摘要
目的:寻找与肿瘤及胚胎组织特异表达蛋白65(cancerandembryoexpressionprotein65,CEP65)相互结合的蛋白分子,为研究CEP65在肿瘤发生中的作用机制提供线索。方法: 以CEP65为诱饵蛋白,通过酵母双杂交方法, 筛选人胚胎组织cDNA表达文库。将获得的阳性克隆cDNA片段分别克隆到原核和哺乳细胞表达质粒,通过GSTpull down和免疫共沉淀对相互作用蛋白作进一步验证。结果: 筛选到能与CEP65相互结合的低密度脂蛋白受体相关蛋白产关联蛋白(lowdensitylipoproteinreceptor relatedprotein associatedprotein1,RAP)。将分别在原核和哺乳细胞中表达的RAP与CEP65进行GSTpull down实验,结果显示,原核表达的GST RAP可以与His CEP65相互结合,而在哺乳细胞共表达的GST CEP65和Myc RAP也能相互结合。结论:RAP可以和CEP65相结合,CEP65有可能通过与RAP相互作用,在肿瘤的浸润、转移及增生中发挥其功能。
Objective:To understand the mechanism of cancer and embryo expression protein 65 (CEP65) in cancer development Methods:CEP65 was usel as a bait protein to isolate the partners of CEP65 from a human placenta cDNA library with yeast two hybrid system. The DNA fragments from positive clone were expressed prokaryotic and mammalian cells respectively, the GST pull-down experiment was performed to ascertain the binding activity. Results:After screening a human placenta cDNA library, the low density lipoprotein receptor-related protein-associated protein 1 (RAP) was isolated and shown to interact with CEP65. GST pull-down assay results indicated that His-CEP65 and GST-RAP expressed by prokaryotic system were immunoprecipitated,and so were Myc-RAP and GST-P65 expressed by mammalian cells . Conclusion: RAP can interact with CEP65 and RAP may be a candidate to mediate the function of CEP65.
出处
《北京大学学报(医学版)》
CAS
CSCD
北大核心
2005年第3期297-301,共5页
Journal of Peking University:Health Sciences
基金
国家自然科学基金(30270685)项目资助
北京市科技计划重大项目(H02022002310)资助
北京大学基因中心项目的资助~~