TAT-p38(AF)融合肽的构建及其对紫外线诱导的p38 MAPK通路激活的抑制作用  

Construction and inhibitory effect of TAT-p38(AF) on UV-induced activation of p38 MAPK pathway

作　　者：张秀娟[1] 刘亚伟[2] 王娟[2] 邓鹏[2] 姜勇[2] 

机构地区：[1]广东医学院,广东湛江524023 [2]南方医科大学广东省功能蛋白质组学重点实验室

出　　处：《山东医药》2010年第19期19-21,共3页Shandong Medical Journal

基　　金：国家自然科学基金面上项目(30572151)

摘　　要：目的构建TAT蛋白转导域介导的p38(AF)MAPK蛋白转导系统,研究该蛋白转导系统对紫外线刺激引起的p38通路激活作用的影响。方法将p38无活性突变体p38(AF)亚克隆至含有TAT蛋白转导域的pET-14b-TAT质粒,原核表达、纯化His-TAT-p38(AF)融合蛋白。激酶实验检测His-TAT-p38(AF)自身磷酸化作用。将该蛋白与NHI/3T3细胞孵育,Western blot法检测该蛋白的蛋白转导功能;紫外线照射后,检测内源性p38及其底物ATF2的磷酸化水平。结果酶切、测序表明质粒构建正确;目的蛋白被正确表达;His-TAT-p38(AF)融合蛋白无自身磷酸化作用;His-TAT-p38(AF)蛋白以浓度依赖性方式高效转导入细胞;His-TAT-p38(AF)的导入抑制紫外线诱导的内源性p38及其底物ATF2的磷酸化。结论TAT蛋白转运系统能高效转导外源蛋白进入真核细胞;p38无活性突变体TAT-p38(AF)抑制紫外线诱导的p38 MAPK通路的激活。Objective To construct inactive p38 mutant（AF）MAPK protein transduction system mediated by TAT PTD and study the effect of the protein transduction system on ultraviolet ray（UV）-induced activation of p38 MAPK pathway.Methods p38（AF） was subcloned into pET-14b-TAT plasmid.His-TAT-p38（AF） fusion proteins were expressed and purified.Kinase assay was performed to detect the autophosphorylation of His-TAT-p38（AF） fusion proteins.The protein transduction effect of the fusion protein was detected by Western blot after incubated with NIH/3T3 cells;the phosphorylation of endogenous p38 MAPK and ATF2 were examined.Results The recombinant plasmid was correctly constructed and the interest proteins were correctly expressed.His-TAT-p38（AF） fusion proteins was not phosphorylated.His-TAT-p38（AF） fusion proteins entered NIH/3T3 cells effectively in a concentration-dependent manner,and the introduction of His-TAT-p38（AF） proteins into NIH/3T3 cells inhibited UV-induced phosphorylation of endogenous p38 MAPK and ATF2.Conclusion The protein transduction system mediated by TAT PTD can effectively delivery the exogenous proteins into the eukaryotic cells.TAT-p38（AF） fusion proteins can partly inhibit the UV-induced activation of p38 MAPK pathway.

关 键 词：P38丝裂原活化蛋白激酶 紫外线 蛋白转导域 

分 类 号：Q291[生物学—细胞生物学]