Cloning and prokaryotic expression of <I>flaB</I> gene from <I>Vibrio alginolyticus</I> strain HY9901, the causative agent of vibriosis in <I>Lutjanus sanguineus</I>

Abstract

To investigate the possibility of flaB as a candidate antigen for vaccine production,primers were designed based on flaB gene sequences published in GenBank. The flaB gene of Vibrio alginolyticus strain HY9901,the causative agent of vibriosis in Lutjanus sanguineus,was amplified by PCR and cloned into pMD19-T vector. Sequence analysis revealed that flaB gene is 1 134 bp and encodes a putative protein of 377 amino acids. The amino acid sequence of FlaB of V. algonilyticus showed highest identity to V.parahaemolytus (92%). The flaB gene was linked into prokaryotic vector pET-32a(+),and the His-FlaB fusion protein with 60.5 ku molecular mass was successfully expressed in E. coli BL21. The soluble recombinant protein was highly expressed under induction conditions of exposure to IPTG (0.4 mmol/L) at 28 ℃ for 10 h and successfully purified on Ni2 +-IDA column. The purified fusion protein was injected into SPF mice to produce anti-FlaB serum. Western blot analysis revealed that the prepared antiserum not only specifically reacts to the FlaB fusion protein,but also specifically reacts to natural total protein extracted from V.alginolyticus. This result indicates that the FlaB may be one of the important protective antigens of V.alginolyticus,which could provide a basis for further study on the immunogenecity of FlaB and vaccine preparation.