Novel Library Assembly Technique for Developing Nanobodies Targeting IPNv VP2 Protein

Infectious pancreatic necrosis virus (IPNv) poses a significant threat to the global salmon farming industry, causing high mortality and economic losses. Given the limitations of traditional antibody therapies in aquaculture, we explored the development of nanobodies targeting the VP2 protein of IPNv. We developed a novel nanobody library generation method using a streamlined assembly protocol based on Type IIS restriction enzymes. This method enabled quick and efficient bacterial display library generation, and concatenated, multi-level cloning through an ingenious design in which the ligation of the first level generates a new restriction site that can be utilized in the subsequent cloning level, allowing for rapid transfer to other vectors. By combining this assembly approach with density gradient-based enrichment and high-throughput screening, we identified nanobodies that specifically recognize IPNv’s VP2 protein. Notably, the P9 clone demonstrated high specificity for IPNv in immunofluorescence assays, highlighting its diagnostic potential. Our method not only accelerates nanobody library generation but also enhances its quality and adaptability, marking a significant advancement in enhancing the responsiveness of nanobody development platforms against emerging pathogen outbreaks.