应用酵母双杂交技术筛选与RANK蛋白新基序IVVY相互作用的蛋白  

Screening of proteins interacting with IVVY motif of RANK protein by yeast two-hybrid technique

作　　者：王顺清[1] 钟雯婷[1] 邓晖[2] 毛平[1] 许艳丽[1] 

机构地区：[1]广州医学院附属市一人民医院血液科,广东广州510180 [2]广州医学院附属市一人民医院输血科,广东广州510180

出　　处：《中国病理生理杂志》2012年第4期730-732,737,共4页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30871101);广州市医药卫生科技重点资助项目(No.2006-ZDi-02)

摘　　要：目的:应用酵母双杂交技术筛选小鼠巨噬细胞中与核因子κB受体激活因子(RANK)蛋白上新基序IVVY相互作用的蛋白,以探寻RANKL/RANK系统中介导破骨细胞形成的RANK下游新信号转导蛋白。方法:应用GAL4酵母双杂交系统3,构建仅包含编码新基序535IVVY538的一小段RANK的cDNA片段为诱饵质粒pGBKT7-IVVY,并与巨噬细胞cDNA文库质粒pGADT7-library共转化AH109酵母,筛选与RANK蛋白新基序IVVY相互作用的蛋白,通过回复性杂交实验验证其可靠性,并对阳性克隆进行测序和基因同源性分析。结果:筛选出4个可能与IVVY基序有相互作用的蛋白,包括Ring1和YY1结合蛋白(RYBP)、ATP结合盒、E2F转录因子和热休克蛋白8,其中表达RYBP的阳性克隆出现频率高、速度快。结论:应用酵母双杂交技术成功地筛选出4个可能与IVVY基序相互作用的候选蛋白,其中RYBP可能性最大。AIM： To explore the new downstream signal transduction proteins mediating osteoclast formation in RANKL/RANK system by the technique of yeast two-hybrid in mouse macrophages.METHODS： Using MatchmakerTM GAL4 Two-Hybrid System 3,a small piece of RANK cDNA which harbored a cDNA fragment coding a novel motif,535IVVY5381,was sub-cloned into pGBKT7 to construct the bait plasmid（named as pGBKT7-IVVY）.The yeast strain AH109 co-transformed with pGBKT7-IVVY and a yeast expression macrophage cDNA library（pGADT7-library） was used to screen the proteins interacting with IVVY motif of RANK.Repeated yeast two-hybridization experiments were conducted to exclude the false positive clones and to verify the interactants.The positive clones were sequenced and analyzed with BLAST in NCBI.RESULTS： Four candidate proteins probably interacting with IVVY were obtained,including Ring1 and YY1 binding protein（RYBP）,ATP-binding cassette,E2F transcription factor and heat-shock protein 8.The clone coding RYBP was screened out frequently and quickly.CONCLUSION： Four candidate interactive proteins are successfully identified using yeast two-hybrid screening.RYBP is the most probable one.

关 键 词：酵母双杂交 RANKL/RANK系统 IVVY基序 相互作用蛋白质 

分 类 号：R733.3[医药卫生—肿瘤]