Tec家族蛋白酪氨酸激酶Itk PH结构域与OS-9蛋白的相互作用  

The Interaction Between Tec Family Protein Tyrosine Kinase Itk PH Domain and OS-9 Protein

作　　者：韩月恒[1] 张文红[1] 张晓光[1] 刘新平[1] 药立波[1] 

机构地区：[1]第四军医大学生物化学与分子生物学教研室,西安710032

出　　处：《中国生物化学与分子生物学报》2003年第4期499-503,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目 (No .3 9870 718;No .3 982 5 113 )~~

摘　　要：用酵母双杂交技术筛选与ItkPH结构域相互作用的蛋白分子 ,以了解Itk的功能及其在T细胞信号转导中的位置与作用 .Itk的PH结构域扩增后克隆入酵母双杂交系统的pLexA载体 ,转化酵母细胞EGY4 8(p8op lacZ) ,经检测PH结构域无自激活作用 ,且对酵母细胞无毒性作用 .用PH结构域作为“钓饵”蛋白 ,在酵母双杂交系统中筛选构建于AD载体的T细胞cDNA文库 .将PH结构域及筛库所得基因片段分别进行融合表达 ,用于体外结合实验 ,进一步证实二者的相互作用 .经营养缺陷选择、诱导筛选和鉴定确证 ,筛库所得的插段约 15 0 0bp的文库质粒为一真阳性克隆 .经blast比较分析为骨肉瘤、横纹肌肉瘤等肿瘤组织中高表达的os 9基因 .体外结合实验也表明 ,ItkPH结构域可与该基因表达产物结合 .Itk的PH结构域可与OS 9蛋白相互作用 .The yeast two hybrid system was used to explore the partner of inducible T cell kinase (Itk) and the role of Itk in T cell signaling were investigated. Itk PH domain fragments were amplified by PCR using cloned Itk as template. The PH domain fragment was then cloned into a pLexA vector, which contained the DNA binding (BD) domain. The recombinant was used as a bait to screen a T cell cDNA library after it was testified to have neither ability of autonomous reporter gene activation, nor yeast cell toxicity. The genes of PH domain and the positive library cDNA fragment were cloned into prokaryotic expression vectors pGEX4T 1 and pET28a, respectively. And using the soluble fusion proteins an in vitro binding assay was employed to verify their interaction. One of the positive clones, sequenced and analyzed in the BLAST, was confirmed to be the gene fragment of OS 9, a protein which exists in many tissues while expressed highly in some sarcomas. The binding assay of the two fusion proteins demonstrated that they interacted with each other in vitro . OS 9 protein may be a potential partner of the Itk PH domain. The significance of the interaction between these two proteins is to be studied.

关 键 词：Tec家族蛋白 酪氨酸激酶 ITK PH结构域 OS-9蛋白 酵母双杂交系统 融合表达 

分 类 号：Q51[生物学—生物化学] Q786