reportor, tat And gfp genes were tandem cloned into vector pET28a. Then pET28a-tat-gfp recombinant was transfeeted into BL21. The recombinant was induced to express fusion protein TAT-GFP by IPTG. The purified protein was incubated with BHK-21 for different time to observe its location in cells by laser scanning confocal fluorescence microscope. At the same time, TAT-GFP and control GFP were injected into mice by caudal vein. After some time, the mice were dissected and the main organs were taken and fixed in paraffin. After 24 h, sections of the fixed tissue samples were made to study the disposition of protein. Results： The DNA sequencing showed that the recombinant plasmid pET28a-tat-gfp was constructed successfully. The purity of acquired protein was 85%. Cell test indicated that TAT-GFP could penetrate plasm membrane and distribute extensively in BHK-21 cell,especially in nucleus. The control protein GFP could not enter the BHK-21 cell. Animal test indicated that TAT could take GFP into many organs and tissues of mice 2 h after ip injection, even passing through blood brain barrier. Conclusion：The results confirmed that the recombinant protein TAT-GFP had the cell-penetrating ability. This work contributes to further research.