Preparation of a Nanobody Specific to Dectin and Its Anti-inflammatory Effects on Fungal Keratitis

Abstract

Objective: To prepare a nanobody specific to dectin 1 and verify its specificity and anti-inflammatory effects on Aspergillus fumigatus keratitis. Methods: The nanobody was selected from a high-quality shark-antibody library constructed with phage-display technology. The nanobody was developed in the expression systems of Escherichia coli. Indirect ELISA was used to determine the specificity of the nanobody to recombinant dectin 1 protein. The potential of the nanobody to be recognized and expressed on the surfaces of cells and corneas was detected by immunofluorescence, and its anti-inflammatory effect on A. fumigatus keratitis was further verified. After infection with A. fumigatus, eyes of C57B L/6 mice were treated with nanobodies. Human corneal epithelial cells (HCECs) were pretreated with nanobodies and then incubated with A. fumigatus. Clinical scores and slit-lamp photography were used to assess disease response in mouse corneas. RT-PCR and ELISA were used to evaluate mRNA and protein expression of IL1β and IL6 in both mouse corneas and HCECs. Results: The nanobody was successfully expressed through microbial system and showed specific high-affinity binding to recombi-nant dectin 1. Furthermore, it exhibited specific binding to dectin 1 expressed on the surfaces of cells and recognized dectin 1 in mouse corneas. Importantly, it reduced clinical scores of A. fumigatus keratitis in mice compared with a PBS-treatment group. In addition, it decreased mRNA and protein expression of IL1β and IL6 in infected corneas and HCECs stimulated with A. fumigatus. Conclusion: These results suggest that this nanobody can bring about anti-inflammatory effects. This highlights the potential of these nanobodies as innovative therapeutic agents in A. fumigatus.