| Enterohemorrhagic Escherichia coli(EHEC) 0157, an emerging pathogen, causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome (HUS) in 5 to 10% of patients. E. coli 0157, produce Shiga toxin 1 or 2 (Stx1 or Stx2, respectively), or both. The source of E. coli O157 is mainly of contaminated food or drinking water . Treatment of infection with E. coli 0157 has been difficult because antibiotics do not change the course of the enteritis of E. coli O157 and may increase the incidence of HUS caused by the pathogen. This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis and release of intracellular Shiga toxins.Stxl and Stx2 are multimeric proteins displaying the classic AB5 structure seen in other bacterial exotoxins. Five Stx B subunits form a pentamer that recognizes globotriaosylceramide (Gb3-Cer) receptors found on many different eukaryotic cells. Upon host cell receptor ligation, the toxin is internalized, the A and B subunits dissociate, and the A subunit's N-glycosidase activity is activated, resulting in the removal of the adenine group from position 4324 in the eukaryotic28S rRNA of the 60S ribosomal subunit.The resulting A subunit-mediated inhibition of protein biosynthesisis cytotoxic to the target cell. This activity is extremely potent, displaying a Vero cell 50% cytotoxic dose in the picograms/milliliter range.Published evidence indicates a closer association between Stx2 expression by STEC and a more severe course of illness. Stx2 is more lethal than Stxl in mice and causes a greater gastrointestinal pathology in rabbits. So any potential STEC vaccine must generate a protective Stx2 immune response. Although animals immunized with Stx2 toxoid preparations are protected against a holotoxin challenge, there are safety concerns associated with using inactivated holotoxins in human vaccines, especially when theincidence of the disease is low. Therefore, if a practical Stx-based acellular STEC vaccine is to be developed, the atoxic Stx2B subunit would be a potential component.In addition to producing Shiga toxins, E. coli O157:H7 has been shown to attach to the cytoplasmic membranes of intestinal epithelial cells, to efface their microvilli, and to cause actin to accumulate beneath sites of bacterial attachment. These features are shared with several other enterohemorrhagic E. coli (EHEC) serotypes and members of the enteropathogenic E. coli (EPEC) group. The eae gene, which has been shown to be necessary for attaching and effacing activity, encodes a 96-kDa outer membrane protein (OMP) which is termed Intimin. And Intimin is necessary for intimate attachment of the bacteria to epithelial cells . Analysis of the nucleotide sequences of the Intimin genes from different EHEC and EPEC strains has shown a high degree of homology in the 5' two-thirds of the genes and a significant degree of heterogeneity in the 3' one-third of the genes. Intimin is reported to be highly immunogenic and there're studys showed that anti-Intimin antibodies can protect animals from colonization with EHEC O157:H7. But the whole molecule of Intimin is too large to produce and the C-terminal fragment about 300 amino acid residue of Intimin (IntiminC300) is special and more suitable for fusion protein as vaccine.In view of these, this study investigated EHEC 0157 vaccine from the following aspects:1. Gene cloning, expression, primary purification and animal immunization of EHEC O157:H7 IntiminC300. The primers were designed and synthesized to amplify the IntiminC300 coding gene eae-c300 by PCR. The target gene was cloned into pET-28a(+) plasmid through Ndel and Xhol restriction site after T-A cloning. Then the recombinant prokaryotic expression plasmid was transformed into E.coli BL21(DE3) and the target protein induced by IPTG was detected by PAGE. After primary purification, IntiminC300 was used to immunize two rabbits. As a result, IntiminC300 of EHEC O157:H7 were successfully expressed in prokaryotic expression system and a high...
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