HNP-3相互作用蛋白的酵母双杂交筛选  

Interaction proteins of HNP-3 by yeast two hybrid system

作　　者：张伟义[1] 邓璐霞[2] 黄宁[2] 朱涛[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,成都610041

出　　处：《重庆医学》2011年第33期3335-3339,I0001,共6页Chongqing medicine

摘　　要：目的筛选胎肾上腺cDNA文库中与人中性粒细胞多肽(HNP-3)成熟肽具有相互作用的蛋白分子。方法通过逆转录-聚合酶链反应(RT-PCR)成功获得HNP-3成熟肽基因,并将其插入酵母表达载体pGBKT7中构建诱饵质粒,转染酵母细胞AH109,而后经缺陷性培养基上培养并观察pGBKT7-HNP-3在AH109中的表达情况,检测诱饵载体有无毒性作用和自激活功能。同时收集胎肾上腺,提取RNA,通过自我检测、分析、报告技术(SMART技术)制备人胎肾上腺cDNA文库。并采用Match-maker LexA酵母双杂交系统从胎肾上腺cDNA文库中筛选与HNP-3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,通过回转实验,谷胱甘肽巯基转移酶沉降技术(GST pull down)以及免疫共沉淀实验再次验证二者的相互作用关系。结果成功构建诱饵质粒pGBK-T7-HNP-3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-3成熟肽相互作用的蛋白——促肾上腺皮质激素受体(ACTH-R)2、钙粘着蛋白关联蛋白1(CTNNB1)、低密度脂蛋白受体相关蛋白5(LRP5)、成对样同源域转录因子2(PITX2)等。最终选定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二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-3成熟肽相互作用蛋白——ACTH-R。Objective To screen proteins binding with human neutrophil peptide 3 （HNP-3） mature peptide from fetal adrenal eDNA library by yeast two hybrid technique. Methods The cDNA fragment encoding HNP-3 mature peptide was amplified by re- verse transcription-polymerase chain reaction（RT-PCR） and constructed into pGBK-T7 vector as the bait plasmid in yeast two hy- brid system 3. The plasmid pGBKT7-HNP-3 was then transformed into the yeast cell AH109,and its toxicity and transcriptionaI activation was tested by both the phenotype assay and the color assay. Subsequently, the RNA from fetal adrenal gland was obtained and then transformed into eDNA library using SMART technology. The fetal adrenal eDNA library was screened with pGBKTT- HNP 3 as bait plasmid by yeast-two hybrid system Matchmaker Lexa. Finally,the positive clone was obtained by PCR and then i- dentified by sequence. Then the interaction between them was determined by GST pull clown in vitro and coimmunoprecipitates ex- periments in vivo. Results Bait and eDNA library have been constructed successfully and transformed into yeast. Then the interac- tion protein was found -- melanocortin 2 receptor（ACTH-R）,melanocortin 2 receptor（ACTH R）,catenin（cadherin-associated pro- tein）, beta I（CTNNB1）, low density lipoprotein receptor-related protein 5 （LRP5）, paired-like homeodomain transcription factor 2 （PITX2）. Therefore,melanocortin 2 receptor（ACTH-R） was determined into the major subjects. And bands which can demonstrate the relationship between HNP-3 and ACTH-R was obtained in GST（Glutathione S-transferase） pull down and coimmunoprecipi- tates experiments. Conclusion ACTH-R can bind to HNP-3 we obtained from fetal adrenal cDNA library and it may play important roles in the function of HNP-3 mature peptide.

关 键 词：α防御素 双杂交系统技术 受体 促肾上腺皮质激素 人α 防御素3 胎肾上腺cDNA文库 

分 类 号：R363[医药卫生—病理学]