连接蛋白36和闭锁小带蛋白1在HeLa细胞的表达及相互关系研究

THE EXPRESSION AND ASSOCIATION OF CONNEXIN 36 WITH ZONULA OCCLUDENS PROTEIN 1 IN HeLa CELLS

目的观察连接蛋白36和闭锁小带蛋白1在HeLa细胞的表达及探讨两者之间的相互关系。方法RT-PCR获得Cx36全长编码区基因片段,与PCR2.1TOPO克隆载体连接测序,亚克隆到pcDNA3真核表达载体,脂质体介导法转染HeLa细胞,G418抗性筛选阳性克隆,Western blotting,双标记免疫荧光和免疫沉淀分析HeLa细胞Cx36和ZO-1表达及关系。结果构建了重组真核表达载体Cx36-pcDNA3,转染的HeLa细胞获得稳定表达Cx36的克隆,Western blotting显示转染的HeLa细胞表达Cx36蛋白。双标记免疫荧光染色显示,HeLa细胞膜之间Cx36呈点状或线状表达,在Cx36表达部位也同样表达ZO-1。免疫沉淀显示细胞裂解液分别与抗ZO-1或抗Cx36抗体共沉淀,抗Cx36或抗ZO-1抗体进行免疫检测,前者在36kD有特异性Cx36蛋白带,后者在220kD处有ZO-1蛋白带。结论转染Cx36的HeLa细胞表达Cx36和ZO-1,两者共表达并且相互结合。

Objective To investigate the expression and association of Cx36 with ZO-1 in Cx36 transfected HeLa cells. Methods RT-PCR, PCR products cloning into PCR2.1TOPO vector, Cx36-pcDNA3 expression vector construction, cell culture, transient transfection using lipofectamine 2000, stable clone screening with G418, Western blotting, double immunofluorescence, and immunoprecipitation （IP） were used. Results Cx36-pcDNA3 expression vector was constructed and transfected into HeLa cells. Using homogenates from Cx36 transfected HeLa cells, Western blotting showed Cx36 band. By immunofluorescence microscopy, Cx36 transfected HeLa cells displayed punctate immunolabeling for Cx36 between cells. Irmmunolabeling for ZO-1 in these ceils exhibited similar distribution to Cx36. By laser scanning confocal microscopy after double labeling with Cx36 and ZO-1 antibody revealed a high degree of Cx36 and ZO-1 colocalization at sites of cell-cell contact. Cx36 transfected HeLa cells were taken for IP with ZO-1 antibody, blots of IP protein probed with Cx36 antibody showed detectin of Cx36. Blots showed detection of ZO-1 after IP with Cx36 antibody from Cx36 transfected HeLa cells, Conclusion Cx36 was expressed in Cx36 transfected HeLa cells, Cx36 colocalizated and associated with ZO-1 in Cx36 transfected HeLa cells.