口蹄疫病毒Asia1型猪源中和抗体的筛选与抗原表位鉴定

doi:10.11843/j.issn.0366-6964.2024.11.036

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (11): 5211-5221.doi: 10.11843/j.issn.0366-6964.2024.11.036

口蹄疫病毒Asia1型猪源中和抗体的筛选与抗原表位鉴定

董开恒¹(), 黄书伦²^,³, 李凤娟²^,³, 李坤²^,³, 刘果¹, 张强²^,³, 包慧芳²^,³, 李洪炫²^,³, 卢曾军²^,³^,*(), 张小丽¹^,*()

1. 甘肃农业大学动物医学院, 兰州 730070
2. 中国农业科学院兰州兽医研究所, 动物疫病防控全国重点实验室, 兰州大学动物医学与生物安全学院, 国家口蹄疫参考实验室, 兰州 730000
3. 甘肃省病原生物学基础学科研究中心, 兰州 730046

收稿日期:2024-01-17 出版日期:2024-11-23 发布日期:2024-11-30
通讯作者: 卢曾军,张小丽 E-mail:1593853715@qq.com;luzengjun@caas.cn;zhxl228@qq.com
作者简介:董开恒(1998-), 男, 甘肃武威人, 硕士, 主要从事动物微生物与免疫及分子生物学研究, E-mail: 1593853715@qq.com
基金资助:
“十四五”国家重点研发项目(2021YFD1800300)

Screening and Identification of the Antigenic Epitope of Pig Neutralizing Antibodies against Foot-and-mouth Disease Virus Serotype Asia 1

Kaiheng DONG¹(), Shulun HUANG²^,³, Fengjuan LI²^,³, Kun LI²^,³, Guo LIU¹, Qiang ZHANG²^,³, Huifang BAO²^,³, Hongxuan LI²^,³, Zengjun LU²^,³^,*(), Xiaoli ZHANG¹^,*()

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. State Key Laboratory for Animal Disease Control and Prevention, National Foot-and-Mouth Diseases Reference Laboratory, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China
3. Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou 730046, China

Received:2024-01-17 Online:2024-11-23 Published:2024-11-30
Contact: Zengjun LU, Xiaoli ZHANG E-mail:1593853715@qq.com;luzengjun@caas.cn;zhxl228@qq.com

摘要/Abstract

摘要：

Asia1型口蹄疫病毒(FMDV)仍然在我国周边国家存在，对我国畜牧业造成长期威胁。本研究旨在利用单个B细胞抗体技术研制Asia1型FMDV的中和性单克隆抗体，以此为工具，鉴定Asia1型FMDV的保护性抗原位点。以FMDV Asia1/JS/05为诱饵抗原，通过流式细胞术分选免疫猪PBMCs中的抗原特异性B细胞，通过巢式PCR扩增单个B细胞IgG抗体重链与轻链可变区基因序列，分别构建IgG抗体重链与轻链表达质粒，将其共转染CHO-S细胞进行抗体表达；通过间接ELISA、间接免疫荧光试验(IFA)、病毒中和试验(VNT)验证抗体反应性和中和活性，利用免疫印迹、中和抗体逃逸突变株筛选鉴定中和抗体所识别的抗原表位类型和抗原表位关键氨基酸。结果显示：获得了5株反应性良好的Asia1型FMDV特异性抗体，其中PD3和PD7为中和抗体，两株中和抗体识别相同表位，关键氨基酸为VP2蛋白72位残基(D)。本研究首次获得Asia1型FMDV特异性猪源中和抗体，鉴定VP2 72D是中和表位的关键氨基酸，进一步丰富了Asia1型FMDV抗原位点信息，为FMDV Asia1型分子疫苗和新诊断检测技术研究的奠定了基础。

关键词: 口蹄疫病毒, Asia1型, 猪源单克隆抗体, 抗原表位

Abstract:

Foot-and-mouth disease virus (FMDV) serotype Asia 1 is still circulating in Asia countries around China, which will be a long-term threat to animal husbandry. The aim of this study was to screen porcine monoclonal neutralizing antibodies against Asia1 FMDV using single B-cell antibody technology, and to identify the antigenic sites recognized by neutralizing antibodies. FMDV Asia1/JS/05 inactivated whole virus particles were used as the bait antigen to sort antigen-specific B cells in PBMCs of immunized porcine by flow cytometry. The sequences of variable regions of the heavy and light chain of the IgG were amplified by nested PCR. The expression plasmids of the IgG heavy and light chain were constructed respectively and co-transfected into CHO-S cells to express the whole IgG antibodies. The reactivity and neutralization ability of expressed antibodies were verified by indirect ELISA, indirect immunofluorescent assay (IFA) and viral neutralizing test (VNT). The epitopes as well as the critical amino acids recognized by the neutralized antibody were identified using Western blot and neutralizing antibody escape mutants screening. Five Asia 1 FMDV-specific antibodies were obtained, of which PD3 and PD7 showed neutralizing ability. These two neutralizing antibodies recognized the same epitope and the key amino acids residue was D 72 of the VP2 protein. In this study, neutralizing antibody against Asia 1 FMDV from pig were obtained for the first time, which provides further information of the protective antigen sites of Asia1 FMDV, and make a basis for the design of molecular vaccine and the development of new tools for diagnosis of FMD Asia 1.

Key words: foot-and-mouth disease virus, serotype Asia 1, porcine monoclonal antibody, antigenic epitope

中图分类号:

S852.659.6

董开恒, 黄书伦, 李凤娟, 李坤, 刘果, 张强, 包慧芳, 李洪炫, 卢曾军, 张小丽. 口蹄疫病毒Asia1型猪源中和抗体的筛选与抗原表位鉴定[J]. 畜牧兽医学报, 2024, 55(11): 5211-5221.

Kaiheng DONG, Shulun HUANG, Fengjuan LI, Kun LI, Guo LIU, Qiang ZHANG, Huifang BAO, Hongxuan LI, Zengjun LU, Xiaoli ZHANG. Screening and Identification of the Antigenic Epitope of Pig Neutralizing Antibodies against Foot-and-mouth Disease Virus Serotype Asia 1[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5211-5221.

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克隆 Clone	中和效力/毒株IC₅₀/Strains
克隆 Clone	Asia1/JS/05	A/WH09	O/Tibet99
PD1	＞50	＞50	＞50
PD2	＞50	＞50	＞50
PD3	5.82	＞50	＞50
PD4	＞50	＞50	＞50
PD5	＞50	＞50	＞50
PD6	＞50	＞50	＞50
PD7	4.50	＞50	＞50
PD8	＞50	＞50	＞50

克隆 Clone	亲本毒株 Parent virus	氨基酸残基 Residue change	频率 Frequency	抗体浓度/ (μg·mL^-1) Antibody concentration
PD3	Asia1/JS/05	VP2 D72E	4/7	200
PD3	Asia1/JS/05	VP2 D72G	3/7	200
PD7	Asia1/JS/05	VP2 D72G	7/7	200

克隆 Clone	中和效力/毒株IC₅₀/Strains
克隆 Clone	Asia1/JS/05	Asia1/JS/05^△VP2D72G
PD3	5.82	＞50
PD7	4.50	＞50

克隆 Clone	BCR类型 BCR Type	V基因 V gene	D基因 D gene	J基因 J gene	互补决定区1 CDR1	互补决定区2 CDR2	互补决定区3 CDR3
PD1	IGH	1-4*02	1*01	5*01	GFTFSSHS	VDTSGRGT	ARHWGRLDV
PD2		1-401 or 1-402	1*01	5*01	GFTFSTAY	IETGNSGT	AKGTMGYYSMDL
PD5		1-402 or 1S201	3*01	5*01	GFTFSSVP	ITISGTGD	RGRSSGLDSSMNL
PD3		1-10*01	2*01	5*01	GFTFSGTH	IDTSGSGT	ATWYRGGWGAMDL
PD7		1-10*01	2*01	5*01	GFTFSTNY	IRTTDQSS	ATRYRGGWGALDL
PD1	IGL	8-16*01	-	2*01	SGSVTTSNY	SGN	ALYKGSGTYTYL
PD2		8-16*01	-	2*01	SGSVTSTNY	KTN	SLYKGSGIAIFI
PD5	IGK	1-11*02	-	201 or 202	QSIDSF	YVS	QHQWEVPYG
PD3		1-1101 or 1D-1101	-	1*01	QSIRSY	GTS	LQRNAVPWT
PD7		1-1101 or 1-1102 or 1-1401 or 1D-1101	-	1*01	QSIRSY	KAS	LQRQVLPWT

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口蹄疫病毒Asia1型猪源中和抗体的筛选与抗原表位鉴定

Screening and Identification of the Antigenic Epitope of Pig Neutralizing Antibodies against Foot-and-mouth Disease Virus Serotype Asia 1

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