酵母双杂交筛选胎肾上腺cDNA文库中HNP-1结合蛋白  

SCREENING THE GENE SEQUENCES OF THE INTERACTION PROTEINS OF HNP-1 FROM HUMAN FETAL ADRENAL CDNA LIBRARY BY YEAST TWO HYBRID SYSTEM

作　　者：杜润滋[1] 邓璐霞[2] 黄宁[2] 罗朝志[1] 

机构地区：[1]四川大学华西医院麻醉科,成都610041 [2]四川大学华西医学中心感染与免疫教研室

出　　处：《现代预防医学》2011年第18期3723-3726,3729,共5页Modern Preventive Medicine

基　　金：CMB基金(98-681);自然科学基金(30470763;30671963)资助

摘　　要：[目的]筛选胎肾上腺cDNA文库中与α防御素HNP-1成熟肽具有相互作用的蛋白分子。[方法]通过聚合酶链反应(PCR)成功获得HNP-1成熟肽基因插入酵母表达载体pGBK-T7中构建诱饵质粒,同时提取胎肾上腺RNA,SMART技术制备人胎肾上腺cDNA文库,并采用Matchmaker LexA酵母双杂交系统从胎肾上腺cDNA文库中筛选与HNP-1成熟肽相互作用的蛋白。最后通过PCR筛选获得阳性克隆并测序,而后经回转实验,GST pull down以及免疫共沉淀再次验证两者的相互作用关系。[结果]成功构建诱饵质粒pGBKT7-HNP-1以及胎肾上腺cDNA文库并在酵母细胞中正确表达,筛选获得HNP-1成熟肽相互作用的蛋白-melanocortin 2 receptor (ACTH-R),CCAAT-enhancer-bind-ing proteins(C/EBP),Tramembrane trafficking protein(TMP21),low density lipoprotein receptor-related protein 6(LRP6)。最终选定促肾上腺皮质激素受体(ACTH-R)作为主要研究对象,且经GST pull down以及免疫共沉淀实验获得了证实两者间具有相互作用的相应的条带。[结论]从胎肾上腺cDNA文库中成功筛选得到α防御素HNP-1成熟肽相互作用蛋白——促肾上腺皮质激素受体。[Objective]To screen proteins binding with α-defensin（HNP-1）mature peptide from placenta cDNA libraty by yeast two hybrid technique.[Methods]The cDNA fragment encoding HNP-1 mature peptide was amplified by polymera-sechain reaction（PCR）and constructed into pGBK-T7 vector as the bait plasmid in yeast two hybrid system 3.Subsequently,the RNA from fetal adrenal gland was obtained and then transformed into cDNA library using SMART technology.The fetal adrenal cDNA library was screened with pGBKT7-HNP-1 as bait plasmid by yeast-two hybrid system Matchmaker Lexa.Finally,the positive clone was obtained by PCR and then identified by sequence.Then the interaction between them was determined by GST pull down in vitro and coimmunoprecipitates experiments in vivo.[Results]Bait and cDNA library have been constructed successfully and transformed into yeast.Then the interaction protein was found--melanocortin 2 receptor（ACTH-R）,CCAAT-enhancer-binding proteins（C/EBP）,Tramembrane trafficking protein（TMP21）,low density lipoprotein receptor-related protein 6（LRP6）.Therefore,melanocortin 2 receptor（ACTH-R）was determined into the major subjects.And bands which can demonstrate the relationship between HNP-1 and ACTH-R was obtained in GST pull down and coimmunoprecipitates experiments.[Conclusion]ACTH-R can bind to HNP-1 we obtained from fetal adrenal cDNA library and it may play important roles in the function of HNP-1 mature peptide.

关 键 词：α防御素 HNP-1 成熟肽 酵母双杂交 胎肾上腺cDNA文库 促肾上腺激素受体 

分 类 号：R34[医药卫生—基础医学]