Preparation of a Nanobody Specific to Dectin 1 and Its Anti-inflammatory Effects on Fungal Keratitis

Abstract

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 recombinant 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.

Biopanning, Heterologous Expression, and Characterization of a Shark-Derived Single-Domain Antibody Fusion Protein against Pancreatic Lipase

“…The eluents of imidazole with different concentrations were identified using SDS-PAGE to determine the elution conditions of the target protein. The corresponding protein was dialyzed with a binding buffer to remove the corresponding imidazole …”

“…The corresponding protein was dialyzed with a binding buffer to remove the corresponding imidazole. 36 2.6. PL-D2S ELISA Affinity Determination.…”

Exploration of Specific Nanobodies As Immunological Reagents to Detect Milk Allergen of β-Lactoglobulin without Interference of Hydrolytic Peptides

“…The apparent affinity of selected Nbs was estimated by performing a saturated ELISA against immobilized BLG. , Generally, 10 μg/mL BLG was coated in microtiter plates. After blocking with 1% gelatin, Nbs with serial dilutions of 100,000, 10,000, 1000, 100, 10, 1, 0.1, 0.01, and 0.001 nM were added.…”

Intestinal phages interact with bacteria and are involved in human diseases

“…The most common bacterial infections of the gastrointestinal tract are diarrhea caused by Clostridioides difficile , dysentery caused by Shigella , gastroenteritis caused by strains of E. coli , cholera caused by V. cholerae , and salmonellosis caused by Salmonella enteritidis . Many studies have assessed the potential of phages for treating gastrointestinal infections caused by E. coli, Enterobacte r, 128 V. cholera e, 129 and C. difficil e. 130 Phage therapy has successfully provided support for the treatment of intestinal infection in diabetic foot infection, 131 lung infection, 132 corneal abscess 133 and other conditions. For example, the Phagoburn (phase I–II clinical trial) project funded by the European Union under the 7th R&D Framework Programme has conducted a large-scale clinical trial of lysophages for the treatment of infected burn wounds.…”