利用酵母双杂交技术筛选与hPOT1相互作用的新蛋白

摘要

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 sereen the interacting protein of human protection of telomeres 1 (hPOT1). Methods:The encoding fragment of hPOTl(300～634 aa) was inserted into pGBKT7 as a bait. SD/-Leu/-Trp nutrient medium and X-o-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-immunopreeipitation and GST pull-down. Conclusion: hPOT1 can interact with NM23B, and the biology of the interaction can be further studied based on this interaction.