Table 1.
Progressively truncated peptide sequences for defining the minimal linear epitope.
Figure 1.
The binding of MAb 1B3 with all 15 reference strains of H. parasuis.
(A) Reactivity of MAb 1B3 with the lysates of all 15 serotype strains of H. parasuis by Dot blot analysis. 1–15: 15 serotype reference strains of H. parasuis, respectively; 16: S. enterica; 17: S. aureus; 18: ETEC; 19: A. pleuropneumoniae. (B) Reactivity of MAb 1B3 with the lysates of all 15 serotype strains of H. parasuis by Western blot analysis. M: PageRuler™ Prestained Protein Ladder; Lane 1–15: 15 serotype reference strains of H. parasuis, respectively; Lane 16: S. enterica; Lane 17: S. aureus; Lane 18: ETEC; Lane 19: A. pleuropneumoniae.
Figure 2.
Identification of bacterial protein reacting with MAb 1B3.
(A) SDS-PAGE of bacterial proteins after IP. The MAb 1G7-HPS HS80 mixture and the MAb 1B3-ETEC mixture were used as the negative controls. Lane 1: MAb 1G7-HPS HS80; lane 2: MAb 1B3-ETEC; lane 3: MAb 1B3-HPS HS80. (B) Identified results of protein spots through MALDI-TOF-MS. Protein scores (n = 152 for MAb 1B3) that were greater than 58 were significant (P<0.05). Protein scores were derived from ion scores as a non-probabilistic basis for ranking protein hits. (C) The amino acid sequence of OppA. The matched peptides with the OppA protein sequence were shown in bold red.
Figure 3.
Identification of the epitope recognized by MAb 1B3.
(A) Detection of the selected phages for antibody binding by Phage ELISA. Fifteen phage clones selected after three rounds of biopanning were analyzed the binding activity to MAb 1B3 by Phage ELISA. Three independent assays were carried out. (B) Sequence comparison of random peptide inserts displayed on the positive phages. The conservative amino acid motifs are boxed.
Figure 4.
Confirmation of the epitope recognized by MAb 1B3.
(A) Reactivity between MAb 1B3 and the truncated OppA protein of H. parasuis. (a) Coomassie brilliant blue stained SDS-PAGE of the truncated OppA protein expression. (b) Reactivity of the truncated OppA protein with MAb 1B3 by Western blot analysis. M: PageRuler™ Prestained Protein Ladder; Lane 1: pET-OppA; Lane 2: empty vector pET-32a. (B) Progressively truncated peptides defined the minimal linear epitope recognized by MAb 1B3 or murine antisera. MAb 1B3 was screened against a series of truncated peptides with progressively deleted amino acid residues from the amino and/or carboxyl termini to determine the minimal linear peptide sequence required for MAb 1B3 binding. (C) Competitive inhibition assay using increasing concentrations of the peptide 1B3-8 and control peptide 1B3-6.
Figure 5.
Amino acid sequence alignments of the epitope regions from the H. parasuis OppA protein.
(A) Sequence alignments of 6 H. parasuis strains from GenBank. The GenBank accession numbers of H. parasuis strains are shown in front of the strains' names. The homologous sequences of different H. parasuis strains corresponding to the identified epitope are highlighted. (B) Sequence alignments of the epitope region from OppA proteins of H. parasuis and other 75 animal bacteria. The sequence corresponding to the region encompassing H. parasuis minimal linear epitope was identified and aligned for a panel of 75 bacterial strains from animals. The defined epitope and their corresponding regions in OppA proteins of bacteria are highlighted.