基于纳米抗体的猪伪狂犬病毒gE蛋白抗体阻断ELISA检测方法的建立

doi:10.1016/j.jia.2023.09.033

摘要/Abstract

摘要：

伪狂犬病（Pseudorabies，PR）是由伪狂犬病病毒（Pseudorabies virus, PRV）引起的猪的一种急性传染病，给养猪业造成严重的经济损失。基因缺失活疫苗的广泛使用有效控制了该病的大范围暴发。尤其是gE基因缺失活疫苗配合其血清学鉴别诊断方法区分疫苗免疫和野毒株感染，为该病在猪群中的净化提供了技术支撑。目前，已有多种以gE蛋白为包被抗原的PRV抗体检测的ELISA方法被应用于野毒感染的筛查。然而，该类ELISA试剂盒大多基于传统抗体研发，其生产工艺复杂，成本高。纳米抗体具有抗原结合力强、耐极端环境、易基因工程改造和体外生产成本低等优点，已广泛用于疫病诊断技术的研发，具有巨大的市场应用前景。然而，目前还没有关于纳米抗体在PRV诊断与治疗中应用的研究报道。本研究用gE重组蛋白免疫双峰驼，并利用噬菌体展示文库筛选出了2株抗gE重组蛋白的特异性纳米抗体。随后，将纳米抗体与辣根过氧化物酶（HRP）融合表达，建立了基于纳米抗体-HRP猪血清PRV-gE抗体阻断ELISA（bELISA）方法。确定以PRV-Nb36-HRP为检测探针建立PRV-gE抗体bELISA，其条件为抗原包被量为每孔200 ng，Nb36-HRP和待检猪血清的稀释度分别为1：320和1：5。bELISA方法的临界值为24.20%，敏感性和特异性分别为96.43%和92.63%。与商品化IDEXX ELISA试剂盒的符合率为93.99%。此外，通过表位分析发现PRV-gE-Nb36识别的构象表位在不同PRV参考株中高度保守。本研究建立了一种基于纳米抗体的操作简单、稳定性高、重复性好、成本低的PRV-gE抗体bELISA检测方法，为PR的监测和净化提供创新型诊断试剂。本研究首次以纳米抗体融合HRP作为检测探针建立PRV-gE抗体bELISA检测方法，无需酶标二抗的使用，简化了生产工艺，节约了生产成本。

Abstract:

Pseudorabies (PR) is an acute infectious disease of pigs caused by the PR virus (PRV) and results in great economic losses to the pig industry worldwide. PRV glycoprotein E (gE)-based enzyme-linked immunosorbent assay (ELISA) has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries. Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent. However, there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies. In the present study, the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel. Then, two nanobodies against PRV-gE were screened from the immunized camel by phage display technique. Subsequently, two nanobody-HRP fusion proteins were expressed with HEK293T cells. The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA (bELISA) to detect anti-PRV-gE antibodies. Through optimizing the conditions of bELISA, the amount of coated antigen was 200 ng per well, and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5. The cut-off value of bELISA was 24.20%, and the sensitivity and specificity were 96.43 and 92.63%, respectively. By detecting 233 clinical pig sera with the developed bELISA and a commercial kit, the results showed that the coincidence rate of two assays was 93.99%. Additionallly, epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains. Simple, great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed. The bELISA could be used for monitoring and eradicating PR.

Key words: nanobody , nanobody-HRP , blocking ELISA , PRV-gE , antibody

. 基于纳米抗体的猪伪狂犬病毒gE蛋白抗体阻断ELISA检测方法的建立[J]. Journal of Integrative Agriculture, 2024, 23(4): 1354-1368.

Huanhuan Lü, Pinpin Ji, Siyu Liu, Ziwei Zhang, Lei Wang, Yani Sun, Baoyuan Liu, Lizhen Wang, Qin Zhao.

A nanobody-based blocking enzyme-linked immunosorbent assay for detecting antibodies against pseudorabies virus glycoprotein E [J]. Journal of Integrative Agriculture, 2024, 23(4): 1354-1368.

参考文献

An T Q, Peng J M, Tian Z J, Zhao H Y, Li N, Liu Y M, Chen J Z, Leng C L, Sun Y, Chang D, Tong G Z. 2013. Pseudorabies virus variant in Bartha-K61-Vaccinated pigs, China. Emerging Infectious Diseases, 19, 1749–1755.

Ao J Q, Wang J W, Chen X H, Wang X Z, Long Q X. 2003. Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA. Journal of Virological Methods, 114, 145–150.

Bannas P, Hambach J, Koch-Nolte F. 2017. Nanobodies and nanobody-based human heavy Chain antibodies as antitumor therapeutics. Frontiers in Immunology, 8, 1603.

Boonham N, Kreuze J, Winter S, van der Vlugt R, Bergervoet J, Tomlinson J, Mumford R. 2014. Methods in virus diagnostics: From ELISA to next generation sequencing. Virus Research, 186, 20–31.

Chen H, Zhang X L, Jin Z Y, Huang L P, Dan H B, Xiao W, Liang J J, Zou S Y, Tang Y. 2020. Differential diagnosis of PRV-infected versus vaccinated pigs using a novel EuNPs-virus antigen probe-based blocking fluorescent lateral flow immunoassay. Biosensors & Bioelectronics, 155, 112101.

Cheng T Y, Magtoto R, Henao-Díaz A, Poonsuk K, Buckley A, Van Geelen A, Lager K, Zimmerman J, Giménez-Lirola L. 2021. Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA. Journal of Veterinary Diagnostic Investigation, 33, 1106–1114.

Duan H, Chen X, Zhao J, Zhu J, Zhang G, Fan M, Zhang B, Wang X, Sun Y, Liu B, Zhou E M, Zhao Q. 2021. 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. Journal of Clinical Microbiology, 59, e0158021.

Gu Z Q, Dong J, Wang J C, Hou C C, Sun H F, Yang W P, Bai J, Jiang P. 2015. A novel inactivated gE/gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge. Virus Research, 195, 57–63.

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Zidek A, Potapenko A, Bridgland A, Meyer C, Kohl S A A, Ballard A J, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature, 596, 583–589.

Kagami L P, das Neves G M, Timmers L F S M, Caceres R A, Eifler-Lima V L. 2020. Geo-Measures: A PyMOL plugin for protein structure ensembles analysis. Computational Biology and Chemistry, 87, 107322.

Kozakov D, Hall D R, Xia B, Porter K A, Padhorny D, Yueh C, Beglov D, Vajda S. 2017. The ClusPro web server for protein-protein docking. Nature Protocols, 12, 255–278.

Lee J Y, Wilson M R. 1979. A review of pseudorabies (Aujeszky’s disease) in pigs. Canadian Veterinary Journal, 20, 65–69.

Li W H, Zhuang D J, Li H, Zhao M P, Zhu E P, Xie B M, Chen J D, Zhao M Q. 2021. Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013. Bmc Veterinary Research, 17, 164.

Lin J X, Li Z L, Feng Z H, Fang Z, Chen J H, Chen W Z, Liang W W, Chen Q. 2020. Pseudorabies virus (PRV) strain with defects in gE, gC, and TK genes protects piglets against an emerging PRV variant. Journal of Veterinary Medical Science, 82, 846–855.

Liu G, Jiang Y, Opriessnig T, Gu K, Zhang H, Yang Z. 2019. Detection and differentiation of five diarrhea related pig viruses utilizing a multiplex PCR assay. Journal of Virological Methods, 263, 32–37.

Liu H, Shi Z, Liu C, Wang P, Wang M, Wang S, Liu Z, Wei L, Sun Z, He X, Wang J. 2020. Implication of the identification of an earlier pseudorabies virus (PRV) strain HLJ-2013 to the evolution of Chinese PRVs. Frontiers in Microbiology, 11, 612474.

Liu H L, Wang Y, Duan H, Zhang A K, Liang C, Gao J M, Zhang C, Huang B C, Li Q Y, Li N, Xiao S Q, Zhou E M. 2015. An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication. Veterinary Microbiology, 181, 252–260.

Liu Q, Wang X, Xie C, Ding S, Yang H, Guo S, Li J, Qin L, Ban F, Wang D, Wang C, Feng L, Ma H, Wu B, Zhang L, Dong C, Xing L, Zhang J, Chen H, Yan R, et al. 2021. A novel human acute encephalitis caused by pseudorabies virus variant strain. Clinical Infectious Diseases, 73, e3690–e3700.

Ma Z Q, Wang T Y, Li Z W, Guo X Y, Tian Y S, Li Y, Xiao S Q. 2019. A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus. Journal of Nanobiotechnology, 17, 96.

Mei Y, Chen Y, Sivaccumar J P, An Z, Xia N, Luo W. 2022. Research progress and applications of nanobody in human infectious diseases. Frontiers in Pharmacology, 13, 963978.

Mettenleiter T C. 2003. Pathogenesis of neurotropic herpesviruses: Role of viral glycoproteins in neuroinvasion and transneuronal spread. Virus Research, 92, 197–206.

Mettenleiter T C. 2020. Aujeszky’s disease and the development of the marker/DIVA vaccination concept. Pathogens, 9, 563.

De Meyer T, Muyldermans S, Depicker A. 2014. Nanobody-based products as research and diagnostic tools. Trends in Biotechnology, 32, 263–270.

Mu Y, Jia C, Zheng X, Zhu H, Zhang X, Xu H, Liu B, Zhao Q, Zhou E M. 2021. A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2. Journal of Nanobiotechnology, 19, 34.

Muyldermans S, Baral T N, Retarnozzo V C, De Baetselier P, De Genst E, Kinne J, Leonhardt H, Magez S, Nguyen V K, Revets H, Rothbauer U, Stijemans B, Tillib S, Wernery U, Wyns L, Hassanzadeh-Ghassabeh G, Saerens D. 2009. Camelid immunoglobulins and nanobody technology. Veterinary Immunology and Immunopathology, 128, 178–183.

van Oirschot J T, Gielkens A L, Moormann R J, Berns A J. 1990. Marker vaccines, virus protein-specific antibody assays and the control of Aujeszky’s disease. Veterinary Microbiology, 23, 85–101.

Pan J, Li Y, Wang T, Chang J, Hao L, Chen J, Peng W, Deng J, Huang B, Tian K 2022. A poly(dimethylsiloxane)-based solid-phase microchip platform for dual detection of pseudorabies virus gD and gE antibodies. Frontiers in Cellular and Infection Microbiology, 12, 912108.

Romero C H, Meade P, Santagata J, Gillis K, Lollis G, Hahn E C, Gibbs E P J. 1997. Genital infection and transmission of pseudorabies virus in feral swine in Florida, USA. Veterinary Microbiology, 55, 131–139.

Salvador J P, Vilaplana L, Marco M P. 2019. Nanobody: Outstanding features for diagnostic and therapeutic applications. Analytical and Bioanalytical Chemistry, 411, 1703–1713.

Sheng Y, Wang K, Lu Q, Ji P, Liu B, Zhu J, Liu Q, Sun Y, Zhang J, Zhou E M, Zhao Q. 2019. Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay. Journal of Nanobiotechnology, 17, 35.

Sun Y, Zhao L, Fu Z F. 2022. Effective cross-protection of a lyophilized live gE/gI/TK-deleted pseudorabies virus (PRV) vaccine against classical and variant PRV challenges. Veterinary Microbiology, 267, 109387.

Vincke C, Gutierrez C, Wernery U, Devoogdt N, Hassanzadeh-Ghassabeh G, Muyldermans S. 2012. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods in Molecular Biology, 907, 145–176.

Vogt N. 2016. Conditional nanobody tools. Nature Methods, 13, 610–611.

Wang F, Wang H. 2022. Nanobody-based assays for the detection of environmental and agricultural contaminants. Methods in Molecular Biology, 2446, 547–554.

Xu W, Yan P, Zhou Z, Yao J, Pan H, Jiang L, Bo Z, Ni B, Sun M, Gao S, Huan C. 2023. HDAC6 triggers the ATM-Dependent DNA damage response to promote PRV replication. Microbiology Spectrum, 11, e0213222.

Yamagata M, Sanes J R. 2018. Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. Proceedings of the National Academy of Sciences of the United States of America, 115, 2126–2131.

Yu S, Li Z F, Li J Z, Zhao S M, Wu S G, Liu H J, Bi X J, Li D Y, Dong J X, Duan S L, Hammock B D. 2021. Generation of dual functional nanobody-nanoluciferase fusion and its potential in bioluminescence enzyme immunoassay for trace glypican-3 in serum. Sensors and Actuators (B: Chemical), 336, 129717.

Zhang P, Lv L, Sun H, Li S, Fan H, Wang X, Bai J, Jiang P. 2019. Identification of linear B cell epitope on gB, gC, and gE proteins of porcine pseudorabies virus using monoclonal antibodies. Veterinary Microbiology, 234, 83–91.

Zhao J, Zhu J, Wang Y, Yang M, Zhang Q, Zhang C, Nan Y, Zhou E M, Sun Y, Zhao Q. 2022. A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus. Virologica Sinica, 37, 922–933.

Zheng H H, Fu P F, Chen H Y, Wang Z Y. 2022. Pseudorabies virus: From pathogenesis to prevention strategies. Viruses, 14, 1638.