基于细胞穿透肽技术的对p38丝裂原活化蛋白激酶蛋白功能的研究.doc

基于细胞穿透肽技术对p38丝裂原活化蛋白激酶蛋白功能的研究 作者：杨丽萍；刘志锋；黎永明；李志杰；姜勇　　(南方医科大学病理生理学教研室/广东省功能蛋白质组学重点实验室，广东 广州 510515) 摘要：目的??构建基于TAT技术的p38 MAPK蛋白转运系统并对导入细胞的融合蛋白的功能进行鉴定。方法 采用亚克隆方法构建重组质粒pHis-TAT-p38和pHis-TAT-p38(AF)无活性突变体，并诱导原核表达纯化融合蛋白；将这两种蛋白加入培养的ECV304细胞，在高渗刺激下，通过检测p38底物ATF2的磷酸化水平，以观察由TAT转导进入细胞His-TAT-p38及其无活性突变体对内源性p38活性的影响。结果 酶切和测序结果表明，载体构建正确；SDS凝胶电泳可见原核表达纯化得到高纯度目的蛋白；Western blot表明，融合蛋白His-TAT-p38及其突变体能够以一种时间和浓度依赖性方式高效转导进入细胞；导入His-TAT-p38蛋白后，结果发现导入的野生型p38可增强内源性p38的功能，而无活性突变体则竞争性抑制了内源性p38 MAPK蛋白的功能，从而阻止或抑制其对内源性底物ATF2的磷酸化，使得信号不能传递下去，进而抑制p38 MAPK通路的活动。结论 成功建立了基于TAT的蛋白转运系统，并证实TAT能够以浓度和时间依赖方式高效转运蛋白进入真核细胞；进入细胞的 His-TAT-p38和His-TAT-p38无活性突变体蛋白均具有较高的生物学活性，在高渗刺激作用下前者可以增加p38磷酸化水平，而后者则显著抑制p38信号通路的活性。 关键词：I型人免疫缺陷病毒转录激活蛋白；p38丝裂原活化蛋白激酶；蛋白转导 中图分类号：Q291??文献标识码：A??文章编号：1673-4254(2006)05-0553-05 Cell-penetrating peptide-based functional study of p38 MAPKYANG Li-ping； LIU Zhi-feng； LI Yong-ming； LI Zhi-jie； JIANG YongDePartment of Pathophysiology and Guangdong Provincial Key Laboratory of Functional Proteomics, Southern Medical University, Guangzhou 510515, ChinaAbstract: Objective To construct a p38 MAPK protein delivery system based on TAT protein and study its functions in eukaryotic cells. Methods Recombinant vectors pHis-TAT-p38 and pHis-TAT-p38(AF) were constructed, and two recombinant proteins, His-TAT-p38 and His-TAT-p38(AF), were expressed and purified in E.coli. The two fusion proteins were then incubated with ECV304 cells, respectively. The phosphorylation of ATF2 was detected to assay the effect of His-TAT-p38 on endogeneious p38 activity after the cells were stimulated by sorbitol. Results The results of restriction enzyme digestion and DNA sequencing showed that the two recombinant vectors were correctly constructed. The recombinant proteins of His-TAT-p38 and His-TAT-p38(AF) were isolated and purified by SDS, and Western blotting suggested that His-TAT-p38 and its mutant with dual phosphorylation sites could enter the cells efficiently in a time- and concentration-dependent manne