猪急性腹泻综合征冠状病毒核衣壳蛋白单克隆抗体的制备及其抗体序列的分析

doi:10.11843/j.issn.0366-6964.2023.06.027

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (6): 2487-2497.doi: 10.11843/j.issn.0366-6964.2023.06.027

猪急性腹泻综合征冠状病毒核衣壳蛋白单克隆抗体的制备及其抗体序列的分析

曹丽艳^1,2, 孔祥雨^1,2, 李想通^1,2, 索学鹏^1,2, 段月月^1,2, 袁聪^1,2, 施磊^1,2, 张宇^1,2, 马国祥^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 730046

收稿日期:2022-10-08 出版日期:2023-06-23 发布日期:2023-06-16
通讯作者: 郑海学,主要从事动物传染病与流行病学研究,E-mail:zhenghaixue@caas.cn;王琦,从事动物病毒感染与免疫研究,E-mail: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;孔祥雨(1995-),女,河南荥阳人,科研助理,主要从事动物病毒感染与免疫研究,E-mail:kongxiangyu0523@126.com
基金资助:
四川省重大专项(重点研发,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(ASTIP2022-34-IUA-07;ASTIP2023-34-IUA-07和CAAS-ASTIP-2022-LVRI)

Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus

CAO Liyan^1,2, KONG Xiangyu^1,2, LI Xiangtong^1,2, SUO Xuepeng^1,2, DUAN Yueyue^1,2, YUAN Cong^1,2, SHI Lei^1,2, ZHANG Yu^1,2, MA Guoxiang^1,2, ZHENG Haixue^2*, WANG Qi^1,2*

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 730046, China

Received:2022-10-08 Online:2023-06-23 Published:2023-06-16

摘要/Abstract

摘要： 猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome coronavirus, SADS-CoV)目前尚未有商品化诊断试剂盒,为填补行业空白,本研究制备了抗SADS-CoV核衣壳(N)蛋白单克隆抗体并对其序列进行了分析。利用原核表达系统表达SADS-CoV N蛋白并进行纯化,纯化的N蛋白作为免疫源免疫BALB/c小鼠,通过细胞融合、筛选及亚克隆,获得5株能够稳定分泌抗SADS-CoV N蛋白的杂交瘤细胞株,将其命名为1C10、4B10、6G1、6F3和6E8;此外,利用套式PCR扩增技术获得了抗体可变区基因序列。间接免疫荧光试验(IFA)结果表明,5株单抗特异性识别Huh7细胞感染的SADS-CoV。ELISA结果表明,5株单抗与纯化的SADS-CoV N蛋白具有良好的反应性,但是,与SADS-CoV反应性分析发现,只有6E8的反应性较好,其余四株均不反应。Western blot结果表明,5株单抗均能特异性识别纯化的以及SADS-CoV感染表达的N蛋白。亚类分型鉴定结果表明,1C10、4B10、6G1和6F3重链为IgG1型,6E8为IgG2a型,它们的轻链为Kappa型。截短表达分析表明,5株单抗识别N蛋白的区域为1-142 aa。总之,本研究制备的抗SADS-CoV N蛋白单克隆抗体为SADS-CoV新型诊断方法的开发以及SADS-CoV N蛋白功能及结构的研究提供了有用的工具。

关键词: 猪急性腹泻综合征冠状病毒, N蛋白, 单克隆抗体, 抗体序列分析

Abstract: There is no commercially diagnostic kits for swine acute diarrhea syndrome coronavirus (SADS-CoV). To fill the gap, in this study, monoclonal antibodies (MAbs) against the SADS-CoV nucleocapsid (N) protein were prepared and the sequences of MAbs were analyzed. The N protein was expressed using prokaryotic expression system. The purified N protein was used as an antigen to immunize Balb/C mice. After cell fusion, screening, and subcloning, five stable secreting MAbs against N protein were obtained, and designated as 1C10, 4B10, 6G1, 6F3 and 6E8,respectively. The variable region gene sequences of MAbs were obtained by nested PCR. Indirect immunofluorescence assay (IFA) results showed that all MAbs could specifically recognize Huh7 infected with SADS-CoV. ELISA results showed that the five MAbs could react with the purified N protein, while only the MAb 6E8 could react with SADS-CoV. The results of western blot showed that the five MAbs specifically recognized with the purified N protein, as well as the native N protein in cells infected with SADS-CoV. Isotyping revealed that the MAbs 1C10, 4B10, 6G1, and 6F3 were of the IgG1 class, and 6E8 was of the IgG2a class, and which have a kappa light chain. Truncated expression analysis showed that the recognition region of N protein of the five MAbs was 1-142aa. Overall, the MAbs against SADS-CoV N protein were promising a useful tool for development of new immunodiagnostic methods and research N protein function and structure.

Key words: swine acute diarrhea syndrome coronavirus (SADS-CoV), nucleocapsid protein, monoclonal antibody, antibody sequence analysis

中图分类号:

S852.659.6

曹丽艳, 孔祥雨, 李想通, 索学鹏, 段月月, 袁聪, 施磊, 张宇, 马国祥, 郑海学, 王琦. 猪急性腹泻综合征冠状病毒核衣壳蛋白单克隆抗体的制备及其抗体序列的分析[J]. 畜牧兽医学报, 2023, 54(6): 2487-2497.

CAO Liyan, KONG Xiangyu, LI Xiangtong, SUO Xuepeng, DUAN Yueyue, YUAN Cong, SHI Lei, ZHANG Yu, MA Guoxiang, ZHENG Haixue, WANG Qi. Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2487-2497.

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猪急性腹泻综合征冠状病毒核衣壳蛋白单克隆抗体的制备及其抗体序列的分析

Preparation and Sequence Analysis of Monoclonal Antibody against the Nucleocapsid Protein of Porcine Acute Diarrhea Syndrome Coronavirus

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