弧菌外膜蛋白OmpK免疫交叉反应性的分析

Immunological Cross-Reactivity Analysis Among Outer Membrane Protein K of Vibrio Species

本研究旨在研究弧菌外膜蛋白（oute rmembrane protein，OMP）OmpK免疫交叉反应的特征，探讨OmpK免疫交叉反应性的分子机制．利用克隆得到5株弧菌的ompK基因，通过比对10种（22株）弧菌的OmpK基因序列发现，ompK基因在弧菌中高度保守，其核苷酸序列的相似性达77％～93％．对副溶血弧菌（V．parahaemolyticus）VPL4—90的ompK基因进行原核表达，利用纯化的OmpK重组蛋白，制备了兔抗OmpK血清．通过westernblot分析发现，OmpK抗血清能够与16种不同的弧菌发生交叉识别反应，其中V．proteolyticus、y．furnissii、V．damsela和V．metschnikovii这4种弧菌OmpK的免疫交叉反应为首次报道，并且证实弧菌OmpK的免疫交叉反应具有种属特异性．通过抗原表位预测和比对发现，弧菌的OmpK具有8个保守的抗原表位，包括7个T细胞表位以及1个T细胞和B细胞的共同表位．流式细胞术分析显示，OmpK抗体能够识别这些相同或相似的抗原表位，提示这些抗原表位可能位于细菌表面．结果表明，这些保守的且位于细胞表面的OmpK抗原表位可能是弧菌OmpK具有免疫交叉反应性的分子基础．

To investigate the immunological cross-reaction feature of outer membrane protein K（OmpK） and to understand the mechanism of the immunological cross-reactivity among OmpK of Vibrio species, the following assays were performed. First, the ompK genes from five Vibrio strains were cloned. Comparison of the ompK gene sequences of ten Vibrio species （twenty-two strains） showed that these sequences were highly conserved and their similarities were 77 %-93 % between V.parahaemolyticus VPIA-90 and other Vibrio species. Second, the ompK gene from V.parahaemolyticus VPL4-90 without the signal peptide coding sequence was cloned and expressed in E. coli Rosetta （DE3）. Purified recombinant OmpK protein was used for the generation of rabbit antisera. To investigate the cross-reaction feature of OmpK, the total protein and bacteria cells of the pathogenic Vibrio species in aquiculture was used for western blot and flow cytometry analysis, respectively. Sixteen different Vibrio strains were shown for the cross-reaction properties against the anti-OmpK sera in the western blot analysis. As far as we know, this is the first report of the cross-reaction among OmpK of V. proteolyticus, V.furnissii, V.damsel and V.metschnikovii. The western blot also indicated that cross-reaction of OmpK of Vibrio species could be Genus-Specificity. By epitope prediction, eight homologous epitopes were found in OmpK and shared by different Vibrio species, including seven epitopes recognized by T cell and one epitope recognized by both B and T cells. The flow cytometry analysis indicated that the anti-OmpK antibodies could recognize the same or the similar epitopes of OmpK which may expose on the surface of cells. The results show that the immunological crosseactivity among OmpK of Vibrio species may be correlative with these homologous epitopes of OmpK located on the cell surface.