Objective Grass carp reovirus genotype Ⅱ (GCRV-Ⅱ) poses a serious threat to the healthy development of grass carp (Ctenoparyngodon idellus) aquaculture in China. This study aims to generate genetically engineered antibodies targeting the VP4 protein of GCRV-Ⅱ by integrating single B cell antibody technology with mammalian expression systems.

Methods The VP4 gene of GCRV-Ⅱ was cloned into a eukaryotic expression vector and expressed in a mammalian system to produce recombinant VP4 protein. New Zealand white rabbits were immunized with the purified protein to elicit antigen-specific memory B cells. Splenic B cells were isolated and subjected to polymerase chain reaction (PCR) amplification of immunoglobulin variable (VH/VL) and constant region genes. Recombinant monoclonal antibodies (mAbs) were expressed in HEK293F cells through transient transfection. Antigen-binding characteristics were systematically evaluated using enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence assay (IF).

Results Through screening the supernatants of isolated single B cells via ELISA, we identified three B-cell clones (1D5, 1F9, and 1H1) with strong binding affinity to the VP4 protein. Their ability to bind VP4 antigens in virus-infected tissues was further confirmed by immunofluorescence (IF) assays. Subsequently, we recombinantly expressed these antibodies and obtained three anti-VP4 monoclonal antibodies (1D5, 1F9, and 1H1), all exhibiting ELISA titers of up to 1∶128000. Western blot analysis demonstrated that the 1D5, 1F9, and 1H1 monoclonal antibodies specifically recognized a 65.4 kDa protein band in GCRV-YX246-infected brain cells, consistent with the theoretical molecular weight of the viral VP4 protein. IF assays further verified that these antibodies could specifically detect viral antigens in GCRV-YX246-infected head kidney tissue sections of grass carp.

Conclusion The study successfully generate and characterize VP4-specific recombinant monoclonal antibodies with high affinity and specificity. The results of the study lays the foundation for the development of rapid diagnostic kits for GCRV-Ⅱ and functional studies of the VP4 protein.