抗口蹄疫病毒IgA抗体的构建表达及其抗病毒活性的分析

doi:10.11843/j.issn.0366-6964.2025.08.036

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (8): 3985-3991.doi: 10.11843/j.issn.0366-6964.2025.08.036

抗口蹄疫病毒IgA抗体的构建表达及其抗病毒活性的分析

刘峰¹^,²(), 李坤¹^,², 章兴赜¹^,², 马雪青¹^,², 孙普¹^,², 李凤娟¹^,², 曹轶梅¹^,², 白兴文¹^,², 付元芳¹^,², 袁红¹^,², 欧阳一凡¹^,², 刘在新¹^,², 卢曾军¹^,², 李平花¹^,²^,*()

1. 中国农业科学院兰州兽医研究所, 兰州大学动物医学与生物安全学院, 动物疫病防控全国重点实验室, 兰州 730000
2. 甘肃省病原生物学基础学科研究中心, 兰州 730046

收稿日期:2024-11-11 出版日期:2025-08-23 发布日期:2025-08-28
通讯作者: 李平花 E-mail:liufeng1534@163.com;lipinghua@caas.cn
作者简介:刘峰(1994-)，男，安徽安庆人，硕士生，主要从事病毒生物学与免疫学研究，E-mail：liufeng1534@163.com
基金资助:
“十四五”国家重点研发计划项目(2021YFD1800304)

Construction and Expression of IgA Antibodies against Foot-and-mouth Disease Virus and Analysis of Its Antiviral Activity

LIU Feng¹^,²(), LI Kun¹^,², ZHANG Xingze¹^,², MA Xueqing¹^,², SUN Pu¹^,², LI Fengjuan¹^,², CAO Yimei¹^,², BAI Xingwen¹^,², FU Yuanfang¹^,², YUAN Hong¹^,², OUYANG Yifan¹^,², LIU Zaixin¹^,², LU Zengjun¹^,², LI Pinghua¹^,²^,*()

1. State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China
2. Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou 730046, China

Received:2024-11-11 Online:2025-08-23 Published:2025-08-28
Contact: LI Pinghua E-mail:liufeng1534@163.com;lipinghua@caas.cn

摘要/Abstract

摘要：

口蹄疫是口蹄疫病毒引起危害猪、牛和羊等主要家畜的一种烈性传染病，为了发展抗口蹄疫病毒的新策略，进行疾病的综合防控，本研究将O型、A型口蹄疫病毒特异性猪BCR文库中筛选获得的5株O型和A型口蹄疫病毒共有克隆型IgA抗体的重链可变区(VH)和轻链可变区(VL)序列分别与猪IgA的重链恒定区基因和轻链(Kappa链)恒定区基因拼接，分别构建5个IgA的重链载体质粒和5个轻链载体质粒，并将其分别共转染CHO-S悬浮细胞，转染7 d后收集上清进行蛋白纯化。纯化后的抗体用SDS-PAGE和Western blot鉴定、并用微量病毒中和试验分析5株IgA抗体中和O型、A型口蹄疫病毒的能力，用间接ELISA方法检测5株IgA抗体与口蹄疫病毒的反应性。结果表明：本研究成功构建质粒并表达了5株抗口蹄疫病毒的IgA抗体，这些抗体对O型和A型口蹄疫病毒均无中和活性，但其中1株抗体(POA-A8)与口蹄疫病毒特异性结合的能力最强，可以用于未来口蹄疫病毒诊断检测方法的建立。本研究为未来抗口蹄疫病毒非中和的分泌型IgA的研究以及病毒诊断方法的建立奠定了基础。

关键词: 口蹄疫病毒, IgA抗体, 构建表达, 抗病毒活性

Abstract:

Foot-and-mouth disease(FMD)caused by foot-and-mouth disease virus (FMDV) is a high infectious disease of pigs, cattle, sheep and other major livestock. In order to develop new strategies against FMDV to carry out comprehensive prevention and control of the disease, the heavy chain variable region (VH) sequences and light chain variable region (VL) sequences of 5 different IgA antibodies against type O and A FMDV were screened from the specific porcine BCR library of type O and A FMDV and linked with the gene sequences of the heavy chain constant region and the light chain (Kappa chain) constant region of porcine IgA, then, five heavy chain plasmids and five light chain plasmids were respectively constructed. The corresponding heavy chain plasmids and light chain plasmids were co-transfected into CHO-s suspension cells, after 7 days post transfection, the transfected supernatants were collected for antibody purification. The purified antibodies were identified by SDS-PAGE and Western blot. The ability of the five antibodies neutralizing types O and A FMDV was analyzed by virus neutralization test. The reactivity of the five antibodies with FMDV was detected by ELISA. Results showed that 5 IgA antibodies were successfully expressed in CHO-s cells and they all could not neutralize type O and A FMDV, but one of them (POA-A8) had the strongest ability to specifically bind with FMDV, which could be used for establishment of FMDV diagnostic methods in the future. This study lays a foundation for research of anti-FMDV nonneutralizing sIgA antibodies and development of diagnostic methods for FMDV in the future.

Key words: FMDV, IgA antibody, construction and expression, antiviral activity

中图分类号:

S852.659.6

刘峰, 李坤, 章兴赜, 马雪青, 孙普, 李凤娟, 曹轶梅, 白兴文, 付元芳, 袁红, 欧阳一凡, 刘在新, 卢曾军, 李平花. 抗口蹄疫病毒IgA抗体的构建表达及其抗病毒活性的分析[J]. 畜牧兽医学报, 2025, 56(8): 3985-3991.

LIU Feng, LI Kun, ZHANG Xingze, MA Xueqing, SUN Pu, LI Fengjuan, CAO Yimei, BAI Xingwen, FU Yuanfang, YUAN Hong, OUYANG Yifan, LIU Zaixin, LU Zengjun, LI Pinghua. Construction and Expression of IgA Antibodies against Foot-and-mouth Disease Virus and Analysis of Its Antiviral Activity[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3985-3991.

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IgA重/轻链载体质粒 Heavy/ light chain plasmid of IgA	载体质粒名称 Vector plasmid	信号肽序列(aa) Signal peptide sequence	可变区编码长度/aa Code length of variable region
重链载体质粒 Heavy chain vector plasmid	POA-AH3	MEFRLHWVVLFALLQDVQG	143
	POA-AH5	MEFRLNWVVLFVLLQGVQG	138
	POA-AH6	MEFRLNWVVLFALLQGVQG	137
	POA-AH7	MEFRLNWVVLFALLQGVQG	132
	POA-AH8	MEFRLNWVVLFALLQGVQG	136
轻链载体质粒 Light chain vector plasmid	POA-AL3	MAWTVLLIGLLAVGSGVDS	127
	POA-AL5	MAWTVLLIGLLAVGSGVES	129
	POA-AL6	MAWTVLLIGLLAVGSGVDS	127
	POA-AL7	MAWTVLLIGLLAVGSGVDS	127
	POA-AL8	MAWTVLLIGLLAVGSGVDS	125

IgA抗体	IC₅₀
IgA抗体	O/BY/CHA/ 2010	O/HK99	O/GSLX	O/Mya/98	O/HN/ CHA/93	O/XJ/CHA/ 2017	A/WH/09	A/GDMM/ CHA/2013	A/AF72
PAO-A3	>50	>50	>50	>50	>50	>50	>50	>50	>50
POA-A5	>50	>50	>50	>50	>50	>50	>50	>50	>50
POA-A6	>50	>50	>50	>50	>50	>50	>50	>50	>50
POA-A7	>50	>50	>50	>50	>50	>50	>50	>50	>50
POA-A8	>50	>50	>50	>50	>50	>50	>50	>50	>50

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抗口蹄疫病毒IgA抗体的构建表达及其抗病毒活性的分析

Construction and Expression of IgA Antibodies against Foot-and-mouth Disease Virus and Analysis of Its Antiviral Activity

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