Abstract：Aeromonas hydrophila is a gram-negative opportunistic pathogen that can result in motile aeromonad septicemia in numerous freshwater fish species. In order to develop a safer vaccine strain for oral immunization, aopB-, aopD- and aroA- triple-deletion mutant was constructed. Firstly, the recombination suicide vector pREaopB/aopD with sacB gene (sucrose-sensitive) and 369 bp-deleted aopB-/aopD- gene (encoding A. hydrophila out membrane protein B, D) was constructed and conjugated with Ah2056. The aopB / aopD deleted strain was selected by two-step method and identified by PCR. Then, the other recombination suicide vector pSUParoA with gentamycin resistance gene and 1290 bp-deleted aroA gene (encoding 5-enolpyruvyl shikimate 3-phosphate synthase) was constructed and conjugated with the aopB-/aopD- deleted strain. The aopB-/aopD-/aroA- deleted strain was selected by gentamycin and identified by PCR. The growth properties and virulence in fish of the aopB-/aopD-/aroA- deleted strain were further characterized. Results showed that the aopB-/aopD-/aroA- deleted mutant was successful constructed. The mutant was lower virulence and aromatic amino acids dependent. The mutant can be potentially used as an oral vaccine strain.
