Identification And Immunological Character Researching Of Enterohemorrhagic Escherichia Coli O157: H7 EspA B Cell Epitope

Enterohemorrhagic Escherichia coli(EHEC) O157:H7 is an enteric pathogen prevailing all over the world since it was first recognized in 1982.It causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome(HUS).The major source of EHEC O157:H7 is contaminated food and drinking water.Treatment of infection with EHEC O157:H7 is always being 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.The adherence of EHEC to intestinal epithelial cells is the first step for the development of this diseases.The EspA(E.coli secreted proteinA) protein makes up a filament structure being part of the typeâ…¢secretion system(TTSS) that delivers effector proteins to the host epithelial cell.Actin polymerization and pedestal formation involve the recruitment of several cytoskeletal proteins to the site of EHEC attachment,The EspA protein plays an important role in the formations of actin polymerization and A/E (attaching/effacing) lesion.The EspA filament is a polymer of the translocator protein EspA,which is likely to be the sole constituent of these hollow filamentous conduits.Polymerization of the EspA filaments is mediated by coiled-coil interactions between EspA subunits.Hybridoma cell lines were obtained by fusing SP2/0 with spleen cells from BALB/c mice immunized with E.coli O157:H7 EspA.Anti-EspA monoclonal antibodies had no effect on bacterial adhesion however they had a marked effect upon E.coli O157:H7-induced host cell actin rearrangement.Furthermore,the mAbs were shown to identification the B cell epitope of EspA by epitope mapping.In a word,the mAbs provide available immunological reagents for novel disease resistance strategies.Also,the EspA epitope region determined here provides a suitable target for knowledge based vaccine design.Methods1.Production and purification of anti-EspA mAbsHybridoma cell lines were obtained by fusing SP2/0 with spleen cells from BALB/c mice immunized with EHE O157:H7 EspA.The mAbs were purified with Protein A-Sepharose from ascites respectively,then their purity were identified by SDS-PAGE and the titer degree of ascites by ELISA.2.Identification the biological activity of anti-EspA mAbsThe affinity constant and the specificity of mAbs were determined and identified by ELISA agglutination assay,Western blotting and laser focusing.The subclasses and isotypes were identifyied by Mouse Monoclonal Antibody Isotyping Kit.The antigenic epitopes were analyzed by the ELISA additivity test.3.Epitope mapping of anti-EspA antibodiesUsing DNASTAR package software analysis the epitope of EspA,tmncated EspA gene was amplified by PCR,cleaved with the restriction enzymes Nde1,Nco1 and Xho1.Epitope mapping was carried out by similar cloning of fragments of this EspA gene transformation into pM 18T and expression into pET28a.Expression was induced by the addition of 1mM IPTG and grown for a further 4h.EspA fragmants were shown by Tricine -PAGE and analysed by Western blotting where fragmants were detected with monoclona anti- EspA antibody.4.Peptides syntheis and identificationSeven overlapping peptides covering EspA were synthesized,possible specific peptides of the EspA specific mAbs were detected in ELISA assay and immunoblotting analysis.Then the interaction between EspA specific mAb and sequences were further identified in immunoblotting analysis.5.Competitive inhibition assayThe method was similar to ELISA.Recombination EspA were coated into 96 well plates at a concentration of 2μg/ml.The mAb 1H10 and epitope E_100-120 were mixed at a certain concentration:mAb 1H10 at a concentration of 100μg/ml,epitope E_100-120 at a concentration of 0.02,0.04,0.2,1,2μg/ml.According to the competitive inhibition percentage,the competitive inhibition of epitope E_100-120 can be reviewed.The formula of inhibitted percentage was as follow:Inhibition percentage=[(the OD₄₉₂ without epitope E_100-120—the OD₄₉₂ with mixed mAb 1H10 and epitope E_100-120)/the OD₄₉₂ without epitope E_100-120]×100%6.Analysis of immunological characterUsing identification peptide combine with BSA,tite of serum was detected by ELISA at the 41th day.These immune 20 Balb/c female mice aged 6 to 8 weeks were separated into four groups:BSA(blank control),purity EspA(positive control 100μg),peptide (negative control)and peptide combination BSA(assay group).7.Immunofluorescence and western blotting assayImmune- fluorescence assay was performed to review the ability of mAb 1H10 and polyclonal sera blocking the actin of hela cells.Results1.Three hybridoma cell lines were named 1H10,2A3 and 3E5 respectively specificity against EspA.The purities of mAbs ascites were 90%through affinity chromatography.2.The results showed that isotypes of mAbs belonged to IgG1κ,IgG1λand IgG2κ. The affinity constants were 3.0×10⁹,2.8×10⁹,1.9×10⁹,respectively.As demonstrated by Western blotting,mAbs specificly reacted with recombinant EspA protein.Using fluorescence microscopy,EHEC O157:H7 of immune fluorescence stain could adhere to the membrace of Hela cell.3.These recombinant EspA segment proteins were produced by transiently transfection into pM18T and expression in pET28a and used as antigens in the SDS-PAGE analyses.The mAb 1H10 was mapped to E2,E3,E4,E5,E6 based on the result of Western-blotting.The results indicated that the epitope recognized by mAb 1H10 was located in 100-120aa,but the mAbs 2A3 and 3E5 were detected to recognize conformational epitopes.4.According to the results above,seven overlapping peptides were synthesized. ELISA and immunoblotting aimed to analyze the specific fragments for anti-EspA mAbs. Therefor,the one epitope Era0-120 recognized by 1H10 was identification what we had expected.This linear epitope 100-120 amino acids was finely localized by the overlapping peptide.5.The epitope E_100-120 could compete the site of which mAb 1H10 react with recombination EspA.It could inhibit the activity of EHEC O157:H7 EspA,and the inhibition percentage was increased by raising dose of epitope E_100-120.6.The conjugate of epitope-BSA immunized to BALB/c mice,it produced high titer antiserum which reached to 1:25600,but antiserum titer of epitope and PBS control were very low.7.Polyclonal sera of epitope E_100-120 and mAb 1H10 dramaticly inhibited EHEC O157-induced FAS.ConclusionsEspA(E.coli secreted proteinA) is recognized to be an important pathogenic factor in EHEC.Here we obtained three anti-EspA monoclonal antibodies and identified a new B cell epitope in EspA 100-120 amino acids which can be recognized by the mAb 1H10.The mAbs 2A3 and 3E5 were respectively determined to recognize conformational epitopes.In a word,these findings may be facilitate to better understand the multipler pathopoiesis of EHEC O157:H7 as well as to the development of vaccine.