CytoTrap酵母双杂交系统筛选p85α蛋白相互作用蛋白  

Screening of interacting proteins with p85α by CytoTrap yeast two-hybrid system

作　　者：李小娜[1,2] 时小燕[1,2] 米小轶[3] 黄传书[3] 刘晋祎[2] 

机构地区：[1]河南大学中药研究所,河南开封475004 [2]第三军医大学军事预防医学院军事毒理学研究所,重庆400038 [3]美国纽约大学环境医学研究所,纽约10987

出　　处：《河南大学学报（医学版）》2015年第2期82-86,90,共6页Journal of Henan University:Medical Science

基　　金：中国博士后科学基金(2013M542496);河南省教育厅科学技术研究重点项目(13A310094)

摘　　要：目的筛选与p85α蛋白发生相互作用的新蛋白质分子。方法 PCR扩增小鼠p85α基因全长,并将此基因重组入pSOS载体,构建诱饵质粒pSOS-p85α,经酶切及测序鉴定构建正确后将其转化酵母菌cdc25Hα,检测其在酵母中的表达及有无自激活作用,并利用CytoTrap酵母双杂交系统筛选与诱饵蛋白p85α相互作用的蛋白质分子。结果成功获得重组诱饵质粒pSOS-p85α,并验证其可在酵母细胞中表达诱饵蛋白pSOS-p85α,且诱饵质粒在此双杂交系统中无自激活。利用此系统筛选出4个能与p85α相互作用的细胞蛋白,分别为v-Ki-ras2克尔斯滕大鼠肉瘤病毒癌基因同族体蛋白KRAS、小鼠着丝粒蛋白K、小鼠多聚嘧啶结合蛋白PTBP1、小鼠雄性激素调节蛋白RP2。结论成功筛选出小鼠p85α蛋白的相互作用蛋白,为进一步研究小鼠p85α蛋白及其相关信号通路的生物学功能提供了新线索。Objective To screen the mouse proteins that interacts with p85α.Methods The recombinant plasmid pSOS-p85αwas constructed by inserting p85αfragment into the bait vector.After sequence verification the plasmid was transformed into competent yeast cells.Then,the expression and self activation of pSOS-p85αprotein was detected in the yeast cells.The mouse proteins interacting with the bait protein pSOS-p85α were screened with CytoTrap yeast two-hybrid system.Results The reconstructed plasmid including mouse p85αgene could express pSOS-p85αfusion protein in the yeast cells without self-activation of the protein.CytoTrap yeast two-hybrid system identified 4mouse proteins that interacted with p85α,including Mus musculus v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog,mouse musculus centromere protein K,mouse musculus polypyrimidine tract binding protein 1and mouse kidney testosterone-regulated RP2.Conclusion Four p85α-binding mouse proteins have been identified using the CytoTrap yeast two-hybrid system,which provides clues for further investigation of the role of p85αprotein and its downstream signal pathway.

关 键 词：p85α蛋白 CytoTrap酵母双杂交 蛋白质相互作用 

分 类 号：R730.2[医药卫生—肿瘤]