中文摘要：

目的设计并构建分别位于融合蛋白氨基端和羧基端的穿透肽TAT表达载体,比较两种融合蛋白的内化差异,并利用TAT将凋亡素VP3转导入细胞内,检测其促凋亡活性。方法荧光显微镜观察His-TAT-EGFP和His-EGFP-TAT融合蛋白内化效率的差异,并利用Western blotting检测内化入细胞的蛋白量。将VP3序列插入到内化效率高的穿透肽载体pET14b-EGFP-TAT中并诱导表达蛋白,观察该蛋白在不同细胞系（HeLa、HEK293、3T3、PC-3）的内化;利用DAPI染色观察该融合蛋白诱导HeLa细胞凋亡后核小体的形成,MTT法检测细胞存活率。结果在蛋白浓度梯度实验中,荧光显微镜观察和Western blotting检测均显示His-EGFP-TAT融合蛋白的内化效率明显高于His-TAT-EGFP融合蛋白。在不同种类细胞中均可观察到His-VP3-EGFP-TAT内化的强荧光颗粒;该蛋白和对照蛋白（His-EGFP-TAT）孵育HeLa细胞48h后,His-VP3-EGFP-TAT孵育细胞中可观察到核小体形成,MTT法检测该组细胞的存活率在500、1000、2000nmol/L蛋白作用下分别为87.23%±2.09%、53.01%±1.79%和42.52%±1.90%,与His-EGFP-TAT孵育细胞存活率（分别为97.21%±1.41%、95.72%±1.26%和94.35%±1.67%）比较差异有统计学意义（P〈0.01）。结论 TAT位于融合蛋白羧基端的蛋白内化活性高于位于氨基端的蛋白,His-VP3-EGFP-TAT融合蛋白可以高效内化进入不同的细胞系,并能有效诱导细胞凋亡。

英文摘要：

Objective To design and construct the fusion expression plasmid with TAT located at N terminal and C terminal of the fusion protein separately,to compare their discrepancy in penetrating activity,and then utilize TAT to transfect VP3 into cells to detect its proapoptotic activity. Methods Observe the differences in the efficiency of internalization between the two fusion proteins His-TAT-EGFP and His-EGFP-TAT with fluorescence microscopy and Western-blot. VP3 sequence was inserted into pET14b-EGFP-TAT,which possessed higher penetrating activity. The purified fusion protein His-VP3-EGFP-TAT was obtained and incubated with several kinds of cells including Hela,HEK293,3T3 and PC-3 to show its penetrating activity. Cell apoptosis was identified with the method of DAPI staining to observe the formation of nucleosome,and the VP3 fusion protein＇s proapoptotic activity was detected with MTT assay. Results In the protein concentration gradient experiment,fluorescence microscopy and Western-blot showed that His-EGFP-TAT had significantly higher penetrating activity than His-TAT-EGFP. When incubated with His-VP3-EGFP-TAT,strong fluorescence of particles might be found in all the different types of cells. After 48h incubation,the nucleosomes were found to form in experimental cells. MTT assay revealed that,compared to control group,the survival rates of Hela cells in the experimental group were reduced to 87.23±2.09%,53.01±1.79% and 42.52±1.90% at different fusion protein concentrations. The difference of survival rate of Hela cells between the two groups was statistically significant （P0.001）. Conclusions TAT at N terminal of the fusion protein His-EGFP-TAT shows higher penetrating activity than that at C terminal of His-TAT-EGFP. The purified protein His-VP3-EGFP-TAT can internalize into different kinds of cells through their membranes and display efficient proapoptotic activity.