Ezrin多克隆抗体制作及应用  

The Production and Application of Ezrin Polyclonal Antibody

作　　者：张宏玲[1,2] 万骏[1,2] 严飞[1,2] 张新胜[1,2] 曹威[1,2] 陶伟[1,2] 黄来强[1,2] 

机构地区：[1]清华大学生命科学学院,北京100084 [2]清华大学深圳研究生院生命与健康学部生物医药研究中心和基因与抗体治疗重点实验室,深圳518055

出　　处：《生物技术通报》2013年第1期129-133,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(30570960;30900749);深圳市重点实验室建设与提升计划

摘　　要：构建Ezrin原核重组表达载体pET-28a(+)-ezrin,将其转化至大肠杆菌BL21(DE3)中,诱导获得Ezrin蛋白,将经镍柱亲和层析纯化后的蛋白分别免疫新西兰大耳兔和昆明小鼠(KM),获得抗血清,采用ELISA和Western blotting,免疫荧光测定其效价和特异性。ELISA对抗血清进行效价测定表明抗血清可与抗原发生特异性免疫反应;通过Western blotting对抗血清进行鉴定表明抗血清可以识别几种细胞株内的特异性条带,其相对分子量为82 kD,与预测分子量相符;免疫荧光试验表明抗血清可以识别细胞内的Ezrin蛋白,且定位情况与文献报道相符。最后通过protein G对抗血清进行了纯化。结果表明用该方法制备的Ezrin多克隆抗体有较高的特异性和灵敏度。Constructed Ezrin prokaryotic expression vector pET-28a （ ＋ ） -ezrin, the recombinant plasmid was transformed into E. coli BL21 （ DE3 ） , induced Ezrin protein was purified through Ni-chelafing affinity chromatography and purified protein was used to immunize New Zealand rabbits and Kunming mice, antiserum was removed. The titer and specificity of anti-sera were determined by ELISA, Western blotting and immunofluorescence. The specificity of the anti-Ezrin antibody was identified by Western blot and immunofluorescence. The results showed that anti-sera antibody reacted with antigen and detected a specific band of 82 kD, and the size was agreed with the predicted molecular mass, localization dectected by inmmunofluorescence was in good agreement with the references. Finally, the anti-surem was purified by protein G. These results suggested that the Ezrin polyclonal antibody revealed high sensitivity and specificity.

关 键 词：原核表达纯化 EZRIN蛋白 动物免疫 抗血清 抗体鉴定 

分 类 号：R392[医药卫生—免疫学]