应用酵母双杂交方法筛选与鉴定组蛋白乙酰基转移酶hTIP60β相互作用蛋白  

Screening and identification of interaction proteins of human TIP60β by yeast two- hybrid system

作　　者：黄轶[1] 曾昭淳[2] 

机构地区：[1]重庆医科大学附属儿童医院临床分子医学中心,重庆400014 [2]重庆医科大学基础医学院生物化学与分子生物学教研室,重庆400016

出　　处：《第三军医大学学报》2010年第14期1495-1498,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30701004)~~

摘　　要：目的克隆人组蛋白乙酰基转移酶TIP60β基因,应用酵母双杂交技术研究筛选与TIP60β发生相互作用的蛋白。方法运用RT-PCR方法从人脑组织克隆TIP60β cDNA,构建酵母双杂交BD诱饵载体pGBKT7-TIP60β,以其为诱饵从成人肝cDNA文库中筛选与之相互作用的阳性克隆,并对阳性克隆进行序列测定和相关生物学分析。结果诱饵载体pGBKT7-TIP60β经双酶切可释放出1443bp DNA片段,与理论值大小一致,测序比对分析表明序列正确。以其为诱饵经酵母双杂交筛选共获得32个克隆,进一步验证与测序分析,最终确认HDAC7A、DMD2、CREB1、BD73、PHF17、AR等9个克隆基因。结论以TIP60β为诱饵利用酵母双杂交系统获得9个基因编码的蛋白,它们很可能与TIP60β转录活性调节功能有关。Objective To construct a yeast expression vector containing human TIP60β gene （a spliced form of HIV-1 tat interactive protein,HTATIP,60×103） and screen its interaction proteins by yeast two-hybrid. Methods Human TIP60β gene fragment was amplified by RT-PCR and cloned into pGBKT7 vector. Using TIP60β as bait,the proteins interacting with TIP60β were screened from a human liver cDNA library. The positive clones were analyzed by bioinformatic methods. Results Human TIP60β cDNA was successfully amplified and cloned into the pGBKT7 vector and indentified by double enzyme digestion and DNA sequencing. Using yeast two-hybrid,32 clones were screened from a human liver cDNA library,and 9 positive clones including histone deacetylase 7A （HDAC7A）,mouse double minute 2 （DMD2）,B-cell CLL/lymphoma 3 （BCL3）,endothelin receptor type A （EDNRA）,androgen receptor （AR）,PHD finger protein 17 （PHF17）,Ataxia Telangiectasia Mutated （ATM）,CAMP responsive element binding protein 1 （CREB1）,orphan steroid receptor BD73 were verified after sequencing. Conclusion The screened interaction proteins with TIP60β by yeast two-hybrid may be involved in the roles of transcriptional regulation of TIP60β.

关 键 词：组蛋白乙酰基转移酶 TIP60β 酵母双杂交 

分 类 号：R394-33[医药卫生—医学遗传学] R341[医药卫生—基础医学]