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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.
Key wordsAeromonas hydrophila      suicide vector pRE112      suicide vector pSUP202      aopB-/aopD-/aroA- mutant      
Received: 18 March 2008     
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http://journal05.magtech.org.cn/Jwk_ny/EN/      OR     http://journal05.magtech.org.cn/Jwk_ny/EN/Y2009/V17/I1/29
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