LIM结构域蛋白KyoT相互作用分子的筛选  

Sreening and Detecting of Proteins Interaction with KyoT

作　　者：李荣[1] 王冀姝[1] 孙强[1] 王键[1] 杨曦[1] 黄红燕[1] 周鹏[1] 韩骅[1] 

机构地区：[1]第四军医大学医学遗传学与发育生物学教研室,西安710032

出　　处：《Acta Genetica Sinica》2002年第2期175-180,共6页

基　　金：国家自然科学基金资助项目 (3 9970 3 76)~~

摘　　要：KyoT为LIM结构域蛋白 ,可与转录因子RBP Jk相互作用。为了研究其功能 ,用酵母双杂交法筛选了与KyoT相互作用的蛋白。以KyoT2为诱饵筛选人淋巴结cDNA文库 ,从 5× 10 6 个克隆中共获得 4 2个阳性克隆。提取质粒进行酶切鉴定 ,获得 2 2个非重复克隆。对此 2 2个克隆进行序列分析 ,共获得 13种基因 ,其中包括已知的KyoT相互作用蛋白RBP Jk和两个未知基因。另一种被筛选到的已知基因是激活状态的信号转导物和转录因子 1活化物的抑制蛋白 (proteininhibitorofactivatedsignaltransducerandactivatoroftranscription 1,PIAS1)。由于KyoT在小鼠睾丸中表达较高 ,因此进一步探讨了KyoT2与PIAS1的相互作用。并首先在酵母中证实KyoT2与PIAS1的相互作用并得到阳性结果。进而在大肠杆菌中表达纯化了KyoT2蛋白 ,并以此为抗原制备了抗KyoT2多克隆抗体 ,再用GST pulldown实验验证了KyoT2和PIAS1在体外的相互作用。成功筛选了KyoT相互作用蛋白 ,发现并验证了KyoT与PIAS1的相互作用 。To study the function of KyoT2 in vivo ,two hybrid yeast, purification of KyoT2 protein,preparation of antibody and GST pulldown methods were used in the experiments. 42 clones were obtained after 5×10 6 clones were screened by four kinds of nutrition limitation and β galactosidase assay,22 clones were obtained after restriction of positive clones. Finally,13 genes were obtained by sequence assay. Two of these were RBP Jk and PIAS1. After they and KyoT2 changed vectors,negative two hybrid yeast was finished. The result was positive;Using KyoT2 protein and antibody GST pulldown of KyoT2 and RBP Jk,KyoT2 and PIAS1 were done,the result was also positive. Therefone KyoT2 interacted with RBP Jk and PIAS1.

关 键 词：LIM蛋白 KyoT PIAS1 酵母双杂交 相互作用分子 转录 筛选 

分 类 号：Q591.2[生物学—生物化学]