Epitope Mapping of Senecavirus A 3A Protein Using Monoclonal Antibodies

Figures

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  Figure 1. Characterization, expression, and purification of SVA 3A protein. (A) Analysis of antigenicity and hydrophilicity of 3A protein using Protean software. (B) The secondary structure of the 3A protein was analyzed by PSIPRED online software. (C, D) The expression in E. coli and purification of 3A protein were analyzed by SDS-PAGE (C) and Western blot (D).

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  Figure 2. Preparation and functional analysis of mAbs. (A) Immunization protocol for mice. (B) BHK-21 cells were infected with SVA-WT at an MOI of 0.1. Cells were harvested and fixed at 24 hpi for IFA analysis using mAbs AG4 and 2F3 as primary antibody. (C) Western blot analysis of the reactivity of mAbs AG4 and 2F3 with the linear 3A protein.

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  Figure 3. Epitope mapping of mAb AG4. (A) Eight overlapping truncated fragments of the 3A protein were expressed in E. coli BL21 and analyzed by Western blot using mAb AG4. (B, C) Identification of the minimal epitope recognized by mAb AG4. The 3A1-20aa fragment was sequentially truncated from the C-terminus (B) and N-terminus (C). (D) Alanine scanning was performed to identify the critical amino acids recognized by mAb AG4, as well as to mutate residue 1S to 1N.

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  Figure 4. Construction of 3A epitope deletion mutants and epitope mapping of mAb 2F3. (A) The B cell epitope of SVA 3A was predicted using the BepiPred 3.0 tool. (B) Schematic diagram of the construction of 3A epitope deletion infectious clones. (C) The constructed infectious clones were transfected into BHK-21 cells for virus rescue. Only the pSVA-Δ3A(75-80) and pSVA-Δ3A(77-86) clones successfully rescued the virus, which were named rSVA-Δ3A(75-80) and rSVA-Δ3A(77-86), respectively. The infection of the rescued virus was detected by mAb 2C7. (D) To identify the epitope recognized by mAb 2F3, BHK-21 cells were infected with the rescued epitope deletion viruses rSVA-Δ3A(75-80) and rSVA-Δ3A(77-86) at an MOI of 0.1. Cells were harvested and fixed at 12 hpi for IFA using mAb 2F3. (E) The rSVA-Δ3A(75-80) virus was serially passaged 12 times in BHK-21 cells, and the genetic stability analysis of the epitope deletion virus rSVA-Δ3A(75-80) was conducted. Nucleic acids were extracted and PCR amplified from passages 2, 4, 6, 8, 10, and 12. The PCR product from the 12th passage was sequenced for validation. (F) Growth curve of SVA WT and rSVA-Δ3A. BHK-21 cells were infected with SVA-WT and…

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  Figure 5. Structural analysis and multiple sequence alignment of SVA 3A. (A) Sequence alignment analysis of the identified epitopes was performed using MEGA 7.0 software. The sequence alignment of the epitope 1SPNEND6 among different SVA isolates (left) and the alignment of the epitope 75QEETEG80 among different SVA isolates (right). (B) The detailed positions of the 3A protein epitopes 1SPNEND6 (red) and 75QEETEG80 (green) were analyzed, respectively. (C) Sequence alignment analysis of 3A proteins of different picornaviruses. The red arrows indicate the key amino acids identified. The red boxes represent the epitope 1SPNEND6 and 75QEETEG80, respectively.