牛冠状病毒单克隆抗体的制备和鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2025-0838

生物技术通报 ›› 2026, Vol. 42 ›› Issue (1): 67-75.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0838

牛冠状病毒单克隆抗体的制备和鉴定

刘余¹^,²(), 刘筱筱²^,³, 郑建豪²^,⁴, 李彬², 何翃闳¹(), 毛立¹^,²()

^1.青藏高原动物遗传资源保护与利用教育部重点实验室西南民族大学畜牧兽医学院，成都 610041
^2.江苏省农业科学院兽医研究所农业农村部兽用生物制品工程技术重点实验室，南京 210014
^3.西北农林科技大学动物医学院，杨凌 712100
^4.南京农业大学动物医学院，南京 210095

收稿日期:2025-08-02 出版日期:2026-01-26 发布日期:2026-02-04
通讯作者: 毛立，男，博士，副研究员，研究方向：预防兽医学；E-mail: mao-li@live.cn
何翃闳，男，博士，副教授，研究方向：动物生殖生理及疾病诊疗；E-mail: honghong3h@126.com
作者简介:刘余，女，硕士研究生，研究方向：动物疫病防控；E-mail: Y18728628336@outlook.com
基金资助:
国家重点研发计划项目(2023YFD1801302);国家重点研发计划项目(2023YFD1801305);江苏省重点研发计划项目(BE2022330);西南民族大学中央高校基本科研业务费专项资金项目(ZYN2024168)

Preparation and Identification of Monoclonal Antibodies against the Bovine Coronavirus

LIU Yu¹^,²(), LIU Xiao-xiao²^,³, ZHENG Jian-hao²^,⁴, LI Bin², HE Hong-hong¹(), MAO Li¹^,²()

^1.Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education, Southwest Minzu University, College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041
^2.Institute of Veterinary Science, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biotechnology Engineering of Ministry of Agriculture and Rural Affairs, Nanjing 210014
^3.College of Animal Medicine, Northwest A&F University, Yangling 712100
^4.College of Animal Medicine, Nanjing Agricultural University, Nanjing 210095

Received:2025-08-02 Published:2026-01-26 Online:2026-02-04

摘要/Abstract

摘要：

目的牛冠状病毒（BCoV）是引发犊牛腹泻、成年牛冬季痢疾及各年龄段牛呼吸道疾病的主要病原体，其引发的疫病在畜牧业中广泛存在且危害严重，传统诊断和防控手段存在局限。制备牛冠状病毒单克隆抗体，为精准诊断冠状病毒研究提供关键材料。方法用纯化病毒免疫BALB/c小鼠，利用细胞融合技术制备杂交瘤细胞，筛选获得稳定分泌特异性抗体的单克隆细胞株。采用间接免疫荧光法（IFA）和间接酶联免疫吸附试验（ELISA）对单克隆抗体进行鉴定和亚类分析。同时构建cpCoV的S蛋白全长及S1、S2亚基真核表达质粒，转染293T细胞，通过IFA和Western-blot方法鉴定单克隆抗体识别的抗原表位区域。结果成功筛选制备了BCoV的单克隆抗体1E1，腹水效价>1∶81 000，亚型为IgG1，其抗体可与BCoV和羊冠状病毒（cpCoV）均发生特异性反应。通过真核表达病毒各结构蛋白及S蛋白各区域，验证1E1抗体识别的抗原表位均位于S蛋白的S1结构域。结论通过制备牛冠状病毒单克隆抗体，获得了能用于检测BCoV、cpCoV的单抗1E1，并鉴定其识别的抗原表位位于S1结构域，分析发现S1蛋白在BCoV和cpCoV同源性为95.4%-99.6%，但有明显差异的位点，本文为检测牛羊冠状病毒提供了新方法，为病毒的基础研究提供新工具。

关键词: 牛冠状病毒, 羊冠状病毒, 单克隆抗体, 抗原表位, 鉴定

Abstract:

Objective Bovine coronavirus (BCoV) is a major pathogen causing diarrhea in calves and winter dysentery in adult cattle, and respiratory diseases in cattle of all ages. The diseases caused by BCoV are widespread and lead to serious harm in the livestock industry. There are limitations in traditional diagnostic and control methods. The development of BCoV-specific monoclonal antibodies aims to facilitate precise diagnostic detection and provide critical experimental materials for coronavirus research. Method BALB/c mice were immunized with purified virus, and hybridoma cells were prepared using cell fusion technology. Monoclonal cell lines stably secreting specific antibodies were screened. The monoclonal antibodies were identified and subclassified by indirect immunofluorescence assay (IFA) and indirect enzyme-linked immunosorbent assay (ELISA). Concurrently the eukaryotic expression plasmids for the full-length S protein and S1, S2 subunits of cpCoV were constructed and then transfected into 293T cells. The viral proteins recognized by monoclonal antibodies were identified by IFA and Western-blot techniques. Result Monoclonal antibody 1E1 was successfully obtained through monoclonal screening. The ascites titer of antibody was >1∶81 000, and the subtype was identified as IgG1. The antibody specifically reacted with both BCoV and cpCoV. Through gene cloning and eukaryotic expression, it was verified that the antigenic epitopes recognized by 1E1 antibody were located in the S1 domain of the S protein. Conclusion By preparing BCoV monoclonal antibodies, the obtained monoclonal antibody 1E1 can be used for identification of both BCoV and cpCoV. The antigenic epitopes recognized by the antibody are verified, and analysis reveals that the homology of S1 protein sequence is 95.4%-99.6%, and has significant differences between BCoV and cpCoV. The result provides a new method for the detection of the two coronaviruses and a new tool for the basic research of BCoV and cpCoV.

Key words: bovine coronavirus, caprine coronavirus, monoclonal antibody, antigen epitope, identification

刘余, 刘筱筱, 郑建豪, 李彬, 何翃闳, 毛立. 牛冠状病毒单克隆抗体的制备和鉴定[J]. 生物技术通报, 2026, 42(1): 67-75.

LIU Yu, LIU Xiao-xiao, ZHENG Jian-hao, LI Bin, HE Hong-hong, MAO Li. Preparation and Identification of Monoclonal Antibodies against the Bovine Coronavirus[J]. Biotechnology Bulletin, 2026, 42(1): 67-75.

图/表 7

表1 引物序列

Table 1 Primer sequences

引物名称 Primer	寡核苷酸序列 Oligonucleotide sequence（5'-3'）	基因长度 Gene length （bp）
cpCoV-S-F	ATGTTTTTGATACTTTTAATTTC	4 092
cpCoV-S-R	GTCGTCATGTGAGGTTTT	4 092
cpCoV-S1-F	ATGTTTTTGATACTTTTAATTTC	2 304
cpCoV-S1-R	TCTACGACTTCGTCTTTTTG	2 304
cpCoV-S2-F	AGACGAAGTCGTAGATCG	1 803
cpCoV-S2-R	GTCGTCATGTGAGGTTTT	1 803

图1 免疫小鼠血清效价检测

Fig. 1 Measurement of serum antibody titer in immunized mice

图2 IFA鉴定单抗1E1与BCoV、cpCoV的反应性

Fig. 2 IFA analysis of the reactivity of mAb 1E1 against BCoV and cpCoV

图3 S基因质粒鉴定结果A：pCAGGS-cpCoV-S-HA PCR扩增； B：pCAGGS-cpCoV-S1-HA PCR扩增； C：pCAGGS-cpCoV-S2-HA PCR扩增。M：DNA分子质量标准；1、2：目的基因的RT-PCR产物

Fig. 3 Identification results of plasmids of S geneA: PCR amplification of pCAGGS-cpCoV-S-HA. B: PCR amplification of pCAGGS-cpCoV-S1-HA. C: PCR amplification of pCAGGS-cpCoV-S2-HA. M: DNA molecular weight standard. 1, 2: RT-PCR products of the target gene

图4 S蛋白表达鉴定A：IFA鉴定结果；B：Western-blot鉴定结果。M：蛋白质分子质量标准；1：HA-BCoV-HE阳性质粒转染细胞；2、3：HA-cpCoV-S重组质粒转染细胞；4、5：HA-cpCoV-S1重组质粒转染细胞；6、7：HA-cpCoV-S2重组质粒转染细胞；8：pCAGGS-HA空载转染细胞；9：293T细胞对照

Fig. 4 Expression and identification of S proteinA: IFA test results. B: Western-blot test results. M: Protein molecular weight standard. 1: Positive plasmid control of HA-BCoV-HE. 2, 3: Western-blot test results of HA-cpCoV-S recombinant plasmid. 4, 5: Western-blot test results of HA-cpCoV-S1 recombinant plasmid. 6, 7: Western-blot test results of HA-cpCoV-S2 recombinant plasmid. 8: Empty pCAGGS-HA; 9: 293T cells control

图5 单抗1E1和6D5 IFA鉴定结果

Fig. 5 IFA identification results of monoclonal antibodies 1E1 and 6D5

图6 BCoV、cpCoV S1蛋白的同源性和差异蛋白分析结果A：基于BCoV、cpCoV S1片段完整氨基酸序列的成对遗传距离热图；黑色三角形：分离株BCoV-NXWZ2301；B：BCoV、cpCoV的S1蛋白序列对比

Fig. 6 Analysis results of homology and differential proteins of BCoV and cpCoV S1 proteinsA: Pairwise genetic distance heatmap based on the complete amino acid sequences of the S1 fragments of BCoV and cpCoV. Black triangle: Isolate BCoV-NXWZ2301. B: Comparison of the S1 protein sequences of BCoV and cpCoV

参考文献 30

[1]	Holmes EC. The emergence and evolution of SARS-CoV-2 [J]. Annu Rev Virol, 2024, 11: 21-42.
[2]	Woo PCY, Lau SKP, Lam CSF, et al. Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus [J]. J Virol, 2012, 86(7): 3995-4008.
[3]	Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses [J]. Nat Rev Microbiol, 2019, 17(3): 181-192.
[4]	Zhong N, Zheng B, Li Y, et al. Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People’s Republic of China, in February, 2003 [J]. Lancet, 2003, 362(9393): 1353-1358.
[5]	Zaki AM, van Boheemen S, Bestebroer TM, et al. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia [J]. N Engl J Med, 2012, 367(19): 1814-1820.
[6]	Su S, Wong G, Shi WF, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses [J]. Trends Microbiol, 2016, 24(6): 490-502.
[7]	Saif LJ. Bovine respiratory coronavirus [J]. Vet Clin N Am Food Anim Pract, 2010, 26(2): 349-364.
[8]	Liang YW, Wang ML, Chien CS, et al. Highlight of immune pathogenic response and hematopathologic effect in SARS-CoV, MERS-CoV, and SARS-cov-2 infection [J]. Front Immunol, 2020, 11: 1022.
[9]	Heckert RA, Saif LJ, Hoblet KH, et al. A longitudinal study of bovine coronavirus enteric and respiratory infections in dairy calves in two herds in Ohio [J]. Vet Microbiol, 1990, 22(2/3): 187-201.
[10]	Decaro N, Campolo M, Desario C, et al. Respiratory disease associated with bovine coronavirus infection in cattle herds in southern Italy [J]. J VET Diagn Invest, 2008, 20(1): 28-32.
[11]	Boileau MJ, Kapil S. Bovine coronavirus associated syndromes [J]. Vet Clin N Am Food Anim Pract, 2010, 26(1): 123-146.
[12]	Fulton RW, Herd HR, Sorensen NJ, et al. Enteric disease in postweaned beef calves associated with Bovine coronavirus clade 2 [J]. J VET Diagn Invest, 2015, 27(1): 97-101.
[13]	Alekseev KP, Vlasova AN, Jung K, et al. Bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences [J]. J Virol, 2008, 82(24): 12422-12431.
[14]	Burimuah V, Sylverken A, Owusu M, et al. Molecular-based cross-species evaluation of bovine coronavirus infection in cattle, sheep and goats in Ghana [J]. BMC Vet Res, 2020, 16(1): 405.
[15]	Mao L, Cai XH, Li JZ, et al. Discovery of a novel Betacoronavirus 1, cpCoV, in goats in China: The new risk of cross-species transmission [J]. PLoS Pathog, 2025, 21(3): e1012974.
[16]	Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975, 256(5517): 495-497.
[17]	Amer HM. Bovine-like coronaviruses in domestic and wild ruminants [J]. Anim Health Res Rev, 2018, 19(2): 113-124.
[18]	Saif LJ, Jung K. Comparative pathogenesis of bovine and porcine respiratory coronaviruses in the animal host species and SARS-CoV-2 in humans [J]. J Clin Microbiol, 2020, 58(8): e01355-20.
[19]	靳双媛, 杜家伟, 王雪妍, 等. 牛冠状病毒S和HE蛋白研究进展 [J]. 中国畜牧兽医, 2024, 51(7): 3118-3127.
	Jin SY, Du JW, Wang XY, et al. Research progress on S and HE proteins of bovine coronaviruses [J]. China Anim Husb Vet Med, 2024, 51(7): 3118-3127.
[20]	Zhu QH, Li B, Sun DB. Advances in bovine coronavirus epidemiology [J]. Viruses, 2022, 14(5): 1109.
[21]	刘筱筱, 余秋蓉, 李霞, 等. 牛冠状病毒家兔源多克隆抗体的制备与应用 [J/OL]. 中国兽医科学, 2025: 1-9. (2025-05-29). .
	Liu XX, Yu QR, Li X, et al. Preparation and application of rabbit polyclonal antibodies against bovine coronavirus [J/OL]. Chin Vet Sci, 2025: 1-9. (2025-05-29). .
[22]	Dong SJ, Sun JC, Mao Z, et al. A guideline for homology modeling of the proteins from newly discovered betacoronavirus, 2019 novel coronavirus (2019-nCoV) [J]. J Med Virol, 2020, 92(9): 1542-1548.
[23]	Li F. Structure, function, and evolution of coronavirus spike proteins [J]. Annu Rev Virol, 2016, 3: 237-261.
[24]	黎梦帆, 李青阳, 宋艳雯, 等. 冠状病毒S蛋白的结构和功能研究进展 [J]. 畜牧兽医学报, 2025, 56(9): 4241-4252.
	Li MF, Li QY, Song YW, et al. Structure and function of coronavirus S proteins [J]. Acta Vet Zootechnica Sin, 2025, 56(9): 4241-4252.
[25]	Jackson CB, Farzan M, Chen B, et al. Mechanisms of SARS-CoV-2 entry into cells [J]. Nat Rev Mol Cell Biol, 2022, 23(1): 3-20.
[26]	Zhu QH, Su MJ, Li ZJ, et al. Epidemiological survey and genetic diversity of bovine coronavirus in Northeast China [J]. Virus Res, 2022, 308: 198632.
[27]	Walls AC, Park YJ, Tortorici MA, et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein [J]. Cell, 2020, 181(2): 281-292.e6.
[28]	Jung J, Bong JH, Kim TH, et al. Isolation of antibodies against the spike protein of SARS-CoV from pig serum for competitive immunoassay [J]. BioChip J, 2021, 15(4): 396-405.
[29]	Millet JK, Jaimes JA, Whittaker GR. Molecular diversity of coronavirus host cell entry receptors [J]. FEMS Microbiol Rev, 2021, 45(3): fuaa057.
[30]	Hulswit RJG, de Haan CAM, Bosch BJ. Coronavirus spike protein and tropism changes [M]//Coronaviruses. Amsterdam: Elsevier, 2016: 29-57.

牛冠状病毒单克隆抗体的制备和鉴定

Preparation and Identification of Monoclonal Antibodies against the Bovine Coronavirus

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	程婷婷, 刘俊, 王利丽, 练从龙, 魏文君, 郭辉, 吴尧琳, 杨晶凡, 兰金旭, 陈随清. 杜仲查尔酮异构酶基因家族全基因组鉴定及其表达模式分析[J]. 生物技术通报, 2025, 41(9): 242-255.
[2]	苏秀敏, 韩文清, 王佼, 李鹏, 王秋兰, 李万星, 曹晋军. 哈茨木霉M408的分离鉴定、生物学特性及对番茄早疫病的生防效果[J]. 生物技术通报, 2025, 41(9): 277-288.
[3]	廉少杰, 唐胜硕, 康传利, 刘磊, 郑德强, 杜帅, 汤丽伟, 张美霞, 刘蔷. 高产银耳多糖酶菌株的分离、鉴定、发酵条件优化及其酶的特性分析[J]. 生物技术通报, 2025, 41(9): 302-313.
[4]	闫梦阳, 梁晓阳, 戴君昂, 张妍, 关团, 张辉, 刘良波, 孙志华. 阿莫西林降解菌的筛选及降解机制研究[J]. 生物技术通报, 2025, 41(9): 314-325.
[5]	张茹, 李一鸣, 张桐溪, 孙占斌, 任清, 潘寒姁. 厚朴中1株高产厚朴酚与和厚朴酚菌株的分离鉴定及其“发汗”工艺优化[J]. 生物技术通报, 2025, 41(8): 322-334.
[6]	李凯月, 邓晓霞, 殷缘, 杜亚彤, 徐元静, 王竞红, 于耸, 蔺吉祥. 蓖麻LEA基因家族的鉴定和铝胁迫响应分析[J]. 生物技术通报, 2025, 41(7): 128-138.
[7]	龚钰涵, 陈兰, 尚方慧子, 郝灵颖, 刘硕谦. 茶树TRB基因家族鉴定及表达模式分析[J]. 生物技术通报, 2025, 41(7): 214-225.
[8]	王轶民, 李莹, 董海涛, 张恒瑞, 常璐, 高田甜, 韩德俊, 吴建辉. SRO家族蛋白在小麦多倍化进程中的演化规律[J]. 生物技术通报, 2025, 41(5): 70-81.
[9]	李文兰, 侯辛未, 李燕, 赵瑞君, 孟昭东, 岳润清. 转基因抗虫耐除草剂玉米LD05纯杂合植株的鉴定及抗性检测[J]. 生物技术通报, 2025, 41(4): 123-133.
[10]	孙天国, 衣兰, 秦旭洋, 乔梦雪, 谷新颖, 韩艺, 沙伟, 张梅娟, 马天意. 大白菜DABB基因家族的全基因组鉴定及盐碱胁迫下的表达分析[J]. 生物技术通报, 2025, 41(4): 156-165.
[11]	王田田, 常雪瑞, 黄婉洋, 黄嘉欣, 苗如意, 梁燕平, 王静. 辣椒GASA基因家族的鉴定及分析[J]. 生物技术通报, 2025, 41(4): 166-175.
[12]	黄金恒, 黄茜, 张家燕, 周新裕, 廖沛然, 杨全. 广金钱草C3H基因家族鉴定及不同品种表达分析[J]. 生物技术通报, 2025, 41(4): 243-255.
[13]	刘爽, 江洲, 赵帅, 赵雷真, 黄峰, 周佳, 屈建航. 一株产蛋白酶细菌的筛选、鉴定及发酵工艺优化[J]. 生物技术通报, 2025, 41(4): 335-344.
[14]	宋姝熠, 蒋开秀, 刘欢艳, 黄亚成, 刘林娅. ‘红阳’猕猴桃TCP基因家族鉴定及其在果实中的表达分析[J]. 生物技术通报, 2025, 41(3): 190-201.
[15]	马天意, 许家佳, 路文婧, 吴艳, 沙伟, 张梅娟, 彭疑芳. ‘金小童’大白菜BrcGASA3基因在盐碱胁迫下的表达分析及抗性鉴定[J]. 生物技术通报, 2025, 41(2): 127-138.