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Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

ISSN号：2095-4352
期刊名称：《中华危重病急救医学》
时间：0
分类：Q555.7[生物学—生物化学] Q78[生物学—分子生物学]
作者机构：[1]Department of Microbiology and Immunology, the Burns Institute, First Hospital Affiliated to the Chinese PLA GeneralHospital, Beijing 100048, P.R. China
相关基金：Acknowledgement： This work was supported, in part, by grants from the National Basic Research Program of China （2005CB522602）, the National Natural Science Foundation （30672178, 30872683, 30800437）, and the National Natural Science Outstanding Youth Foundation of China （30125020）.

作者： Liping Yang Yongming Yao Zhiyong Sheng Xiaomei Zhu Yong Jiang[1]

关键词： P38丝裂原活化蛋白激酶, 真核细胞, 给药系统, 人类免疫缺陷病毒, 信号转导通路, 蛋白质转导, 时间依赖性, 穿透, Human immunodeficiency virus-1, transactivator of transcription, p38 mitogen-activated protein kinase, protein transduction： sorbitol

中文摘要：

客观 p38 激活 Mitogen 的蛋白质 kinase (MAPK ) 是各种各样的小径的一个交叉中心。在这研究，蛋白质 transduction 系统基于人的免疫不全抄写的病毒(HIV )-1 transactivator (梭织) ，它是进房间的外国蛋白质的有效交货肽，被采用在真核细胞的房间学习 p38 MAPK 功能。方法 p38 和它的主导的否定形式， p38AF，正确地被构造进 pET-His-TAT 向量证实 recombinant plasmids 通过限制酶消化并且 DNA 定序是有根据的。二蛋白质， His-TAT-p38 和 His-TAT-p38AF，被 SDS 页在 Escherichia 表示并且净化 coli。然后，他们分别地并且乐意地与 ECV304 房间被孵化 transduced 进在一个时间依赖者和剂量依赖者举止的房间。房间被山梨糖醇刺激。激活抄写因素(ATF ) 2 phosphorylation 水平用西方的污点被检查估计内长的 p38 的活动。与控制相比结果，当 His-TAT-p38AF 禁止了它时， His-TAT-p38 在刺激山梨糖醇的 ECV304 房间增加了水平 ofATF2 phosphorylation，这被发现当 His-TAT-p38AF 禁止了它时，显示 p38 MAPK 蛋白质交货系统基于梭织成功地被构造。TAT-p38 和它的主导的否定形式在 transduction 以后拥有了高生物的活动进 ECV304 房间由梭织蛋白质交货系统。结果显示出熔化与的那 p38AF 梭织能部分地禁止内长的 p38 信号小径的 transduction，并且另外的小径可能调整 p38 phosphorylation。我们的学习提供一条新奇小径禁止 p38 信号小径并且建立一个新方法学习 p38 的结论工作。

英文摘要：

Objective p38 Mitogen-activated protein kinase （MAPK） is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus （HIV）-1 transactivator of transcription （TAT）, which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor （ATF） 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

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