Development of monoclonal antibodies for GoAstV-2 VP27 protein and precise mapping of linear antigenic epitopes

Since 2016, a novel astrovirus identified as goose astrovirus (GoAstV), is classified into genotypes 1 and 2 (GoAstV-1 and GoAstV-2). GoAstV-2 has caused a severe outbreak of visceral gout in goslings characterized by extensive visceral urate deposition and kidney swelling, resulting in substantial financial losses to the goose farming industry in China. The GoAstV-2 VP27 protein encoded by ORF2 contains neutralizing antigenic epitopes, thus representing a key candidate for the development of diagnostic reagents and epitope vaccines. This study aimed to prepare the monoclonal antibody (mAb) against GoAstV-2 VP27 and identify its epitope. The GoAstV-2 VP27 protein was expressed and purified using a prokaryotic expression system, followed by immunization of BALB/c mice. We employed hybridoma technology to generate a stable monoclonal antibody-secreting cell line targeting GoAstV-2 VP27, which was named 11-39B and characterized as IgG2b with kappa light chain. Furthermore, the mAb 11-39B specifically bound to GoAstV-2 as confirmed by Western blotting (WB), immunofluorescence assay (IFA) and immunohistochemistry (IHC), and potently neutralized GoAstV-2 infection in vivo in a dose-dependent manner. For epitope mapping, sequential truncations of the GoAstV-2 VP27 protein were constructed by eukaryotic expression and tested by WB. The results indicated that peptide ⁶⁶¹SLKTS⁶⁶⁵ was the minimal epitope recognized by mAb 11-39B. Homology and structural analyses demonstrated that the epitope was situated on the surface of the VP27 protein and exhibited high conservation among GoAstV-2 strains but exhibited significant differences in the GoAstV-1 serotype. Our study contributes to a better understanding of the GoAstV-2 VP27 antigenic region and provides a basis for establishing epitope-based GoAstV-2 diagnostic methods and vaccine development.