酵母双杂交文库筛选及hTRIP15与Y盒结合蛋白-1相互作用的探讨

Yeast-two-hybrid Screening and Identification of Interaction Between hTRIP15and Y Box Binding Pro tein-1

为探讨甲状腺素受体相互作用蛋白(hTRIP15)是否与其他核蛋白发生相互作用,以hTRIP15作为诱饵,用酵母双杂交技术筛选人肝cDNA文库,在高严格度选择培养基(SD-Trp-Leu-Ade-His)上长出的克隆773个,再经β-gal表达和PCR等技术进一步缩小范围,经Blastn搜寻最终显示2个重复克隆编码一种共调节辅因子——Y盒结合蛋白-1(YB-1).将插入序列与全长YB-1比较发现进入ORF内230bp,插入序列编码82aa,该蛋白称为YB-1-p,经蛋白体外翻译及GST-pulldown试验证实YB-1-p与hTRIP15发生相互作用.提示hTRIP15可通过与YB-1相互作用,参与调控MHCII及TSH受体等甲状腺细胞重要基因表达.

To explore the interaction of human thyroid receptor interacting prote in 15(hTRIP15)with other important nuclear molecules,a huma n liver cDNA library was screened using hTRIP15as a bait.After 773positive clones grew up on high strin gent (SD-Trp-Leu-Ade-His)culture plates,further narrowing was done with different techniques,includi ngβ-gal expression,PCR and so on.Using Blastn search,two positive clones that encode a coregulator-Y box bind ing protein-1(YB-1)were found.By comparison with full-length mRNA of YB-1,the insertion of the positive c lones encoded a portion of 82amino acids,called as YB-1-p.The interac-tion of YB-1-p and hTRIP15was identi fied using protein translation in vitro and GST pulldown assay.It sug-gests that hTRIP15regulate expression of key thyroid genes,such as HMC I I antigen and TSH receptor,by interacting with YB-1.