杀鲑气单胞菌溶血素和菌毛蛋白的重组表达及其免疫保护效果研究

Study on the Immune Protective Effects of Recombinant Hemolysin and Pilin Against Aeromonas salmonicida

为探究杀鲑气单胞菌溶血素和菌毛蛋白能否作为亚单位疫苗候选抗原,本研究针对前期分离的杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida subsp.masoucida,ASM),利用原核表达系统对其进行重组表达,免疫虹鳟后检测其免疫保护效果与诱导的免疫应答特征。Western-blotting结果显示,虹鳟抗灭活ASM血清可与2种重组蛋白发生阳性反应,说明其具有较好的免疫原性。免疫保护实验结果显示,重组溶血素、重组菌毛蛋白及两者混合物免疫对虹鳟的相对免疫保护率(Relative Percentage Survival,RPS)分别为64.6%、37.5%和75.0%;ELISA检测发现,重组溶血素和重组菌毛蛋白单独免疫以及两者混合免疫均能有效诱导虹鳟产生抗各抗原的特异性抗体,在免疫2周后特异性抗体水平均显著高于对照组(P>0.05),其中混合免疫组虹鳟血清中抗重组溶血素抗体水平显著高于重组溶血素单独免疫组,但其诱导产生的抗重组菌毛蛋白抗体水平与重组菌毛蛋白单独免疫组无显著性差异;荧光定量PCR检测结果显示,3个免疫组中IL-6、IL-8、TCR及CD4基因的表达水平均显著高于PBS对照组,其中重组溶血素与重组菌毛蛋白共免疫组中IL-6、CD4、MHC-Ⅱ及IgM基因表达水平显著高于其他免疫组。研究结果表明,重组溶血素对虹鳟具有较好的免疫保护效果,且提示重组菌毛蛋白具有开发成为佐剂分子的潜在价值,该结果可为杀鲑气单胞菌亚单位疫苗的研制及应用提供了重要参考。

In order to explore the potential of hemolysin and pilin of Aeromonas salmonicida to be used as subunit vaccine candidate antigens,this study focused on the previously isolated A.salmonicida subsp.masoucida(ASM),using the prokaryotic expression system to recombine and express them separately,and to detect the immune protection effect and induced immune response characteristics after immunizing rainbow trout.Western blotting results showed that the anti-ASM whole bacteria serum of rainbow trout could react with the recombinant hemolysin(rH)and recombinant pilin(rP),indicating that it had good immunogenicity.The results of immune protection experiments showed that the relative percentage survival(RPS)of recombinant hemolysin,pilin,and mixture of hemolysin and pilin on rainbow trout were 64.6%,37.5%,and 75.0%,respectively.ELISA test found that immunization with rH and rP alone and the combined immunization of the two could effectively induce rainbow trout to produce specific antibodies against each antigen.And after 2 weeks of immunization,the specific antibody level was significantly higher than that of control group(P>0.05).Among them,the level of anti rH antibodies in the serum of rainbow trout in the mixed immunization group was significantly higher than that in the recombinant hemolysin alone immunization group,but the level of anti recombinant pilin protein induced by it was not significantly different from that in the recombinant pilin alone immunization group.Meanwhile,RT-qPCR detection results showed that the expression levels of IL-6,IL-8,TCR and CD4 genes in the three immunization groups were significantly higher than that in the PBS control group.Among them,the expression levels of IL-6,CD4,MHC-Ⅱand IgM genes in the group immunized with recombinant hemolysin and recombinant pilin were significantly higher than those in other immunization groups.These results demonstrate that rH has a better immune protection effect on rainbow trout,and suggests that the pilus protein has the potential value of developing as an adjuvant molecule.This result provides an important reference for the development and application of the subunit vaccine of A.salmonicida.