猪急性腹泻综合征冠状病毒核衣壳蛋白新型线性B细胞表位的鉴定

doi:10.11843/j.issn.0366-6964.2025.04.033

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (4): 1854-1864.doi: 10.11843/j.issn.0366-6964.2025.04.033

猪急性腹泻综合征冠状病毒核衣壳蛋白新型线性B细胞表位的鉴定

曹丽艳¹^,²(), 孔祥雨¹^,², 袁聪¹^,², 段月月¹^,², 马国祥¹^,², 施磊¹^,², 张宇¹^,², 万颖¹^,², 李想通¹^,², 王娅婷¹^,², 杜煜¹^,², 郑海学²^,*(), 王琦¹^,²^,*()

1. 中国农业科学院都市农业研究所, 成都 610213
2. 中国农业科学院兰州兽医研究所, 兰州 730030

收稿日期:2024-05-22 出版日期:2025-04-23 发布日期:2025-04-28
通讯作者: 郑海学,王琦 E-mail:caoliyan@caas.cn;zhenghaixue@caas.cn;qiwang@caas.cn
作者简介:曹丽艳(1983-), 女, 甘肃陇西人, 助理研究员, 博士, 主要从事动物病毒感染与免疫研究, E-mail: caoliyan@caas.cn
基金资助:
四川省科技计划项目(重点研发项目，2022YFN0008)；四川省杰青项目(21JCQN0175)；中国农业科学院青年英才项目(王琦)；中国农业科学院创新工程(ASTIP2024-34-IUA-07)

Identifcation of a Novel Linear B-cell Epitope in the Nucleocapsid Protein of Swine Acute Diarrhea Syndrome Coronavirus

CAO Liyan¹^,²(), KONG Xiangyu¹^,², YUAN Cong¹^,², DUAN Yueyue¹^,², MA Guoxiang¹^,², SHI Lei¹^,², ZHANG Yu¹^,², WAN Ying¹^,², LI Xiangtong¹^,², WANG Yating¹^,², DU Yu¹^,², ZHENG Haixue²^,*(), WANG Qi¹^,²^,*()

1. Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu 610213, China
2. Lanzhou Veterinary Research Institute of Chinese Academy of Agriculture Science, Lanzhou 730030, China

Received:2024-05-22 Online:2025-04-23 Published:2025-04-28
Contact: ZHENG Haixue, WANG Qi E-mail:caoliyan@caas.cn;zhenghaixue@caas.cn;qiwang@caas.cn

摘要/Abstract

摘要：

猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome coronavirus, SADS-CoV)是一种蝙蝠来源的新型猪肠道冠状病毒，可引起新生仔猪腹泻、呕吐、脱水而死亡。SADS-CoV核衣壳蛋白(N)是病毒复制中最保守的结构蛋白，具有良好的抗原性。前期研究获得的5株抗SADS-CoV N蛋白的单克隆抗体(1C10、4B10、6G1、6F3和6E8)，其中6E8与SADS-CoV的反应性最强。在本研究中，利用pET32a作为表达载体构建N蛋白的截短及缺失突变体质粒，经诱导表达后，用6E8进行抗原抗体反应。Western blot结果表明，6E8识别的抗原表位为69-QKGQRK-74。同源性分析结果显示，该表位在不同SADS-CoV毒株和蝙蝠冠状病毒HKU2中完全一致；在猪传染性胃肠炎病毒(TGEV)和猫感染性腹膜炎病毒(FIPV)中发现相似的表位(VKGQRK)，仅有一个氨基酸的差异；而在其他冠状病毒中同源性较低。特异性试验中，6E8不能与猪流行性腹泻冠状病毒(PEDV)和TGEV发生发应，尽管6E8识别表位与TGEV仅有一个氨基酸的差异，这说明69-Q是6E8识别表位的关键氨基酸。进一步试验发现，当6E8与SADS-CoV孵育后再感染Huh7细胞，能增强SADS-CoV的感染，出现抗体依赖性增强(antibody-dependent enhancement, ADE)现象。总之，SADS-CoV N蛋白新型B细胞表位的鉴定，将为设计SADS-CoV新型疫苗和开发表位相关的诊断试剂提供新的见解。

关键词: 猪急性腹泻综合征冠状病毒, N蛋白, 6E8单克隆抗体, 表位鉴定

Abstract:

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was a new bat-origin swine enteric coronavirus that can cause diarrhea, vomiting, dehydration and death in newborn piglets. Nucleocapsid (N) protein is the most conserved structure protein, and has a good antigenicity. In the previous study, five monoclonal antibodies (mAbs, 1C10, 4B10, 6G1, 6F3 and 6E8, respectively) against the SADS-CoV N protein were obtained, among which 6E8 showed the strongest reactivity with SADS-CoV. In this study, truncated and deletion mutant plasmids of N protein were constructed using pET32a expression vector. The 6E8 epitope was located to amino acids 69-QKGQRK-74 by testing the 6E8 for reactivity with series of N truncation and deletion using Western blot. The 6E8 epitope was highly conserved among different SADS-CoV isolated strains and bat coronavirus HKU2. A similar epitope (VKGQRK) was found in porcine transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV), with one amino acid difference; however, it showed low homology with other coronaviruses. Specific tests showed that the 6E8 could not be reacted with porcine epidemic diarrhea virus (PEDV) and TGEV, despite only one amino acid different in the epitope recognized by 6E8 and TGEV, indicating that 69-Q was the key amino acid for 6E8 epitope. Further experiments showed that the 6E8 had a capacity to enhance the infection of SADS-CoV in Huh7 cells when the 6E8 incubated with SADS-CoV before infection, which was called antibody-dependent enhancement (ADE). In summary, the identification of the novel B-cell epitope in the SADS-CoV N protein provides new insights for designing novel SADS-CoV vaccines and developing epitope-related diagnostic reagents.

Key words: swine acute diarrhea syndrome coronavirus (SADS-CoV), nucleocapsid protein, 6E8 monoclonal antibody (mAb), epitope identification

中图分类号:

S852.659.6

曹丽艳, 孔祥雨, 袁聪, 段月月, 马国祥, 施磊, 张宇, 万颖, 李想通, 王娅婷, 杜煜, 郑海学, 王琦. 猪急性腹泻综合征冠状病毒核衣壳蛋白新型线性B细胞表位的鉴定[J]. 畜牧兽医学报, 2025, 56(4): 1854-1864.

CAO Liyan, KONG Xiangyu, YUAN Cong, DUAN Yueyue, MA Guoxiang, SHI Lei, ZHANG Yu, WAN Ying, LI Xiangtong, WANG Yating, DU Yu, ZHENG Haixue, WANG Qi. Identifcation of a Novel Linear B-cell Epitope in the Nucleocapsid Protein of Swine Acute Diarrhea Syndrome Coronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1854-1864.

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参考文献 13

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基因Genes	引物序列(5′→3′) Sequence
N (1-376 aa)^a	Forward: CGCGGATCCATGGCCACTGTTAATTGG
R1 (1-142 aa)^b	Reverse: CCGCTCGAGCTAAACAACAGTCAGGTCT
B1 (1-95 aa)	Reverse: CCGCTCGAGCTAGAAAGGAGCATCTGCGTGAG
B2 (43-142 aa)	Forward: CGCGGATCCAGAAATGGAGTTCCGACAGG
C1 (64-142 aa)	Forward: CGCGGATCCAAACGCTGGCGAATGCAAAAAGG
C2 (84-142 aa)	Forward: CGCGGATCCTACCTTGGTACTGGTCCTCA
D1 (69-142 aa)	Forward: CGCGGATCCCAAAAAGGCCAACGTAAAG
D2 (75-142 aa)	Forward: CGCGGATCCGATCAGCCTTCTAACTGGC
D3 (80-142 aa)^c	Forward: CGCGGATCCTGGCACTTTTATTACCTTGG
E (1-68 aa)^a	Reverse: CATTCGCCAGCGTTTTTGTTC
E (75-142 aa)^b	Forward: GATCAGCCTTCTAACTGGC
F (1-74 aa)^a	Reverse: TCTTTACGTTGGCCTTTTTGC
F (84-142 aa)^b	Forward: TACCTTGGTACTGGTCCTCA

毒株名称 Virus names	GenBank登录号 Accession numbers
SADS-CoV	MK651076、MG605087、MG605090、MG605091、MT747188、MG605088、MG557844、MG605089、MT199599、MT199597、MK994937、MT199598、MT199600、MT199601、MH615810和OK078018
Bat coronavirus HKU2	EF203067
TGEV	AJ271965
PEDV	AF353511
PDCoV	KX022605
SARS-CoV	ON032815
IBV	AY319651
Bovine coronavirus	NC_003045
Canine repiratory coronavirus	KX432213
Equnine coronavirus	EF446615
Human coronavirus 229E	NC_002645
MERS-CoV	KF192507
PHEV	KY994645
Rat coronavirus	JF792617
Turkey coronavirus	EU022526
Camel alphacoronavirus	NC_028752
FIPV	A22378

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猪急性腹泻综合征冠状病毒核衣壳蛋白新型线性B细胞表位的鉴定

Identifcation of a Novel Linear B-cell Epitope in the Nucleocapsid Protein of Swine Acute Diarrhea Syndrome Coronavirus

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