酵母双杂交技术分析耳聋基因功能的实验研究  

Experimental Study of Yeast Two-hybrid Technique to Explore the Deafness Genes’ Functions

作　　者：肖自安[1] 谢鼎华[1] 房海燕[2] 黄亮群[2] 施小六[3] 夏家辉[2] 

机构地区：[1]中南大学湘雅二医院耳鼻咽喉科和听力研究室,长沙410011 [2]中南大学医学遗传学国家重点实验室 [3]中南大学湘雅二医院消化内科

出　　处：《听力学及言语疾病杂志》2004年第4期235-238,T002,共5页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金资助课题 (编号 :3 0 0 0 0 0 94;3 0 0 70 80 7)

摘　　要：目的　在耳聋基因功能的蛋白质相互作用研究领域建立酵母双杂交实验技术 ,探讨第三代酵母双杂交系统在该领域的应用价值。方法　利用第三代MATCHMAKERGAL4酵母双杂交系统 ,以最常见的耳聋基因GJB2(Cx2 6 )为诱饵 ,以人胎脑cDNA文库为猎物 ,钓取Cx2 6的相互作用蛋白质的阳性克隆 ,DNA测序阳性克隆并行生物信息学分析。结果　从胎脑文库筛选到Cx2 6互作蛋白的 5 6个阳性克隆 ,对其中 2 6个克隆分析认定为 11个独立克隆 ,DNA测序发现 3个克隆是已克隆基因 ,8个克隆是新基因序列。结论　第三代MATCHMAKERGAL4酵母双杂交系统是筛选Cx2 6相互作用蛋白质的一个可靠的实验手段 。Objective To set up the experimental method of the yeast two-hybrid technique and to explore the feature of the Matchmaker Gal4 Two-hybrid System 3 in the research field of interaction proteins of deafness genes.Methods The most common deafness gene GJB2 (Cx26) was used to be bait and the human fetal brain cDNA library be prey, to screen the clones of the interaction proteins with Cx26 by the Matchmaker Gal4 Two-hybrid System 3. The library plasmid DNA was sequenced, and analyzed by bioinformatics techniques.Results 56 clones of the interaction proteins with Cx26 were screened out from the human fetal brain cDNA library. 11 different clones were identified from 26 in 56 clones by endonuclease spectrum of HaeIII and AluI. The inserts of 3 in 11 clones were partial sequences of genes that had been cloned and 8 in 11 clones were sequences of new genes.Conclusion The Matchmaker Gal4 Two-hybrid System 3 is an excellent research means to screen the interaction proteins with Cx26. The yeast two-hybrid technique can be used in the research field of protein interactions of deafness genes’ function.

关 键 词：酵母双杂交技术 耳聋基因 克隆 

分 类 号：R764.43[医药卫生—耳鼻咽喉科]