Screening And Identification Of EHEC O157:H7Protective Antigen

Backgrounds Enterohemorrhagic Escherichia coli (EHEC) O157:H7is an important zoonostic pathogen, management of patients is only supportive, because antimicrobial agents may increase the risk of HUS. Research the vaccine for human is the best way to control EHEC O157:H7settled and breeding in the gut of animals, and to prevent human from infection and complications.Objectives Pathogenesis of EHEC O157:H7are adherence factors, toxins, hemolysin and so on. These molecules and their segments are the available targets of protective antigens. In this study, the bioinformatic analysis will be used to screen the conservation various ORF sequence which are different from the non-pathogenic E.coli str.K12MG1655strain. This screening aims to cover these major epidemic strains from different areas, and the ORF sequences encoded the extracellular proteins and proteins located on the outer membrane. E. coli prokaryotic expression system was used to prepare these target proteins. To discriminate immunodominant antigen, adhesion and invasion on cells and immune protective efficacy in animal model were used, and the immune mechanism was studies subsequently. This study provided useful foundation on the development of novel vaccine.Methods The genome sequences of MG1655、EDL933、Sakai、EC4115、TW14359and Xuzhou21were found at NCBI website. Xuzhou21as the contrast, mauve was used twice to screen pathogenic island, the Xuzhou21ORF sequences had high homology with EDL933、Sakai、EC4115、TW14359but did not exist in MG1655. Subcellular location was done with Psortb v3.0(Gram negative), an on-line analysis software.27extracellular proteins,33proteins located on the outer membrane and314uncertain proteins were obtained.12genes that relevant to adhesion were selected from the previous results. E.coli prokaryotic expression system was used to obtain engineering strains contains antigen gene, and induced by IPTG to express. The expressed product was purified by Ni-IDA agarose and the purity was up to95%. Proteins were detected by SDS-PAGE and western blot, and immunized on BALB/c mice, PBS as the controls. Titers of mice serum IgG and the subtye of IgG against target proteins were detected by ELISA. Mice were infected with lethal dose EHEC O157:H7to select effective antigens and would be infected more high dose EHEC O157:H7. The discriminate of adhesion on cell and the bacteria in excretion were used on Paa, Z0390, and Z4191. And the cell aggregation function of Z0390and Z1211was verificated simply on the basis of analysis to structrual domain.Results7strains contained target proteins and5target proteins were obtained, and all of the five proteins could protect mice from EHEC0157:H7infecting. By challenging more dose of EHEC0157:H7, Paa and Z0390still could protect part of mice under100MLD EHEC0157:H7infection. Moreover, Paa could increase the bacteria in excretion and inhibit90%of EHEC0157:H7adhereing to Hep-2cell. Z0390and Z1211strains showed cell autoaggregation conspicuously.Conclusions All of the above results proved target proteins obtained from the method were effective in preventing or cure EHEC0157:H7infection, and worth to further study.