弧菌外膜蛋白OmpU的免疫交叉反应性和交叉保护性

Immunological cross-reactivity and cross-protection of outer membrane protein OmpU among Vibrio species

摘要:

[目的] 通过对弧菌外膜蛋白OmpU的克隆、表达以及免疫学特性分析,明确外膜蛋白OmpU是否为弧菌的共同抗原,并具有免疫交叉反应性和交叉保护性。[方法] 对弧菌外膜蛋白ompU基因进行克隆和生物信息学分析。分别制备副溶血弧菌、溶藻弧菌、创伤弧菌、拟态弧菌和霍乱弧菌的OmpU重组蛋白抗血清,对OmpU的免疫交叉反应特性以及抗原表位定位情况进行比较分析。以霍乱弧菌的OmpU重组蛋白免疫小鼠后,再以多种弧菌进行攻毒,分析其交叉免疫保护作用。[结果] 外膜蛋白OmpU在弧菌种内和种间相似性分别为73.0%-100%和58.6%-89.0%,并至少存在9个保守的B细胞抗原表位。OmpU重组蛋白抗血清在弧菌种内和种间均产生显著的免疫交叉反应,识别弧菌中分子量35-40 kDa的同源蛋白。副溶血弧菌ATCC17802、创伤弧菌ATCC27562和拟态弧菌ATCC33653来源的OmpU重组蛋白抗体能识别供试菌株,提示这些菌株的OmpU抗原表位定位于细胞表面。OmpU重组蛋白对免疫后的小鼠具有交叉免疫保护作用,攻毒实验后小鼠相对存活率(RPS)为43.0%-100%。[结论] 上述结果表明,外膜蛋白OmpU是弧菌中一种保守的共同抗原,具有免疫交叉保护性,可以作为弧菌广谱疫苗的候选抗原。

Abstract:

[Objective] To study the immunological cross-reactivity and cross-protection characteristics of OmpU in Vibrio species. [Methods] The ompU genes from 10 Vibrio strains were cloned, sequenced, followed by bioinformatics analysis. Western blot and whole-cell ELISA assay were used respectively to determine immunological cross-reaction feature and subcellular location of OmpU with rabbit serum against recombinant OmpU from V. parahaemolyticus ATCC17802, V. alginolyticus ATCC33787, V. vulnificus ATCC27562, V. mimicus ATCC33653 and V. cholera Vb0. Finally, the cross-protective property of recombinant OmpU (V. cholera-derived) was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in mice. [Results] The similarities of OmpU proteins of Vibrio ranged from 73.0 to 100% intra-species, and from 58.6 to 89.0% inter-species. Furthermore, homologous epitopes were found in OmpU and shared by different species of Vibrios. Western blot of rabbit serum against recombinant OmpU showed cross-recognition intra- and inter-species. Bands were observed ranging from 35 to 40 kDa. Whole-cell ELISA assay further confirmed that the antiserum of recombinant OmpU from V. parahaemolyticus ATCC17802, V. vulnifgicus ATCC27562 and V. mimicus ATCC33653 recognized the tested Vibrio species, implying that epitopes of OmpU were located on the cell surface. Recorded relative percent survival of the vaccinated group varied from 43.0 to 100%, showing that mice were protected from Vibrio infection after immunization with OmpU protein. [Conclusion] OmpU was a conserved antigen among tested Vibrio species and might be a universal vaccine candidate for the prevention of Vibriosis.