Preparation and epitope identification of a novel monoclonal antibody against 3A protein of Senecavirus A

Abstract

Senecavirus A (SVA) infection causes vesicular disease in pigs and acute death of newborn piglets. Frequent gene mutation and recombination events lead to difficulties for SVA prevention and eradication, especially impeding the development of effective vaccine or treatment drug. SVA nonstructural 3 A protein plays an important role in viral replication and various immune evasion pathways, which makes it a potential therapeutic target. In this study, prokaryotic 3 A protein was successfully expressed in Escherichia.coli BL21 (DE3) system and purified with Ni-affinity chromatography. Western blotting results showed 3 A protein specifically reacted with serum of SVA infected pig, indicating that nonstructural 3 A protein was a potential diagnostic maker for SVA serological testing, especially for differentiating infected from vaccinated animals. In addition, specific monoclonal antibody (mAb) 5A7 against 3 A protein was also prepared. Indirect immunofluorescence assay and Western blotting assay showed mAb 5A7 specifically reacted with SVA cultured in IBRS-2 cells. To characterize the epitope of mAb 5A7, serial truncated peptides of 3 A protein were prepared. Western blotting assay showed the epitope of mAb 5A7 was ⁵NDDTPVDEALGR¹⁶. Bioinformatic studies revealed that the epitopes are located on the extrinsic membrane domain of 3 A protein with good antigenicity. To sum up, SVA 3 A protein and its specific mAb 5A7 were successfully prepared in this study, which will contribute to biological function study of 3 A protein and the pathogenic mechanism of SVA, as well as the diagnosis and prevention of this disease.