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‐(G 4 S) 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‐(G 4 S) 3 ‐flaB in Escherichia coli BL21(DE3) was confirmed by SDS‐PAGE. Western blot analysis showed that the fusion protein FlaA‐(G 4 S) 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‐(G 4 S) 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‐(G 4 S) 3 ‐FlaB produced specific antibodies and were highly resistant to infection by virulent V. alginolyticus. Conclusions:  The fusion gene flaA‐(G 4 S) 3 ‐flaB from V. alginolyticus strain HY9901 was cloned by gene SOEing and was expressed in E. coli. This fusion protein FlaA‐(G 4 S) 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‐(G 4 S) 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.