CAJ | 학술논문

非洲爪蟾Paraxial Protocadherin(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(glutathione S transferase,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150 ku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.
비주조섬Paraxial Protocadherin(PAPC)시일개재조섬Spemann조직자특이표체적막단백.타재조섬원장운동계단적회취연신운동화체절발생계단적체절변계형성,이급조기은포적형태발생화세포특화과정중도유중요적작용.위료연구PAPC기인재조기배태발육과정중적표체급기생물학공능,수요제비PAPC항체.응용곡광감태S-전이매(glutathione S transferase,GST)표체계통표체GST-PAPC융합단백,친화순화후용이면역신서란대백토,획득PAPC다극륭항체.면역인적분석발현,이1∶3000희석적해다극륭항체위일항시,능구재전염료전장PAPC질립적HEK293T세포적단백질추제물중,특이지식별출150 ku적인적조대.동시,GST-PAPC융합단백가이경쟁성억제해항체대전장PAPC질립전염세포적단백질추제물적특이성조대.용1∶500희석적해항체위일항진행면역형광분석시,발현,PAPC다극륭항체능구식별재HEK293T세포중과표체이급조섬동물겁세포중과표체적PAPC단백,형광신호정위재세포막상.면역인적분석증명,PAPC항체능구식별조섬배태중내원표체적PAPC단백.
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 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 recognized 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.