Construction And Screening Of Human Fab Phage Antibody Library Against The Toxic Fragment (stx2a₁) Of Shiga ToxinⅡ From EHEC O157:H7

Shiga toxins (stx) are produced by enterohemorrhagic Escherichia coli (EHEC). Infections with EHEC can lead to a variety of gastrointestinal and systemic complications, such as bloody and non-bloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS). However, an epidemiological study indicated that treatment of EHEC O157:H7 infection cannot involve antibiotics because they can sometime increase the incidence of HUS due to the pathogen.Stxs have been divided into 2 subtypes, stx1 and stx2. Stx2 is usually more strongly associated with clinical disease than stx1 when consider from the standpoint of both epidemiological and experimental studies. Furthermore, the two types of toxins are AB5 holotoxins that are comprised of a single enzymatically active A subunit (32 kDa) that non-covalently associates with multiple B subunits (7.6 kDa). The A subunit that acts as an N-glycosidase removes an adenosine residue from the 28S rRNA of the 60S ribosome, inhibits protein synthesis, and this in turn leads to cell death. The B subunit is responsible for binding to target cells via its glycolipid receptor, the globotriaosyl ceramide (Gb3) or Gb4 cellular receptor. The crystal structure of stx2 shows the A subunit to consist of the A1 portion (28 kDa) covalently associated with the A2 portion (4 kDa) through a disulfide bond. The A2 peptide forms anα-helix and traverses the pore of the B-pentamer. The tyrosine residue (A77) of the A1 portion forms the catalytic site. Despite decades of research, we still lack an effective prevention and therapy for EHEC infections, and novel therapeutic strategies have yet to be found. Thus, it is important to investigate the A subunit, especially of the toxic active fragment, i.e. the A1 subunit of the shiga toxin. The humoral immune response evoked by Stx2A1 can more effectively prevent EHEC O157:H7 infections, with the antibody raised to Stx2A1 having a better specificity against the toxin. However, the use of rodent antibodies in a therapeutic setting has the disadvantage that repeated administrations of such monoclonal antibodies often result in a human anti-mouse (HAMA) immune response. So producing human antibodies had become an emergency problem. An alternative approach to these humanization techniques is the use of epitope guided selection (EGS) in recently years. Firstly, the Fd or Lc gene of a murine anti-Stx2a1 antibody was paired with a repertoire of human light chain or heavy chain, and displayed on the filamentous phage forming a hybrid phage antibody library. The selected human light chain or heavy chain were in turn paired with a repertoire of human Lc or Fd fragments forming a human phage antibody library, then human Fab antibodies that binds to an epitope of the original rodent antibody are isolated. In the study, we constructed a murine Fab phage antibody library against the toxic fragment (stx2a1) of Shiga ToxinⅡfrom EHEC O157:H7 and adopted EGS for humanization murine Fab phage antibody library, it would lay a solid foundation for its further application in the therapy of infection with shiga-like toxin from EHEC O157:H7.There are three parts of our study as below:Part 1: Cloning, expression and purification a truncated fragment (stx2a1) of the shiga-like toxin 2A1 subunit of EHEC O157:H7Objective: To express and purify the shiga-Like toxin 2A1 subunit of EHEC O157:H7. Methods: The full-length sequence of stx2A1 was analyzed using DNAstar. The 15 amino acid residues of the carboxyl terminal of stx2A1 were truncated. Stx2a1 was PCR-amplified from the E. coli strain 99A021 using a conventional method. The recombinant plasmid was transformed into competent E. coli BL21 (DE3). Transformed bacteria was grown in LB medium containing ampicillin (100μg/ml), at 37°C. When the OD600 reached 0.8, the cultures were divided and induced with 0.1 mM isopropylβ-D-thiogalactoside (IPTG) at 37°C. The expressed Stx2a1 was purified by Ni-affinity chromatography. The purified production was identified by SDS-PAGE, Western blot and ELISA analysis respectively. Results: DNA sequencing of stx2a1 showed it had no amino-acid mutation. SDS-PAGE showed that Stx2a1 was expressed and that its mass was 25 kDa, in accordance with prediction. The study on Stx2a1 expression was showed that there was a higher level of soluble expression when Stx2a1 was induced at 16°C. The expression level reached up to 30% of the total cellular protein, about 1.12 g/l culture and the purified Stx2a1 had a purity of 98%. Purified Stx2a1 can strongly react with S1D8, a monoclonal antibody raised against the A subunit of stx2 in Western blot and ELISA. Conclusion: A truncated fragment (stx2a1) of the shiga-like toxin 2A1 subunit of EHEC O157:H7 was successfully expressed and purified. This fragment (stx2a1) provides a good antigen for screening the humanized Fab phage library.Part 2: Construction and screening mouse Fab phage antibody library against shiga toxin 2 of EHEC O157:H7Objective: To construct and screen mouse Fab phage antibody library against shiga toxin 2 of EHEC O157:H7. Methods: BALB/c mice were immunized with inactivated Stx2 of EHEC O157:H7, and the antibody light chainкgenes and heavy chain Fd genes from the spleen cells were amplified by RT-PCR. After restrictive digestion the light chainкgenes and heavy chain Fd genes were inserted into the phagemid vector pComb3x successively and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Stx2 of EHEC O157:H7 was constructed by infection of helper phage M13K07. The specific antibody against Stx2 of EHEC O157:H7 was obtained after selected with native Stx2. The characteristics of combinding with antigens were identified by Western blot and the positive clones were sequenced. Results: The Fab phage antibody library with 1.56×107 volume was constructed and three positive clones which specifically recognized the native shiga toxin 2 were isolated. Sequence analysis of the three positive clones showed that the variable heavy domains (VH) and variable light domains (VL) were highly homologous with the registered murine Ig heavy chain V region sequences and kappa light chain sequences, respectively. Conclusion: Fab phage antibody library was successfully constructed and specific antibodies against Stx2a1 of EHEC O157:H7 were obtained.Part 3: Selection and expression of human Fab phage antibody against Stx2a1 of EHEC O157:H7Objective: To screen and express the antibody against Stx2a1 from a human Fab phage antibody library. Methods: The human Lc and Fd repertorie genes were amplified by RT-PCR from PBMC in normal persons, and human Lc genes cloned into the phagemid vector pComb3x with murine Fd gene against Stx2a1 to construct the human-mouse hybrid Fab antibody library. The antibody gene recombinant percentage and diversity were identified by colony counting, plasmid digestion and colony sequence analysis, respectively. Purified Stx2a1 was used as antigen to screen the displayed phage hybrid antibody library rescued by helper phage M13K07 for three rounds. The positive clones were selected and identified by western bloting. The human Fd genes were cloned into the pComb3x- hLc to construct the human Fab antibody library. Identified by colony counting, plasmid digestion and colony sequence analysis by the same methods as the human-mouse hybrid Fab antibody library. Results: A human Fab antibody library was constructed with 90% Fd gene recombinant. After 3 rounds panning with Stx2a1 two clones were selected by western bloting. Conclusion: Human anti-Stx2a1 Fab antibody was successfully screened using guided selection.