纳米抗体及其在兽医领域的研究现状

doi:10.11843/j.issn.0366-6964.2023.08.004

摘要/Abstract

摘要： 纳米抗体是一种衍生于骆驼科和鲨鱼类动物的单域抗体，与传统抗体相比，它具有分子量小、结构稳定、易于改造和表达、低免疫原性、高抗原结合性、组织穿透性强等优点，独特的分子特性使其在诊断和治疗各种疾病及检测方面展现出良好应用前景。本文总结了纳米抗体的结构、特性、文库类型、筛选与表达策略，及其在动物疫病预防、诊断和治疗，动物产品食源性病原体和药物残留检验中的研究现状，并对纳米抗体的局限性和未来发展方向进行分析。

关键词: 纳米抗体, 特性, 动物疫病防控, 动物产品检验

Abstract: Nanobody is a kind of single-domain antibody derived from Camelidae and sharks. Compared with the traditional antibody, nanobody has the characteristics of small molecular weight, stable structure, easiness of modification and expression, low immunogenicity, high antigen affinity, strong tissue penetration and so on. Its unique molecular characteristics make it promising for application for diagnosis and treatment of various diseases and assays. This review summarized the structure, characteristics, library types, screening and expression strategies of nanobody and research status in the prevention, diagnosis and treatment of animal disease, as well as the detection of foodborne pathogens and drug residues in animal products. The limitations and future development of nanobody are also analyzed.

Key words: nanobody, characteristics, animal diseases prevention and control, animal products detection

中图分类号:

S852.65

胡湘云, 曹艳红, 吕玲燕, 刘征, 黄发才, 吴柱月, 肖正中. 纳米抗体及其在兽医领域的研究现状[J]. 畜牧兽医学报, 2023, 54(8): 3164-3172.

HU Xiangyun, CAO Yanhong, LÜ Lingyan, LIU Zheng, HUANG Facai, WU Zhuyue, XIAO Zhengzhong. Nanobodies and Their Research Status in Veterinary Field[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3164-3172.

参考文献 58

[1]	HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMANS S, et al.Naturally occurring antibodies devoid of light chains[J].Nature, 1993, 363(6428):446-448.
[2]	GREENBER A S, AVILA D, HUGHES M, et al.A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks[J].Nature, 1995, 374(6518):168-173.
[3]	NGUYEN V K, HAMERS R, WYNS L, et al.Camel heavy-chain antibodies:Diverse germline VHH and specific mechanisms enlarge the antigen-binding repertoire[J].EMBO J, 2000, 19(5):921-930.
[4]	GOVAERT J, PELLIS M, DESCHACHT N, et al.Dual beneficial effect of interloop disulfide bond for single domain antibody fragments[J].J Biol Chem, 2012, 287(3):1970-1979.
[5]	VATTEKATTE A M, SHINADA N K, NARWANI T J, et al.Discrete analysis of camelid variable domains:sequences, structures, and in-silico structure prediction[J].PeerJ, 2020, 8:e8408.
[6]	DESCHACHT N, DE GROEVE K, VINCKE C, et al.A novel promiscuous class of camelid single-domain antibody contributes to the antigen-binding repertoire[J].J Immunol, 2010, 184(10):5696-5704.
[7]	MUYLDERMANS S.Nanobodies:Natural single-domain antibodies[J].Annu Rev Biochem, 2013, 82:775-797.
[8]	LI C, ZHAN W Q, YANG Z L, et al.Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody[J].Cell, 2022, 185(8):1389-1401.e18.
[9]	LIU W S, SONG H P, CHEN Q, et al.Recent advances in the selection and identification of antigen-specific nanobodies[J].Mol Immunol, 2018, 96:37-47.
[10]	ZAVRTANIK U, LUKAN J, LORIS R, et al.Structural basis of epitope recognition by heavy-chain camelid antibodies[J].J Mol Biol, 2018, 430(21):4369-4386.
[11]	AL QARAGHULI M M, FERRO V A.Analysis of the binding loops configuration and surface adaptation of different crystallized single-domain antibodies in response to various antigens[J].J Mol Recognit, 2017, 30(4):e2592.
[12]	BAO G F, TANG M, ZHAO J, et al.Nanobody:A promising toolkit for molecular imaging and disease therapy[J].EJNMMI Res, 2021, 11(1):6.
[13]	GOLDMAN E R, LIU J L, ZABETAKIS D, et al.Enhancing stability of camelid and shark single domain antibodies:an overview[J].Front Immunol, 2017, 8:865.
[14]	SALVADOR J P, VILAPLANA L, MARCO M P.Nanobody:Outstanding features for diagnostic and therapeutic applications[J].Anal Bioanal Chem, 2019, 411(9):1703-1713.
[15]	PARDON E, LAEREMANS T, TRIEST S, et al.A general protocol for the generation of nanobodies for structural biology[J].Nat Protoc, 2014, 9(3):674-693.
[16]	LIU B Y, YANG D W.Easily established and multifunctional synthetic nanobody libraries as research tools[J].Int J Mol Sci, 2022, 23(3):1482.
[17]	王加利, 和似琦, 康子茜, 等.噬菌体抗体展示技术及其在抗新冠病毒抗体发现中的应用[J].生物技术通报, 2022, 38(5):248-256.WANG J L, HE S Q, KANG Z X, et al.Antibody phage display technology and its application in the discovery of anti-SARS-CoV-2 Antibodies[J].Biotechnology Bulletin, 2022, 38(5):248-256.(in Chinese)
[18]	LIU Y K, HUANG H.Expression of single-domain antibody in different systems[J].Appl Microbiol Biotechnol, 2018, 102(2):539-551.
[19]	DE GREVE H, VIRDI V, BAKSHI S, et al.Simplified monomeric VHH-Fc antibodies provide new opportunities for passive immunization[J].Curr Opin Biotechnol, 2020, 61:96-101.
[20]	VIRDI V, CODDENS A, DE BUCK S, et al.Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection[J].Proc Natl Acad Sci U S A, 2013, 110(29):11809-11814.
[21]	VIRDI V, PALACI J, LAUKENS B, et al.Yeast-secreted, dried and food-admixed monomeric IgA prevents gastrointestinal infection in a piglet model[J].Nat Biotechnol, 2019, 37(5):527-530.
[22]	VANMARSENILLE C, ELSEVIERS J, YVANOFF C, et al.In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter[J].PLoS One, 2018, 13(9):e0204222.
[23]	BAKSHI S, GARCIA R S, VAN DER WEKEN H, et al.Evaluating single-domain antibodies as carriers for targeted vaccine delivery to the small intestinal epithelium[J].J Control Release, 2020, 321:416-429.
[24]	ALZOGARAY V, URRUTIA M, BERGUER P, et al.Characterization of folding-sensitive nanobodies as tools to study the expression and quality of protein particle immunogens[J].J Biotechnol, 2019, 293:17-23.
[25]	LI H Z, DEKKER A, SUN S Q, et al.Novel capsid-specific single-domain antibodies with broad foot-and-mouth disease strain recognition reveal differences in antigenicity of virions, empty capsids, and virus-like particles[J].Vaccines (Basel), 2021, 9(6):620.
[26]	MA Z Q, WANG T Y, LI Z W, et al.A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus[J].J Nanobiotechnol, 2019, 17(1):96.
[27]	王天宇.基于猪流行性腹泻病毒S蛋白纳米抗体的阻断ELISA方法的建立[D].杨凌:西北农林科技大学, 2019.WANG T Y.Establishment of blocking ELISA method based on nanobody against porcine epidemic diarrhea virus S protein[D].Yangling:Northwest A&F University, 2019.(in Chinese)
[28]	DU T F, ZHU G, WU X P, et al.Biotinylated single-domain antibody-based blocking ELISA for detection of antibodies against swine influenza virus[J].Int J Nanomed, 2019, 14:9337-9349.
[29]	朱家宏.基于非洲猪瘟病毒P72蛋白纳米抗体的竞争ELISA检测方法的建立[D].杨凌:西北农林科技大学, 2020.ZHU J H.Establishment of a competitive ELISA detection method based on African swine fever virus p72 protein nanobodies[D].Yangling:Northwest A&F University, 2020.(in Chinese)
[30]	MU Y, JIA C Y, ZHENG X, et al.Correction to:A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2[J].J Nanobiotechnol, 2021, 19(1):66.
[31]	樊杰.猪传染性胃肠炎病毒N蛋白纳米抗体的制备和基于纳米抗体竞争ELISA的建立[D].杨凌:西北农林科技大学, 2021.FAN J.Preparation of nanobodies against transmissible gastroenteritis virus N protein and development of the nanobody-based competitive ELISA[D].Yangling:Northwest A&F University, 2021.(in Chinese)
[32]	DUAN H, CHEN X, ZHAO J K, et al.Development of a nanobody-based competitive enzyme-linked immunosorbent assay for efficiently and specifically detecting antibodies against genotype 2 porcine reproductive and respiratory syndrome viruses[J].J Clin Microbiol, 2021, 59(12):e01580-21.
[33]	王坤.基于禽流感病毒NP蛋白纳米抗体建立鸡血清中禽流感抗体检测的竞争ELISA[D].杨凌:西北农林科技大学, 2020.WANG K.Establishment of a competitive ELISA for detection of avian influenza antibody in chicken serum based on avian influenza virus NP protein nanobody[D].Yangling:Northwest A&F University, 2020.(in Chinese)
[34]	SHENG Y M, WANG K, LU Q Z, et al.Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay[J].J Nanobiotechnol, 2019, 17(1):35.
[35]	陈天祥.禽戊型肝炎病毒ORF2截短蛋白纳米抗体的制备和竞争ELISA检测方法的建立[D].杨凌:西北农林科技大学, 2021.CHEN T X.Preparation of nanobodies against a truncated protein of avian HEV ORF2 and development of a competitive ELISA[D].Yangling:Northwest A&F University, 2021.(in Chinese)
[36]	JI P P, ZHU J H, LI X X, et al.Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus[J].J Nanobiotechnol, 2020, 18(1):44.
[37]	周子恒.牛病毒性腹泻病毒纳米抗体ELISA检测方法的初步研究[D].石河子:石河子大学, 2019.ZHOU Z H.Preliminary study on ELISA method of bovine viral diarrhea virus nanobody[D].Shihezi:Shihezi University, 2019.(in Chinese)
[38]	ZHU M, GONG X, HU Y, et al.Streptavidin-biotin-based directional double nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1[J].J Transl Med, 2014, 12:352.
[39]	LU Q Z, LI X X, ZHAO J K, et al.Nanobody-horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays[J].J Nanobiotechnol, 2020, 18(1):7.
[40]	YANG S L, LI L, YIN S H, et al.Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus[J].Appl Microbiol Biotechnol, 2018, 102(20):8931-8942.
[41]	YANG S L, YUAN L, SHANG Y J, et al.Selection and characterization of CSFV-Specific single-domain antibodies and their application along with immunomagnetic nanobeads and quantum dots[J].Biomed Res Int, 2020, 2020:3201630.
[42]	WANG D, YANG S L, YIN S H, et al.Characterization of single-domain antibodies against foot and mouth disease virus (FMDV) serotype O from a camelid and imaging of FMDV in baby hamster kidney-21 cells with single-domain antibody-quantum dots probes[J].BMC Vet Res, 2015, 11:120.
[43]	高小龙, 胡湘云, 付向晶, 等.新城疫病毒F蛋白纳米抗体的筛选及活性鉴定[J].畜牧兽医学报, 2016, 47(8):1645-1651.GAO X L, HU X Y, FU X J, et al.Screening and characterization of VHH against newcastle disease virus fusion protein[J].Acta Veterinaria et Zootechnica Sinica, 2016, 47(8):1645-1651.(in Chinese)
[44]	GAO X L, HU X Y, TONG L N, et al.Construction of a camelid VHH yeast two-hybrid library and the selection of VHH against haemagglutinin-neuraminidase protein of the Newcastle disease virus[J].BMC Vet Res, 2016, 12:39.
[45]	ZHANG L, WANG L Z, CAO S S, et al.Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages[J].Vet Res, 2021, 52(1):25.
[46]	李阔阔.猪繁殖与呼吸综合征病毒Nsp9纳米抗体在体内抗病毒感染的初步验证[D].杨凌:西北农林科技大学, 2019.LI K K.Preliminary validation of swine reproductive and respiratory syndrome virus Nsp9 nanobody for antiviral infection in vivo[D].Yangling:Northwest A&F University, 2019.(in Chinese)
[47]	FIORAVANTI A, VAN HAUWERMEIREN F, VAN DER VERREN S E.Structure of S-layer protein Sap reveals a mechanism for therapeutic intervention in anthrax[J].Nat Microbiol, 2019, 4(11):1805-1814.
[48]	TU Z, CHEN Q, LI Y P, et al.Identification and characterization of species-specific nanobodies for the detection of Listeria monocytogenes in milk[J].Anal Biochem, 2016, 493:1-7.
[49]	HE Y X, REN Y R, GUO B, et al.Development of a specific nanobody and its application in rapid and selective determination of Salmonella enteritidis in milk[J].Food Chem, 2020, 310:125942.
[50]	高杨.沙门菌PhoN蛋白单克隆抗体和纳米抗体的制备及初步应用[D].扬州:扬州大学, 2021.GAO Y.Preparation and application of different types of antibodies against Salmonella PhoN protein[D].Yangzhou:Yangzhou University, 2021.(in Chinese)
[51]	HU Y Z, SUN Y, GU J X, et al.Selection of specific nanobodies to develop an immuno-assay detecting Staphylococcus aureus in milk[J].Food Chem, 2021, 353:129481.
[52]	JI Y W, LI X, LU Y L, et al.Nanobodies based on a sandwich immunoassay for the detection of Staphylococcal enterotoxin B free from interference by protein A[J].J Agric Food Chem, 2020, 68(21):5959-5968.
[53]	SUN T Q, ZHAO Z Q, LIU W T, et al.Development of sandwich chemiluminescent immunoassay based on an anti-Staphylococcal enterotoxin B nanobody-alkaline phosphatase fusion protein for detection of Staphylococcal enterotoxin B[J].Anal Chim Acta, 2020, 1108:28-36.
[54]	TANG X Q, CATANANTE G, HUANG X R, et al.Screen-printed electrochemical immunosensor based on a novel nanobody for analyzing aflatoxin M1 in milk[J].Food Chem, 2022, 383:132598.
[55]	高海岗.孔雀石绿纳米抗体的制备及其免疫层析检测方法的建立与应用[D].扬州:扬州大学, 2021.GAO H G.Preparation of malachite green nanobody and establishment and application of immuno-chromatography detection method[D].Yangzhou:Yangzhou University, 2021.(in Chinese)
[56]	翟圆君.骆驼天然单域重链抗体库的构建及抗莱克多巴胺纳米抗体的初步筛选[D].郑州:郑州大学, 2017.ZHAI Y J.Construction of camelid single-domain antibody library and Perliminayry biopanning of nanobody against ractopamine[D].Zhengzhou:Zhengzhou University, 2017.(in Chinese)
[57]	翟艳芳.天然纳米抗体库的构建及抗盐酸克伦特罗纳米抗体的初步筛选[D].郑州:郑州大学, 2020.ZHAI Y F.Construction of natural nanobody library and preliminary screening of anti-clenbuterol nanobody[D].Zhengzhou:Zhengzhou University, 2020.(in Chinese)
[58]	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.