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

Abstract

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

CLC Number:

S852.659.6

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

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