关键词: 弧菌, 弧菌外膜蛋白K, 单克隆抗体

Abstract: 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.

Key words: Vibrio, outer membrane protein K, monoclonal antibody