Single-B-cell cloning and recombinant antibodies generation to analyze the antigenicity of porcine reproductive and respiratory syndrome virus nonstructural protein 12

The prevalence and variation of porcine reproductive and respiratory syndrome virus (PRRSV) in China are increasing. The rapid preparation of essential antibodies will effectively reveal the antigenicity, epitopes, and intracellular distribution of viral proteins. Single-B-cell antibody technology is a novel method for screening diverse functional monoclonal antibodies (mAbs). Herein, we successfully expressed PRRSV nonstructural protein 12 (Nsp12) in suspension-cultured Chinese hamster ovary (CHO) cells. Using single-B-cell antibody technology, we utilized fluorescence-activated cell sorting to collect individual immune B cells and prepared single-cell reverse transcription-polymerase chain reaction to clone the variable region of immunoglobulin heavy chain (IgH) and immunoglobulin light chain (IgK). Two recombinant mAbs were generated via transient transfection of CHO cells with the corresponding expression plasmids of IgH and IgK. A novel linear epitope (¹⁰⁴YEFTGNGEDW¹¹³) of Nsp12 was identified using mAb_1N14. This epitope was conserved in lineages 1, 5, and 8 of PRRSV-2 and was located on the surface of the Nsp12 spatial structure. The amino acid mutation in Nsp12 of lineage 3 PRRSV-2 affected the antigenicity of this linear epitope. A conserved conformational epitope was identified using mAb_2S18, and the spatial structure of Nsp12 showed high similarity between PRRSV-1 and different lineages of PRRSV-2. During PRRSV infection, Nsp12 was distributed in the cytoplasm and accumulated in the nucleus. Overall, antigenicity analysis and novel epitope identification contributed to the in-depth exploration of the biological function of Nsp12 and will facilitate the development of detection assays and antiviral strategies.