抗Erbin特异性单克隆抗体的制备及鉴定  

Preparation and identification of specific monoclonal antibody against Erbin

作　　者：于鸣[1] 张浩[1] 施明[1] 张之勇[2] 胡美茹[1] 郭宁[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]山东省医学科学院基础医学研究所,济南250000

出　　处：《军事医学科学院院刊》2009年第5期421-423,478,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家"863"计划项目(2006AA02A245);国家"973"计划项目(2006CB504305)资助

摘　　要：目的:制备抗Erbin单克隆抗体。方法:应用基因工程技术构建含Erbin PDZ序列的原核表达载体,转化大肠杆菌BL21(DE3)以获得重组蛋白的表达;采用镍离子亲和层析柱纯化重组蛋白;通过质谱鉴定重组蛋白;利用纯化的重组蛋白免疫小鼠,通过细胞融合技术制备单克隆抗体。结果:构建了原核表达载体pET-28a(+)-Erbin PDZ,获得了重组蛋白在工程菌中的高效表达。通过对以包涵体形式存在的重组蛋白进行洗涤、溶解、复性和纯化,获得了纯度在90%以上的重组蛋白。经质谱鉴定确证表达的重组蛋白为Erbin PDZ。用重组蛋白免疫小鼠后,经细胞融合、筛选及鉴定,获得了高效价的单克隆抗体。讨论:该抗体可识别天然状态下的Erbin蛋白,可用于ELISA、免疫荧光和免疫沉淀实验。抗Erbin单克隆抗体的制备为研究Erbin的功能奠定了基础。Objective：To prepare the monoclonal antibody against Erbin. Methods： Molecular cloning technique was used to construct the prokaryotic expression plasmid containing Erbin PDZ cDNA. The plasmid was transformed to E. coli BL21 （ DE3 ） to obtain the expression of recombinant protein that was to be purified by Ni-NTA column chromatography and identified by MALDI-TOF-MS analysis. The purified protein was used to immunize BALB/c mice and the monoclonal antibody was prepared by hybridoma technique. Results： Prokaryotic expression plasmid pET-28a （ ＋ ）-Erbin PDZ was con- structed. The recombinant protein was expressed in E. coli efficiently, isolated in inclusion bodies, and purified by refold- ing and Ni-NTA column chromatography. The purity of the recombinant protein was 〉 90%. MALDI-TOF-MS confirmed that the protein was Erbin PDZ. The monoclonal antibody was obtained by immunizing mice with the recombinant protein. Monoclonal antibody at high titer was selected and identified. Conclusion：The monoclonal antibody can recognize natural Erbin protein. It can be used in ELASA, immuno-fluorescence and immuno-precipitation. The monoclonal antibody against Erbin will be an important tool for further exploration of Erbin functions.

关 键 词：ERBIN 重组蛋白类 抗体 单克隆 原核表达 克隆 分子 

分 类 号：Q78[生物学—分子生物学]