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| Design and Recombinant Production of Nanobodies Targeting Middle East respiratory syndrome coronavirus |
| ZHANG Yan-Ru1,2, WANG Tian-Tian2, XU Hui-Fang2, WANG Hao2, WANG Han2, WU Jun2, LI Xiang-Chen1,*, LIU Bo2,* |
1 College of Animal Science and Technology·College of Veterinary Medicine, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China;
2 Laboratory of Advanced Biotechnology, Academy of Military Medical Sciences, Academy of Military Sciences, Beijing 100071, China |
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Abstract Middle east respiratory syndrome coronavirus (MERS-CoV), first identified in 2012, primarily infects dromedary camels (Camelus dromedarius) and humans (Homo sapiens). Nanobodies have garnered significant attention due to their unique structural and functional advantages. This study focuses on the molecular design, recombinant expression in engineered yeast, purification, and biological activity evaluation of nanobodies targeting MERS-CoV. Based on previously screened nanobodies Nb1 and Nb2, this study amplified the corresponding nanobody genes and constructed eukaryotic expression vectors for monovalent nanobodies (Nb1, Nb2) as well as a bispecific nanobody (Nb3). High-expression yeast (Pichia pastoris) strains were subsequently selected. Considering the physicochemical properties of nanobodies, a purification protocol was established incorporating affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography to obtain tag-free monovalent and bispecific nanobodies.The binding capabilities of the nanobodies to MERS-CoV antigens were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). Additionally, pseudovirus-based neutralization assays were conducted to verify antiviral activity. High-purity, tag-free preparations of Nb1, Nb2, and Nb3 were successfully obtained. Among them, Nb3 exhibited the highest antigen-binding activity, with an efficient concentrations of half-maximal (EC50) of 8.419 nmol/L. Notably, Nb2 showed the strongest neutralizing activity in Huh-7 cells, with an inhibitory concentrations of half-maximal (IC50) of 1.126 nmol/L.In conclusion, this study successfully developed 3 kinds of nanobodies targeting MERS-CoV and provides a valuable reference for the optimization of nanobody design and yeast-based production. The findings also lay a technical foundation for the development of nanobody-based therapeutics against MERS-CoV.
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Received: 24 February 2025
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Corresponding Authors:
*liubo7095173@163.com; xcli863@zafu.edu.cn
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