HLH缺失型Id2候选相互作用蛋白  

A novel class of HLH-deleted Id2 interacting proteins

作　　者：伍志强[1] 孟元光[2] 赵亚力[1] 杨洁[1] 田丽媛[1] 司艺玲[1] 韩为东[1] 

机构地区：[1]解放军总医院分子生物室,北京100853 [2]解放军总医院妇产科,北京100853

出　　处：《军事医学科学院院刊》2007年第4期317-321,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然基金项目资助(30670809)

摘　　要：目的:筛选可以与螺旋-环-螺旋(HLH)结构域缺失的Id2相互作用的蛋白。方法:用重叠延伸PCR方法将Id2中的HLH结构域缺失,并插入到pGBKT7载体,构建BD:Id2-DBM-δHLH融合诱饵质粒;构建MCF-7细胞的dscDNA文库;采用共转化方法进行1d2-DBM-δHLH相互作用蛋白的酵母双杂交筛选,采用PCR方法扩增阳性克隆中的AD:cDNA序列并测序;将获得的AD:cDNA质粒分别与BD:Id2-DBM-δHLH诱饵质粒共转化酵母进行配对验证。结果:酵母双杂交方法共筛选到19个阳性克隆,PCR方法在这19个克隆中共扩增到28条片段,序列测定证实含18个不同的基因,对其中的13个进行配对验证后证实了其中8个与HLH缺失型Id2相互作用,这8个基因分别是:UXT、VIM、KRT7、FHL2、SEI1、PCBP1、SIVA和LSM2。结论:本研究首次利用HLH缺失型Id2作为诱饵,利用酵母双杂交技术筛选识别了一族新的Id2相互作用蛋白,为进一步研究Id2的功能调控以及非HI.H依赖的功能活性奠定基础。Objective： To screen the helix-loop-helix （HLH）-independent Id2 interaction proteins. Metbods：HLH domain was deleted from the complete coding Id2 cDNA sequence by the sequential PCR amplification, and the HLH-deleted Id2 was inserted into pGBKT7 vector to construct BD： Id2-DMB-δHLH bait plasmid. Double-strand cDNA library was generated from MCF-7 cells. The cotransformation assay was used to screen the interacting proteins in yeast two-hybrid system. The AD： cDNA harboring in the positive colonies was amplified and sequenced. The interacting proteins with Id2-DMB- δHLH were verified by direct yeast two-hybrid assays. Results：Twenty-eight PCR products with different sizes derived from 19 positive clones were obtained by yeast two-hybrid screening, which represented 18 different genes. Eight genes out of 13/18 AD： cDNA proteins were confirmed to interact with BD： Id2-DMB-δHLH protein in yeast cells. The eight genes were UXT, VIM, KRTT, FHL2, SEI1, PCBP1, SIVA and LSM2. Conclusion：A novel class of Id2 interacting proteins was identified using HLH-deleted Id2 as bait in yeast two-hybrid screen system. These results provide essential clues for further investigation of the functional regulation of Id2 and identification of HLH-independent Id2 functions.

关 键 词：ID2 螺旋-环-螺旋构型 酵母菌 双杂交系统技术 蛋白相互作用 

分 类 号：Q75[生物学—分子生物学]