利用可控表达Cyclin B1的单克隆细胞株寻找与Cyclin B1结合的蛋白  

Finding Proteins Interacting with Cyclin B1 Using Monoclone Cell Line Expressing Cyclin B1 Induced by Tetracycline

作　　者：何乐亚[1] 魏欣[1] 徐丰[1] 闵江[1] 刘鹭[1] 李国东[1] 傅寅佳[1] 陶德定[1] 胡俊波[1] 龚建平[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院胃肠外科,武汉430030

出　　处：《华中科技大学学报（医学版）》2012年第5期534-536,540,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家"973"计划资助项目(No.2009CB521802);国家自然科学基金资助项目(No.81171927;30872472;30973496;30800569)

摘　　要：目的利用已经构建好的四环素诱导细胞周期素B1(Cyclin B1)表达的单克隆细胞株,探索可能与Cyclin B1相互作用的蛋白。方法加入四环素的单克隆细胞株能表达带有Myc和His标签的Cyclin B1。将未诱导和用四环素诱导48h的细胞裂解后,分别加入10μg Myc的单克隆抗体,用免疫沉淀法(immune precipitation,IP)沉淀可能和Cyclin B1相作用的蛋白,将蛋白混合物行聚丙烯酰胺凝胶电泳,用考马斯亮蓝染色法和银染法明确差异蛋白,再用质谱(massspectrometry,MASS)行蛋白鉴定。结果用质谱鉴定出的蛋白有细胞周期素依赖蛋白激酶1(Cyclin-dependent kinase1,CDK1),细胞周期素依赖蛋白激酶2(Cyclin-dependent kinase 2,CDK2),有丝分裂阻滞缺失样蛋白1(Mitotic arrest deficient-like protein 1,MAD1-like 1,MAD1L1)和热休克蛋白8(heat shock 70kD protein 8isoform 1)。结论除CyclinB1和CDK1直接相互作用外,Cyclin B1与CDK2、MAD1L1和热休克蛋白8都可能有直接或间接的相互作用。Objective To find proteins interacting with Cyclin B1 using the monoclone cell line expressing Cyclin B1 induced by tetracycline.Methods The monoclone cell line could express Cyclin B1 with a Myc and His tag after tetracycline was added.Cells uninduced or induced by tetracycline for 48 h were both lysed,then 10 μg Myc monoclonal antibody was added.Immune precipitation（IP）was used to detect proteins which might interact with Cyclin B1.The protein mixture were run in SDS-PAGE,and the proteins were confirmed by mass spectrometry（MASS） after different bands was cut from SDS-PAGE by coomassie blue staining and silver staining.Results The proteins confirmed by MASS were cyclin-dependent kinase 1（CDK1）,CDK2,mitotic arrest deficient-like protein 1（MAD1L1） and heat shock 70kD protein 8 isoform 1.Conclusion Besides the known direct interaction between Cyclin B1 and CDK1,direct or indirect interaction might exist between Cyclin B1 and CDK2,MAD1L1 or heat shock 70kD protein 8 isoform 1.

关 键 词：细胞周期素B1 四环素 免疫沉淀 质谱 

分 类 号：Q7[生物学—分子生物学]