Production And Biological Characterization Of Neutralizing MAb Based On B Cell Epitope Peptide Of Shiga Toxin Ⅱ From EHEC O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, an emerging pathogen, infection has become the public health problem and been generally paid close attention in the world, because of its large-scale break-out epidemic. It is possible that EHEC O157:H7 is took as bacteriological weapon and biological terror warfare agent in future, due to its infectivity and diversity of route of transmission. Treatment of EHEC O157:H7 infection has been difficult because antibiotics could induce bacteriolysis, and then Shiga toxins are released in intracellular and increase the incidence of HUS caused by the pathogen. This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis and release of intracellular Shiga toxins.EHEC O157:H7 produce Shiga toxin 1 or 2 (Stx1 or Stx2, respectively), or both. After Stx ingresses blood circulation, it can damage target tissue and result in the serious complications such as uremic syndrome hemolytic (HUS) and thromb thrombocytopenia purpura (TTP). Toxicity of Stx2 is as 1000 times as Stx1,so Stx2 is the major factor, because it result in incidence of HUS in children and old people and the death rate is 50% among them.Researchers are trying to develope other medicine such as antibody, analog of isoreceptor and vaccine against Stx for therapy and prevention of the infection of the pathogen, respecting antibiotics may increase the incidence of HUS caused by the pathogen. Monoclonal antibody(MAb) against Stx2 is superiority in the urgent therapy after EHEC O157:H7 infection. It is reported that MAbs against Stx2A and StxB can neutralize the toxicity of Stx2 in certain extent.Selection of target antigens is very important for preparing neutralizing MAb against toxins. It was blind, large work load, and difficult to obtain neutralizing MAbs, if MAbs were prepared by holoantigens. It is possible to preparing neutralizing MAb which target to active region of Stx2 by synthetical B cell epitope peptides. At present, the crystal structure of the compound of Stx2 and its ligand adenines has been resolved, and the active site of Stx2 is definite, which is the seventy-seventh amino acids, Ganimalon, of Stx2 A1 fragment.The crystal structure of Stx2 and software of predicting B cell epitope peptide are favourable for the prediction of B cell epitope peptide of Stx2.In view of these, we have predicted the protective B cell epitope peptide including the active region of A1 fragment of Stx2, based the crystal structure of Stx2 and software of predicting B cell epitope peptide.We have prepared MAb and Fab antibodies against the active region of Stx2 by the protective B cell epitope peptide and evaluated the binding affinity and biological activity of the antibodies. The study is including four sections.1. Prediction of B cell epitope peptide of Stx2 and evaluation of immunogenicityThe first, the crystal structure of the compound of Stx2 and its ligand adenines was search in the struture data bank of NCBI. The second, four peptides from Stx2A1 were selected according to the principle predicting B cell epitope.Then hydrophilicity, surface accessibility, ductility , secondary structure, secondary structure of four peptides have evaluated by DNAStar software .The homology alinement of pre-selected four peptides was done by BLAST. The last, the sequences of four cell epitope peptides were decided, which are P1(53aa~80aa), P2(162aa~188aa), P3(28aa~49aa) and P4(100aa~114aa). Four B cell epitope peptides were synthesized and coupled with the Keyhole limpet hemocyanin(KLH). Japanese rabbits were immuned with four peptides coupled with KLH, respectively. The result of indirect ELISA shows that four peptides have immunogenicity. The result of comparison of the titer of the four anti-serums against native Stx2 is P2>P1>P3>P4. The result of Dot ELISA shows that anti-serum can react with peptides, KLH and native Stx2. The result of Western blot shows four antiserum can react with Stx2A subunit. The results indicate that predicted 4 peptides are sure B cell epitope peptides. After immuned with the four B cell epitope peptides, the Balb/c mouse were administered the native Stx2 by intraperitoneal injection for screening protective B cell epitope peptides. It is indicated that the B cell epitope peptide, named as P1(52aa~80aa) and including active rengion of Stx2 , has protective effectiveness.2. Production and biological characterization of neutralizing MAb based on B cell epitope peptide of Shiga ToxinⅡfrom EHEC O157:H7 Currently, there is not specific therapeutic drug against Stx2. It is available to develop neutralizing monoclonal antibodies against Stx2 in the urgent therapy after EHEC O157:H7 infection. In this section, we have prepared and identificated neutralizing monoclonal antibodies targeting to the active region of Stx2 with B cell epitope peptide P1. The first, The Balb/c mouse were immuned with the B cell epitope peptide P1 coupled KLH, monoclonal hybridoma cell lines secreting MAbs against Stx2 were obtained quickly to apply the hybridoma technique, the semisolid culture containing methylium cellulose and indirect ELISA assay. The result is that 7 hybridoma cell lines lasting secreting MAbs against native Stx2 were screened from 1700 monoclonal hybridoma cell lines. The result of caryotype analysis shows that chromosome number of hybridoma cell stains 1F2, 18C3, 1G1and 6B3 is 106, 100, 98 and 104, respectively, and approximation of the summation of chromosome number of a splenic cell and a myeloma cell, which demonstrate that they are the fusion body of a splenic cell and a myeloma cell. However, chromosome number of hybridoma cell lines 9D7, 8D4and 11D3 is 140, 144and 142, respectively, about 40 more than summation of chromosome number of a splenic cell and a myeloma cell and approximation of the summation of chromosome number of two splenic cell and a myeloma cell. The result of Dot ELISA and Western Blot demonstrate that the MAb 1F2, 18C3, 1G1and 6B3 are specific MAb against A subunit of Stx2.and the MAb 8D4, 11D3 and 9D7 have cross reaction with KLH. The reason is maybe that a monoclonal cell stain has fused chromatosome of two splenic cells secreting two different kinds of MAb, against Stx2 and KLH.Subclass of MAb 1F2, 3B6, 1G1and 18C3 is IgG1κand the affinity affinity of them is 1.7×10-9, 6.3×10-8, 5.7×10-7and 9×10-7. It is demonstrated that 1F2, 18C3, 1G1 and 6B3 have effect to neutralize toxicity of Stx2 by Vero cells cytotoxicity assay test in vitro and eliminating toxic material test against Balb/c mouse in vivo, which of them, MAb 1F2 is the most valuable and can be used for constructing Fab gene engineering antibody.3. Construction, analysis of gene and structure of Fab gene engineering antibody against toxicity active region of Stx2The mouse MAbs are confined to apply in clinical therapy, because of their heterology. In this section, MAb is reconstructed to Fab antibody with gene engineering technology. The first, DNA sequences of of 1F2 antibody was fished from 1F2 hybridoma cells by one step RT-PCR. Then,κchain and Fd chain are inserted Pcomb3X, which is the Fab antibody expression vector, DNA sequences of Fab was sequenced , translated to amino acid sequence and analyzed by the software of NCBI and modeled tertiary structure by SWISS-MODEL online.The results show that theκchain gene was 648bp, and encode 216amino acids, and its molecular weight is 23882.52Da, and that the Fd chain gene was 636 and encode 212 amino acids, and its molecular weight is 22436.31Da. The Fab fragment is highly homology with the Fd heavy chain fragment andκlight chain gene of murine Ig. The amino acid sequences of Fab had typical features of Fd heavy chain fragment andκlight chain of murine Ig. The variable region ofκchain is 1~106 amino acids, its CDR1,CDR2 and CDR3 regions are 24aa~34aa,52aa~54aa and 91aa~98aa, respectively.The variable region of Fd chain is 1~108 amino acids, its CDR1,CDR2 and CDR3 regions are 20aa~27aa, 45aa~52aa and 91aa~100aa, respectively; The modeled tertiary structures ofκchain and Fd chain had typical features the tertiary structures of murine Ig. The analytic results of gene and structure of Fab indicate that Fab is a new murine antibody.4. Expression and identification of biological activity of Fab antibody against toxicity active region of Stx2The recombinant expression vector Pcomb3X-1F2Fab was transformed into E.coli XL-blue strain. After screened on SB plates with Ampicillin and identified by endonuclease digestion and PCR, the positive strain was induced by IPTG.. The location of expression products were determinated by ELISA and SDS-PAGE. At last, interested protein was purified by protein L resin and purified product was identified by SDS-PAGE, Dot ELISA and Western blot assay. The ability of Stx2-specific Fab antibody to neutralize Stx2 was examined both with the Vero cell cytotoxicity assay in vitro and with eliminating toxic material test against Balb/c mouse in vivo. The resulit is that the Fab antibody was successfully expressed and secreted from the bacteria containing the recombinant plasmid after induction by IPTG, and expression products mainly existed in periplasmic space of E.coli. The results of SDS-PAGE and Western blot assay showed that molecular weight of interested protein was approximately 23 000, which is consist with predicted molecular weight ofκchain and Fd chain, and the band can bind with Stx2. The engineering Fab antibody against Stx2 was successfully constructed and secretorily expressed in E.coli. The expression product has good specicity and desirable affinity to relative antigen. In the same time, Stx2A1-specific Fab antibody has been shown to neutralize the tocxicity of Stx2 in vitro and in vivo in murine.In a word,antibody will play a key role in prevention and therapy of EHEC O157:H7 infection. It is important to study anti-Stx2 antibodies against EHEC O157:H7.It is no doubt that the therapeutic antibodies against EHEC O157:H7 will benefit the people in all over the world in future.