Screening and Interaction Analysis of Shark-Derived Nanobodies against Crayfish Major Allergen Pro c 2

Pro c 2 (arginine kinase) is a major allergen in crayfish (<i>Procambarus clarkii</i>). Shark-derived variable domains of new antigen receptors (VNARs) have advantages in developing allergen detection and immunotherapy. This study constructed a VNAR domain library from <i>Chiloscyllium plagiosum</i> immunized with Pro c 2. Three VNARs (VNAR-11, VNAR-20, and VNAR-29) against Pro c 2 obtained by screening the library were expressed in the HEK293F cells, fusing with the immunoglobulin (Ig) G1 Fc fragment (VNAR-Fc-11, VNAR-Fc-29, and VNAR-Fc-20). The VNAR-Fc fusions bound to Pro c 2 with an affinity <i>K</i>_D ranging from 0.2131 ∼ 465.3 μM, with the ability to inhibit patients' IgE binding to Pro c 2. VNAR-20 and VNAR-29 displayed more stable binding with Pro c 2 during molecular dynamics simulation. The binding sites of the VNARs are distributed in the conserved IgE epitopes of arginine kinase. These achievements indicate the application potential of VNARs in allergen detection and allergy therapeutics.