Tec家族蛋白酪氨酸激酶ItkPH结构域的克隆鉴定及其在酵母双杂交系统中自激活作用的检测

Cloning and Identification of Tec Family Protein Tyrosin Kinase Itk PH Domain and Detection of its Self activation in the Yeast Two hybrid System

Tec家族蛋白酪氨酸激酶Itk特异地表达于T细胞 ,对T细胞的成熟与活化等具有很大影响 ,因而可能在T细胞的信号转导过程中处于重要位置。我们利用PCR的方法从含Itk基因全长cDNA的pME18S EMT质粒中扩增出其PH结构域基因片段 ,并将其克隆入酵母双杂交载体pLexA ,经测序鉴定正确 ,转化酵母细胞EGY48(p8op lacZ ) ,挑取阳性克隆 ,利用液相法和平板法检测 ,证实PH结构域无自激活作用 ,且对宿主菌无毒性作用 ,可用于T细胞cDNA文库的筛选 ,捕捉与之相互作用的蛋白。

Tec family protein tyrosine kinase Itk is expressed specifically in T cells and plays an important role in T cells differentiation and activation In order to fish out PH domain interacted protein for Itk,we amplified its PH domain cDNA fragment from the plasmid pME18S emt,which contains the full length of Itk cDNA by PCR Then we constructed two pLexA vectors,which would express Itk PH domain fusion protein,either bearing three N terminal prolines as a linker or not,with DNA binding domain of transcriptional regulator in yeast cells After sequencing,the right recombinants were transformed into yeast cell EGY48(p8op LacZ) respectively The β galactosidase activity of the positive clones were tested using both liquid assay and plate assay Experimental result suggests that PH domain has neither ability of autonomous reporter gene activation,nor yeast cell toxicity,so that it could be used in screening of T cell cDNA library to trap the protein interacting with it