Centrobin与BRCA2蛋白间相互作用及其细胞定位

为分析乳腺癌易感基因2（breast cancer susceptibility gene 2, BRCA2）蛋白与中心体BRCA2相互作用蛋白（centromal BRCA2 interacting protein， centrobin）间相互作用及其细胞定位的关系，探讨二者功能上的联系，本研究采用哺乳细胞双杂交实验检测体内结合并初步判定BRCA2分子上的结合区域；免疫共沉淀实验进一步验证其体内结合活性，GST-pulldown法检测其体外结合活性，免疫组织化学染色观测BRCA2蛋白的细胞定位及在有丝分裂各期centrobin的细胞定位.结果显示,BRCA2与centrobin间存在体内和体外结合，且BRCA2分子的结合区域主要位于2　393～2　952氨基酸残基处;外源表达BRCA2定位于中心体，在有丝分裂各时相centrobin均定位于中心体. BRCA2与centrobin在体内形成复合物，并存在直接物理结合作用，二者存在细胞空间定位的一致性.该结果为进一步研究BRCA2在中心体复制中的调控作用提供了线索.

Interaction and Localization of Centrobin and BRCA2 in Centrosome

To investigate the protein protein interactions and cellular spatial location between breast cancer susceptibility gene 2(BRCA2) and centromal BRCA2 interacting protein (centrobin), as well as their functional association, the mammalian two-hybrid assay was performed to determine the binding in vivo and BRCA2 potent binding domain; the co-immunoprecipitation and GST pulldown methods were used to confirm this binding in vitro and in vivo. The cellular location of BRCA2 and dynamic distribution of centrobin have been observed with immunohistochemistry staining. The results showed that the binding in vivo and in vitro between BRCA2 and centrobin was mediated via binding region (2　393～2　952 amino acid residues) of BRCA2. In both exogenous BRCA2 and mitotic phase centrobin were overlap expressed in an overlapping way in centrosome.　These results indicated that the direct interaction of BRCA2 and centrobin occurs in both in vivo and in vitro, BRCA2 may regulate centrosome duplication and mitosis, probably through its interacting proteins such as centrobin.