1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定

doi:10.11843/j.issn.0366-6964.2022.07.020

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (7): 2239-2251.doi: 10.11843/j.issn.0366-6964.2022.07.020

1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定

陈孝军^1,2, 马俊^1,2, 陈熊男², 梁祎凡², 高琦^1,2, 黄钊^1,2, 许润达^1,2, 郑佳琛^1,2, 郑泽中^1,2, 张桂红^1,2,4, 王衡^1,2,3*, 龚浪^1,2*

1. 华南农业大学非洲猪瘟防控技术研究中心, 广州 510642;
2. 国家非洲猪瘟区域实验室(广州), 广州 510642;
3. 华南农业大学兽医学院广东省临床重大疾病综合防控重点实验室, 广州 510642;
4. 华南农业大学国家生猪种业工程技术研究中心, 广州 510642

收稿日期:2021-10-25 出版日期:2022-07-23 发布日期:2022-07-23
通讯作者: 龚浪,主要从事动物传染病研究,E-mail:gonglang@scau.edu.cn;王衡,主要从事兽医病理学研究,E-mail:wangheng2009@scau.edu.cn
作者简介:陈孝军(1996-),男,硕士生,主要从事动物传染病研究,E-mail:xiaojunchen@stu.scau.edu.cn;马俊(1996-),女,硕士生,主要从事兽医病理学研究,E-mail:junma@stu.scau.edu.cn
基金资助:
广东省重点领域研发计划资助(2019B020211003)；茂名实验室科研启动项目(2021TDQD002)国家自然科学基金专项项目(31941004)；财政部和农业农村部：国家现代农业产业技术体系

Identification of the B Cell Epitopes Recognized by a Monoclonal Antibody against the p30 Protein of African Swine Fever Virus

CHEN Xiaojun^1,2, MA Jun^1,2, CHEN Xiongnan², LIANG Yifan², GAO Qi^1,2, HUANG Zhao^1,2, XU Runda^1,2, ZHENG Jiachen^1,2, ZHENG Zezhong^1,2, ZHANG Guihong^1,2,4, WANG Heng^1,2,3*, GONG Lang^1,2*

1. Research Center for African Swine Fever Prevention and Control, South China Agricultural University, Guangzhou 510642, China;
2. African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou 510642, China;
3. Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
4. National Engineering Research Center for Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China

Received:2021-10-25 Online:2022-07-23 Published:2022-07-23

摘要/Abstract

摘要： 旨在研究非洲猪瘟病毒(ASFV) p30蛋白的B细胞抗原表位，本研究制备了p30蛋白的单克隆抗体(mAb)，并以该单克隆抗体为工具进行B细胞抗原表位定位。首先，通过原核表达及Ni柱亲和纯化获得p30蛋白，将纯化蛋白免疫BALB/c小鼠进行杂交瘤细胞制备，通过间接酶联免疫吸附试验(iELISA)筛选出阳性杂交瘤细胞，并以细胞表达的方法制备单克隆抗体；采用间接免疫荧光试验(IFA)和蛋白质免疫印迹(Western blot)对单克隆抗体的特异性进行鉴定。利用IEDB表位预测软件对p30蛋白B细胞抗原表位进行预测，根据预测结果对CP204L基因进行截短表达，利用IFA、Western blot和iELISA对其抗原表位进行鉴定。最后，利用噬菌体十二肽库对制备的p30单克隆抗体进行4轮生物淘选，筛选多肽表位，并与上述基因截短表达筛选方法进行比对。特异性鉴定结果显示，该单克隆抗体能成功识别感染猪肺泡巨噬细胞的ASFV；基因截短表达筛选结果表明,其识别的抗原表位区域为⁸⁴M～K¹⁴²；噬菌体淘选试验结果表明,¹¹⁶TSSFETLFE¹²⁴为本试验制备的单克隆抗体所识别的p30蛋白抗原表位核心序列，该结果进一步缩小了表位分布范围。本研究制备了p30蛋白的1株单克隆抗体，并对其抗原表位进行鉴定，为血清学诊断试剂的研发和p30蛋白功能研究奠定基础。

关键词: 非洲猪瘟病毒, p30蛋白, 单克隆抗体, 生物淘选, 抗原表位

Abstract: To explore the B cell epitopes of African swine fever virus (ASFV) p30 protein, the monoclonal antibody (mAb) of p30 protein was prepared, and it was used as a tool for screening its B cell epitopes. First of all, p30 protein was obtained by prokaryotic expression system and purified by Ni affinity method. BALB/c mice were immunized with purified protein and hybridoma cells were prepared. Positive hybridoma cells were screened by indirect enzyme linked immunosorbent assay (iELISA). The mAb was then prepared by cell expression method. The specificity of mAb was identified by indirect immunofluorescence assay (IFA) and Western blot. Then, IEDB epitope prediction software was used to predict the B cell epitopes of p30 protein. According to the prediction results, CP204L gene was truncated to express in prokaryotic cells. IFA, Western blot and iELISA were used to identify the epitopes. Finally, a phage-displayed 12-mer peptide library was used to conduct four rounds of biological panning for the p30 monoclonal antibody, and identify the specific epitope of p30, which was further compared with the truncated protein mapping described above. The results of specific identification showed that the mAb could successfully identify ASFV in porcine alveolar macrophages. The results of truncated protein mapping showed that the epitopes region recognized by the mAb of p30 protein was ⁸⁴M-K¹⁴². The biopanning experiment indicated that ¹¹⁶TSSFETLFE¹²⁴ was a core domain of the B cell linear epitope of p30 protein, which further narrowed the range of the epitope distribution. Altogether, a mAb against ASFV p30 protein was prepared, and the epitopes were identified, which provided a reference for the development of serological diagnostic reagents and lay a foundation for the study of p30 protein function

Key words: African swine fever virus, p30 protein, monoclonal antibody, biopanning, epitope

中图分类号:

陈孝军, 马俊, 陈熊男, 梁祎凡, 高琦, 黄钊, 许润达, 郑佳琛, 郑泽中, 张桂红, 王衡, 龚浪. 1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定[J]. 畜牧兽医学报, 2022, 53(7): 2239-2251.

CHEN Xiaojun, MA Jun, CHEN Xiongnan, LIANG Yifan, GAO Qi, HUANG Zhao, XU Runda, ZHENG Jiachen, ZHENG Zezhong, ZHANG Guihong, WANG Heng, GONG Lang. Identification of the B Cell Epitopes Recognized by a Monoclonal Antibody against the p30 Protein of African Swine Fever Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2239-2251.

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1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定

Identification of the B Cell Epitopes Recognized by a Monoclonal Antibody against the p30 Protein of African Swine Fever Virus

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