| Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an enteric pathogen prevailing all over the word since it was first recognized in 1982. It causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome (HUS). EHEC O157:H7, produce Shiga toxin 1 or 2 (Stx1 or Stx2, respectively), or both. The major source of EHEC O157:H7 are contaminated food and drinking water .Treatment of infection with EHEC O157:H7 has been difficult because antibiotics can not change the course of the enteritis of EHEC O157:H7 and may increase the incidence of HUS caused by the pathogen .This unexpected effect has been proposed to be mediated by antibiotic-induced bacteriolysis and release of intracellular Shiga like toxins. Therefore, the effective immunization of vaccine provides a promising way against EHEC O157:H7 infection.Increasing concern about severe side-effects due to the etiologic agents of EHEC O157:H7 whole cell and common antigens with E.coli has led to decreased acceptability of EHEC O157:H7 whole cell vaccination. Thus research is currently focused on the expression of candidate antigens for vaccine development for EHEC O157:H7.E. coli secreted protein A(EspA),intimin and Shiga like toxinⅡ(Stx2) are protective antigens. Taking our laboratory progress into consideration, we propose to develop a multivalent genetic engineering EHEC O157:H7 vaccine based on EspA, intimin and Stx. Aiming at constructing fusion genes with EspA fragment,the C-terminal fragment about 300 amino acid residue of Intimin (IntiminC300) gene and Stx2B subunit,obtaining the multicomponent fusion protein .Purified the recombinant fusion protein and then immunized the Balb/c mice by hypodermic injection with target protein as well as other compared vaccine protocols. The study provides a certain extent of experimental evidence for a new form of EHEC O157:H7 vaccine. The study has been involved in the following aspects:1. Constructing and primary purification of the fusion protein EspA-IntiminC300(EI).1.1 The primers were designed and synthesized to amplify the eaeC300 gene and espA gene by PCR. Having constructed the prokaryotic expression plasmid pET-28a(+)-espA, the eaeC300 gene was cloned with a linker coding sequence and then inserted into plasmid pET-28a(+)-espA through BamHI and XhoI restriction site. The recombinant plasmid pET-28a(+)-espA-eaeC300 was transformed into E.coli BL21(DE3) and the target protein induced by IPTG was detected by SDS-PAGE and western blotting . After primary purification, two rabbits were immunized with EI protein. As a result, fusion protein EI were successfully expressed in prokaryotic expression system and a high titer antiserum (1:32) against EI protein was gained.1.2 Study on biological function of EI fusion protein1.2.1 Constructing the in vitro adherent cell model of O157. After O157 had been incubated in HEPES-DMEM to OD600nm≈0.6, HeLa cell adherence grown on the cover glass was incubated with the bacterium together for 4 hours with 5%CO2 at 37℃. Finally, adherence effect was observed by Giemsa's staining and electron microscope.1.2.2 Study on the anti-adherence effect of EI antibody. EI antibody and O157 bacterium was added into HeLa cell well at the same time. HeLa cells were washed with PBS after incubinated 4h at 37℃,5%CO2 and inoculated on LB plate by diluted, counted the colony. Statistic analysis showed the effect of EI antibody block adherence was not significantly different compared with O157. EI antibody could not block the adhesion of bacterium and cell.1.2.3 Detecting the adherence between O157 and HeLa cell by immunofluorescence to check the immune reaction of anti-EI antibody. Using rabbit anti-EI as the first antibody, and FITC-conjugated goat anti-rabbit IgG as the second antibody, the immune fluorescence stain showed that the rabbit antiserum could react with O157.1.2.4 Detecting the adherence between O157 and HeLa cell by Fluorescent actin staining(FAS) to check the action polymerization. Result show that rabbit anti-EI antibody could effect the formation of"attaching and effacing"(A/E) lesion.2. Constructing and primary purification of the fusion protein EspA-IntiminC300-Stx2B(EIS). The gene fragments espA-eaeC300 and Stx2B coding gene stx2b were cloned respectively by PCR. We combined EI fragment with stx2b gene by SOE PCR (splicing by overlap extension), which constructed a fused gene form espA-eaeC300- stx2b. The espA-eaeC300-stx2b gene was constructed into prokaryotic expression vector pET-28a(+) by restriction endonucleases NcoI and XhoI, the positive recombinant was transformed into E.coli BL21(DE3) and the target protein induced by IPTG was detected by SDS-PAGE and western blotting . As a result, fusion protein EIS were successfully expressed in prokaryotic expression system and the preliminarily purified fusion protein has been gotted.3. Immunoprotection test of recombination fusion protein EI and EIS to host mammal. Balb/c mice were injected 3 times in the subscapular region with the preliminarily purified fusion protein EI and EIS. After the first vaccination, the two booster vaccinations were given every week thereafter. 7 days after each booster vaccination, mice were phlebotomized from the tail and the special IgG titer of the sera were determined by ELISA. 10 days after the second booster vaccination. Mice were infected with O157 live bacterium and the protective efficacy was observed thereafter. Result show that titer of antiserum of the mice to EI and EIS increased evidently, the fusion protein EIS has certain protective efficacy, immunoprotection rate was 92%. In the test of death of Balb/c caused by conteracting toxic substances with O157 ultrasonic supernatant, EIS immunoprotection rate was 67%.In a word, the EI and ESI recombination fusion protein were expressed and an cell adherence model of O157 has been constructed successfully. The anti-EI and anti-EIS had immunoprotection effect by different means. There is no question that these vaccines against EHEC O157:H7 may benefit mankind all over the world in the future.
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