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Construction of a fusion flagellin complex and evaluation of the protective immunity of it in red snapper ( Lutjanus sanguineus )
Author(s) -
Liang H.Y.,
Wu Z.H.,
Jian J.C.,
Liu Z.H.
Publication year - 2012
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2012.03267.x
Subject(s) - flagellin , biology , vibrio alginolyticus , gene , microbiology and biotechnology , virology , genetics , bacteria , vibrio
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.