连结蛋白36和闭锁小带蛋白1的PDZ结构域关系

Relationship between connexin 36 protein and PDZ domain of zonula occludens protein 1

目的:探讨连结蛋白36(Cx36)和闭锁小带蛋白1(ZO-1)相互作用的PDZ结构域。方法:构建pGEX-3X GST-PDZ重组载体在DH5α细菌表达,加IPTG产生融合蛋白,经纯化,免疫印迹分析Cx36与ZO-1相互作用的PDZ结构域,PVDF膜洗脱后抗GST和考马斯亮蓝染色。结果:构建了pGEX-3X GST-PDZ重组载体,与转染Cx36的HeLa细胞孵育,免疫印迹显示Cx36与ZO-1的PDZ1结构域结合。而对照组Cx43与ZO-1的PDZ2结构域结合。实验组在相对分子质量为40 000-42 000有蛋白带,提示GST-PDZ融合蛋白表达。与鼠视网膜组织孵育免疫印迹显示Cx36与ZO-1的PDZ1结构域结合,而对照组Cx43与ZO-1的PDZ2结构域结合,相同膜洗脱后抗GST抗体和考马斯亮蓝染色显示GST-PDZ融合蛋白表达。结论:Cx36与ZO-1的PDZ1结构域结合。

Objective： To investigate the interaction between connexin 36 （Cx36） and PDZ domains of zonula occludens 1 （ZO-1）. Methods： PDZ domains fusion protein linked to GST in pGEX-3X recombinant vectors were expressed in E. coli DHSα （induced by IPTG）. The purified protein was examined with Western blotting. The same PVDF membrane was stripped, reprobed with anti-GST and stained with coomassie blue. Results： pGEX-3X recombinant vectors were constructed. Cx36 transfected HeLa cells incubated with ZO-1 PDZ domains showed that Cx36 bound to PDZ1 domain of ZO-1. In contrast, Cx43 transfected HeLa cell incubated with ZO-1 PDZ showed that Cx43 bound to PDZ2 domain of ZO-1. Membranes were stripped and reprobed with anti-GST, showing one band migrated at 40 000-42 000. Stained with coomassie blue, the membrane revealed the equal loading of GST-PDZ fusion proteins. Cx36 also bound to PDZ1 domain of ZO-1 in mouse retina and Cx43 bound to PDZ2 domain of ZO-1 in mouse brain. Fusion protein was detected in the membranes with anti-GST and coomassie blue. Conclusion： Cx36 can interact with PDZ1 domain of ZO-1.