BIK和SOCS3相互作用的筛选和鉴定

Isolation and identification of SOCS3 interacting with BIK

目的利用酵母双杂交技术筛选促凋亡因子BIK的相互作用蛋白。方法利用PCR在成人肝cDNA文库中扩增BIK全长ORF，构建pDBLeu—BIK载体。转化MaV203，检测细胞毒性和自激活作用，确定His基础表达的3AT浓度，依次转化成人肝cDNA文库。在营养缺陷型培养基上挑去三阳性克隆，进行回转验证，以及利用GST—pull down、CO—IP在体内外进行验证。结果成功构建pDBLeu—BIK载体，转化酵母细胞MaV203，无细胞毒性、无自激活作用，3AT浓度为25mmol／L。经回转实验、GST—pulldown、CO—IP证实了BIK和细胞因子信号通路抑制因子3（SOCS3）之间具有相互作用。结论BIK和SOCS3之间的相互作用可能是凋亡调控的新机制。

Objective To screen proteins in human adult liver cDNA library interacting with BCL2 - interacting killer（BIK） by yeast two - hybrid technique. Methods BIK was amplified by PCR in human adult liver cDNA library. The recombinant vector pDBLeu - BIK was constructed and transformed into yeast MaV203, and the expression of GAIA BD - BIK fusion protein was dectected by Western blotting. The self- activation was examined, and 3 - amino - 1,2,4 - triazole （3AT） concentration required to titrate basal HIS expression level was determined. The human adult liver cDNA library was screened sequentially. The clones that grew on SC - Leu - Trp - His - Ura plates was employed to test if they could activate His, Ura and LacZ report genes. The plasmids from those clones that could activate the 3 report genes were isolated, then cotransformed with pDBLeu - BIK, and verified with GST - pull down and co - IP after sequencing. Results The bait vector pDBLeu - BIK was successfully constructed and expressed correctly in MaV203. One clone suppressor of cytokine signaling 3 （SOCS3） was obtained and could interact with BIK v/a cotransformation, GST - pull down and co - IP. Conclusions The interaction between BIK and SOCS3 may be a new mechanism of apoptosis regulation.