弧菌外膜蛋白OmpK多肽免疫原制备及其免疫原性研究

Preparation and Immunogenicity Study of Vibrio ConservedPeptide Immunogens from OmpK

外膜蛋白OmpK在弧菌属中保守性大于79%,是有良好应用前景的诊断和疫苗抗原,但弧菌属保守并且暴露在膜外的OmpK肽段有限,多肽免疫原免疫效果相比完整蛋白可能更具优势。采用Protein蛋白序列分析软件分析弧菌外膜蛋白OmpK,筛选出2条在弧菌属内广泛保守,同时亲水性和表面暴露性较好的多肽P1和P2。将合成的多肽与BSA蛋白偶联制备成多肽免疫原,免疫BALB/c小鼠,制备抗血清。间接ELISA结果显示,2条多肽免疫原抗血清对OmpK蛋白的效价均达到1∶81000,对弧菌菌体的效价P1-BSA免疫血清高于P2-BSA免疫血清。通过Western blot分析发现,多肽的抗血清均能与重组OmpK蛋白(27 kDa)以及测试的7株副溶血弧菌和1株创伤弧菌全菌蛋白发生免疫反应,P1多肽抗血清与2株气单胞菌,P2多肽抗血清与3株肠杆菌,存在不同程度的交叉反应。结果表明,OmpK的P1和P2多肽是弧菌共有的抗原表位,免疫血清对菌体识别较弱的原因有待进一步研究。

The homology of outer membrane protein OmpK in Vibrio is more than 79%,and is a promising diagnostic and vaccine antigen.However,the conserved and surface-exposed epitopes on OmpK are usually very limited and may have advantages over intact protein as immunogen.In this study,the protein sequence of OmpK of Vibrio was analyzed by bioinformatics software(Protein),two conserved and hydrophilic peptides P1 and P2 were selected and studied for their immunogenicity.Peptide immunogens were prepared by coupling the synthesized polypeptide with carrier protein BSA,and BALB/c mice were immunized to prepare antiserum.Indirect ELISA results showed that the antisera titer of the two peptide immunogens against OmpK protein both reached 1∶81 000.The titer of P1-BSA against Vibrio spp.was higher than the titer of P2-BSA against Vibrio spp.Western blotting showed that the antiserum of the two peptides both strongly reacted with the recombinant OmpK protein(27 kDa)and whole bacterial protein of seven strains of V.parahaemolyticus and one strain of V.vulnificus.Furthermore,antiserum against P1 crossly reacted with two strains of Aeromonas and antiserum against P2 cross-reacted with three strains of Enterobacteriaceae.The results indicate that P1 and P2 polypeptides are the conserved epitopes of Vibrio,and further study is urged to understand the low binding of antiserum against the Vibrio cells.