Construction and evaluation of a synthetic <scp>VNAR</scp> library for targeted antigen screening

Sharks produce a unique antibody type, whose variable domain (VNAR) is the smallest known antibody fragment. VNARs possess high affinity, stability, and tissue penetration, making them attractive for biomedical applications. In this study, a synthetic VNAR library with extensive CDR3 sequence diversity was constructed through a three-step PCR amplification process of VNAR gene fragments, which were subsequently cloned into the pR2 phagemid vector. The resulting library achieved a size of 1.17 × 10¹⁰ colony-forming units (CFU). Using green fluorescent protein (GFP) and hemoglobin as coating proteins, six GFP-specific VNARs and two hemoglobin-specific VNARs were screened from the constructed synthetic library using phage display technology. The binding affinities and physicochemical stability of these VNARs were evaluated through ELISA and BLI assays, revealing that the GFP-specific VNARs (14A-4C, 15A-5C, 14-3H, and 14-1A) and the hemoglobin-specific VNAR (13F-1A) exhibited nanomolar-level affinities, comparable to those derived from immune libraries. Hence, the synthetic VNAR library developed in this study holds great potential for biological research and applications, providing a versatile platform for screening VNARs specific to various antigens.