酵母双杂交系统筛选Mps1相互作用蛋白

Screening interactive proteins Mps1 by yeast two hybride system

目的:对前期筛出的与Mps1可能有相互作用的两种蛋白用酵母双杂交回转,验证与Mps1有相互作用的蛋白,为染色体分离机制研究提供线索,并为蛋白质相互作用网络提供资料。方法:应用酵母双杂交技术,将前期以pDBLeu-Mps1为诱饵质粒筛选成人肝脏cDNA文库,得到的Leu+LacZ+阳性克隆进一步回转酵母进行验证,即将文库质粒与诱饵质粒一对一重新转入酵母体内对相互作用进行验证。并对验证后的阳性克隆的外源片段进行测序及同源性分析。结果:用同源性分析,从人胚胎cDNA文库筛选到的与M ps1有相互作用的两个蛋白在酵母双杂交回转实验中仅有一个为阳性,另一个相互作用为阴性。结论:应用酵母双杂交系统从人胚胎cDNA文库中筛选并初步验证了与Mps1有相互作用的蛋白,为Mps1蛋白的功能研究奠定了基础。

objective：To screen and identify the interactive proteins of Mps1 and provide clues for the studies on Mps1 function on the chromosome segregation.Methods：The human embryonic cDNA library was screened with pDBLeu-Mps1 as bait plasmid by yeast two-hybrid system.And two Leu＋ and LacZ＋ positive yeast clones were obtained,which were contransformed to the MaV203 along with pDBLeu-Mps1 plasmid one to one by yeast simultaneous contransformation so as to identify the protein interactions again. The inserted fragments of identiyied positive clones were sequence and analyzed with BLAST in NCBI. Results： Only one positive clone identified one to one yeast simultaneous cotransformation, and the other was negative. The positive clone fragment was MAD1 protein by BLAST analysis in NCBI.Conclusion：To screen and initially identify the interactive protein of.Mps1 this research is a foundation for the study on the founctions of Mps 1.