弧菌外膜蛋白OmpK单克隆抗体的制备及其特性

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

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。

This study aimed to prepare and characterize monoclonal antibodies(Mab)against outer membrane proteinK(OmpK)of Vibrio.We injected Balb/c mice with OmpK of the Vibrio parahaemolyticus wild-type strain B expressedby a prokaryotic expression system.Spleen cells from the immunized mice were fused with SP2/0tumor cells.Then thehybrid cell lines which can stably secrete monoclonal antibody were screened out by using the limited dilution methodand indirect enzyme linked immunosorbent assay(ELISA)method.Ascites was produced in the mice and then purifiedby saturated ammonium sulfate precipitation and Protein G affinity chromatography.Finally,we obtained two hybridomacell lines namely OmpK-Mab-4B7and OmpK-Mab-3C5,which can stably secrete anti OmpK monoclonal antibody.TheMab subtypes secreted by both hybrid tumor cell lines were IgG1and their titers reached1:128000.The sensitivity IC50of3C5and4B7antibodies were2.5and5.0μg/mL,respectively.The results of Western blotting showed that the Mab couldcombine with the outer membrane proteins of12strains Vibrio parahaemolyticus(A,B,C,D,E,F,G,H,I,J,ATCC17802,and ATCC33847),3(A,B,and C)of4Vibrio alginolyticus strains and1Vibrio anguillarum strain(MVM).The monoclonalantibodies can be used for basic research and rapid detection of OmpK.