非洲爪蟾PAPC多克隆抗体的制备及其特异性鉴定(英文)

Preparation and Characterization of Polyclonal Antibody Against Xenopus PAPC

非洲爪蟾ParaxialProtocadherin(PAPC)是一个在爪蟾Spemann组织者特异表达的膜蛋白.它在爪蟾原肠运动阶段的汇聚延伸运动和体节发生阶段的体节边界形成,以及早期听泡的形态发生和细胞特化过程中都有重要的作用.为了研究PAPC基因在早期胚胎发育过程中的表达及其生物学功能,需要制备PAPC抗体.应用谷胱甘肽S-转移酶(glutathioneStransferase,GST)表达系统表达GST-PAPC融合蛋白,亲和纯化后用以免疫新西兰大白兔,获得PAPC多克隆抗体.免疫印迹分析发现,以1∶3000稀释的该多克隆抗体为一抗时,能够在转染了全长PAPC质粒的HEK293T细胞的蛋白质抽提物中,特异地识别出150ku的印迹条带.同时,GST-PAPC融合蛋白可以竞争性抑制该抗体对全长PAPC质粒转染细胞的蛋白质抽提物的特异性条带.用1∶500稀释的该抗体为一抗进行免疫荧光分析时,发现,PAPC多克隆抗体能够识别在HEK293T细胞中过表达以及爪蟾动物极细胞中过表达的PAPC蛋白,荧光信号定位在细胞膜上.免疫印迹分析证明,PAPC抗体能够识别爪蟾胚胎中内源表达的PAPC蛋白.

Xenopus Paraxial Protocadherin （PAPC）, which was initially identified in a screen for genes present in the Spemann organizer of Xenopus embryos, is required for gastrulation, somitogenesis and otic vesicle formation. In order to investigate its function in various developmental events, an antibody was prepared which could specifically recognize Xenopus PAPC. Glutathione S transferase （GST） expression system was used to express the fusion protein GST-PAPC. Rabbits were immunized with GST-PAPC fusion protein and the anti-PAPC polyclonal antibody was obtained. In dilution of 1 ： 3 000 the antibody recognized a specific band in Western blotting analysis of FL-PAPC transfected HEK 293T cells lysates, which could be specifically blocked by pre-adsorption of prokaryotic expressed GST-PAPC fusion protein. Furthermore, by using immunofluorescence analysis the polyclonal antibody recoanized membrane-bound PAPC in FL-PAPC transfected 293T cells and Xenopus animal cap cells. By Western blotting analysis, the endogenous 150 ku PAPC protein was detected in Xenopus embryos using the anti-PAPC antibody. Take together it could be concluded that a polyclonal antibody specifically against Xenopus PAPC was developed.