两种方式制备原核表达含TAT蛋白转导域融合蛋白穿膜能力比较.docVIP

两种方式制备原核表达含TAT蛋白转导域融合蛋白穿膜能力比较 　 作者：许逊，蔺昕，田雨香，招倩倩，季宝菊，刘静，夏圣，王胜军，许化溪，邵启祥 【摘要】 　　目的： 联合应用多种技术评价不同方式制备的TAT转导结构域eGFP目的蛋白的穿膜效率及亚细胞定位情况，以期建立有效、稳定的高效穿膜蛋白制备方法。方法： 诱导表达、纯化包涵体形式融合蛋白，分别进行直接脱盐和透析复性，并与细胞共育，然后通过流式细胞术、激光共聚焦显微镜及免疫印迹法评价融合蛋白穿膜能力。结果： 通过透析复性制备的融合蛋白穿膜效率极低；而纯化后直接脱盐的融合蛋白经流式细胞术、激光共聚焦显微镜及免疫印迹等方法分析，表明均具有高效穿膜能力。结论： 两种方式制备的融合蛋白均能穿膜，但透析复性制备的蛋白穿膜效率低，且不易定位于细胞核中，影响进一步对该目的蛋白在胞核中生物学行为的研究；而脱盐的变性蛋白具有高效的穿膜效率。 【关键词】 转导结构域； 透析； 复性； 包涵体； 穿膜效率 　　［Abstract］Objective: To evaluate the efficacy and subcellular localization of the fusion protein eGFPinterest protein including protein transduction domain of TAT by multitechnology and to obtain the more effective cellular uptake protein for the further research of its biological function.Methods： Induced the Expression of fusion protein, and extracted and purified it from the inclusion bodies. Jurkat cells exposure to fusion protein which was directly desalted and refolded via dialysis, respectively. Flow cytometry analysis, confocal microscope observation and Western blot detection were used to evaluate the efficacy of the fusion protein to transduce into the cell cytoplasm. Results： The results of the detected by flow cytometry, confocal microscope and Western blot indicated that the fusion protein which desalted by PD10 column could penetrate Jurkat cells more efficiently, contrasty, the renaturated protein via dialysis had low activity. Conclusion： Both of the fusion proteins treated with two different methods could translocate through the cell membranes，but the dialyzed fusion protein had low efficiency and didn′t locate into cell nucleus, would disbennifit for the following research work about the biologic activity and behavior of the fusion protein on transcriptional level. 　　［Key words］protein transduction domain; dialysis; refolding; inclusion bodies; efficacy of transduce through the cells membrance 通过细胞穿透肽进行蛋白转导是体外蛋白传递的一种有效方法。最常用的蛋白转导肽就是HIVTAT基因的第一个外显子编码的第48～56位氨基酸残基，其具有核定位和蛋白转导结构域（protein transduction domain，PT