人间隙连接蛋白31相互作用蛋白Annexin Ⅱ的筛选与证实

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间隙连接蛋白31(connexin31,Cx31)是间隙连接蛋白(connexin)家族的一员,目前对于Cx31的功能及其调节方式知之甚少.采用固相多肽合成的方法合成Cx31羧基端一个多肽片段(250~266),经HPLC纯化后偶联到匙孔蜮血蓝蛋白,免疫新西兰雄兔后采血检测、并纯化、经蛋白质印迹、细胞免疫荧光染色、免疫沉淀证实得到的抗体为特异性抗Cx31的抗体.运用制备的抗Cx31多克隆抗体免疫沉淀,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离,蛋白质条带回收,蛋白质胶块酶解,Q-TOF质谱分析,数据库扫描筛选可能相互作用蛋白,运用抗体pull-down实验,筛选到可能相互作用蛋白annexinⅡ,经过免疫共沉淀、细胞免疫共定位等实验证实annexinⅡ与Cx31相互作用.
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