Cloning, Expression And Immunoprotection Of Enterohemorrhagic Escherichia Coli O157:H7 Tir

1.Background and ObjectiveEnterohemorrhagic Escherichia coli(EHEC)O157:H7,an emerging pathogen,is a common cause of severe hemorrhagic colitis(HC),thrombotic thrombocytopenic purpura(TTP) and hemolyticuremic syndrome(HUS) in human.EHEC O157:H7 can attach intimately to host cell membranes and efface microvilli and cytoplasm in a pattern referred to as an attaching-and-effacing(A/E) lesion.Treatment of infection with E.coli O157:H7 has been difficult because antibiotics do not change the course of the enteritis of E.coli O157:H7 and may increase the incidence of HUS.This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis and release of intracellular Shiga toxins.To date, there is no vaccine for clinic.Research EHEC O157:H7 vaccine for humans would be of great benefit to counter bioterrorism and help to prevent the spread of the disease in children and the elderly.EHEC O157:H7 virulence factors are translocation intimin receptor(Tir),Intimin, Shiga toxin and Haemolysin(Hly) and so on.Tir is membrane protein encoded by locus of enterocyte effacement(LEE).It is inserted into the epithelial cell plasma membrane by the typeâ…¢secretion system.Once translocated into the host cell,Tir then functions as the receptor for intimin,which is an integral outer membrane protein of EHEC.Tir-intimin binding attaches EHEC to the intestinal cell surface and triggers actin cytoskeletal rearrangements beneath adherent EHEC,resulting in pedestal formation and an attaching-and-effacing(A/E) lesion.Blocking intimin and Tir combinat- ion is significance to avoid A/E lesion in infection first stage.Tir was predicted for its structure and function by bioinformatics tools on line. Tir was amplified by PCR using the DNA sequence as template,and the truncated sequence was cloned into the vector pET-30a(+),and expressed the fusion protein in E.coli BL21/DE3.The experiment of Immunoprotectional was done to identify the purified protein of pET-3Oa(+)-tir transformants.2.Methods2.1 Bioinformatics analysis of TirDNA and protein sequences were analyzed at NCBI website.Protein analysis was done with Expasy,Phobius and sequence analysis tools(http://tools.immuneepitope. org/tools).2.2 Cloning,expression and purification of TirTir was amplified.The PCR product and expression vector plasmid were digested by Ndeâ… and Xhoâ… ,then linked and the recombinant plasmid was identified by PCR, sequencing and digestion by restriction endonucleases.The PET-30a(+)-tir recombinant was transformed into E.coli BL21,and expression was induced by IPTG..The expressed product was analyzed by SDS-PAGE and purified by Ni-IDA agarose.The immunoreactivity of the recombinant protein was demonstrated by Western-blotting using the 6×His monoclonal antibody as first antibody,and horse radish peroxidase sheep-mices IgG as the second antibody.2.3 Immunization of mices and detection of Tir specific antibody by indirect ELISABALB/c mices were chose to subcutaneous immunization,subcutaneous immunization control,intranasal immunization,intranasal immunization control group, 14 mice per group.The Tir protein was used to immunize BALB/c mice.Mice sera and fecal were collected after the third immunization to evaluate for their specific IgG and IgA antibodies by indirect ELISA. 2.4 The mices were orally challengedThe mice were orally challenged with 0.3ml 1×10¹⁰CFU/ml live E.coli O157:H7, and the mortality was recorded.While the alive mices were put to death,the nephridial and colon tissue were sliced after orally challengeing Fifteen days.3.Results3.1 Bioinformatics analysis of TirThe length of the tir is 1,677bp,with the initiation codon ATG and the termination codon TAA.The conceptual molecular mass of Tir is 58022.4Da.The protein contains three functional domains in the primary structure.There are 22.76%αhelixes,17.38%βsheets and 59.86%loops in the secondary structure.In addition, the protein includes two transmembrane structures and twenty linear B-cell epitopes.3.2 Cloning,expression and purification of TirPCR,sequencing and digestion with restriction endonucleases all confirm that the recombinant vector was constructed successfully.The expressed product and purified product were identified by SDS-PAGE,and the molecular weight was same as the predictive value.3.3 Immunization of mites and detection of Tir specific antibody by indirect ELISAIn Enzyme linked Immunosorbent Assay(ELISA),both intranasal and subcutaneous immunizations with Tir induced high titer specific IgG antibody.The titer of serum IgG antibody induced by intranasal and subcutaneous immunization is 3200 and 6400 respectively.The titer of serum IgA antibody induced by intranasal immunization is 6400,more than subcutaneous immunization.The fecal IgA antibody titer induced by intranasal immunization is 16 more than subcutaneous immunization too.3.4 The mices were orally challengedThe results of protective efficacy after challenge showed that the protective ability of subcutaneous immunization group is no statistical significance compare with control group(P=0.704),and intranasal immunization group possessed a protective ability than control group(P=0.033).4.ConclusionTir molecule is potential vaccine candidate for preventing EHEC disease.The prokaryotic expressing plasmid pET-30a(+)-tir was successfully constructed. The fusion protein of Tir was expressed and purified.The specific IgG and IgA antibody of sera from mice were detected by ELISA method.The IgA antibody of fecal from mice was detected too.The intranasal immunization group possessed a protective ability than control group.Our study is helpful for preparation of investigation of vaccine to EHEC O157:H7.