应用酵母双杂交系统筛选与TRF1相互作用的蛋白质

Screening proteins that interact with telomeric repeat binding factor 1 via yeast two-hybrid system

目的:应用酵母双杂交系统筛选与端粒重复序列结合蛋白1(TRF1)相互作用的蛋白质,探讨TRF1在HeLa细胞有丝分裂期的调控机制。方法:应用酵母双杂交技术,以pGBKT7-TRF1为诱饵质粒筛选人HeLa细胞cDNA文库,并对筛选结果进行测序及同源性分析。运用免疫共沉淀、体外沉降实验验证SAM68与TRF1是否能够相互结合。结果:酵母双杂交结果共发现5个可能与TRF1存在相互作用的蛋白,包括人SAM68蛋白、Cullin1蛋白、Plk1蛋白、Hsp70蛋白以及ATF5蛋白。体内免疫共沉淀实验和体外沉降实验证实SAM68能够与TRF1在体内与体外相互结合。结论:SAM68可能是一个新的TRF1结合蛋白,TRF1有可能参与了有丝分裂期的调控。

Aim：To screen and identify the interactive proteins of TRF 1 and provide clues for the studies on TRF 1 func-tion in mitosis.Methods：The human HeLa cell cDNA library was screened with pGBKT 7-TRF1 as bait plasmid by yeast two-hybrid system, and positive yeast clones were obtained , which were sequenced and analyzed with Blast in NCBI .The in vivo immunoprecipitation and the in vitro pulldown experiments were used to identify the interaction between SAM 68 and TRF1.Results：Five proteins,human SAM68,Cullin1,Plk1,Hsp70 and ATF5,that may interact with TRF1 were found.In＆amp;nbsp;vivo immunoprecipitation and in vitro pulldown experiments showed that SAM 68 could interact with TRF1.Conclusion：SAM68 is a novel TRF1 interacting protein;TRF1 may play an important role in the functional regulation of mitosis .