人缝隙连接蛋白30互作蛋白的筛选及初步鉴定  

Screening and identification interaction proteins of connexin 30

作　　者：贺定华[1] 冯永[1] 梅凌云[1] 贺楚峰[1] 

机构地区：[1]中南大学湘雅医院耳鼻咽喉科教研室,长沙410008

出　　处：《中华耳鼻咽喉头颈外科杂志》2009年第9期758-761,共4页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：基金项目：国家863计划（2007AA022445）;国家自然科学基金（30470954、30671150）;“十一五”国家攻关计划（2007BA118813）;高等学校博士学科点专项基金（20060533043）

摘　　要：目的通过筛选和鉴定缝隙连接蛋白30（connexin30，Cx30）的互作蛋白，探讨互作蛋白对Cx30功能的影响。方法构建Cx30．C—pGEX-4T-2-GST融合表达载体，纯化Cx30-C—GST融合蛋白和谷胱甘肽-S-转移酶（glutathione—S—transferase，GST），以纯化蛋白在胎儿脑组织裂解蛋白中沉降互作蛋白，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS—PAGE）分离互作蛋白，切取互作蛋白形成的差异蛋白带进行质谱分析，通过NCBInr数据库筛选互作蛋白，应用免疫荧光染色共定位进行初步鉴定。结果共筛选到4个互作蛋白，即Keratin16、Camk2b、Tubulinbeta-3以及alpha—tubulin，Cx30与Keratin16和Tubulinbeta-3存在免疫共定位。结论Keratin16、Camk2b、Tubulinbeta-3及alpha—tubulin是Cx30互作蛋白，可能在蛋白的组装，运输及控制缝隙连接通道开关等方面影响Cx30的功能。Objective To explore interaction proteins affect functions of connexin 30 （Cx30） by screening and identification interaction proteins of Cx30. Methods The fusion expression vecto of CX30-C- terminal functional domain-pGEX-4T-2-GST was constructed, and then, fusion protein and GST were purified. They were incubated with the proteins of the foetus brain tissue disruption to pull down interaction proteins. The interaction proteins were separated by SDS-PAGE. Differeal straps were cut to enzymolysis to prepare for mass chromatographic analysis, and then to index and screen interaction proteins in NCBInr database. The interaction proteins were identified by immunolocalization. Results The four interaction proteins of Cx30 were screened in the foetus brain tissue, as follow, Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin. Cx30 was proved to coexist with Keratin 16 and Tubulin beta-3. Conclusions Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin are the interaction proteins of Cx30. The interaction proteins affect the assembly, intracellular transport, and channel switch of Cx30.

关 键 词：听觉丧失 连接蛋白类 谷胱甘肽转移酶 

分 类 号：R686[医药卫生—骨科学]