非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定

doi:10.11843/j.issn.0366-6964.2023.01.026

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (1): 281-292.doi: 10.11843/j.issn.0366-6964.2023.01.026

非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定

齐艳丽^1,2,3, 刘桃雪^1,2,3, 于海深^1,2,3, 张超^1,2,3, 鲁维飞^1,2,3, 王江^1,2,3, 褚贝贝^1,2,3*, 张改平^1,2,3*

1. 河南农业大学农业部动物生化与营养重点开放实验室, 郑州 450046;
2. 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;
3. 河南农业大学动物免疫学国际联合研究中心, 郑州 450046

收稿日期:2022-06-15 出版日期:2023-01-23 发布日期:2023-01-17
通讯作者: 褚贝贝,主要从事动物生物化学研究,E-mail:chubeibei@henau.edu.cn;张改平,主要从事动物免疫学研究,E-mail:zhanggaip@126.com
作者简介:齐艳丽(1998-),女,河南南阳人,硕士生,主要从事动物生物化学研究,E-mail:1570304118@qq.com
基金资助:
国家自然科学基金(31941001)；河南省教学改革研究项目(2021SJGLX351)

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope

QI Yanli^1,2,3, LIU Taoxue^1,2,3, YU Haishen^1,2,3, ZHANG Chao^1,2,3, LU Weifei^1,2,3, WANG Jiang^1,2,3, CHU Beibei^1,2,3*, ZHANG Gaiping^1,2,3*

1. Key Laboratory of Animal Biochemistry and Nutrition of Ministry of Agriculture and Rural Affairs, Henan Agricultural University, Zhengzhou 450046, China;
2. Key Laboratory of Animal Growth and Development, The Education Department of Henan Province, Henan Agricultural University, Zhengzhou 450046, China;
3. International Joint Research Center of National Animal Immunology, Henan Agricultural University, Zhengzhou 450046, China

Received:2022-06-15 Online:2023-01-23 Published:2023-01-17

摘要/Abstract

摘要： 旨在制备非洲猪瘟病毒(ASFV)p54蛋白的特异性单克隆抗体。本研究利用大肠杆菌表达系统表达p54蛋白，免疫BALB/c小鼠，取其脾细胞与SP2/0细胞进行细胞融合。利用纯化的p54蛋白作为包被抗原，采用间接ELISA方法筛选获得阳性杂交瘤细胞。经4次亚克隆后，取杂交瘤细胞上清进行单克隆抗体亚型鉴定，利用体内诱生法制备单克隆抗体并进行纯化。间接ELISA方法检测单克隆抗体的效价，利用交叉反应性试验、间接免疫荧光试验和蛋白印迹对所获单克隆抗体的特异性进行鉴定。根据预测的p54蛋白二级结构，采用逐步截短法分析鉴定单克隆抗体识别的抗原表位区域，并在p54的三级结构中进行标注。结果显示：成功筛选了6株分泌p54单克隆抗体的杂交瘤细胞，分别命名为28G12-1、31G7-1、31G7-2、35F10-1、35F10-2、38D3-1。其中28G12-1、31G7-1、31G7-2重链为IgG2a型，35F10-1、35F10-2、38D3-1重链为IgG1型；轻链均为κ链。单克隆抗体的最低效价为1∶25600，与猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪流行性腹泻病毒、猪细小病毒、猪急性腹泻综合征冠状病毒、猪圆环病毒2型不发生交叉反应，特异性强，所获抗体均识别p54蛋白C端127—146aa肽段。本研究成功获得了ASFV p54蛋白和p54单克隆抗体，并鉴定了6株单克隆抗体的抗原表位，为p54蛋白功能研究和ASFV新型表位疫苗研究奠定了基础。

关键词: 非洲猪瘟病毒, p54蛋白, 原核表达, 单克隆抗体, 间接ELISA, 抗原表位

Abstract: The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times’ subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all κ chains. The lowest titer of mAb was 1∶25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine.

Key words: African swine fever virus, p54 protein, prokaryotic expression, monoclonal antibodies, indirect ELISA, epitope identification

中图分类号:

S852.659.1

齐艳丽, 刘桃雪, 于海深, 张超, 鲁维飞, 王江, 褚贝贝, 张改平. 非洲猪瘟病毒p54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(1): 281-292.

QI Yanli, LIU Taoxue, YU Haishen, ZHANG Chao, LU Weifei, WANG Jiang, CHU Beibei, ZHANG Gaiping. Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 281-292.

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非洲猪瘟病毒p54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 p54 Protein and Identification of the Antigenic Epitope

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