Two novel conserved linear B-cell epitopes identified in the S2 subunit of the infectious bronchitis virus spike protein

Fig. 1. Expression and purification of the S860–1088 protein. (A) Sequence similarity matrix of the S2 subunit region (860–1088 aa) from five major IBV lineages (GI-1, GI-7, GI-13, GI-19 and GI-28) isolated in China. The color scale indicated similarity percentages. (B) Agarose gel electrophoresis of PCR products. Lane 1: amplified DNA fragment encoding S860–1088 amino acids. Lane 2: pET-28a vector digested with BamH I and Xho I. (C) SDS-PAGE analysis of recombinant S860–1088 protein expression. Left panel: protein purification steps. Lane M: protein marker; Vector: empty vector control; Flow-through: unbound proteins; Wash buffer: wash fraction; Elution buffer: purified target protein. Right panel: SDS-PAGE analysis of protein expression in bacterial lysates. Lane M: protein marker; Lane 1: empty vector control; Lane 2: pellet after sonication; Lane 3: supernatant after sonication; Lane 4: purified recombinant S860–1088 protein. (D) WB confirmation of recombinant S860–1088 protein identity. The left panel was probed with anti-His antibody, and the right panel with anti-YN antiserum. Lane M: protein marker. Lane 1: empty vector control; Lane 2: purified recombinant S860–1088 protein.

Fig. 2. Production of mAbs. (A) IFA showing reactivity of sera from three immunized mice against CEK cells with or without IBV infection. Mouse sera served as primary antibodies, and Alexa Fluor 488-conjugated anti-mouse IgG (H + L) as secondary antibody. Scale bar: 100 μm.

Fig. 3. Identification of linear epitopes recognized by mAbs using WB and IFA. (A-C) Truncation scheme for S860–1088 (left) and corresponding WB showing recognition of GST-tagged fragments expressed in Vero E6 cells (right). GAPDH was used as an internal control. "+" indicates positive recognition; "-" indicates no recognition. Primary antibodies: mAbs 6A6, 6E2, 6A1and 6A9; secondary antibody: HRP-conjugated goat anti-mouse IgG. (D) IFA assessment of mAbs recognition of minimal epitopes. Epitopes 1040KWWND1044 and 1046KHELPDF1052 were cloned into pCMV-FLAG-GST vector and transfected into VERO cells. GST alone served as negative control. Primary antibodies: prepared mAbs; secondary antibody: Alexa Fluor 488-conjugated anti-mouse IgG (H + L).

Fig. 4. Bioinformatics analysis of epitopes. (A) Schematic diagram of IBV spike protein domains. S1-NTD: N-terminal domain of S1 subunit; S1-CTD: C-terminal domain of S1 subunit; S1/S2: cleavage site between S1 and S2 subunits; CH: central helix; FP: fusion peptide; HR1: heptad repeat 1; HR2: heptad repeat 2. (B) Analysis of antigenicity, hydrophobicity, and surface probability of S860–1088 using the Protean software. (C) Mapping of the epitopes on the three-dimensional structure of the full-length spike protein. The epitope 1040KWWND1044 is shown in yellow, while epitope 1046KHELPDF1052 is shown in purple.

Fig. 5. Evaluation of epitopes conservation and mAbs cross-reactivity among different IBV strains. (A) Amino acid sequences of the corresponding epitope from different lineages of IBV were aligned using Geneious Prime software, and sequence conservation logos were generated using WebLogo. The y-axis shows information content (in bits), indicating the degree of conservation at each position. (B) Effect of amino acid variation at residue 1044 on monoclonal antibody recognition. Vero E6 cells were harvested 24 h after transfection, and the reactivity of mAbs 6A6 and 6E2 against different amino acid variants was analyzed by WB, with GAPDH as an internal control. (C) Epitope sequences of three representative IBV strains. "+" indicates a positive reaction; "-" indicates a negative reaction. (D) IFA showing the reactivity of mAbs against different IBV strains. Cells infected at an MOI of 0.1 were fixed at 36 hpi and stained with antibodies against spike (green) and nucleocapsid (N, red) proteins. Scale bar: 100 μm. (E) WB analysis of mAbs reactivity against different IBV strains. Cells infected at an MOI of 0.1 were harvested and lysed at 24 hpi. mAbs reactivity against different strains was…