Evaluation of immunogenicity, protective immunity on aquaculture pathogenic vibrio and fermentation of vibrio alginolyticus flagellin flac protein

Abstract

Background: Vibrio are the main pathogenic bacteria in aquaculture. The flagellin protein C (FlaC) of Vibrio alginolyticus has good immunogenicity and the prospect of potential application in a vaccine. Objectives: We aimed to evaluate the immunogenicity, protective immunity, and prokaryotic expression fermentation of V. alginolyticus FlaC protein for the vaccine in aquaculture. Material and Methods: A molecular cloning method was used to construct the expression strain of FlaC protein, and the protein was purified with Ni-affinity chromatography. Polyclonal antiserum was prepared via mice immunized with the FlaC protein. The Western blot and enzyme-linked immunosorbent assay (ELISA) were used to check the specificity and titre of the antiserum. ELISA and pull-down assay detected the interaction between FlaC protein antiserum and Vibrio. The immune protection function of FlaC protein was detected with mice actively immunized with FlaC protein and challenged by V. alginolyticus and V. parahaemolyticus. The optimal expression conditions for FlaC protein were detected using an L9(34) orthogonal design model. Results: The expression strain of FlaC protein was obtained successfully, and purified FlaC protein was prepared using a mice polyclonal antibody. The FlaC protein antiserum held a high specificity, and the titre was 1:3200. The antiserum directly interacted with V. alginolyticus and V. parahaemolyticus, and the FlaC protein demonstrated a significant immune protection function (50%) against V. alginolyticus infection and some immune protection function (41.66%) against V. parahaemolyticus. The optimal expression conditions for FlaC protein included a strain OD600 value of 0.8, final isopropyl-β-d-thiogalactoside (IPTG) concentration of 0.1 mmol/L, an inducing time of 8 hours, and an inducing temperature of 28°C. Conclusions: This study showed that the FlaC protein possesses a significant immunogenicity and immune protection effect and obtained the optimal fermentation conditions. It is expected to be a potential vaccine against V. alginolyticus and V. parahaemolyticus.