猪流行性腹泻病毒S蛋白纳米抗体的筛选与鉴定

doi:10.11843/j.issn.0366-6964.2021.09.022

摘要/Abstract

摘要： 猪流行性腹泻病（porcine epidemic diarrhea，PED）是一种高度接触性肠道传染性疫病，主要危害1周龄以内的仔猪，仔猪感染死亡率高达100%，是目前危害世界养猪业的主要疫病之一。本研究旨在制备针对猪流行性腹泻病毒S蛋白的特异性纳米抗体并鉴定其结合活性。作者原核表达并纯化PEDV S1蛋白，将纯化后的PEDV S1重组蛋白免疫双峰驼，第4次免疫后分离其外周血淋巴细胞，提取淋巴细胞RNA，反转录得到cDNA，通过巢式PCR扩增VHH片段，并构建至pCANTAB-5E载体中，电转化至TG1感受态细胞，得到VHH噬菌体抗体展示文库；随后，对构建的噬菌体抗体展示文库进行救援和3轮富集，利用噬菌体展示技术从中筛选针对PEDV S蛋白纳米抗体，通过ELISA验证筛选的纳米抗体的特异性和结合力。通过Western blot和间接免疫荧光验证纳米抗体与PEDV的结合活性。结果显示：成功表达并纯化PEDV S1蛋白，经4次免疫后，双峰驼血清中的特异性抗体效价达到了1∶256 000。构建的噬菌体展示文库的库容量为2.1×10⁷，阳性率85%；对噬菌体展示文库3轮的淘选富集后，最终筛选出6株氨基酸序列不同的纳米抗体，ELISA结果显示，6株纳米抗体均对PEDV S1重组蛋白具有良好的结合力与特异性。随后验证了Nb3能够与PEDV结合，表明其具有良好的活性。成功筛选到针对PEDV S1蛋白的特异性纳米抗体，所筛选纳米抗体有望用于PED的诊断和治疗，同时为PEDV的致病机制研究提供抗体材料。

关键词: 猪流行性腹泻病毒, 结构蛋白S, 纳米抗体, 噬菌体展示技术

Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease, which mainly harms piglets less than 1 week of age, and the infection mortality rate of piglets is up to 100%, and it is a major disease that harms the world's pig industry. We aimed to prepare a specific nanobody against porcine epidemic diarrhea virus (PEDV) S protein and to identify its binding activity. The prokaryotic expression and purification of PEDV S1 protein were performed, and the purified recombinant PEDV S1 protein was used to immunize Bactrian camel. The peripheral blood lymphocytes were isolated, the lymphocyte RNA was extracted, and the cDNA was obtained by reverse transcription. The VHH fragment was amplified by nested PCR, then was constructed into pCANTAB-5E vector and transformed into TG1 competent cells, the VHH phage antibody display library was obtained. The constructed phage antibody display library was rescued and enriched for three rounds. Phage display technology was used to screen the PEDV S protein nanobodies from the library. The specificity and binding force of the selected specific nanobodies were verified by ELISA. The binding activity of the nanobody to PEDV was verified by Western blot and indirect immunofluorescence. Results were as follows:PEDV S1 protein was successfully expressed and purified and the titer of specific antibodies in the serum of Bactrian camels reached 1:256 000 after four immunizations of the PEDV S1 protein. The capacity of the library was 2.1×10⁷, and the positive rate was 85%. After three rounds of screening and enrichment of the display library, six nanobodies with different amino acid sequences were screened out. ELISA results showed that all of them have good binding ability and specificity for PEDV S1 recombinant protein. Subsequently, it was verified that Nb3 can bind to PEDV virus, indicating that it has good activity. The specific nanobodies against PEDV S1 protein were successfully screened. The screened nanobodies are expected to be used for the diagnosis and treatment of PED, and provide antibody materials for the study of the pathogenic mechanism of PEDV.

Key words: porcine epidemic diarrhea virus, structural protein S, nanobody, phage display technology

中图分类号:

王天宇, 李志伟, 杨婷, 董林芳, 马志倩, 边巴次仁, 肖书奇, 李爽. 猪流行性腹泻病毒S蛋白纳米抗体的筛选与鉴定[J]. 畜牧兽医学报, 2021, 52(9): 2589-2598.

WANG Tianyu, LI Zhiwei, YANG Ting, DONG Linfang, MA Zhiqian, BIANBA Ciren, XIAO Shuqi, LI Shuang. Screening and Identification of Nanobodies against Porcine Epidemic Diarrhea Virus S Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2589-2598.

参考文献 30

[1]	LIANG W, ZHOU D N, GENG C, et al. Isolation and evolutionary analyses of porcine epidemic diarrhea virus in Asia[J]. PeerJ, 2020, 8:e10114.
[2]	ZHNAG L P, LIU X S, ZHANG Q L, et al. Biological characterization and pathogenicity of a newly isolated Chinese highly virulent genotype GIIa porcine epidemic diarrhea virus strain[J]. Arch Virol, 2019, 164(5):1287-1295.
[3]	GUO J H, FANG L R, YE X, et al. Evolutionary and genotypic analyses of global porcine epidemic diarrhea virus strains[J]. Transbound Emerg Dis, 2019, 66(1):111-118.
[4]	LEE D K, PARK C K, KIM S H, et al. Heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in Korea[J]. Virus Res, 2010, 149(2):175-182.
[5]	ZANG Y, TIAN Y, LI Y G, et al. Recombinant Lactobacillus acidophilus expressing S1 and S2 domains of porcine epidemic diarrhea virus could improve the humoral and mucosal immune levels in mice and sows inoculated orally[J]. Vet Microbiol, 2020, 248:108827.
[6]	YU J, CHAI X L, CHENG Y, et al. Molecular characteristics of the spike gene of porcine epidemic diarrhoea virus strains in Eastern China in 2016[J]. Virus Res, 2018, 247:47-54.
[7]	SUN D B, FENG L, SHI H Y, et al. Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein[J]. Vet Microbiol, 2008, 131(1-2):73-81.
[8]	HOU Y X, LIN C M, YOKOYAMA M, et al. Deletion of a 197-amino-acid region in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets[J]. J Virol, 2017, 91(14):e00227-17.
[9]	SUZUKI T, TERADA Y, ENJUANES L, et al. S1 subunit of spike protein from a current highly virulent porcine epidemic diarrhea virus is an important determinant of virulence in piglets[J]. Viruses, 2018, 10(9):467.
[10]	TSAI K J, DENG M C, WANG F I, et al. Deletion in the S1 region of porcine epidemic diarrhea virus reduces the virulence and influences the virus-neutralizing activity of the antibody induced[J]. Viruses, 2020, 12(12):1378.
[11]	HSUEH F C, LIN C N, CHIOU H Y, et al. Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan[J]. Transbound Emerg Dis, 2020, 67(1):417-430.
[12]	HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMNS S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature, 1993, 363(6428):446-448.
[13]	MUYLDERMANS S, BARAL T N, RETAMOZZO V C, et al. Camelid immunoglobulins and nanobody technology[J]. Vet Immunol Immunopathol, 2009, 128(1-3):178-183.
[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]	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.
[16]	BANNAS P, HAMBACH J, KOCH-NOLTE F. Nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics[J]. Front Immunol, 2017, 8:1603.
[17]	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.
[18]	SUN R Q, CAI R J, CHEN Y Q, et al. Outbreak of porcine epidemic diarrhea in suckling piglets, China[J]. Emerg Infect Dis, 2012, 18(1):161-163.
[19]	LI W T, LI H, LIU Y B, et al. New variants of porcine epidemic diarrhea virus, China, 2011[J]. Emerg Infect Dis, 2012, 18(8):1350-1353.
[20]	LIN C M, SAIF L J, MARTHALER D, et al. Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains[J]. Virus Res, 2016, 226:20-39.
[21]	QI M P, ZAMBRANO-MORENO C, PINEDA P, et al. Several lineages of porcine epidemic diarrhea virus in Colombia during the 2014 and 2016 epidemic[J/OL]. Transboundary and Emerging Diseases, 2020, doi:10. 1111/tbed. 13914.
[22]	DUBEY A, DAHIYA S, ROUSE B T, et al. Perspective:Reducing SARS-CoV2 infectivity and its associated immunopathology[J]. Front Immunol, 2020, 11:581076.
[23]	CUSTÓDIO T F, DAS H, SHEWARD D J, et al. Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2[J]. Nat Commun, 2020, 11(1):5588.
[24]	WANG L Z, ZHANG L, HUANG B C, et al. A nanobody targeting viral nonstructural protein 9 inhibits porcine reproductive and respiratory syndrome virus replication[J]. J Virol, 2019, 93(4):e01888-18.
[25]	BAO F X, WANG L X, ZHAO X X, et al. Preparation and characterization of a single-domain antibody specific for the porcine epidemic diarrhea virus spike protein[J]. AMB Express, 2019, 9(1):104.
[26]	DELFIN-RIELA T, ROSSOTT M, ALVEZ-ROSADO R, et al. Highly sensitive detection of Zika virus nonstructural protein 1 in serum samples by a two-site nanobody ELISA[J]. Biomolecules, 2020, 10(12):1652.
[27]	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.
[28]	LU Q, LI X, ZHAO J, et al. Nanobody-horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays[J]. J Nanobiotechnol, 2020, 18(1):7.
[29]	DU T, ZHU G, WU X, 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.
[30]	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.