Characterization of a Shiga toxin 1-neutralizing recombinant Fab fragment isolated by phage display system.
Shiga toxin (Stx)-producing Escherichia coli (STEC) causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Molecular structural analysis of Stx-neutralizing monoclonal antibody (mAb) will be helpful for development of therapeutics and prophylactics for STEC infection. In this study, we cloned the genes of Stx 1-neutralizing mAb, termed 5-5B from hybridoma cells by phage display system and characterized its recombinant Fab (rFab) fragment. 5-5B rFab fragment reacted with Stx1, but not with Stx2 and bovine serum albumin (BSA). It also showed the neutralizing activity against the cytotoxicity of Stx1. These results imply that 5-5B rFab fragment is functionally identical to parent mAb. The variable heavy (VH) and light domains were found to be highly homologous with the derived germ line sequences. As for VH domain, the complementarity determining regions (CDRs) showed higher replacement/substitution mutation ratio than that in the frame work regions. Among the regions, CDR2 showed the most frequent nucleotide and amino acid substitutions. These results suggest that heavy chain CDR2 may mainly be associated with the 5-5B function, that is neutralizing cytotoxicity of Stx1.
Inoue
Kazuyuki
K
Department of Pharmaceutical Science, Akita University Hospital, Hondo, Japan.
Itoh
Kunihiko
K
Nakao
Hiroshi
H
Takeda
Tae
T
Suzuki
Toshio
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002292:Carcinoma, Renal Cell / Q000473:pathology; D002417:Cattle; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D005798:Genes, Bacterial; D006801:Humans; D006825:Hybridomas / Q000166:cytology; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007680:Kidney Neoplasms / Q000473:pathology; D007928:Lethal Dose 50; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D018675:Toxicity Tests
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D012710:Serum Albumin, Bovine