Surface display of the protective antigen FlaB of Vibrio anguillarum in Lactobacillus plantarum PO23 and its immune response in Paralichthys olivaceus.

Vibrio anguillarum caused extensive economic losses to the aquaculture industry. As known, vaccines have the advantages of low toxicity and high safety to the environment. Notably, Lactobacillus has been identified as a highly-adhesive intestinal microorganism that can effectively stimulate mucosal immune responses, and could be used as an antigen delivery vehicle of oral vaccine. In this study, highly-adhesive L. plantarum PO23 (LP-PO23) which was isolated from the intestine of Paralichthys olivaceus was used to construct a surface-displayed live vaccine containing the protective antigen FlaB of V. anguillarum. Specifically, the gene of FlaB was recombined with LP-PO23 transglycosylase gene containing anchoring domain LysM (lp_3014, GenBank accession no.: CCC80071.1) as well as LP-PO23 cell surface protein gene containing anchoring motif LPXTG (lp_2578, GenBank accession no.: CCC79729.1) to construct recombinant plasmids pMG36e-LysM-FlaB and pMG36e-FlaB-LPXTG, named pFLY and pFLP respectively. Moreover, pMG36e, pFLY and pFLP were transformed into LP-PO23 by electroshock to obtain three recombinant L. plantarum strains named LP-PO23-e, LP-PO23-1, LP-PO23-2 respectively. SDS-PAGE, Western-blotting (WB) were carried out to verify the expression of LysM-FlaB and FlaB-LPXTG. Immunofluorescence (IF) and Immunoelectron Microscopy (IEM) were applied to confirm the surface location of LysM-FlaB and FlaB-LPXTG. The feed containing LP-PO23-1 and LP-PO23-2 at a concentration of 1 × 10 cfu/g were prepared and used for oral immunization. IF and qPCR assays showed that the LP-PO23-1 and LP-PO23-2 could effectively colonize the intestine of the flounder, meanwhile, the activities of intestinal mucus enzymes acid phosphatase, alkaline phosphatase and total superoxide dismutase exhibited dynamic changes after oral immunization. Moreover, the expression of MHCII, TNFα, IL-1β and Ig M genes in the intestine and specific Ig M antibodies in the serum up-regulated significantly after oral immunization. The results demonstrated that LP-PO23-1 could induce strong immune response of flounder, which indicated that LP-PO23-1 was a promising vaccine candidate against V. anguillarum infection.