利用酵母双杂交系统筛选NKD1结合蛋白质  

Screening of naked cuticle homolog 1 interacting proteins by yeast two-hybrid system

作　　者：刘迁 于华裔 沈颖 王婉怡 王林林 韩晓东[2] LIU Qian;YU Huayi;SHEN Ying;WANG Wanyi;WANG Linlin;HAN Xiaodong(Wujin Hospital Affiliated with Jiangsu University/The Wujin Clinical College of Xuzhou Medical University,Jiangsu Changzhou 213017,China;College of Life Sciences,Inner Mongolia Agriculture University,Inner Mongolia Hohhot 010018,China)

机构地区：[1]江苏大学附属武进医院/徐州医科大学临床学院,江苏常州213017 [2]内蒙古农业大学生命科学学院,内蒙古呼和浩特010018

出　　处：《现代肿瘤医学》2022年第5期757-762,共6页Journal of Modern Oncology

基　　金：国家自然科学基金面上项目(编号:81872275);江苏省卫生健康委科研基金面上项目(编号:M2020002)。

摘　　要：目的:酵母双杂交技术筛选结肠癌细胞中与裸角质层同源蛋白1(naked cuticle homolog 1,NKD1)相互结合的蛋白质,从而进一步了解NKD1。方法:以结肠癌细胞SW480的cDNA为模板,克隆NKD1基因,并将其成功构建在pGBKT7诱饵载体上。利用酵母双杂交技术从结肠癌SW480细胞文库进行筛选。结果:初步筛选获得13个阳性克隆菌株。经测序,在数据库中BLAST比对后,共确定为8种不同基因编码的蛋白片段。回转实验进一步验证了这8种不同基因编码的蛋白均能够激活酵母双杂交系统中的His3、Ade2和LacZ报告基因。这8种不同基因中的4种基因编码的蛋白片段为YWHA蛋白家族成员,其中2种都是编码YWHAE蛋白片段。在结肠癌HCT116细胞中通过免疫共沉淀实验进一步验证,NKD1与YWHAE在结肠癌细胞中可以相互结合。结论:结肠癌细胞中,证实YWHAE是NKD1结合蛋白质,通过调控胰岛素的敏感性,进而调控结肠癌细胞对葡萄糖的摄入。Objective:To screen the naked cuticle homolog 1(NKD1) interacting proteins by yeast two-hybrid system, and clarify the biological function of NKD1.Methods:NKD1 gene was cloned from the cDNA of SW480 cells and successfully constructed into pGBKT7 bait vector.Yeast two-hybrid technology was used to screen from colon cancer SW480 cell library.Results:Thirteen positive clones were obtained.After sequencing and blast comparison in the database, eight different protein coding genes were identified.Rotation experiments further verified that these eight protein coding genes could activate the His3,Ade2 and LacZ reporter genes in yeast two-hybrid system.Four of them encode YWHAE protein and its homologous protein.In colon cancer HCT116 cells, NKD1 and YWHAE could bind to each other by immunoprecipitation assay.Conclusion:NKD1 can interact with YWHAE in colon cancer cells to regulate glucose intake by regulating insulin sensitivity.

关 键 词：结肠癌 酵母双杂交 肿瘤标志物 裸角质层同源蛋白1 免疫共沉淀 

分 类 号：R735.35[医药卫生—肿瘤]