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Screening and Interaction Analysis of Shark-Derived Nanobodies against Crayfish Major Allergen Pro c 2
Yang Yang1, Xin-Rong He2, Si-Yang He3
1College of Environment and Public Health, Xiamen Huaxia University, 288 Tianma Road, Xiamen 361024, Fujian, China.
Abstract:
Pro c 2 (arginine kinase) is a major allergen in crayfish (Procambarus clarkii). 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 Chiloscyllium plagiosum 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 KD 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.
Insights
Shark-derived VNARs show promise for detecting crayfish allergens and developing new allergy treatments. These novel antibodies effectively bind to Pro c 2, inhibiting allergic reactions.
Area of Science:
- Biotechnology and Immunology
- Allergen immunotherapy
- Protein engineering
Background:
- Pro c 2, a major allergen in crayfish (Procambarus clarkii), triggers allergic reactions in susceptible individuals.
- Shark-derived variable domains of new antigen receptors (VNARs) offer unique advantages for developing diagnostic and therapeutic tools against allergens.
- Existing methods for allergen detection and immunotherapy require innovative solutions for improved efficacy and specificity.
Purpose of the Study:
- To construct and screen a VNAR domain library from Chiloscyllium plagiosum immunized with Pro c 2.
- To express and characterize Pro c 2-specific VNAR-Fc fusion proteins for potential therapeutic applications.
- To evaluate the binding affinity, inhibitory capacity, and stability of VNARs against Pro c 2.
Main Methods:
- Construction of a VNAR domain library from Chiloscyllium plagiosum immunized with Pro c 2.
- Screening of the library to identify Pro c 2-specific VNARs (VNAR-11, VNAR-20, VNAR-29).
- Expression of VNAR-Fc fusion proteins in HEK293F cells and characterization of their binding affinity (KD) and inhibitory potential against patient IgE.
Main Results:
- Three VNARs (VNAR-11, VNAR-20, VNAR-29) were identified and expressed as VNAR-Fc fusion proteins.
- VNAR-Fc fusions demonstrated binding to Pro c 2 with dissociation constants (KD) ranging from 0.2131 to 465.3 μM.
- The VNARs successfully inhibited IgE binding to Pro c 2, with VNAR-20 and VNAR-29 showing enhanced stability in molecular dynamics simulations.
Conclusions:
- VNARs derived from Chiloscyllium plagiosum exhibit significant potential for the detection of crayfish allergen Pro c 2.
- The characterized VNARs can inhibit IgE binding to Pro c 2, indicating their therapeutic applicability in managing crayfish allergy.
- These findings highlight the promise of VNAR technology in advancing both diagnostic and therapeutic strategies for food allergies.
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