Cloning and sequencing of FlaA gene of Vibrio harveyi and construction of its eukaryotic expression recombinant plasmid

Vibrio harveyi is a kind of conditioned pathogen of ten found in the marine fishery aquaculture. In this study, a pair of degenerate primers were designed according to the homologous sequences of the FlaA genes from Genbank to amplify the FlaA gene of V. harveyi TS-628 strain, and the suitable PCR product was clonedin to a pMD 18-T vector. After sequencing and analyzing, the FlaA gene was found to contain 1 140 bp, which encodes 379 deducedamino acids. Presumably the FlaA gene would encode a protein of 40.6 kDa based on DNA-deducedam inoacid sequence. When compared with homologs in other vibrios from Genbank, the FlaA gene of V. harveyi revealed the greatest homology with that of V. choler ae(79.5%). The comparison also indicated that these polypeptides were typically conserved in amino and carboxy-termini, while the central regions were more diverse and there were no cysteine residues in the flag ellin. Then apair of specific primers, with a short nucleotide sequence encoding Flagtag, were designed according to the obtained sequence of the FlaA genes to amplify the FlaA of V. harveyi. The PCR product was clonedinto eukaryotic expression vector pcDNA 3.1(+) and the positive clone was chosen and identified through the digestion analysis and sequencing. A eukaryotic expression recombinant plasmid, pcDNA-FlaA-tag, containing the FlaA gene of polar f lagellin in V. harveyi was constructed, which would be a foundation for fur ther study on its DNA vaccine.