Development and epitope mapping of monoclonal antibodies against group a porcine rotavirus VP6 protein

Porcine rotavirus (PoRV) is a major causative agent of viral diarrhea in piglets. Its VP6 protein, the most abundant structural component of the viral particle, is highly conserved and immunogenic, making it an ideal target for developing broad-spectrum diagnostics and investigating cross-protective immunity. In this study, the PoRV VP6 recombinant protein was successfully produced and purified using a prokaryotic expression system. The recombinant VP6 protein was successfully expressed in E. coli and purified, with its purity assessed by RP-HPLC (61.43%). Four hybridoma cell lines (1D9, 2E7, 3G11, and 5E3) stably secreting monoclonal antibodies (mAbs) against VP6 were established via hybridoma technology. These mAbs exhibited strong reactivity and high specificity, as confirmed by indirect ELISA, Western blot, and indirect immunofluorescence assays. Furthermore, employing a strategy of serial truncation and alanine scanning, we identified the four linear B-cell epitopes on the VP6 protein: ⁴³NDFQTGG⁴⁹, ⁵¹GNLPIRNWTF⁶⁰, ²⁰⁸EHVVPLR²¹⁴, and ²³⁷INSAD²⁴¹. Conservation analysis revealed that the epitopes recognized by mAbs 1D9 and 3G11 were highly conserved across geographically distinct strains, whereas those recognized by mAbs 5E3 and 2E7 displayed notable region-specific characteristics. Molecular docking simulations demonstrated that the mAbs formed stable complexes with their corresponding epitopes primarily through hydrogen bonding, electrostatic interactions, and hydrophobic forces, with mAb 5E3 exhibiting the highest affinity (Docking Score: -208.4). The mAbs obtained in this study, together with the precise mapping of their epitopes, provide crucial tools and a foundation for advancing PoRV serological detection methods and studying the antigenic structure of the VP6 protein.