人早胚发育相关蛋白ZNF268的抗体制备  

Antibody Preparation of Human Early Embryonic Development Associated Protein ZNF268

作　　者：郭玉静[1] 许均华[1] 赵舟宙[1] 朱晨刚[1] 

机构地区：[1]武汉大学生命科学学院,湖北武汉430072

出　　处：《氨基酸和生物资源》2007年第2期55-58,共4页Amino Acids & Biotic Resources

摘　　要：目的:获得纯化抗原用于制备ZNF268的多克隆抗体。方法:PCR扩增目的基因片Spacer区序列,亚克隆入融合蛋白表达载体pET28a+,构建了重组质粒pET28a+/SPA。然后将该重组质粒转化E.coliDE3(Rosseta),IPTG诱导表达,SDS-PAGE分离,获纯化融合蛋白6His-SPA。用6His-SPA免疫家兔,颈动脉取血,分离血清,从血清中获得ZNF268特异的多克隆抗体。结论:通过构建融合蛋白重组表达质粒pET28a+/SPA,用获得的初步纯化融合蛋白6His-SPA,制备了特异的ZNF268的多克隆抗体6His-SPA Ab。To get large amounts of pure antigens to raise specific antibodies and to perform quantifications, spacer region of ZNF268 was amplified by PCR reaction, and then digested and pET28a. We then identified recombinants pET28a ＋ SPA by BamH Ⅰ and Xho Ⅰ digesti ligated to expression vector on. The expression of fusion proteins were induced by adding isopropyl - thiogalactoside（IPTG） . Several clones showed high - level expression of fusion proteins. Insoluble protein was isolated from E. coli DE3 （Rosseta）and the fusion protein was recovered and purified from a preparative （2mm） SDS - PAGE. The polyarcrylamide gel containing the fusion proteins 6His - SPA was used to immunize rabbit from which polyclonal antibodies was prepared. The purified fusion protein 6His - SPA was used to test the specificity of the polyclonal antibody 6His - SPA Ab. Fusion protein constructed between 6His and spacer region was expressed in E. coli DE3 （Rosseta）. Rabbit antibodies were raised against the fusion proteins6His -SPA. 6His -SPA Ab was found to be specific antibody.

关 键 词：ZNF268 锌指蛋白 多克隆抗体 抗体制备 

分 类 号：Q503[生物学—生物化学]