一种新的中心体蛋白质TACP1与有丝分裂激酶Nek2A相互作用的研究  

Interaction between a novel centrosomal protein TACP1 and mitotic kinase Nek2A

作　　者：朱园园[1] 蓝建平[1] 余建[1] 来晓瑜[1] 曾芬芳[1] 黄河[1] 

机构地区：[1]浙江大学医学院附属第一医院,浙江杭州310003

出　　处：《浙江大学学报（医学版）》2007年第4期337-342,共6页Journal of Zhejiang University(Medical Sciences)

基　　金：国家重点基础研究发展计划(973计划)资助项目(2002CB713704);国家自然科学基金资助项目(30570941)

摘　　要：目的:研究一种新的中心体蛋白TACP1蛋白与有丝分裂激酶Nek2A在有丝分裂期的相互作用关系。方法:在大肠杆菌中表达和纯化的Nek2A305-446蛋白与293T细胞中表达的TACP1蛋白,进行体外pull-down实验;TACP1和Nek2A两种质粒共转染293T细胞,用TACP1多抗进行免疫共沉淀实验;Hela细胞共转TACP1和Nek2A,利用间接免疫荧光的方法在荧光显微镜下观察两种蛋白的亚细胞定位。结果:TACP1条带可以出现在大肠杆菌中表达和纯化的Nek2A305-446泳道中,而对照组中无TACP1条带。TACP1抗体可以将细胞裂解液中的Nek2A蛋白连同TACP1蛋白一起沉淀下来,而对照组免疫前的兔血清则不能将TACP1或Nek2A沉淀下来。在Hela细胞有丝分裂中期,TACP1和Nek2A都呈点状定位于中心体,两者信号叠加后也重合。结论:中心体蛋白TACP1在体内、体外能与有丝分裂激酶Nek2A相互作用,两者在细胞有丝分裂期都定位于中心体。Objective： To study interaction between a novel centrosomal protein TACP1 and mitotic kinase Nek2A. Methods： Nek2A^305-446 protein was expressed and purified in E. coli and TACP1 protein was expressed in transfected 293T cells. Pull-down assay was used to examine the interaction between Nek2A^305-446 and TACP1. TACP1 and Nek2A complex was tested by coimmunoprecipitation assay with polyclonal anti-TACP1 antibody. The localization of those two proteins in Hela cells was verified by immunofluorescence. Results： TACP1 was pulled down by Nek2A^305-446 protein but not by GST control. Nek2A was co-precipitated with TACP1 protein by polyclonal anti-TACP1 antibody but not by pre-immunization serum. The Immunofluorescence test showed that these two proteins formed a complex at centrosome during mitosis. Conclusion：Centrosomal protein TACP1 is a novel interacting protein with Nek2A, both of which are localized in centrosome during mitosis.

关 键 词：有丝分裂 HELA细胞 中心体 中心体蛋白 Nek2A TACP1 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]