肿瘤抑制蛋白质P53单克隆抗体的制备及其免疫活性分析  

Preparation and binding activity of monoclonal antibody against P53 protein

作　　者：武军华[1] 魏文清[2] 贾培媛[1] 赵宇[1] 王晨宇[1] 刘晶[2] 王玉霞[1] 

机构地区：[1]军事医学科学院毒物药物研究所军事毒理及生化药理研究室,北京100850 [2]北京军区总医院,北京100700

出　　处：《中国药理学与毒理学杂志》2011年第5期479-482,共4页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(30973562);国家重大新药创制专项(2009ZX09103-614);国家重点基础研究发展计划资助项目(2010CB933904)~~

摘　　要：目的制备可特异性地识别肿瘤抑制蛋白质P53的单克隆抗体。方法应用重组人野生型P53蛋白为免疫抗原,采用经典的细胞融合技术制备单克隆抗体。亲和层析纯化抗体蛋白;ELISA测定抗体滴度。纯化的抗体作用于含有不同p53基因型的MDA-MB-231〔p53(mutant)〕和〔H1299,p53(null)〕肿瘤细胞系,Western印迹、免疫组化和免疫荧光染色法检测抗体与内源性P53蛋白的结合特异性。结果对筛选到的3株单克隆抗体1P15,2P37和3P40,进行了亲和层析技术纯化。SDS-PAGE结果显示,纯化后的3株抗体纯度均在90%以上;ELISA滴度测定结果显示,3株单抗的滴度均在1∶6000以上。免疫印迹结果表明,3株抗体在p53检测中具有较高的灵敏度,其中3P40的灵敏度最高,可以达到5 ng。Western印迹、免疫组化以及免疫荧光染色结果显示,抗体与细胞内源性P53蛋白的结合特异性,说明3P40可以特异性地结合细胞总蛋白提取物以及细胞中内源性P53蛋白,而在检测不表达P53蛋白的细胞系样品时,没有非特异性结合。结论成功制备3株对P53具有高亲和力的单克隆抗体,其中3P40的抗体滴度和灵敏度最佳。OBJECTIVE To prepare monoclonal antibodies specially recognized P53 protein.METHODS Recombinant human wild-type P53 protein expressed in E.coli.BL21 was used as antigen.Monoclonal antibodies were prepared and identified via the classic protocol of monoclonal antibody preparation.Identified monoclonal antibodies were purified by affinity chromatography.Antibody titer was determined by enzyme linked immunosorbent assay（ELISA）.The specific binding activity of antibody was detected by Westen blotting and immunohistochemistry.RESULTSThree antibodies,1P15,2P37 and 3P40,against P53 protein were prepared and purified by affinity chromatography.The purity of antibodies was higher than 90% shown by SDS-PAGE.The titers of antibodies were more than 1∶6000 determined by ELISA.All antibodies could recognize P53 protein by Western blotting,in which 3P40 had the sensitivity of 5 ng for P53.Two tumor cell lines with different p53 gene type（MDA-MB-231,p53 mutant;H1299,p53 null） were used to determine the binding activity of antibody 3P40 by Western blotting and immunohistochemistry.The result indicated that endogenous P53 protein in tumor cells could be identified by 3P40.CONCLUSIONThree monoclonal antibodies against P53 are prepared.Antibody 3P40 is the most sensitive and specific one to P53.

关 键 词：肿瘤抑制蛋白质P53 单克隆抗体 酶联免疫吸附分析 

分 类 号：R392.1[医药卫生—免疫学]