| Enterohemorrhagic Escherichia coli (EHEC) O157, an emerging pathogen, causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome (HUS). E. coli O157, produce Shiga toxin 1 or 2 (Stx1 or Stx2, respectively), or both. The major source of E. coli O157 is contaminated food or drinking water. Treatment of E. coli O157 infection is difficult because antibiotics do not change the course of it′s and may increase the incidence of HUS caused by the pathogen. This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis such as vaccines and antibody. It is very significant to this disease.The interplay between bacterium and ruminant host probably has its main role in promoting infection and pathopoiesis. The adherence of EHEC on intestinal epithelial cells is believed to be the first step for developing these diseases. The adhesion is mainly mediated through the type III secretion system (TTSS). EHEC employ a TTSS to deliver effector virulence proteins translocated intimin receptor (Tir) and Tir-cytoskeleton coupling protein (TccP) into host cells in order to produce'attaching and effacing'(A/E) lesions. TccP is a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. Once translocated, TccP directly binds and activates neural Wiskott-Aldrich syndrome protein (N-WASP) to stimulate actin polymerization, leading to pedestal formation. TccP is present in the sequenced EHEC genomes Z3072 is located at the 5′end of a cryptic prophage, CP-933U, have identical proline-rich repeats. TccP as only the second type III EHEC effector protein after Tir that is required for EHEC-induced actin polymerization and A/E lesion formation. The aim of this study was to investigate prokaryotic expression, purification of TccP Protein from EHEC O157:H7, preparation of its antiserum and application.First, the gene of tccP DNA was amplified from EHEC O157:H7 by PCR, and then tccP DNA was inserted into prokaryotic expression vector pET-28a(+) for the IPTG-induced expression in E. coli BL21(DE3). The expression product fused with 6his at C-terminal was analyzed by Western blotting,and purified by using Ni2+-NTA ion exchange resin. The purity of TccP protein was analyzed by SDS-PAGE. Results showed that EHEC O157:H7 tccP DNA was obtained , and the expression plasmid pET-28a(+)-tccP was constructed successfully. Western blotting analysis showed that TccP with 37kD molecular weight was expressed in E. coli. The purity of the recombined TccP was more than 95% after purification using Ni2+-2NTA ion exchange resin.The rabbit antibody against TccP was prepared by immunizing two New Zealand white rabbits using the purified TccP as immunogen. The rabbit antibody against TccP was prepared successfully and its titer was about 1:640 000. Western blotting analysis showed that the antibody cold bind to the expressed TccP protein specifically. The obtained antibody was used for samples assay by double immunodiffusion, ELISA, Western blotting, and immunofluorescence staining. Western blotting showed that the antibody specifically recognized about 37kD protein. Immunofluorescence staining showed green fluorescence mainly in the site HeLa cell of bacterial adhesion. The immunofluorescence staining showed that the rabbit antiserum against recombinant TccP protein could react with O157.To investigate the intracellular localization of TccP after bacterial attachment to HeLa cells, using the approaches of fluoro immuno-cytochemistry staining and TccP polyclonal antibody. The TccP protein was expressed in E.coli and rabbit against TccP was prepared and application successfully, highly purified expression product and prepared polyclonal antibody provide the necessary material for further study. |
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