A型口蹄疫病毒多表位纳米融合蛋白制备及全自动磁微粒CLIA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.12.027

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (12): 6257-6270.doi: 10.11843/j.issn.0366-6964.2025.12.027

A型口蹄疫病毒多表位纳米融合蛋白制备及全自动磁微粒CLIA抗体检测方法的建立

郑雪莹¹(), 王培¹(), 孙伟珊², 赵浩军¹, 殷雨晴¹, 李佳¹, 程汝佳¹, 刘海莹¹, 王大猛¹, 穆伟¹, 李达¹, 陈锡俊², 郑博君¹^,*(), 梅力¹^,*()

1. 北京市动物疫病预防控制中心, 北京 102629
2. 北京测易生物科技有限公司, 北京 102629

收稿日期:2025-02-28 出版日期:2025-12-23 发布日期:2025-12-24
通讯作者: 郑博君,梅力 E-mail:798620457@qq.com;763758941@qq.com;lee_klmmyt@126.com;meili0816@126.com
作者简介:郑雪莹(1989-)，女，山东茌平人，高级兽医师，硕士，主要从事动物传染病实验室检测技术研究，E-mail: 798620457@qq.com
王培(1989-)，女，山东金乡人，兽医师，硕士，主要从事动物传染病实验室检测技术研究，E-mail: 763758941@qq.com
第一联系人：
郑雪莹和王培为同等贡献作者
基金资助:
北京市动物疫病预防控制中心综合保障项目(实验室运转与动物疫病监测)(11000024Y000002861241)

Preparation of Multi-epitope Nanofusion Protein for Foot-and-Mouth Disease Virus Serotype A and Development for Antibodies Detection of a Full-Automated Magnetic Particle Chemiluminescent Immunoassay (CLIA)

ZHENG Xueying¹(), WANG Pei¹(), SUN Weishan², ZHAO Haojun¹, YIN Yuqing¹, LI Jia¹, CHENG Rujia¹, LIU Haiying¹, WANG Dameng¹, MU Wei¹, LI Da¹, CHEN Xijun², ZHENG Bojun¹^,*(), MEI Li¹^,*()

1. Beijing Center for Animal Disease Control and Prevention, Beijing 102629, China
2. Beijing CeYi Biotechnology Co., Ltd., Beijing 102629, China

Received:2025-02-28 Online:2025-12-23 Published:2025-12-24
Contact: ZHENG Bojun, MEI Li E-mail:798620457@qq.com;763758941@qq.com;lee_klmmyt@126.com;meili0816@126.com

摘要/Abstract

摘要：

本研究旨在制备A型口蹄疫病毒(FMDV-A)多表位纳米融合蛋白并建立磁微粒CLIA抗体检测方法，为FMDV的临床监测和防控提供技术支撑。以表达的FMDV-A型多表位融合蛋白及其多克隆抗体为基础建立FMDV-A型磁微粒CLIA抗体检测方法，优化反应条件，并与商品化试剂盒同时进行临床样品的检测，比较两种方法的符合率。结果显示：本研究最终成功制备了高可溶性FMDV-A型多表位纳米融合蛋白及其特异性多克隆抗体，多克隆抗体效价为1∶5 120，并根据竞争法免疫原理建立了FMDV-A型磁微粒CLIA抗体检测方法。SA-Beads最佳浓度为0.5 mg·mL^-1，生物素标记的FMDV-A型重组蛋白抗原最佳工作浓度为5 μg·L^-1，AE标记的多克隆抗体最佳工作浓度为2 μg·L^-1；最佳上样体积为10 μL，最佳反应时间为15 min。敏感性试验结果表明，该方法的敏感性为97.47%，敏感性好。特异性试验结果表明，与其他相似病原没有交叉反应，特异性强。重复性试验结果说明变异系数CV值均小于5%，具有良好的稳定性和重复性。临床试验结果表明，与中国农科院兰州兽医研究所LPB-ELISA方法比较，总符合率为96.76%，为国内FMDV-A的研究与快速诊断建立了技术基础。本研究成功制备FMDV-A型毒株的VP1多表位融合纳米颗粒蛋白，并获得其高效价多克隆抗体，应用该蛋白和多克隆抗体建立了FMDV-A型磁微粒CLIA抗体检测方法。

关键词: A型口蹄疫病毒, 铁蛋白, CLIA, 磁微粒化学发光, 纳米颗粒

Abstract:

This experiment was conducted to prepare a multi-epitope nanofusion protein of foot-and-mouth disease virus serotype A (FMDV-A) and to establish a magnetic particle CLIA antibody detection method, which was to provide technical support for clinical immunization monitoring, prevention and control of FMDV. In this study, a magnetic particle chemiluminescent immunoassay (CLIA) for detecting antibodies against foot-and-mouth disease virus serotype A (FMDV-A) was established based on the expressed multi-epitope fusion protein of FMDV-A and its polyclonal antibody. Reaction conditions were optimized, and clinical samples were simultaneously tested using this method and commercial kits to compare the coincidence rate between the two methods. Finally, a highly soluble FMDV-A multi-epitope nanofusion protein and specific polyclonal antibody with a titer of 1∶5 120 were successfully prepared. A magnetic particle CLIA for FMDV-A antibodies detection was established based on the principle of competitive immunoassay. The optimal concentration of streptavidin magnetic beads (SA-Beads) was 0.5 mg·mL^-1, the optimal concentration of biotin-labeled recombinant FMDV-A protein antigen was 5 μg·L^-1, and the optimal concentration of acridinium ester (AE)-labeled polyclonal antibody was 2 μg·L^-1. The optimal sample volume was 10 μL, and the optimal reaction time was 15 minutes. The sensitivity test results show that the sensitivity of this method is 97.47%, and the sensitivity is good. No cross-reactivity was observed with other clinically similar pathogens, demonstrating strong specificity. The results of repeatability test demonstrated that coefficient of variation (CV) values were less than 5%, which had good stability and repeatability The results of the clinical trials indicated that the overall coincidence rate was 96.76% when compared with the LPB-ELISA method developed by Lanzhou Veterinary Research Institute. This research has established a technical foundation for the research and rapid diagnosis on type A foot-and-mouth disease in China. This study successfully prepared a VP1 multi epitope fusion nanoparticle protein of FMDV-A strain and obtained its high titer polyclonal antibody. The protein and polyclonal antibody were used to establish a detection method for FMDV-A magnetic particle CLIA antibody.

Key words: foot-and-mouth disease virus type A, ferritin, CLIA, magnetic particles chemiluminescence, nanoparticle

中图分类号:

S855.3

郑雪莹, 王培, 孙伟珊, 赵浩军, 殷雨晴, 李佳, 程汝佳, 刘海莹, 王大猛, 穆伟, 李达, 陈锡俊, 郑博君, 梅力. A型口蹄疫病毒多表位纳米融合蛋白制备及全自动磁微粒CLIA抗体检测方法的建立[J]. 畜牧兽医学报, 2025, 56(12): 6257-6270.

ZHENG Xueying, WANG Pei, SUN Weishan, ZHAO Haojun, YIN Yuqing, LI Jia, CHENG Rujia, LIU Haiying, WANG Dameng, MU Wei, LI Da, CHEN Xijun, ZHENG Bojun, MEI Li. Preparation of Multi-epitope Nanofusion Protein for Foot-and-Mouth Disease Virus Serotype A and Development for Antibodies Detection of a Full-Automated Magnetic Particle Chemiluminescent Immunoassay (CLIA)[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6257-6270.

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毒株名称 Strain name	GenBank登录号 GenBank Number	序列位置/bp Sequence location	序列(5′→3′) Sequence
A/AF72/A22	MT447399.1	64102	ACACAAGTCCAAAGACGCCAGCACACCAATGTCGGCTTC
		408447	TCCACAGGTAATGCAGGCAGACGGGGTGATCTAGGGTCT
A/HuBWH/CHA/2009	JF792355.1	268318	CCCCATGGAGCACCTGAGGCAGCACTGGACAACACGAGCAACCCCACTGCT
		388444	TACAACGGGACTAGCAAGTACTCTGCGCCTGCAACACGGCGAGGTGACTTGGGGTCT
A/GDMM/CHA/ 2013	KF450794.1	1636	ACCACCGCCACCGGGGAATCAGCAGACCCTGTCACAACCACCGTTGAGAACTACGGTGGCGAGACACAAGTACAGCGGCGTTACCACACCGACGTCGGCTTCTTAATGGACAGGTTCGTGCAGATCAAGCCTGTGGGCCCCACACATGTCATTGACCTCATGCAGACACACCAACACGGGCTGGTGGGCGCCATGTTGCGCGCGGCCACCTACTA CTTTTCTGATCTTGAGATTGTGGTGAACCACACGGGTAACCTAACGTGGGTACCCAATGGAGCACCCGAGGCAGCACTGCAAAACACGAGCAACCCCACTGCTTACCACAAAGCGCCGTTCACGAGGCTTGCGCTCCCCTACACCGCGCCACACCGCGTGCTGGCAACTGTGTACAGTGGGACGAGCAAGTACTCCGCACCTCAAAACCGGCGAGGTGACTCGGGTCCTCTCGCGGCGAGACTCGCTGCACAGCTCCCTGCCTCCTTCAACTTCGGTGCAATTCGGGCCACGGAGATCCGCGAACTCCTTGTGCGCATGAAGCGCGCCGAGCT CTACTGCCCCAGGCCACTGTTGGCGGTGGAGGTGTCGTCGCAAGACAGACACAAGCAGAAAATCATTGCCCCTGCAAAACAACTCCTG

毒株名称 Strain name	GenBank登录号 GenBank Number	序列位置/bp Sequence location	优化后序列(5′→3′) Optimized sequence
A/AF72/A22	MT447399.1	64-102	ACGCAAGTCCAGCGCCGCCAGCATACCAACGTCGGTTTC
		408-447	AGCACTGGTAACGCTGGTCGTCGCGGTGATCTGGGTTCT
A/HuBWH/CHA/2009	JF792355.1	268-318	CCACATGGTGCACCGGAAGCCGCACTGGACAACACTTCCAACCCGACTGCC
		388-444	TACAACGGCACGTCCAAATACTCCGCTCCGGCAACCCGTCGTGGTGATCTGGGTTCT
A/GDMM/CHA/ 2013	KF450794.1	1-636	ACCACCGCGACGGGCGAGAGCGCAGACCCTGTAACCACTACTGTAGAAAACTACGGTGGTGAAACCCAAGTACAGCGTCGCTATCATACCGACGTGGGCTTTCTGATGGATCGTTTCGTCCAGATCAAACCGGTCGGTCCGACCCACGTGATCGACCTGATGCAGACTCACCAACACGGTCTGGTTGGTGCCATGCTGCGTGCTGCAACTTACTACTTTAGCGACCTGGAGATCGTTGTGAACCATACGGGTAACCTGACCTGGGTACCGAACGGCGCCCCAGAAGCTGCCCTGCAGAATACTTCTAACCCGACGGCATACCACAAAGCGCCATTCACCCGCCTGGCTCTGCCGTATACCGCGCCGCATCGTGTGCTGGCGACTGTTTACTCTGGTACCTCTAAGTACTCCGCTCCGCAAAACCGTCGTGGTGACTCCGGTCCTCTGGCAGCACGTCTGGCTGCGCAGCTGCCGGCCTCTTTTAACTTCGGCGCTATCCGTGCTACTGAAATTCGTGAGCTGCTGGTACGTATGAAACGCGCTGAACTGTACTGTCCGCGTCCGCTGCTGGCTGTTGAGGTTAGCTCCCAGGACCGTCACAAACAGAAGATTATCGCCCCAGCAAAACAGCTGCTG

项目 Items	内容 Contents
GenBank登录号 GenBank No.	AY072939.1
优化前序列(5′→3′) The sequence before optimization	ATGTTATCAAAAGACATCATTAAGTTGCTAAACGAACAAGTGAATAAGGAAATGAACTCTTCCAACTTGTATATGAGCATGAGTTCTTGGTGCTATACCCATAGCTTAGACGGCGCGGGGCTTTTCTTATTTGACCATGCGGCTGAAGAATACGAGCATGCTAAAAAGCTTATCATCTTTTTGAATGAAAACAATGTGCCTGTGCAATTGACTAGCATCAGCGCCCCTGAGCATAAGTTTGAAGGTTTGACTCAAATTTTCCAAAAAGCCTATGGACATGAGCAACACATCAGCGAGTCTATTAATAATATCGTCGATCACGCCATAAAAAACAAAGATCATGCGACTTTCAATTTCTTGCAATGGTATGTGGCTGAACAGCATGAAGAAGAAGTGCTTTTCAAGGATATTTTGGATAAAATTGAGTTGATTGGTAATGAAAACCATGGCTTGTATTTGGCTGATCAGTATGTCAAAGGGATCGCTAAAAGCAGGAAATCT
截短优化后序列(5′ →3′) The sequence after truncation and optimization	GACATTATCAAGCTGCTGAACGAACAAGTTAACAAGGAAATGCAATCCTCCAACCTGTACATGAGCATGAGCTCTTGGTGTTATACTCACTCTCTGGACGGTGCAGGCCTGTTCCTGTTTGATCACGCTGCCGAAGAATATGAACACGCCAAAAAACTGATCATCTTCCTGAACGAAAATAACGTTCCGGTTCAGCTGACCTCCATTTCTGCCCCGGAACATAAATTCGAGGGTCTGACCCAAATCTTCCAGAAAGCATATGGTCACGAGCAGCATATCTCCGAATCTATCAACAACATCGTGGACCACGCGATTAAGAATAAAGACCACGCCACTTTTAACTTCCTGCAATGGTACGTAGCTGAACAGCACGAAGAGGAAGTACTGTTCAAAGACATTCTGGACAAGATTGAACTGATCGGTAACGAAAATCACGGTCTGTATCTGGCAGACCAATACGTCAAAGGTATCGCGAAATCTCGCAAGAGC

优化项 Optimization items	反应浓度Reaction concentration			加样量/μL Sampling volume	孵育时间/min Incubation time
优化项 Optimization items	链霉亲和素磁珠/(mg·mL^-1) SA-Beads	标记抗原/(μg·L^-1) Labeled antigen	AE标记抗体/(μg·L^-1) AE labeled antibody	加样量/μL Sampling volume	孵育时间/min Incubation time
1	0.10	1.0	1	5	5
2	0.25	2.5	2	10	10
3	0.50	5.0	4	15	15
4	1.00	10.0	8	20	20

项目 Item		LPB-ELISA方法检测的阳性样品数 Number of positive samples detected by LPB-ELISA method
本研究试验方法 Experimental method	阳性样品数 Number of positive samples	193
	阴性样品数 Number of negaitive samples	5
	总样品数 Total samples	198
敏感性(阳性检出率/%) Sensitivity (Positive conformity rate)		97.47

A型口蹄疫病毒多表位纳米融合蛋白制备及全自动磁微粒CLIA抗体检测方法的建立

Preparation of Multi-epitope Nanofusion Protein for Foot-and-Mouth Disease Virus Serotype A and Development for Antibodies Detection of a Full-Automated Magnetic Particle Chemiluminescent Immunoassay (CLIA)

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项目 Item		LPB-ELISA方法LPB-ELISA method
项目 Item		阳性样品数 Number of positive samples	阴性样品数 Number of negaitive samples	总样品数 Total samples
本研究试验方法 Experimental method of this study	阳性样品数Number of positive samples	390	6	396
	阴性样品数Number of negaitive samples	10	88	98
	总样品数Total samples	400	94	494
阳性符合率/% Positive conformity rate		97.50
阴性符合率/% Negative conformity rate		93.62
总符合率/% Total consistent rate		96.76

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