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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 investigate the possible interaction between Cyclin B1 and Cyclin B1 by using the established tetracycline-induced monoclonal cell line expressing cyclin B1. Methods Cyclin B1 with Myc and His tags was expressed in a tetracycline-containing monoclonal cell line. After un-induced and tetracycline-induced cell lysis for 48h, monoclonal antibodies (10μg Myc) were separately added and immunoprecipitation (IP) was used to precipitate proteins that may interact with Cyclin B1. The protein mixture was polyacrylamide gelled Gel electrophoresis, differentiated proteins were identified by Coomassie brilliant blue staining and silver staining, and protein identification was performed by mass spectrometry (MASS). Results The proteins identified by mass spectrometry were Cyclin-dependent kinase 1 (CDK1), Cyclin-dependent kinase 2 (CDK2), Mitotic mitotic-like protein 1 arrest deficient-like protein 1, MAD1-like 1, MAD1L1) and heat shock 70 kD protein 8 isoform 1. Conclusion Cyclin B1 may have direct or indirect interaction with CDK2, MAD1L1 and HSP7 except for the direct interaction between CyclinB1 and CDK1.