Construction And Immune Efficacy Of Recombinant Lactobacillus Casei Expressing FlaA And FlaB Of Aeromonas Veronii

Aeromonas veronii is an important aquatic pathogen and causes huge economic losses to the aquaculture.Therefore,it is necessary to develop aquaculture vaccine.Due to the large number of restricting vaccination factors,new oral vaccines are developed.The selection of suitable antigens have great significance for the development of aqua vaccines.Flagellin of bacteria can specifically bind to TLR5 and induce the release of cytokines from the organism,which can be used in the development of vaccines.This experiment is mainly divided into the following two parts :In the first experiment,the flaA gene of A.veronii TH0426 strain was cloned,and the prokaryotic expression system and two recombinant Lactobacillus casei were constructed.They were cell surface-expressed Lc-MCS-flaA and secretory-expressed Lc-pPG-flaA respectively.The expression of protein was verified by Western blotting and indirecte immunofluorescence.The results showed that the nucleotide sequence of flaA amplified by PCR was 900 bp.The Western blotting band was clear and the protein weight was 33 kDa.The fluorescence signal of Lc-MCSflaA was obvious by inverted microscope.In order to evaluate the immunoprotection of recombinant L.casei,Carassius auratus was chosen as experimental animal.Carassius auratus were fed the feed wrapped with the recombinant L.casei induced by lactose and sodium alginate.At 0 d before immunization and 7d,16 d,25d and 34 d after immunization,the tissue and blood were collected to determine immune-related indexes.The colonization of recombinant L.casei was detected and the experimental group was set up separately for 7d,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.The levels of IgM,ACP,AKP,SOD,IL-10,IL-1β,TNF-α and IFN-γ were detected by ELISA in serum.The results showed that all the indexes were improved after oral immunization.Carassius auratus immunized by oral immunization could produce high level of IgM and various immune indicators.The leukocyte phagocytosis activity and differentiation ability were enhanced.The transcriptional levels of IL-10,IL-1β,TNF-α,and IFN-γ cytokines in the tissues showed the recombinant L.casei stimulated the mucosa through colonization of the intestine to increase the transcription level of cytokines,thereby enhancing immunity.The results showed the colonization of recombinant L.casei was remakable in instine after 28 days and indirected 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 the second experiment,the flaB gene of A.veronii TH0426 strain was successfully cloned,and the expression and purification of FlaB protein was induced by constructing a prokaryotic expression system,which was proved to be immunogenic by mouse anti-A.veronii TH0426 serum.Based on L.casei as exogenous protein delivery carrier,two recombinant L.casei(Lc-pPG-flaB and Lc-MCS-flaB)were constructed and verified by gene and protein levels.The results showed that both Lc-pPG-flaB and Lc-MCS-flaB can express FlaB protein,and Lc-MCS-flaB can anchor the target protein on the cell surface.The determination methods of immunoassay and immunological indexes are the same as the first experiment.The results showed Lc-MCS-flaB and Lc-pPG-flaB can induce high levels of IgM produced by organism,and Lc-MCS-flaB can induce higher levels of IgM.The recombinant L.casei can effectively induce humoral immunity and increase serum immunological indexes.The leukocyte phagocytosis activity and differentiation ability were enhanced.The results of qRT-PCR showed the recombinant L.casei can effectively increase the expression of immune-related cytokines in the tissues of Carassius auratus immunized,and the colonization ability of them was better.The relative protection rates of Lc-pPG-flaB and Lc-MCS-flaB were respectively 45% and 61.5%.The recombinant L.casei could effectively relieve the acute infection caused by A.veronii,and also play a role in purifying water quality.In conclusion,four recombinants L.casei were successfully constructed in this study.Oral immunization can induce humoral immune responses,inducing high levels of IgM and upregulate immune-related enzyme activities,and promoting leukocyte differentiation and enhance phagocytic activity.The transcription level of cytokines in each tissue also showed an uptrend.The recombinant L.casei can effectively protect the Carassius auratus and improve immunity.The results provide a theoretical basis for the development of oral lactic acid bacteria vaccines for aquaticulture.