Journal of Food Science and Biotechnology
Abstract
[Objective ] Asprosin (ASP ),an adipokine produced from fibrillin- 1 (FBN 1) cleavage,is closely linked to type 2 diabetes mellitus and obesity when its level is abnormal,demonstrating significant effect on human health.Therefore,monitoring the ASP level is crucial.The author aims to screen the nanobodies specifically binding to ASP via phage display technology,offering a new antibody component for the research on ASP.[Method ] The antigenic epitopes of ASP were predicted by the online tools ABCpred and NovoPro,and then coupled with bovine serum albumin (BSA ) to form the coating antigen (ASP-Epitope-BSA ).After five rounds of bio-panning,the nanobodies specifically binding to ASP were screened from a nanobody phage display library.The selected nanobodies were induced for expression in Escherichia coli.The half median effect concentration (EC 50) values with ASP-Epitope-BSA and whole ASP antigen were determined,and the specificity was evaluated.Finally,homology modeling and molecular docking were performed to study the binding mode between the nanobody and ASP.[Result ] One ASP-specific nanobody (VHH- 19) was successfully screened out,with the induced expression level of 2.4 mg/dL in the LB culture with Escherichia coli.Performance evaluation showed that VHH- 19 had EC 50 values of (87.49±0.27) ng/mL and (128.94±0.48) ng/mL for ASP-Epitope-BSA and whole ASP antigen,respectively,demonstrating good specificity.Homology modeling and molecular docking revealed that ASP was adsorbed in an active pocket formed by the CDR 1 and CDR 3 regions of VHH- 19,forming a sandwich-like binding mode.The Try 116,Gly 126,Lys 115,and Glu 124 of ASP form hydrogen bonds and electrostatic interactions with Glu 107,Arg 31,and Arg 33 of VHH- 19,respectively.[Conclusion ] This study rapidly obtained the ASP-specific nanobody VHH- 19 by bio-affinity panning based on a natural nanobody phage display library.
Publication Date
7-15-2025
First Page
31
Last Page
40
DOI
10.12441/spyswjs.20250305002
Recommended Citation
LIANG, Yun; HUANG, Yi; and HE, Qinghua
(2025)
"Preparation of Human Asprosin Nanobodies Based on a Natural Nanobody Phage Display Library,"
Journal of Food Science and Biotechnology: Vol. 44:
Iss.
7, Article 4.
DOI: 10.12441/spyswjs.20250305002
Available at:
https://spsw.spyswjs.cnjournals.com/journal/vol44/iss7/4
References
［1］ ROMERE C，DUERRSCHMID C，BOURNAT J，et al.Asprosin，a fasting-induced glucogenic protein hormone ［J］.Cell，2016，165（3）：566-579.
［2］ DUERRSCHMID C，HE Y L，WANG C M，et al.Asprosin is a centrally acting orexigenic hormone ［J］.Nature Medicine，2017，23（12）：1444 -1453.
［3］ HOFFMANN J G，XIE W，CHOPRA A R.Energy regulation mechanism and therapeutic potential of asprosin ［J］.Diabetes，2020，69（4）：559-566.
［4］ LU Y L，YUAN W W，XIONG X W，et al.Asprosin aggravates vascular endothelial dysfunction via disturbing mitochondrial dynamics in obesity models ［J］.Obesity，2023，31（3）：732-743.
［5］ ZHANG L，CHEN C，ZHOU N，et al.Circulating asprosin concentrations are increased in type 2 diabetes mellitus and independently associated with fasting glucose and triglyceride［J］.Clinica Chimica Acta，2019，489：183-188.
［6］ 谢丽娟，张媛媛，冯宜娟，等.白脂素生物学功能及其与代谢性疾病关系研究进展 ［J］.生物技术，2023，33（4）：530-535.XIE L J，ZHANG Y Y，FENG Y J，et al.Research progress on biological function of asprosin and its role in metabolic diseases ［J］.Biotechnology，2023，33（4）：530-535.（in Chinese ）
［7］ YIN T T，CHEN S，ZENG G H，et al.Angiogenesis-browning interplay mediated by asprosin-knockout contribute s to weight loss in mice with obesity ［J］.International Journal of Molecular Sciences，2022，23（24）：16166.
［8］ MISHRA I，XIE W R，BOURNAT J C，et al.Protein tyrosine phosphatase receptor δ serves as the orexigenic asprosin receptor［J］.Cell Metabolism，2022，34（4）：549-563.
［9］ 杨硕，任明.白脂素在心血管疾病中的作用 ［J］.国际心血管病杂志，2024，51（2）：74-76.YANG S，REN M.Research progress of asprosin and cardiovascular disease ［J］.International Journal of Cardiovascular Disease，2024，51（2）：74-76.（in Chinese ）
［10］ HUANG Q Q，CHEN S，XIONG X W，et al.Asprosin exacerbates endothelium inflammation induced by hyperlipidemia through activating IKK β-NF- κBp65 pathway［J］.Inflammation，2023，46（2）：623-638.
［11］ PELTOMAA R，BARDERAS R，BENITO-PEÑA E，et al.Recombinant antibodies and their use for food immunoanalysis ［J］.Analytical and Bioanalytical Chemistry，2022，414（1）：193-217.
［12］ MISHRA I，DUERRSCHMID C，KU Z Q，et al.Asprosin-neutralizing antibodies as a treatment for metabolic syndrome ［J］.eLife，2021，10：e63784.
［13］ JOHNSON D E.Biotherapeutics：challenges and opportunities for predictive toxicology of monoclonal antibodies ［J］.International Journal of Molecular Sciences，2018，19（11）：3685.
［14］ RAMON A，ALI M，ATKINSON M，et al.Assessing antibody and nanobody nativeness for hit selection and humanization with AbNatiV ［J］.Nature Machine Intelligence，2024，6（1）：74-91.
［15］ ZHAO F C，SHI R R，LIU R X，et al.Application of phage-display developed antibody and antigen substitutes in immunoassays for small molecule contaminants analysis：a mini-review ［J］.Food Chemistry，2021，339：128084.
［16］ MUYLDERMANS S.Nanobodies：natural single-domain antibodies ［J］.Annual Review of Biochemistry，2013，82：775-797.
［17］ XU J L，XU K，JUNG S，et al.Nanobodies from camelid mice and llamas neutralize SARS-CoV- 2 variants ［J］.Nature，2021，595（7866）：278-282.
［18］ KUNZ P，ORTALE A，MÜCKE N，et al.Nanobody stability engineering by employing the ΔTm shift；a comparison with apparent rate constants of heat-induced aggregation ［J］.Protein Engineering，Design and Selection，2019，32（5）：241-249.
［19］ XIONG L，ZHANG X Q，XU Y，et al.Anti-idiotypic VHH mediated environmentally friendly immunoassay for citrinin without mycotoxin ［J］.Food and Agricultural Immunology，2020，31（1）：968-984.
［20］ 孙卓雅，王梦翔，宋金星，等.非洲猪瘟病毒 p10蛋白和p49蛋白的生物信息学分析及表位预测 ［J］.江苏农业科学，2024，52（15）：195-202.SUN Z Y，WANG M X，SONG J X，et al.Bioinformatics analysis and epitope prediction of p 10 and p49 proteins of African swine fever virus ［J］.Jiangsu Agricultural Sciences，2024，52（15）：195-202.（in Chinese）
［21］ PÍREZ-SCHIRMER M，ROSSOTTI M，BADAGIAN N，et al.Comparison of three antihapten VHH selection strategies for the development of highly sensitive immunoassays for microcystins ［J］.Analytical Chemistry，2017，89（12）：6800 -6806.
［22］ BEVER C R S，MAJKOVA Z，RADHAKRISHNAN R，et al.Development and utilization of camelid VHH antibodies from alpaca for 2，2’，4，4’-tetrabrominated diphenyl ether detection ［J］.Analytical Chemistry，2014，86（15）：7875 -7882.
［23］ 何婉钧，崔愷，张西倩，等.驼源天然噬菌体纳米抗体文库的构建及抗 CD22纳米抗体的筛选 ［J］.细胞与分子免疫学杂志，2025，41（3）：254-261.HE W J，CUI K，ZHANG X Q，et al.Construction of a camel-derived natural phage nanobody display library and screening of anti-CD 22 nanobodies ［J］.Chinese Journal of Cellular and Molecular Immunology，2025，41（3）：254-261.（in Chinese ）
［24］ 张雨欣，陈雨，张凤，等.基于噬菌体展示的阿达木单抗特异性结合肽筛选及其在药物检测中的应用 ［J］.药学学报，2025，60（3）：762-770.ZHANG Y X，CHEN Y，ZHANG F，et al.Screening of adalimumab-specific binding peptides based on phage display and its application to drug detection ［J］.Acta Pharmaceutica Sinica，2025，60（3）：762-770.（in Chinese ）
［25］ 高艳春，王艳归，张玉秀，等.靶向幽门螺杆菌 UreB鲨鱼纳米抗体的筛选与鉴定 ［J］.中国海洋药物，2024，43（4）：35-41.GAO Y C，WANG Y G，ZHANG Y X，et al.Screening and characterization of shark VNARs targeting Helicobacter pylori UreB［J］.Chinese Journal of Marine Drugs，2024，43（4）：35-41.（in Chinese ）
［26］ WATERHOUSE A，BERTONI M，BIENERT S，et al.SWISS-MODEL：homology modelling of protein structures and complexes ［J］.Nucleic Acids Research，2018，46（1）：W296-W303.
［27］ LASKOWSKI R A，MACARTHUR M W，MOSS D S，et al.PROCHECK：a program to check the stereochemical quality of protein structures ［J］.Journal of Applied Crystallography，1993，26（2）：283-291.
［28］ BINBAY F A，RATHOD D C，GEORGE A A P，et al.Quality assessment of selected protein structures derived from homology modeling and alphafold ［J］.Pharmaceuticals，2023，16（12）：1662.
［29］ SHUKLA A，GUNDAMPATI R K，RAJASEKHAR C，et al.Homology modeling and molecular docking of heme peroxidase from Euphorbia tirucalli：substrate specificity and thiol inhibitor interactions ［J］.Journal of Molecular Liquids，2016，220：383-394.
［30］ WANG J W，KANG G B，YUAN H B，et al.Research progress and applications of multivalent，multispecific and modified nanobodies for disease treatment ［J］.Frontiers in Immunology，2021，12：838082.
［31］ SHI M S，CHEN T，WEI S P，et al.Molecular docking，molecular dynamics simulations，and free energy calculation insights into the binding mechanism between VS-4718 and focal adhesion kinase ［J］.ACS Omega，2022，7（36）：32442 -32456.