凋亡蛋白Daxx与TRAF3相互作用的筛选及相互作用位点的鉴定  

Isolation and identification of TRAF3 interacting with Daxx via TRAF domain

作　　者：张翠莉[1] 成海恩[2] 王应雄[3] 

机构地区：[1]武警四川总队医院检验科,四川乐山614000 [2]浙江大学医学院附属第二医院检验科,浙江杭州310007 [3]重庆医科大学公共卫生学院遗传学教研室,重庆400016

出　　处：《第四军医大学学报》2007年第9期826-830,共5页Journal of the Fourth Military Medical University

摘　　要：目的:利用酵母双杂交技术在成人肝文库中筛选能与死亡结构域相关蛋白(Daxx)相互作用的蛋白,并用哺乳动物细胞中进行了免疫共沉淀验证,以进一步了解Daxx的功能.方法:利用PCR在成人肝cDNA文库中扩增Daxx全长ORF,构建pDBLeu-Daxx载体.转化MaV203,检测细胞毒性和自激活作用,确定His基础表达的3AT浓度.依次转化成人肝cDNA文库.在营养缺陷型培养基上挑取三阳性克隆.进行回转实验和免疫共沉淀验证,并利用β-gal分析进行了相互作用结构域的鉴定.结果:筛库转化效率达到5.8×106个克隆.筛选到了一株能与Daxx相互作用的阳性克隆,DNA序列分析和同源检索表明该阳性克隆为肿瘤坏死因子受体相关因子3(TRAF3).通过回转及co-IP证实了TRAF3能与Daxx相互作用,β-gal分析发现TRAF3通过TRAFDomain与Daxx相互作用.结论:TRAF3通过TRAFDomain与Daxx相互作用,可能参与了Daxx的调控和功能,为进一步了解Daxx作用的分子机制奠定了基础.AIM： To screen proteins that can interact with death domain-associated protein （Daxx） by yeast two-hybrid technique in human adult liver cDNA library, and verify the result with coimmunoprecipitation in mammal cells for further understanding the function of Daxx. METHODS： Full-length cDNA of Daxx was obtained by PCR amplification from adult liver cDNA library, then they were cloned into pDBLeu vector according to their open read- ing frame （ORF）. The bait vector was transformed into yeast MaV203 cell and the self-activation effect was assayed by measuring the expression of HIS3. Then the human adult liver cDNA library was screened sequentially, and the clones which could grow on SC-Leu-Trp-His ＋ 3AT plate and SC-Leu-Trp-Ura plate, and also could activate X-gal were picked up. The positive clones were retransformed into yeast and verified by co-immunoprecipitation. The interactive domain was identificated by β-gal analysis. RESULTS： A positive clone which could interact with Daxx was obtained. The positive clone was proved as turnout necrosis factor receptor （TNFR） by DNA sequence analysis and homology searching on NCBI. TRAF3 was confirmed to be able to interact with Daxx by co-transforming TRAF3 and Daxx into yeast MaV203 and co-imnnnunoprecipitation analysis in HepG2. β-gal analysis indicated that TRAF3 interacted with Daxx via TRAF domain. CONCLUSION： TRAF3 may be involved in the regulation of Daxx, which provides us a clue for further understanding the function of Daxx.

关 键 词：凋亡 死亡结构域相关蛋白 TRAF3 酵母双杂交 

分 类 号：Q51[生物学—生物化学] R349.5[医药卫生—基础医学]