title: Identification of antigenic epitopes of the SapA protein of Campylobacter fetus using a phage display peptide library.
abstract: In this study, we immunized mice with prokaryotically expressed recombinant surface layer protein, SapA, of Campylobacter fetus, generated hybridomas secreting mouse monoclonal antibodies (mAb) targeting SapA, and purified the mAb A2D5 from mouse ascites using saturated ammonium sulfate solution. The mAb A2D5, coated onto ELISA plates, was used to screen the phage random 12-peptide library through three rounds of panning. Following panning, 15 phage clones were randomly chosen and tested for reactivity with mAb A2D5 by indirect ELISA. Single-stranded DNA from positive clones was sequenced and compared with the sequence of SapA to predict the key epitope. ELISA and/or Western blot analyses further validated that synthetic peptides and recombinant peptide mimotopes all interact with mAb A2D5. Nine of ten positive phage clones identified by screening were sequenced successfully. Seven clones shared the same sequence HYDRHNYHWWHT; one had the sequence LSKNLPLTALGN; and the final one had the sequence SGMKEPELRSYS. These three sequences shared high homology with SapA J05577 in the region GNEKDFVTKIYSIALGNTSDVDGINYW, in which the underlined amino acids may serve as key residues in the epitope. ELISA and/or Western blot analyses showed that mAb A2D5 not only interacted with the four synthetic peptide mimotopes, but also with 14 prokaryotically expressed recombinant peptide mimotopes. The mimotopes identified in this study will aid future studies into the pathological processes and immune mechanisms of the SapA protein of C. fetus.
authors.affiliation: Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 15000, PR China.
authors.forename: Hailing
authors.initials: H
authors.lastname: Zhao
authors.forename: Shenye
authors.initials: S
authors.lastname: Yu
authors.forename: Huifang
authors.initials: H
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Si
authors.forename: Chunlai
authors.initials: C
authors.lastname: Wang
authors.forename: Siguo
authors.initials: S
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001428:Bacterial Vaccines / Q000276:immunology; D015153:Blotting, Western; D002168:Campylobacter fetus / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D011485:Protein Binding
chemical_list: D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D000939:Epitopes; D019151:Peptide Library