抗人大肠癌P-gp Fab抗体的制备、纯化及初步鉴定  

Production,Purification and Identification of the Fab Antibody against Human Colorectal Cancer P - gp

作　　者：王清[1] 蒋葵[2] 李俊[1] 张学梅[1] 

机构地区：[1]大连大学医学院医学研究中心,辽宁大连116622 [2]大连医科大学附属第二医院肿瘤内科,辽宁大连116023

出　　处：《生物技术》2013年第3期65-69,共5页Biotechnology

基　　金：国家"十一五"科技支撑计划项目("科研用核心试剂产业化应用示范";No.2009BAK61B04)资助

摘　　要：目的:构建可溶性抗人大肠癌P-gp Fab抗体原核表达载体,表达、纯化Fab抗体并鉴定其生物活性和所识别的抗原表位。方法:以实验室制备纯化的人大肠癌P-gp21作为抗原从噬菌体Fab抗体库中经5轮淘选,筛选出阳性克隆菌株,切去gⅢ蛋白基因再次转化大肠杆菌XL1-Blue。酶切鉴定后阳性单克隆经IPTG诱导表达,经Protein L亲和柱纯化Fab抗体后,通过SDS-PAGE、Western Blot和ELISA法检测目的蛋白并鉴定其生物活性和所识别的抗原表位。结果:成功构建可溶性抗人大肠癌P-gp Fab抗体原核表达载体,成功表达48kDa的Fab抗体,经纯化后Fab抗体的浓度为150mg/L。经鉴定该抗体所识别的抗原表位为位于紧邻ATP结合区的跨膜肽段10肽:ANDAAQVKGA。结论:成功制备、原核表达并纯化了单克隆抗人大肠癌P-gp的Fab抗体,该Fab抗体的获得有望应用于P-gp功能的研究以及过表达P-gp的肿瘤诊断和干预。Objective： This study was designed to engineer soluble prokaryotic expression vector which can express anti - human colorectal cancer P - gP21 Fab antibody, to express and purify Fab antibody, and identify its bioactivity and the antigen epitope. Method： The specific expression anti - human colorectal cancer P - gP21 Fab antibody clones were screened from the Fab antibody phage display library which had been prepared. Then the phagemid DNA from the positive clone was digested with Spe I and Nhe I to delete g HI which belongs to the bacteriophage. Then the recombinant was transformed to the XL1 - Blue. The verified clone was induced by IPTG to express Fab antibody on the more optimistic condition. After purified by Hi Trap Protein L the Fab antibody was verified by SDS - PAGE, Western Blot and ELISA. Result：The soluble product was identified as the Fab antibody with 48kDa molecular weight against human colorectal cancer P - gP21 by Western Blot. After purification with Hi Trap Protein L,the concentration of Fab antibody reaches to 150mg/L. The recognition of antigen epitope of the Fab antibody was ANDAAQVKGA. Conclusion： The soluble anti - human colorectal cancer P - gP21 Fab antibody had been successfully expressed and purified. The Fab antibody may be used for the diagnosis and intervention of the P - gp which over - expressed by tumor tissues and the function research of P - gp.

关 键 词：原核表达 大肠癌 P—gp FAB抗体 

分 类 号：R392.12[医药卫生—免疫学]