Abstract:[Objective] Asprosin (ASP), an adipokine produced from fibrillin-1 (FBN1) cleavage, is closely linked to type 2 diabetes mellitus and obesity when its level is abnormal, demonstrating significant effect on human health. Therefore, monitoring the ASP level is crucial. The author aims to screen the nanobodies specifically binding to ASP via phage display technology, offering a new antibody component for the research on ASP. [Method] The antigenic epitopes of ASP were predicted by the online tools ABCpred and NovoPro, and then coupled with bovine serum albumin (BSA) to form the coating antigen (ASP-Epitope-BSA). After five rounds of bio-panning, the nanobodies specifically binding to ASP were screened from a nanobody phage display library. The selected nanobodies were induced for expression in Escherichia coli. The half median effect concentration (EC50) values with ASP-Epitope-BSA and whole ASP antigen were determined, and the specificity was evaluated. Finally, homology modeling and molecular docking were performed to study the binding mode between the nanobody and ASP. [Result] One ASP-specific nanobody (VHH-19) was successfully screened out, with the induced expression level of 2.4 mg/dL in the LB culture with Escherichia coli. Performance evaluation showed that VHH-19 had EC50 values of (87.49±0.27) ng/mL and (128.94±0.48) ng/mL for ASP-Epitope-BSA and whole ASP antigen, respectively, demonstrating good specificity. Homology modeling and molecular docking revealed that ASP was adsorbed in an active pocket formed by the CDR1 and CDR3 regions of VHH-19, forming a sandwich-like binding mode. The Try116, Gly126, Lys115, and Glu124 of ASP form hydrogen bonds and electrostatic interactions with Glu107, Arg31, and Arg33 of VHH-19, respectively. [Conclusion] This study rapidly obtained the ASP-specific nanobody VHH-19 by bio-affinity panning based on a natural nanobody phage display library.
