一种新型免疫毒素IAB-TAT-BH3的表达、纯化及初步鉴定  

Expression,purification and preliminary identification of a novel immunotoxin IAB-TAT-BH3

作　　者：范鸣[1] 滕云飞[1] 虞丽平[1] 杜冰[1] 何苗壮[1] 钱旻[1] 任华[1] 

机构地区：[1]华东师范大学生命科学学院,上海200241

出　　处：《现代免疫学》2013年第5期353-359,共7页Current Immunology

基　　金：上海市科委生物医药领域重点科技攻关专项(074319104)资助;国家自然科学基金青年科学基金项目(31000346)资助

摘　　要：利用能与CA125结合的间皮素最小活性片段IAB与跨膜肽TAT和凋亡肽BH3连接后,插入至质粒pET32a(+)中,构建成免疫毒素IAB-TAT-BH3融合表达载体pET32a(+)-IAB-TAT-BH3。将此重组质粒及对照组质粒pET32a(+)-IAB-BH3分别转化E.coli BL21(DE3),表达后经western blot验证,获得了相应的可溶性蛋白表达产物。表达菌的裂解上清经镍柱亲和层析、超滤脱盐后,分别收集到高纯度且浓度均大于250μg/ml的融合蛋白。经MTT检测,这两种融合蛋白针对细胞表面表达有受体蛋白CA125的癌细胞并具有抑制其增殖的作用。其中IAB-TAT-BH3与对照IAB-BH3相比,具有更强抑制细胞增殖的作用。IAB-TAT-BH3这一新型免疫毒素的表达、纯化及初步鉴定,为CA125阳性癌细胞的靶向治疗研究奠定了基础。A novel immunotoxin IAB-TAT BH3 was constructed by transmembrane peptide TAT, apoptotic peptide BH3 and lAB, which is the minimum active fragment of mesothelin binding to CA125 on cell surface. The fusion recombinant gene was cloned into vector pET32a（＋） to allow soluble expression of IAB-TAT-BH3 which has a thioredoxin（Trx） and six-histone tags. The IAB BH3 served as control immunotoxin. These three recombinant proteins were induced to express in Escherichia coli BL21（DE3） and detected by Western blot. The fusion proteins with high purity were obtained and the concentrations of them were more than 250μg/ml after purification through His-bind Resin, uhrafihration and desalination processes. Finally, the cytotoxicity of IAB TAT-BH3 and lAB- BH3 was detected by MTT assay. Both two immunotoxins can inhibit the prolifer- ation of cancer cells. IAB TAT-BH3 showed much more inhibition of the proliferation of cancer cells than the control immuno- toxin IAt3 BH3. Expression, purification and preliminary identification of the novel immunotoxin IAB-TAT-BH3 may lay a foundation for potential application to targeting therapy of cancer cells expressing of CA125.

关 键 词：CA125 免疫毒素 表达 纯化 

分 类 号：R392[医药卫生—免疫学]