Construction of an Enhanced Phage Display Library of Artificially Randomized vNARs from Cloudy Catshark (Scyliorhinus Torazame) and Screening against Miamiensis Avidus.

Single-domain antibodies (sdAbs), particularly variable new antigen receptors (vNARs) from cartilaginous fish, represent promising tools for therapeutic and diagnostic applications due to their small size, high stability, and binding versatility. In this study, we developed a semi-synthetic, open reading frame (ORF)-enriched vNAR library from the cloudy catshark (Scyliorhinus torazame) and optimized it for efficient expression in Escherichia coli through codon optimization. To improve the precision of selection, a β-lactamase-based ORF filtering system and a trypsin-sensitive helper phage packaging system were incorporated to minimize non-functional clones and helper phage contamination. The resulting vNAR library exhibited high diversity and functionality, as demonstrated by successful biopanning against both model (hen egg white lysozyme) and pathogenic (Miamiensis avidus) targets. Screening against M. avidus resulted in the isolation of vNAR-displaying phage clones with robust and specific surface-binding activity, as validated by ELISA and live-cell binding assays. These results highlight the utility of synthetic vNAR libraries in developing specific molecular binders and suggest their potential application in controlling scuticociliatosis in aquaculture.