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
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.
张燕茹, 王甜甜, 徐惠芳, 王昊, 王涵, 吴军, 李向臣, 刘波. 靶向中东呼吸综合征冠状病毒纳米抗体设计与重组制备[J]. 农业生物技术学报, 2025, 33(10): 2299-2310.
ZHANG Yan-Ru, WANG Tian-Tian, XU Hui-Fang, WANG Hao, WANG Han, WU Jun, LI Xiang-Chen, LIU Bo. Design and Recombinant Production of Nanobodies Targeting Middle East respiratory syndrome coronavirus. 农业生物技术学报, 2025, 33(10): 2299-2310.
[1] Alhakeem R F, Midgley C M, Assiri A M, et al.2016. Exposures among MERS Case-Patients, Saudi Arabia, January-February 2016[J]. Emerging Infectious Diseases, 22(11): 2020-2022. [2] Arnau J, Lauritzen C, Petersen G E, et al.2006. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins[J]. Protein Expression and Purification, 48: 1-13. [3] Chan J F, Lau S K, To K K, et al.2015. Middle East respiratory syndrome coronavirus: Another zoonotic Betacoronavirus causing SARS-like disease[J]. Clinical Microbiology Reviews, 28: 465-522. [4] Chen Z S, Huang H C, Wang X, et al.2025. Influenza A virus H7 nanobody recognizes a conserved immunodominant epitope on hemagglutinin head and confers heterosubtypic protection[J]. Nature Communications, 16: 432. [5] Frenken L G, van der Linden R H, Hermans P W, et al.2000. Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae[J]. Journal of Biotechnology, 78: 11-21. [6] Fuchs S M, Raines R T.2005. Polyarginine as a multifunctional fusion tag[J]. Protein Science: A publication of the Protein Society, 14: 1538-1544. [7] Hamers-Casterman C, Atarhouch T, Muyldermans S, et al.1993. Naturally occurring antibodies devoid of light chains[J]. Nature, 363: 446-448. [8] Hansman G S, Kher G, Svirina A D, et al.2024. Development of a broad-spectrum therapeutic Fc-nanobody for Human noroviruses[J]. Journal of Virology, 98: e0070724. [9] Kosobokova E N, Skrypnik K A, Kosorukov V S.2016. Overview of fusion tags for recombinant proteins[J]. Biochemistry. Biokhimiia, 81: 187-200. [10] Kurek D V, Lopatin S A, Varlamov V P.2009. Prospects of application of the chitin-binding domains to isolation and purification of recombinant proteins by affinity chromatography: A review[J]. Prikladnaia Biokhimiia i Mikrobiologiia, 45: 5-13. [11] Li C, Tang Z, Hu Z, et al.2018. Natural single-domain antibody-nanobody: A novel concept in the antibody field[J]. Journal of Biomedical Nanotechnology, 14: 1-19. [12] Li F.2015. Receptor recognition mechanisms of coronaviruses: A decade of structural studies[J]. Journal of Virology, 89: 1954-1964. [13] Ma S, Zhang D, Wang Q, et al.2024. Structure defining of ultrapotent neutralizing nanobodies against MERS-CoV with novel epitopes on receptor binding domain[J]. PLoS Pathogens, 20: e1012438. [14] Pajęcka K, Nielsen C W, Hauge A, et al.2014. Glutamate dehydrogenase isoforms with N-terminal (His)6- or FLAG-tag retain their kinetic properties and cellular localization[J]. Neurochemical Research, 39: 487-499. [15] Randolph T W.2012. The two faces of His-tag: Immune response versus ease of protein purification[J]. Biotechnology Journal, 7: 18-19. [16] Rossotti M A, Bélanger K, Henry K A, et al.2022. Immunogenicity and humanization of single-domain antibodies[J]. The FEBS Journal, 289: 4304-4327. [17] Salvador J P, Vilaplana L, Marco M P.2019. Nanobody: Outstanding features for diagnostic and therapeutic applications[J]. Analytical and Bioanalytical Chemistry, 411: 1703-1713. [18] Tai W, Zhang X, Yang Y, et al.2022. Advances in mRNA and other vaccines against MERS-CoV[J]. Translational Research: The Journal of Laboratory and Clinical Medicine, 242: 20-37. [19] Terpe K.2003. Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems[J]. Applied Microbiology and Biotechnology, 60: 523-533. [20] van Doremalen N, Miazgowicz K L, Milne-Price S, et al.2014. Host species restriction of Middle East respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4[J]. Journal of Virology, 88: 9220-9232. [21] Van Heeke G, Allosery K, De Brabandere V, et al.2017. Nanobodies® as inhaled biotherapeutics for lung diseases[J]. Pharmacology & Therapeutics, 169: 47-56. [22] Zaki A M, van Boheemen S, Bestebroer T M, et al.2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia[J]. The New England Journal of Medicine, 367: 1814-1820. [23] Zhao G, He L, Sun S, et al.2018. A novel nanobody targeting Middle East respiratory syndrome coronavirus (MERS-CoV) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against MERS-CoV[J]. Journal of Virology, 92(18): e00837-18.
