Cloning, Expression And Immunological Characterization Of Eae Gene Of Enterohemorrhagic Escherichia Coli O157

Enterohemorrhagic Escherichia coli, EHEC O157 infection cause bloodydiarrhea or nonbloody diarrhea,hemorrhagic colitis(HC) and hemolytic-uremicsyndrome (HUS), EHEC O157:H7 remains substantial challenges to public healthdespite a research effect that has grown substantially since the first identification ashuman pathogens in 1982. In resent years, the study focus on virulence factors andimmunogenicity of EHEC O157. Treatment of infection with O157 has been difficultbecause antibiotics do not change the cause of enteritis and may increase the incidenceof HUS. As the various options of preventing O157 infection are considered it isimportant to develop a vaccine strategy. There is a variety of data showed that thecrucial protective antigen in potential EHEC vaccine is the Stx and intimin.. An idealbroad-spectrum EHEC vaccine should probably engender both systemic immunityagainst Stx and local intestinal immunity against intimin and other intestinalcolonization factors.The eae gene, which has been shown to be necessary for attaching and effacingactivity, encodes a 94-to 97-kDa outer membrane protein (OMP) which is termedintimin, Intimin is the primary adhesin of Escherichia coli O157:H7. Intimin (Eae)have been shown to play a central role in the formation of A/E lesions by EHECfeaturing intimate adherence of the bacteria to the epithelial cells. The receptor bindingdomain of intimin molecules are localized to the C-terminal 280 amino acids ofintimin (Int280). The role of intimin suggest the use of this antigen as a potentialvaccine component. Shiga toxin (Stx), which are encoded on a bacteriophage insertedinto the chromosome, is thought to be required for the severe clinical manifestations ofEHEC infection, such as hemorrhagic colitis and HUS. Stx produced by EHEC in thegastrointestinal tract is thought to cross the epithelial cell barrier and enter the systemiccirculation, where it can damage endothelial cells. and thereby cause injury tosensitive organs, such as the kidneys and brain. There are two main types of Stxproduced by EHEC, Stx1 and Stx2, both of which are exotoxins comprised of active(A) and binding (B) subunits. The A peptide contains the enzymatic activity. The Bpentamer binds the toxin to a specific glycolipid receptor. The nontoxic B subunit ofStx were proved to be a good antigen for the vaccine. In this experiment,Eae-stx1/2B fusion protein and O157 Δler/ stx(pBR322::stx1/2B::eae) recombinant strain were constructed using crucialprotective antigen genes of EHEC O157:H7, eae and stxB. The Immunologicalcharacterization and protective efficacy of Eae-stx1/2B fusion protein and O157Δler/ stx(pBR322::stx1/2B::eae)were evaluated with mice. To construct eae-stx1/2B fusion gene, the DNA fragment encoding C-terminal280 amino acids(Int280) of intimin was fused in frame with the stx1/2B gene whichwas inserted in pET-28a vector. A recombinant plasmid pET 28::eae-stx1/2B. wascreated and then transformed into BL21(DE3).The fusion protein was expressed inE.coli(BL21)efficiently with IPTG. Analysis of SDS-PAGE and thin layer scanningresults showed that amount of expressed fusion protein was 50.67% in total of bacteriaprotein. The western blotting results indicated the fusion protein was expressedsuccessfully. Because the fusion protein was consisted of eae Int280,stx1B,stx2Bantigens, it can evoke immune responses against stx1B,stx2B and Int280. Theeae-stx1/2B fusion protein expressed in E.coli could offer an important ingredient thatshould be consider in construction of recombinant EHEC O157 vaccine.To make a better and safer vaccine that can induce protective immunity againstboth the bacteria and stx-eae, a new vaccine candidate, O157?ler/ stx(pBR322::stx1/2B::eae), was constructed.Afull–length eae genes were proliferated,the PCR products were inserted into a recombinant vector, pBR322::stx1/2B,and thenthe resultant plasmid pBR322::stx1/2B::eae was transformed into a ler and stx deletionmutant of EHEC O157:H7,generating O157?ler/stx(pBR322::stx1/2B::eae). Theexpression of eae was measured by ELISA. Results showed the expression level ofeae is higher than the control strain,DH5α..The supernatant of O157?ler/ stx(pBR322::stx1/2B::eae)and EHEC O157H:7 EDL933 was inoculated into Verocells, The result indicated that O157?ler/ stx(pBR322::stx1/2B::eae)had nocytotoxins to Vero cells. Mices were intragastrically inoculated with a dose of 5×109CFU O157?ler/ stx(pBR322::stx1/2B::eae)and EHEC O157:H7 EDL933 strain,respectively. Mices were observed daily for clinical signs of disease .After inoculationof O157?ler/ stx(pBR322::stx1/2B::eae), Mices gained weight normally andexperienced no clinical signs. In contrast, Mices exhibited weight loss and all dead insix days. This result demonstrated that O157?ler/ stx(pBR322::stx1/2B::eae)had nopathogenicity to mices. Adherence assays showed that a small but reproducibledifference in the number of adherent bacteria was observed. The characteristic mightbe help to bacterial colonization but not develop intestinal lesions. After oralinoculation of O157?ler/ stx(pBR322::stx1/2B::eae), it maintained a levels ofbacterial colonization in mice for the 10 days experimental time period. Theseresults demonstrate that O157?ler/ stx(pBR322::stx1/2B::eae)might be an goodattenuated vaccine candidate. To assess the effect of Eae-Stx1/2B on EHEC infection, adherence assays andprotection against the challenges of EHEC O157 are done. Anti-Eae-Stx1/2B sera (ornormal sera as controls) are added to EHEC bacteria suspended in adherence media,adherence decrease are microscopically observed using GIEMSA, This indicated thatpolyclonal sera have the capacity to block bacterial binding to HEp-2 cells. Afterintraperitoneal and intranasal inoculation with Eae-Stx1/2B, the titer of the serumanti-Eae-Stx1/2B IgG antibody for the immunized mices was measured by ELISA.High titer polyclonal anti-Eae-Stx1/2B sera are elicited upon intraperitoneal injectionof Eae-Stx1/2B into mice. Testing of antibody titer are shown that polyclonalantibodies can be generated from day 7 and reached their peak on day 21,and thendecreased. After challenging with EHEC O157:H7 EDL933,the mices, injected i.p.two times or i.n. three times with purified Eae-Stx1/2B,displayed a significantreduction in the fecal shedding of EHEC O157 versus the unimmunized mice. Thesurvive rate is higher than the unimmunized mice .Weight in immunized micerecovered more quickly than unimmunized mice. These results suggests thatimmunization with Eae-Stx1/2B can induce an immune response to provide protectionfrom a challenge with wild-type E. coli O157:H7. To study on the immune response following immunizations O157?ler/ stx(pBR322::stx1/2B::eae)strain. Sera IgG antibodies and fecal IgA antibodies weredetected by ELISA. Antibody analysis results for immunized mice showed specificanti-EHEC IgG antibodies were detected in mice sera immunized with O157?ler/ stx(pBR322::stx1/2B::eae). They became detectable on day 7 after postimmunization,and reached their peak on day 14. High levels of specific EHEC fecal IgA antibodywere induced by immunizations intragastrically with O157?ler/ stx(pBR322::stx1/2B::eae)This indicated that protection to both systemic and localimmunization may be achieved. After protection against the challenges of EHEC O157,the survive rate of the mice was noted, the fecal shedding of EHEC O157:H7 werecompared and body weight were measured . Results indicated that O157?ler/ stx(pBR322::stx1/2B::eae)recombinant strain induced protective effects against EHEC...