Identification And Characterization Of Putative Virulent Genes Of Avianpathogenic E.Coli Imt5155

Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) manifested as pericarditis, parahepatitis, peritonitis, septicemia and so on, meningitis are continuously increasing in present. For the various serotypes of APEC,176O antigens,103K antigens,83H antigens, and antibotic resistance of bacteria, the difference of areas and species, it is difficult to eradicated for animal husbandry to serious harm, and is also a zoonotic pathogens.The mechanisms involved in the pathogenesis are not completely understood. To identify putative new virulence genes of avian pathogenic Escherichia coli (APEC) strain, suppression subtractive hybridization (SSH) was performed between an APEC strain IMT5155and the human uropathogenic E. coli (UPEC) strain CFT073which was proved non-pathogenic to chicken. The tester (IMT5155) and driver (CFT073) genomic DNAs were digested with RsaI. The tester DNA was then subdivided into two portions, each of which was ligated with a different adaptor provided. Two hybridizations were performed. The, entire population of molecules was then subjected to PCR to amplify the tester-specific sequences. The PCR amplification product was cloned into TOPO TA vetor and transformed into E. coli TOP10competent cells.96subtractive clones were picked and further analyzed using the Southern blot assay with digoxigenin-labeled genomic DNA of the strain CFT073and the non-pathogenic E. coli strain K-12/MG1655.34DNA fragments were negative for the probe of CFT073and MG1655, which were sequenced. These sequence were analysed on NCBI, it indicated that6fragments present in the genome of CFT073and K-12/MG1655. Thereafter,28fragments were found present only in the genome of the APEC strain IMT5155and not in the genomes of the other two strains. These DNA fragments, containing putative virulence genes, encoded different proteins that homology to proteins involved in various aspects of cellular surface structure, molecular synthesis, energy metabolism regulation, immune, transport systems, conjugal transfer protein and others of unknown function.According to the published genomic sequence in the NCBI,12pairs of PCR primers for IMT5155specific DNA fragments were designed and used for detection of the distribution of these fragments in222E.coli strains from different sources, regions, groups and times. The results showed that these12DNA fragments were widely distributed in different type of E.coli strains, yet were absent among the UPEC CFT073and avirulent strain MG1655. Moreover, fragments D1, E9and F11were more linked to NMEC, which imply that these genes might be virulence factors in the NMEC. However, the prevalences of other fragments such as B11in the APEC were higher than other E. coli.Putative virulence factor F11was amplified from genomic DNA of APEC IMT5155by polymerase chain rection (PCR), then the amplified fragment was cloned into pMD18T vector for sequence. The resulted plasmid pMD18T-F11was digested with BamHl and Xhol, then the772bp of the F11product was cloned into the expression plasmid vector pET-28a(+) in the proper orientation into the site between BamHI and HindⅢ of pET-28a (+) via restriction endonuclease BamHI and XhoI. The recombinant was transformed into the BL21(DE3) and induced to express by1.0mM IPTG at37℃.The expression product was identified by SDS-PAGE and a band with33kDa as expected was obtained. Western blot revealed that the recombinant protein was reactive with rabbit serum against APEC IMT5155, suggesting that the protein is immunogenic.