Construction Of A AscV Isogenic Knockout Aeromonas Hydrophila J-1 Mutant And Study On Omp Of Aeromonas Hydrophila

Aeromonas hydrophila, a Gram-negative bacterium, is pathogenic in fish and many other animals(including human). It is known that Aeromonas hydrophila has many virulence factors such as hemolysin, enterotoxin, extracellular protease, outer membrane protein(OMP) and S-layer,etc. Type III secretion system(TTSS) is associated with pathogenicity in many bacteria. In this work,the TTSS AscV gene isogenic knockout mutant of Aeromonas hydrophila J-1 was constructed and the OMPII of eighteen Aeromonas hydrophila strains were studied.According to the published nucleotide sequence of AscV gene of the type III secretion system in Aeromonas hydrophila, a pair of primers were designed and synthesized. Then a 710 bp DNA fragment was PCR amplified from the genome DNA of Aeromonas hydrophila J-l and cloned into pMD-18T vector. The recombinant plasmid, named pMD-A, was transformed into E.coli strain DH5a and was sequenced. The sequence was aligned with BLAST and the result suggested that it shared 96 % ,86 % and 83 % sequence consistency with Aeromonas hydrophila strain 3, Aeromonas salmonicida and Aeromonas sobria respectively.This work is essential to study the function of type III secretion system in Aeromonas hydrophila.ln tested thirty-nine Aeromonas hydrophila strains, the AscV gene was detected in thirty-seven pathogenic strains and was absent in the other two non- pathogenic strains.Using pMD-A and pJP5603, the recombinant suicide plasmid pYYAscV was constructed. Then it was transformed into the wild type Aeromonas hydrophila strain J-l, which possesses the full length AscV gene of type III secretion system(TTSS). The recombinant suicide plasmid could not replicate in AhJ-1. Five resultant colonies grown on the plates of kanamycin were obtained. Analysed by PCR with three pairs of different primers, an isogenic knockout mutant strain, named AW-1 â–³ascv, was confirmed. The mutant strain had similar growth characteristics to the wild type strain. On the other hand,the main virulence factors like ECPase and hemolysin etc. could not be detected in the mutant strain and the mutant strain showed reduced pathogenicity to the mice. The results showed that TTSS is necessary in the pathogenic process of AhJ-1.A pair of primers were designed and synthesized according to the published nucleotide sequence of porin II gene. A 1008 bp DNA fragment was PCR amplified from eleven of eighteen different Aeromonas hydrophila strains. The major outer membrane proteins(MOMP) from the eighteen strains were prepared by treatment with N-lauroylsarcosinate detergent combined with concentration and purification under ultracentrifuging. The profiles of MOMP SDS-PAGE revealed that most strains have similar protein patterns and the above eleven strains had the 39kD protein. The immunobloting revealed that the 39kD protein had strong positive reaction with the serum against Aeromonas hydrophila J-l. It is suggested that OMPII has a good immunogenicity and is common in most of Aeromonas hydrophila strains.