Construction And Characterization Of β Hemolysin-Deficient Strain J-1 Of Aeromonas Hydrophila

Aeromonas hydrophila is an important zoonosis bacterium which inflicts severe losses to aquaculture. It can induce general septicemia and local infection in mollusca, fresh water fish, amphibian, creeper, bird and mammal. Red recombination system has the ability of restraining host's exonuclease to prevent the degradation of exogenous linearity DNA, and it also can start a high-performance double-strand break repair and recombination. The recombination rate of this systerm is much higer. Red recombination contributes a lot for gene improvement and gene function investigation in the coming Post genome Era.βhemolysin gene is one of the main virulence facter of Aeromonas hydrophila. In the present study,βhemolysin gene was complified from the genomic DNA of Aeromonas hydrophila J-1 stain by polymerase chain reaction (PCR). Then the complified fragment was cloned into pMD18-T vector plasmid. The recombinant plasmid was verified by restrietion endonuclease analysis and nucleotide sequencing. Nucleotide sequenceing analysis revealed one open reading frame of 1482 bp inβhemolysin gene of Ah J-1 strain, which encod a protein of 493 aminoacids. The accession number of this aequence to GenBank is DQ408263. Sequence comparision with other publishedβhemolysin gene of Aeromonas hydrophila hydrophila showed that the homology of nucleotide acids was indentical.Plasmid pKD46 encodes three kinds of proteins: Gam, Bet and Exo. Gam inhibits the host RecBCD exonuclease V, so Bet and Exo can gain access to DNA ends to promote recombination. The plasmid pKD46 was transformed into Ah J-1 by electroporation. Ah J-1 harbouring pKD46 can express recombinase when induced by L-arabinose.In this syudy, we generated pcr products bu using primers with 40-nt extensions that are homologous to regions adjacent to theβhemolysin gene fragment to be inactivated and template plasmid pKD4 carring chloramphencol resistance gene flanked by FRT (Flp recognition target) sits. The PCR products were transformed into Ah J-1 by electroporation. The strains expressing kanamycin resistance gene were selected by Km agar. Aβhemolysin gene knocked out mutant strain of Ah J-1 was constructed; the virulence and biochemical characteristic of the Ah J-1 mutant strain were identified. The result showed that the virulence ofβhemolysin gene deficient strain was much lower than that of parent strain, the LD₅₀ of mutant strain is 2.3×10⁹, but and the biochemistry characteristics of mutant strain was stable.To evaluate the possibility of the mutant strain as the candidate of vaccine strain, inbrediz-week-old ICR mice were immunized by theβhemolysin gene eliminated strain. Semi-quantitative RT-PCR analysis was performed to detect the expression of IL-4,IL-6,IFN-γgene of the vaccinated mice. The results showed that the IL-4,IL-6,IFN-γmRNA expression was increased in immunized group, and the immunopotective potency was 80% in immunizd group after challenge by 5 LD₅₀ Ah J-1 parent strain. The quantity of PCR products was paralled with quantty of the primary template, the results demonstrated that the protection was the effect of response type of both Th1 and Th2. We also detected the protected rate of theβhemolysin-deficient strain.