| The damage of ascites disease in aquatic animals to aquaculture industry, caused by Vibrio mimicus(V.mimicus), is quite serious, but there is no vaccine to prevent the disease. In our previous studies, we found that OmpU protein of V.mimicus was a conserved protective antigen with60%immunoprotective rate to experimental mice. And then we identified several mimotopes of the protein by phage-displayed peptide library, but the immune efficacy of the multi-epitope tandem of the protein remain unclear. The objective of the study is to design and evaluate a multi-epitope tandem of OmpU against V.mimicus infection in Grass carp(Ctenopharyngodon idellus), in order to develop a multi-epitope peptide whose immunoprotective efficacy would be better as compared to OmpU protein.Firstly, a multi-epitope tandem was optimally designed based on the sequences of mimotopes and the tandem design principle. The six mimotopes were tandemly arranged by AAY(Ala-Ala-Tyr) spacer in different ways, then the practicality of the designed series were analysed by the DNAStar Protean program. The results showed the optimized connection order of mimotopes was C25-AAY-C17-AAY-C60-AAY-C20-AAY-C66-AAY-C24, in which each epitope maintained its independent antigenicity and possessed a better antigen index. The best series of the multi-epitope peptide was transformed into gene sequence in which the restriction enzyme sites and the termination codon were added, and the rare codons were also be replaced, then a tandemly arranged multi-epitope gene named6epis gene was designed and cloned into the plaimid pMAL-c4x.Secondly, the multi-epitope tandem was mass-produced for evaluating its immune efficacy to experimental Grass carp.The recombinant expression plasmid named pET-32a-6epis was constructed and transformed into Escherichia coli Rosetta to express,then the recombinant protein His-6EPIS was purified by Ni-Charged Resin affinity chromatography and analysed by SDS-PAGE and Western blot. The results showed the purified protein retained the antigenicity of natural protein with95%pureness and1.5mg/mL concentration.Lastly, the experimental Grass carp were respectively immunized with recombinant multi-epitope tandem protein, recombinant OmpU protein, tag protein and inactivated vaccine,the immune efficacy of these immunogens to immunized fish were evaluated by detecting the immune-related gene expression level,the serum antibody level and the immunoprotective level.The results showed that:(1) Fish immunized with r6EPIS displayed a clear time-dependent mRNA expression pattern of the IgM gene,which gradually increased from3d.p.ito28d.p.i,both in spleen and head kidney. After challenge, IgM mRNA expression level in immunized fish was rapidly up-regulated, and reaching respectively54-(in spleen) and28-fold (in head kidney) of the control on the7th day(p<0.01).The similar kinetics of IgM mRNA expression both in spleen and head kidney were found in OmpU group,inactivated vaccine group and tag protein group.Two-way ANOVA results indicated that, at the unitary level, IgM mRNA expression both in head kidney and spleen of r6EPIS group was significant higher than that in OmpU group,inactivated vaccine group and tag protein group(p<0.05),but there was no significant differences between these three groups(p>0.05).(2) Up-regulation of the pro-inflammatory cytokines IL-1β and TNF-α genes mRNA expression both in spleen and head kidney significantly increased respectively at one day after primary immunization and one day after boosting compared to the un-immunized control(p<0.001).But the difference was not significant after challenge(p>0.05).At unitary level, the proinflammatory cytokines mRNA expression of6EPIS group were significantly higher than other three groups (p<0.01),but the unitary expression levels of OmpU group and inactivated vaccine group,with no significant difference between them(p>0.05),were significantly higher than the tag protein group.(3) Anti-inflammatory cytokine IL-10mRNA expression displayed minor down-regulation both in spleen and head kidney of immunized fish.However, IL-10mRNA expression level significantly increased after challenge(p<0.01).The inflammatory cytokine unitary transcription level of6EPIS group,OmpU group and inactivated vaccine group were significantly higher than tag protein group,but the differences between the three groups was not significant.(4) r6EPIS could well induce the fish to produce serum antibodies with1:16titer detected by agar gel immunodiffusion test on the28th day post-immunization. Meanwhile,the serum antibody titer of OmpU group and inactivated vaccine group was1:4and1:8.However,the specific antibodies to6EPIS and V.mimicus were not detected in tag protein group serum.(5) The immunized fish were protected against lOLDso V.mimicus challenge on the30th day post-immunization, the RPS of recombinant multi-epitope tandem, recombinant OmpU protein and inactivated vaccine were86.67%,66.67%and76.67%, respectively.In conclusion, the multi-epitope tandem of OmpU protein,which could induce remarkable immune responses and provide excellent protection against challenge with V.mimicus in fish,is an ideal candidate for multi-epitope vaccine development... |
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