溶藻弧菌转运蛋白TolB的免疫原性与免疫保护性

Immunogenicity and Immunoprotection of Translocation Protein B(TolB) in Vibrio alginolyticus

旨在研究溶藻弧菌(Vibrio alginolyticus)HY9901转运蛋白Tol B作为疫苗候选抗原的可能性,根据已发表的全基因组序列,设计特异性引物PCR扩增tol B的基因全长序列。序列分析显示,该基因(Gen Bank登录号JQ846501)全长1 353 bp,共编码450个氨基酸残基。BLAST分析发现,溶藻弧菌tol B基因与其他已知弧菌的tol B具有较高的同源性,序列保守,可作为共同抗原候选蛋白。用原核表达的tol B蛋白免疫SPF级小鼠,制备多克隆抗体,ELISA效价达1∶40 000。免疫印迹表明鼠抗tol B血清能与诱导后的重组蛋白发生特异反应。为进一步研究tol B对石斑鱼(Epinephelus awoara)的免疫保护性,用Tol B蛋白两次免疫石斑鱼,ELISA检测发现,免疫石斑鱼血清的抗体效价在第4周达到峰值(1∶2 048)。攻毒实验结果表明Tol B对石斑鱼的免疫保护率为76%。结果表明,溶藻弧菌转运蛋白Tol B具有较好的免疫原性和免疫保护性,可作为弧菌亚单位疫苗的候选抗原。

To investigate the possibility of translocation protein B （ TolB ） from Vibrio alginolyticus HY9901 as a candidate antigen for vaccine production, the full length tolB gene was amplified by PCR and designed specific primers according to the whole genome sequence of V. alginolyticus published in GenBank. Sequence analysis revealed that tolB gene was 1 353 bp and encoded a putative protein of 450 amino acids （ GenBank accession number ： JQ846501 ） . BLAST analysis showed that the tolB of V. alginolyticus had high genetic relationship with other known Vibrio, its sequence was conservative, and might be a common antigen candidate protein. The TolB protein of prokaryotic expression was injected into SPF mice to prepare polyclonal antibody, and its titer of ELISA reached 1 ： 40 000. Western blot indicated that TolB serum reacted specifically with the induced recombinant protein. Further the TolB protein was used as an antigen to immunize grouper （ Epinephelus awoara ） twice, and ELISA detection found that the antibody titer of the immunized grouper increased to the highest （ up to 1 ： 2 048 ） in the fourth week. Challenge assay test showed that the immunoprotection rate of TolB to E. awoara was 76%. Therefore the TolB of V. alginolyticus has solid immunogenicity and immunoprotection, and it can be used as a candidate antigen for vaccine of Vibrio subunits.