弧菌外膜蛋白OmpK单克隆抗体的制备及其特性

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。

Preparation and Characterization of Monoclonal Antibodies against Vibrio Outer Membrane Protein OmpK

This study aimed to prepare and characterize monoclonal antibodies (Mab) against outer membrane protein K (OmpK) of Vibrio. We injected Balb/c mice with OmpK of the Vibrio parahaemolyticus wild-type strain B expressed by a prokaryotic expression system. Spleen cells from the immunized mice were fused with SP2/0 tumor cells. Then the hybrid cell lines which can stably secrete monoclonal antibody were screened out by using the limited dilution method and indirect enzyme linked immunosorbent assay (ELISA) method. Ascites was produced in the mice and then purified by saturated ammonium sulfate precipitation and Protein G affinity chromatography. Finally, we obtained two hybridoma cell lines namely OmpK-Mab-4B7 and OmpK-Mab-3C5, which can stably secrete anti OmpK monoclonal antibody. The Mab subtypes secreted by both hybrid tumor cell lines were IgG1 and their titers reached 1:128 000. The sensitivity IC50 of 3C5 and 4B7 antibodies were 2.5 and 5.0 μg/mL, respectively. The results of Western blotting showed that the Mab could combine with the outer membrane proteins of 12 strains Vibrio parahaemolyticus (A, B, C, D, E, F, G, H, I, J, ATCC17802, and ATCC33847), 3 (A, B, and C) of 4 Vibrio alginolyticus strains and 1 Vibrio anguillarum strain (MVM). The monoclonal antibodies can be used for basic research and rapid detection of OmpK.