利用酵母双杂交技术筛选与hPOT1相互作用的新蛋白

Screening the Novel Interacting Proteins of hPOT1 by Yeast Two-Hybrid System

目的:利用酵母双杂交系统筛选与人POT1(human protection of telomeres 1,hPOT1)相互作用的蛋白。方法:以hPOT1的300～634氨基酸片段为诱饵,在人乳腺cDNA文库中筛选能与hPOT1相互作用的蛋白质;运用营养缺陷型培养基和X-α-Gal实验排除假阳性,并对阳性克隆进行序列测定和比对。结果:经过酵母双杂交筛选,发现7个与hPOT1相互作用的蛋白;选取NM23B与hPOT1通过GST-pull down和免疫共沉淀进行进一步的验证,结果证明它们确实存在相互作用。结论:hPOT1能与NM23B发生相互作用,在此实验基础上可以进一步研究NM23B与hPOT1相互作用的生物学意义。

Objective：To screen the interacting protein of human protection of telomeres 1（hPOT1）.Methods：The encoding fragment of hPOT1（300～634 aa） was inserted into pGBKT7 as a bait.SD/-Leu/-Trp nutrient medium and X-α-Gal were used to exclude the false positive.The positive clones were sequenced and blast.Results：Seven proteins were found to interact with hPOT1,and one protein named NM23B was selected and further confirmed interacting with hPOT1 by co-immunoprecipitation and GST pull-down.Conclusion：hPOT1 can interact with NM23B,and the biology of the interaction can be further studied based on this interaction.