酵母双杂交筛选与分化抑制因子Id1′相互作用的蛋白

Screening of the protein interacting with inhibitor of differentiation 1′ using yeast two hybrid method

目的 筛选成人肺组织文库中与分化抑制因子Id1′相互作用的蛋白。方法 构建重组诱饵质粒pHybLex Zeo Id1′ ,转化入酵母细胞EGY48 pSH18 3 4,检测重组质粒有无非特异激活报告基因 ;通过筛选成人肺组织文库 ,获取真阳性文库质粒 ,进行测序和同源性比较 ,获得真阳性克隆。结果 经测序证实 ,重组诱饵质粒pHybLex Zeo Id1′构建成功 ;将重组质粒转化入酵母细胞EGY48 pSH18 3 4,经检测无非特异激活功能。进一步通过对文库进行筛选 ,获得一个真阳性克隆 ,通过测序和同源性比较证实该阳性克隆是酪氨酸蛋白激酶Fyn。结论 分化抑制因子Id1′与Fyn在酵母细胞中存在相互作用。

Objective To obtain the protein interacting with inhibitor of differentiation1′(Id1′). Methods The recombinant bait plasmid pHybLex/Zeo Id1′ was constructed and transformed into yeast strain EGY48/ pSH18 34 to test pHybLex/Zeo Id1′ for non specific activation. Adult human lung cDNA libraries were screened to obtain true positive library plasmid. The true positive library clone was obtained by sequencing and basic local alignment sequence tool (BLAST). Results The recombinant bait vector, named as pHybLex/Zeo Id1′, was confirmed by sequencing. pHybLex/Zeo Id1′ was transformed into yeast strain EGY48/pSH18 34 and the transformants had no autonomously activated reporter genes. One true positive clone, obtained by screening of the adult human lung cDNA libraries, was confirmed to be Fyn by sequencing and BLAST. Conclusion Id1′ can interact with Fyn.