Suppression Subtractive Hybridization Analysis Of Aeromonas Hydrophila Strains And Cloning, Expression And Activity Analysis Of GneJ Gene Of Ah J-1

Aeromonas hydrophila (Ah) is an ubiquitous waterborne opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in fish, and also a cause of diseases in other aquatic and terrestrial animals. Suppression subtractive hybridization is a technology to find putative virulent genes based on Suppressive PCR. It has been used in comparation between bacteria, screening of unknown gene of pathogeny and molecule evolution.1. To identify unique DNA fragments associated with Aeromonas hydrophila strains, suppression subtractive hybridization (SSH) was used. The genome of low pathogenic Ah strains MR-1 was subtracted from the genome of high pathogenic strain J-1, resulting in the identification of 18 specific fragments. Sequence homology analysis was done. Fragments identified included: adherence correlation fragments, cell division fragments, transcription regulators, metabolize correlation fragments and so on. 15 fragments show high homology to Aeromonas.sp and Vibrio.sp, function of the other 3 fragments remain unknown. The results suggested that there were differences between the highly pathogenic and low pathogenic Ah strains, and SSH could be used in search of virulence of Ah.2. 4 putative virulent fragments acquired by suppression subtractive hybridization (SSH) between high virulent strain J-1 and low virulent strain MR-1 of Ah, including gne,Bvgs,DsbC and FtsY. Their distributions were detected by PCR. The results showed that three of the primers could distinguish virulent strains and avirulent ones efficiently. These results showed that the combination of SSH and PCR would be useful for the search of virulent genes and their distribution detection.3. The gneJ gene is identified to encode a UDP N-Acetylgalactosamine 4-Epimerase by database comparison and experimental validation. The enzyme catalyzes the inter conversion of UDP-GlcNAc and UDP-GalNAc. To study gneJ gene, it was amplified by PCR with the total DNA of as template and was cloned into plasmid pET32a(+) at BamHI-EcoRI sites. After the recombinant plasmid was transformed into E.coli BL21 and induced by IPTG, the protein was expressed as high as 26% of the total cell protein and dominantly as soluble protein form. Enzyme activity assay shows the recombinant protein is a UDP N-Acetylgalactosamine 4-Epimerase. The recombinant protein was detected by Western blot with Ah J-1 antiserum. The results revealed that in vitro expression of the fragment of gneJ had the critical antigenitic epitopes of Ah J-1.The mice were immunized with the recombinant protein. Challenged with germ free culture supernatant of Ah J-1 and J-1 strain (50LD₅₀) respectively, the relative percentage survival (RPS) to the immunized mice was 60%.Itsuggests that gneJ gene commonly presents in the pathogenic Ah and perhaps plays an important role in pathogenesis. The above research lays a foundation for study on function of the gne gene in Aeromonas hydrophila.