副溶血弧菌SH112株OmpA蛋白的高效表达及免疫学特性

Expression and immunological characterization of the OmpA protein from Vibrio parahaemolyticus strain SH112

摘要:

[目的]我们前期研究表明副溶血弧菌SH112株的OmpA蛋白在该菌的致病过程中发挥重要作用，是亚单位疫苗研制的潜在靶标抗原。本研究进一步对ompA（VPA1186）基因进行克隆表达，并研究其免疫学特性。[方法]扩增去除信号肽序列的成熟外膜蛋白OmpA的基因片段，定向克隆至表达载体，基因测序后对其编码蛋白质进行生物信息学分析。重组蛋白His-OmpA经纯化后，免疫ICR小鼠制备鼠多抗血清。Western blotting检测该蛋白的免疫原性及鼠多抗血清的特异性。动物实验验证其免疫保护率。[结果]成功表达分子量约为40.0 kDa的重组蛋白His-OmpA。制备的鼠多抗血清ELISA效价可达1：50000以上。Western blotting检测结果显示，该血清可与His-OmpA蛋白、总外膜蛋白和全菌蛋白发生特异性反应，说明所表达的目的蛋白保持原蛋白的免疫原性。此外，该高免血清可与其他主要血清型的副溶血弧菌发生特异性交叉反应，而与其他非副溶血弧菌菌株无交叉反应，表明该血清特异性较高，且提示OmpA蛋白可能是副溶血弧菌属的共同保护性抗原。小鼠免疫保护实验结果表明，该蛋白可提供约35%的免疫保护率。[结论]OmpA蛋白可作为诊断副溶血弧菌感染和亚单位疫苗研制的靶蛋白，为进一步开展该蛋白的功能研究提供了参考。

Abstract:

[Objective] OmpA protein (VPA1186) of Vibrio parahaemolyticus SH112 stain plays an important role in pathogenesis and can be a potential vaccine candidate against V. parahaemolyticus infection. We expressed and to immunologically characterized OmpA protein from strain SH112. [Methods] The ompA gene from strain SH112 was amplified by PCR method and cloned into express vector. The coding protein was analyzed according to the sequencing analysis. The protein was expressed in Escherichia coli BL21(DE3). The mouse anti-OmpA antiserums were generated by immunization of ICR mice with the recombinant protein purified by Ni-NTA. The immunogenicity and the specificity of OmpA were detected by Western blotting analysis. The vaccine protective efficacy of OmpA was verified by animal challenge experiment. [Results] The 40 kDa recombinant protein His-OmpA was successfully expressed. ELISA result shows that the titer of antiserum was above 1:50000. Moreover, Western blotting results reveals the antiserum reacted not only specifically with the purified His-OmpA protein but also with outer membrane proteins and whole-cell proteins of V. parahaemolyticus, suggesting that the expressed protein remained the immunogenicity of original OmpA protein. In addition, Western blotting result reveals that the antiserum reacted specifically with about 36 kDa proteins from four other V. parahaemolyticus strains with major serotypes in domestic, but not reacted with other non-V. parahaemolyticus strains, suggesting the antiserums have a high specificity and the protein maybe a common protective antigen in different V. parahaemolyticus isolates. We further showed that OmpA conferred protective effect as about 35% of mice survived V. parahaemolyticus infection. [Conclusion] Our findings indicate that OmpA protein could play important roles in development of diagnostic test and may serve as candidate vaccine against V. parahaemolyticus infection.