Construction of a fusion flagellin complex and evaluation of the protective immunity of it in red snapper (Lutjanus sanguineus)

Aims: The main aims of this study were to construct a bivalent subunit vaccine containing flagellin flaA gene and flagellin flaB gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of the fusion protein FlaA-(G4S)3-FlaB as a vaccine candidate for red snapper (Lutjanus sanguineus). Methods and Results: Flagellin gene flaA and flaB of V. alginolyticus were linked by gene SOEing (gene splicing by overlap extension) technology. The expression of the fusion gene flaA-(G4S)3-flaB in Escherichia coli BL21(DE3) was confirmed by SDS-PAGE. Western blot analysis showed that the fusion protein FlaA-(G4S)3-FlaB, which was purified by affinity chromatography on Ni-NTA resin, had positive reaction with mouse anti-FlaA serum and mouse anti-FlaB serum, respectively. The immunoprotection of FlaA-(G4S)3-FlaB as a bivalent subunit vaccine was investigated in red snapper model by enzyme-linked immunosorbent assay (ELISA) and challenge test. Red snapper vaccinated with FlaA-(G4S)3-FlaB produced specific antibodies and were highly resistant to infection by virulent V. alginolyticus. Conclusions: The fusion gene flaA-(G4S)3-flaB from V. alginolyticus strain HY9901 was cloned by gene SOEing and was expressed in E. coli. This fusion protein FlaA-(G4S)3-FlaB is a good protective antigen of V. alginolyticus and should be considered as an effective vaccine candidate against infection by V. alginolyticus in red snapper. Significance and Impact of the Study: Two flagellin genes, flaA and flaB, which are independent in structure and function, were first linked together by gene SOEing technology. The finding that red snapper did adequately respond to the fusion protein FlaA-(G4S)3-FlaB injection made it a promising candidate for vaccine treatment. To develop effective vaccine candidates against V. alginolyticus, more attention should be given to these immunogenic flagellins.