Identification of a novel Haemophilus parasuis-specific B cell epitope using monoclonal antibody against the OppA protein

Abstract

Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched (469)KTPAEAR(475) of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis.

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.