非洲猪瘟病毒p30蛋白单克隆抗体制备及其抗原表位鉴定

doi:10.11843/j.issn.0366-6964.2023.08.026

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (8): 3415-3423.doi: 10.11843/j.issn.0366-6964.2023.08.026

非洲猪瘟病毒p30蛋白单克隆抗体制备及其抗原表位鉴定

刘桃雪^1,2,3, 苏冰倩^1,2,3, 齐艳丽^1,2,3, 郭江涛^1,2,3, 刘忠虎^1,2,3, 褚贝贝^1,2,3, 王江^1,2,3*, 曾磊^1,2,3*

1. 河南农业大学动物医学院, 郑州 450046;
2. 河南农业大学, 农业农村部动物生化与营养重点实验室, 郑州 450046;
3. 河南农业大学, 河南省动物生长发育调控重点实验室, 郑州 450046

收稿日期:2022-10-21 出版日期:2023-08-23 发布日期:2023-08-22
通讯作者: 王江,主要从事动物生物化学与分子生物学研究,E-mail:wangjiang@henau.edu.cn;曾磊,主要从事动物生物化学与分子生物学研究,E-mail:zenglei2021918@163.com
作者简介:刘桃雪(1995-),女,河南商丘人,硕士生,主要从事动物生物化学与分子生物学研究,E-mail:1002691439@qq.com;苏冰倩(1994-),女,河南安阳人,博士生,主要从事基础兽医学研究,E-mail:1126575812@qq.com。
基金资助:
河南农业大学青年英才项目；河南省教学改革项目（2021SJGLX351）

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p30 Protein and Identification of the Antigenic Epitope

LIU Taoxue^1,2,3, SU Bingqian^1,2,3, QI Yanli^1,2,3, GUO Jiangtao^1,2,3, LIU Zhonghu^1,2,3, CHU Beibei^1,2,3, WANG Jiang^1,2,3*, ZENG Lei^1,2,3*

1. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China;
2. Key Laboratory of Animal Biochemistry and Nutrition, Henan Agricultural University, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou 450046, China;
3. Key Laboratory of Animal Growth and Development of Henan, Henan Agricultural University, Zhengzhou 450046, China

Received:2022-10-21 Online:2023-08-23 Published:2023-08-22

摘要/Abstract

摘要： 为制备非洲猪瘟病毒（ASFV） p30蛋白的单克隆抗体，以原核表达的重组p30蛋白为免疫原免疫6~8周龄雌性BALB/c小鼠，3次免疫后分离小鼠脾细胞并将其与SP2/0细胞进行融合，通过间接ELISA方法筛选。结果获得2株能稳定分泌抗ASFV p30蛋白单克隆抗体的杂交瘤细胞株，分别命名为2G10-2和6D2-1。抗体效价分别为1：12 800和1：3 200。亚型鉴定结果显示，2株单克隆抗体的重链均属于IgG，轻链均为κ型。Western blot和间接免疫荧光试验结果显示，2株单克隆抗体与p30蛋白均具有良好的结合活性。通过Western blot检测，2株单克隆抗体能有效结合截短重组p30蛋白的170~194位氨基酸，证明其抗原识别区域为第170~194位氨基酸。本研究制备了p30蛋白的2株单克隆抗体，并对其抗原表位进行鉴定，为ASFV p30蛋白结构和功能的研究及血清学诊断试剂的研发奠定基础。

关键词: 非洲猪瘟病毒, p30蛋白, 原核表达, 单克隆抗体, 抗原表位

Abstract: This study is aimed to prepare monoclonal antibodies against African swine fever virus (ASFV) p30 protein. Five 6-8 weeks female BALB/c mice were immunized with prokaryotic recombinant p30 protein as immunogen. Spleen cells were isolated and fused with SP2/0 cells after 3 times of immunization. Two hybridoma cell lines, named 2G10-2 and 6D2-1, which could stably secrete monoclonal antibodies against ASFV p30 protein were obtained. The antibody titers were 1:12 800 and 1:3 200, respectively. The results of subtype identification showed that the heavy chain of the two monoclonal antibodies belonged to IgG, and the light chain was κ type. The results of Western blot and indirect immunofluorescence assay showed that both monoclonal antibodies had good binding activity to p30 protein. Western blot analysis showed that the two monoclonal antibodies could effectively bind the 170-194 amino acids of the recombinant p30 protein, which proved that the antigenic recognition region was the 170-194 amino acids. In this study, two monoclonal antibodies against p30 protein were prepared and their epitopes were identified, which laid a foundation for the study of the structure and function of ASFV p30 protein and the development of serological diagnostic reagents.

Key words: African swine fever virus, p30, prokaryotic expression, monoclonal antibody, epitope of antigen

中图分类号:

S852、659.1

刘桃雪, 苏冰倩, 齐艳丽, 郭江涛, 刘忠虎, 褚贝贝, 王江, 曾磊. 非洲猪瘟病毒p30蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(8): 3415-3423.

LIU Taoxue, SU Bingqian, QI Yanli, GUO Jiangtao, LIU Zhonghu, CHU Beibei, WANG Jiang, ZENG Lei. Preparation of the Monoclonal Antibody against the African Swine Fever Virus p30 Protein and Identification of the Antigenic Epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3415-3423.

参考文献 26

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非洲猪瘟病毒p30蛋白单克隆抗体制备及其抗原表位鉴定

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p30 Protein and Identification of the Antigenic Epitope

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