酵母双杂交技术筛选与蛋白激酶Wee1相互作用的蛋白

Screening of proteins interacting with protein kinase Wee1 by yeast two hybrid system

目的应用酵母双杂交技术筛选出与Wee1蛋白激酶相互作用的候选分子,为进一步研究Wee1的分子功能提供理论基础。方法利用分子克隆的方法重组酵母双杂交质粒p GBKT7-Wee1,并验证其在酵母中的毒性、自激活能力和表达。利用酵母双杂交技术从人卵巢c DNA文库中筛选出与Wee1蛋白相互作用的蛋白,并在酵母中重新验证其相互作用。结果成功构建p GBKT7-Wee1诱饵质粒,验证了其无毒性及自激活能力,且可以在酵母中表达,可进一步进行酵母双杂交的筛选工作。结论利用酵母双杂交系统筛选出与Wee1相互作用的候选分子30个,为揭示蛋白激酶Wee1可能通过与其他蛋白相互作用进而调控细胞周期进程。

Objective To screen out the candidate molecules interacting with Wee1 protein kinase by yeast two hybrid technique,and to provide theoretical basis for further study of the molecular function of Wee1. Methods The yeast two hybrid plasmid p GBKT7-Wee1 was cloned by molecular cloning method,and its toxicity,self-activation ability and expression in yeast were verified. Screening of proteins interacting with Wee1 from human ovary c DNA library and the interaction between them was verified by yeast two hybrid technique. Results The bait plasmid p GBKT7-Wee1 was successfully constructed,and the plasmid had no toxicity and self-activation ability,and the bait plasmid could be expressed in yeast. Conclusion 30 candidate molecules interacting with Wee1 are screened by yeast two hybrid system,which reveales that protein kinase Wee1 might regulate cell cycle progression by interacting with other proteins.