用酵母双杂交系统鉴定DNA-PKcs磷酸化簇区域和DNA-PKcs单链抗体anti-DPK3-scFv间的相互作用  

Identification of Interaction Between DNA Dependent Protein Kinase Catalytic Subunit(DNA-PKcs) Phosphorylation Cluster and DNA-PKcs scFv Antibody anti-DPK3-scFv by Yeast Two-Hybrid Assay

作　　者：吴成林[1] 刘晓丹 王欲晓[1] 杜丽[2] 付凯飞[1] 周平坤[2] 周丽君[1] 

机构地区：[1]海军总医院中心实验科,北京100048 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《生物技术通讯》2016年第3期335-339,共5页Letters in Biotechnology

基　　金：国家自然科学基金(81172119;81273311)

摘　　要：目的:构建DNA依赖蛋白激酶催化亚单位(DNA-PKcs)磷酸化簇区域的酵母双杂交诱饵载体及针对该区域的单链抗体anti-DPK3-scFv的猎物蛋白的表达载体,并进行活性和相互作用鉴定。方法:应用PCR方法扩增DNA-PKcs磷酸化簇区域和anti-DPK3-scFv并将它们分别克隆至诱饵载体p GBKT7和猎物载体p GADT7中,经酶切和测序鉴定正确后,用LiAc法转化到酵母菌株AH109中,鉴定诱饵蛋白和猎物蛋白的毒性和自激活活性,并通过酵母双杂交方法验证DNA-PKcs磷酸化簇区域和anti-DPK3-scFv的相互作用。结果:扩增出DNA-PKcs磷酸化簇区域和anti-DPK3-scFv并分别将它们克隆至p GBKT7和p GADT7载体中,转化酵母细胞发现它们对酵母细胞的生长均无毒害作用,自激活活性分析显示诱饵质粒和猎物质粒均无自激活活性,并证实DNA-PKcs磷酸化簇区域能够与anti-DPK3-scFv在体外直接相互作用。结论:构建了DNA-PKcs磷酸化簇的诱饵质粒p GBKT7-DPC,为后续筛选与DNA-PKcs磷酸化簇区域相互作用的蛋白奠定了实验基础。Objective：To construct yeast two-hybrid bait vector for DNA dependent protein kinase catalytic subunit（DNA-PKcs） phosphorylation cluster and prey vector for DNA-PKcs phosphorylation cluster scFv antibody antiDPK3-scFv,determine the activity and interaction of bait and prey proteins.Methods：The fragments of DNA-PKcs phosphorylation cluster and anti-DPK3-scFv were amplified using PCR and cloned into pGBKT7 and pGADT7 separately.After identified by enzyme digestion analysis and DNA sequencing,the bait and prey plasmids were transformed into the yeast strain AH109 by LiAc method,and the toxicity and self-activation of bait and prey proteins in yeast cells were tested.Furthermore,the interaction between DNA-PKcs phosphorylation cluster and anti-DPK3-scFv was analyzed using yeast two-hybrid assay.Results：DNA-PKcs phosphorylation cluster and anti-DPK3-scFv were amplified successfully and cloned into pGBKT7 and pGADT7 respectively.The bait and prey plasmids were transformed into yeast cells and proved to have no toxicity and self-activation in yeast cells.Moreover,DNA-PKcs phosphorylation cluster could interact with anti-DPK3-scFv directly in vitro.Conclusion：The bait plasmid for DNA-PKcs phosphorylation cluster named pGBKT7-DPC was successfully constructed and lays the foundation for further research on screening the proteins interacted with DNA-PKcs phosphorylation cluster.

关 键 词：酵母双杂交 DNA依赖蛋白激酶催化亚单位(DNA-PKcs) 磷酸化簇 anti-DPK3-scFv 相互作用 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]