| Aeromonas veronii is responsible for causing opportunistic pathological conditions in humans.It causes various diseases in fish due to which the fish industry incurs huge economic losses annually.Due to problems of antibiotic resistance,and the rapidity with which the infection spreads among fishes,vaccination remains the most effective strategy to combat this infection in fish populations.Among numerous virulence factors associated with bacterial virulence,outer membrane proteins have been widely evaluated for their vaccine potential owing to their surface exposure and related role in pathogenicity.In this study,OmpAⅠ gene from Aeromonas veronii was cloned.By molecular biology techniques,we have constructed the Lactobacillus/E.coli shuttle expression vector,which could express OmpAⅠgene.Following,recombinant Lactobacillus Lc-pPG-1-OmpAⅠof surface-displayed expressing and Lc-pPG-2-OmpAⅠ of secreted expression based on Lactobacillus casei as expressed vector with exogenous gene were constructed and its expression was verified by western blotting and immunofluorescence.Our results demonstrated that the nucleotide sequence of OmpAⅠ gene was 1000 base pairs,which express about 45 kDa protein has possessed well reaction-ogenicity.The indirect immunofluorescence test also showed that the expressed protein was displayed on the cell surface of Lc-pPG-1-OmpAⅠ.To identify whether the recombinant strains have the ability to induce immune responses,carassius aumtus were inoculated with the recombinant strains via orogastric route.In addition,recombinant Lactobacillus casei Lc-pPG-1-OmpAⅠ and Lc-pPG-2-OmpAⅠwere induced by lactose and blend with alginate coated particles bait.The immune protocol was administered on three consecutive days,a booster immunization was given after two weeks.For evaluate the production of parameters in serum and organism,serum and organism of carassius aumtus were collected before immunization and 1 d,7 d,12 d,17 d,21 d,31 d and 38 d days after immunization.The colonization potential of recombinant Lactobacillus casei in carassius aumtus intestines,intestinal lavage was collected before immunization and 1 d,3 d,5 d,7 d,12 d,16 d and 20 d days after immunization.Subsequently,we evaluated immunization effect of recombinant Lactobacillus casei after challenge.To assess immune responses,specific IgM,ACP,SOD and lysozyme activity in serum were determined by ELISA.The results suggested that the fish immunized with recombinant Lactobacillus casei could produce high level IgM in the serum.And the activity of ACP,SOD and lysozyme were up-regulated.The results indicated that recombinant Lactobacillus could induce humoral immunity.Simultaneously,the expression levels of some immunerelevant genes were assessed by quantitative real-time PCR.The results revealed that recombinant strains significantly enhanced the expression of immune-related genes IL-10,IL-1β,IFN-γ and TNF-α in the head-kidney and spleen.Furthermore,the expression of immune-associated genes was up-regulated in the intestinal of immunized fish.These results suggest that recombinant strains can influence immune responses,enhance disease protection,and stimulate immune-related gene expression.In conclusion,we have constructed the recombinant Lactobacillus casei,which could express OmpAⅠgene.The level of IgM,ACP,SOD and lysozyme in the serum,the pro-inflammatory cytokines were highly up-regulated in fish immunized with recombinant Lactobacillus casei.Hence,the recombinant Lactobacillus casei have capacity to induce immune response in carassius aumtus,and laying the foundation for the development of recombinant lactic acid bacteria oral preparations against Aeromonas veronii. |
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