Construction And Immune Efficacy Of Recombinant Lactobacillus Casei Expressing Malt Of Aeromonas Veronii

Aeromonas veronii is an important aquatic pathogen that causes enormous economic losses to the aquaculture industry.Therefore,it is necessary to develop aquatic vaccines.It is great significance to select the appropriate antigen for the development of new aquatic oral vaccine Malt can specifically induce the release of cytokines,which can be used in the development of vaccines.In this study,the Malt gene of A.veronii TH0426 was cloned and the prokaryotic expression system and two recombinant Lactobacillus casei strains were constructed.They were Lc-MCS-Malt and Lc-pPG-Malt,protein expression was verified by Western blotting and indirect immunofluorescence.The results showed that the 1314 bp Malt gene fragment was amplified by PCR and the band was clear by Western blotting.The molecular weight of the protein was 45 kDa.The fluorescent signal of Lc-MCS-Malt was observed by fluorescence microscopy.In order to evaluate the protective effect of recombinant L.casei,Carassius auratus should be used as the experimental animal and fed the feed with mixed sodium alginate after the induction of recombinant L.casei.At 0 days before immunization,7 d、16 d、25 d and 34 d after immunization,the tissues and blood were collected to detect the immune-related indexes.The colonization of recombinant L.casei was detected and the experimental group was set up separately for 7 d,after fasting for 28 days,prosogaster,mid-gut and hind-gut were collected for colony counting;Carassius auratus were challenged by A.veronii for testing immune protection rate.Serum IgM,ACP,AKP,SOD,IL-10,IL-1β,TNF-α and IFN-γ were measured by ELISA.The results showed that all the indexes increased after oral immunization.IgM and various immune indexes were induced by orally immunized Carassius auratus,while phagocytic activity and differentiation ability of leucocytes were enhanced.The results showed that recombinant L.casei could stimulate the mucosa through the colonization of intestine and make the transcriptional level of cytokines rise upward,so as to improve the transcription level of IL-10,IL-1β,TNF-α and IFN-γ.Immunity and colonization also showed that after 28 days of fasting,recombinant L.casei were still existed in the intestine,indicating that its colonization ability was good.After observation of challenge for 28 days,it was found that recombinant L.casei could effectively relieve the acute infection caused by A.veronii.To a certain extent,the recombinant L.casei play a protective role.In conclusion,two kinds of recombinant L.casei,Lc-MCS-Malt and Lc-pPG-Malt were successfully constructed in this experiment.After oral immunization,humoral immune response and high level of IgM could be induced,immune-related enzyme activities were up-regulated,while promoting leukocyte differentiation and enhancing its phagocytic activity.And the transcriptional level of cytokines in all tissues also showed an upward trend.Recombinant L.casei could effectively protect Carassius auratus.The results provide a theoretical basis for the development of oral lactic acid bacteria vaccine for aquaculture.