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制备弧菌外膜蛋白K(outer membrane protein K,Omp K)单克隆抗体并对其特性进行研究。以原核表达系统表达野生株Vibrio parahaemolyticus B的Omp K免疫Balb/c小鼠,取免疫小鼠脾细胞与肿瘤细胞SP2/0进行细胞融合,采用有限稀释法和间接酶联免疫吸附测定法筛选出能够稳定分泌单克隆抗体的杂交瘤细胞株,小鼠体内诱导制备腹水,用饱和硫酸铵沉淀法和亲和层析柱纯化抗体。最终获得能稳定分泌抗Omp K的两株单克隆抗体杂交瘤细胞株Omp K-Mab-4B7、Omp K-Mab-3C5,2株杂交瘤细胞系所分泌的Mab亚类均为Ig G1,效价达1∶128 000,抗体3C5、4B7的敏感度IC50分别为2.5、5.0μg/m L。Western blotting结果显示单克隆抗体可以与本实验室12株副溶血性弧菌(V.parahaemolyticus A、B、C、D、E、F、G、H、I、J、ATCC17802、ATCC33847)的外膜蛋白有不同程度的结合反应,与4株溶藻弧菌中的3株(V.alginolyticus A、B、C)外膜蛋白能够较好的结合,与1株鳗弧菌(V.anguillarumMVM)外膜蛋白也有轻微的结合反应,实验制备的单克隆抗体可用于弧菌Omp K的基础研究和快速检测。
The monoclonal antibody against outer membrane protein K (Omp K) was prepared and its characteristics were studied. Balb / c mice were immunized with Omp K expressing wild-type strain Vibrio parahaemolyticus B in prokaryotic expression system. The splenocytes from immunized mice were fused with tumor cells SP2 / 0, and then were selected by limiting dilution and indirect enzyme-linked immunosorbent assay The hybridoma cell line capable of stably secreting monoclonal antibodies was used to induce the preparation of ascites in mice and the antibody was purified by saturated ammonium sulfate precipitation and affinity chromatography. Finally, two McAb hybridoma cell lines Omp K-Mab-4B7 and Omp K-Mab-3C5 which can stably secrete anti-Omp K were obtained, and the Mab subclasses secreted by two hybridoma cell lines were all Ig G1 The price of 1:128 000, the antibody 3C5,4B7 sensitivity IC50 were 2.5,5.0μg / m L. Western blotting showed that the monoclonal antibodies could bind to the outer membrane of 12 strains of V. parahaemolyticus A, B, C, D, E, F, G, H, I, J, ATCC17802 and ATCC33847 The proteins had different degree of binding reaction, which could bind well with the outer membrane proteins of three strains of V. alginolyticus A, B and C, and had the best binding ability with one strain of V. anguillarum MVM Membrane protein also has a slight binding reaction, the monoclonal antibody experimentally prepared for Vibrio Omp K basic research and rapid detection.