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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: To construct prokaryotic expression vector of soluble anti-human colorectal cancer P-gp Fab antibody, express and purify Fab antibody and identify its biological activity and identified antigenic epitopes. Methods: Purified human colorectal cancer cell line P-gp21 was used as an antigen in 5-round panning of phage Fab antibody library. The positive clones were screened and the gⅢ protein gene was excised and transformed into E.coli XL1-Blue again. After digestion, the positive monoclonal antibody was induced by IPTG. The Fab antibody was purified by Protein L affinity column. The target protein was detected by SDS-PAGE, Western Blot and ELISA, and its biological activity and identified epitope were identified. Results: Prokaryotic expression vector of soluble anti-human colorectal cancer P-gp Fab was successfully constructed, and the Fab antibody of 48 kDa was successfully expressed. The concentration of Fab antibody after purification was 150 mg / L. The epitope recognized by this antibody was identified as the transmembrane peptidyl 10 peptide: ANDAAQVKGA located immediately adjacent to the ATP binding domain. CONCLUSION: Fab antibody was successfully prepared, prokaryotic expressed and purified from monoclonal anti-human colorectal cancer P-gp. The production of Fab antibody is expected to be used in the study of P-gp function and tumor diagnosis and intervention of overexpression of P-gp.