非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用

doi:10.11843/j.issn.0366-6964.2020.05.018

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (5): 1074-1082.doi: 10.11843/j.issn.0366-6964.2020.05.018

非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用

白晨雨¹, 王同燕², 赵少若¹, 王衡³, 郝丽影¹, 李雪锋², 白露露², 邓跃², 王孟月², 邓均华¹, 李向东², 张桂红^3*, 田克恭^1,2*

1. 洛阳普泰生物技术有限公司, 洛阳 471003;
2. 国家兽用药品工程技术研究中心, 洛阳 471003;
3. 国家非洲猪瘟区域实验室(广州), 广州 510642

收稿日期:2019-11-06 出版日期:2020-05-25 发布日期:2020-05-16
通讯作者: 张桂红,主要从事兽医传染病学研究,E-mail:guihongzh@scau.edu.cn;田克恭,主要从事动物疫病诊断与防控技术研究,E-mail:Tiankg@263.net
作者简介:白晨雨(1990-),女,河南驻马店人,主要从事兽用诊断试剂研究,E-mail:plkchenyu@163.com;王同燕(1981-),女,河南南阳人,主要从事基因工程疫苗研究,E-mail:tyanwang@163.com
基金资助:
洛阳市重大科技专项；万人计划青年拔尖人才；广东省非洲猪瘟科技应急防控专题（2019B020211003）

Preparation of Monoclonal Antibodies against Recombinant p62 Protein of African Swine Fever Virus and Its Preliminary Application

BAI Chenyu¹, WANG Tongyan², ZHAO Shaoruo¹, WANG Heng³, HAO Liying¹, LI Xuefeng², BAI Lulu², DENG Yue², WANG Mengyue², DENG Junhua¹, LI Xiangdong², ZHANG Guihong^3*, TIAN Kegong^1,2*

1. Luoyang Putai Biotechnology Co., Ltd., Luoyang 471003, China;
2. National Research Center of Veterinary Medicine, Luoyang 471003, China;
3. African Swine Fever Regional Laboratory of China(Guangzhou), Guangzhou 510642, China

Received:2019-11-06 Online:2020-05-25 Published:2020-05-16

摘要/Abstract

摘要： 为制备非洲猪瘟病毒（ASFV）p62蛋白的特异性单克隆抗体，并初步应用于感染组织样品中ASFV抗原的免疫组化（IHC）检测，本研究以杆状病毒表达的非洲猪瘟病毒重组p62蛋白免疫BALB/c小鼠，取其脾细胞与骨髓瘤细胞进行融合获得杂交瘤细胞。结果显示：基于纯化的p62蛋白建立的间接ELISA方法对杂交瘤细胞进行筛选和亚克隆，获得了18株可稳定分泌抗非洲猪瘟病毒p62蛋白单克隆抗体的杂交瘤细胞株。经IFA检测，制备的单克隆抗体均与非洲猪瘟病毒反应，且不与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型等猪源常见病毒反应，特异性良好。抗体识别蛋白的鉴定结果显示，3株MAbs识别p35蛋白，15株MAbs识别p15蛋白。14株MAbs重链亚类为IgG1型，4株MAbs重链亚类为IgG2a，轻链均为κ链。利用18株MAbs对ASFV感染猪的肺、扁桃体、淋巴结等组织进行IHC检测，结果显示5株MAbs均能够与感染ASFV的组织发生特异性的免疫反应。本研究获得的非洲猪瘟病毒p62蛋白单克隆抗体可为非洲猪瘟病毒免疫学检测方法的建立及p62蛋白的结构功能等基础研究提供重要的生物材料。

关键词: 非洲猪瘟病毒, p62蛋白, 单克隆抗体, 免疫组化

Abstract: The objective of this study was to prepare monoclonal antibodies against p62 protein of African swine fever virus (ASFV), and apply it to immunohistochemistry (IHC) detection of ASFV antigen in infected tissue samples. BALB/c mice were immunized with purified recombinant p62 protein expressed by baculovirus system and hybridoma cells were obtained by fusion of spleen cells and myeloma cells. An indirect ELISA based on the purified p62 protein was developed. Eighteen hybridoma cell lines stably secreting MAbs against the p62 protein were harvested after screening and subcloning. The results of indirect immunofluorescence assay (IFA) showed that all of the monoclonal antibodies could react with ASFV and did not react with swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus and porcine circovirus 2 with good specificity. Three MAbs could recognize ASFV p35 protein, and 15 MAbs recognize p15 protein. Among of them, 14 MAbs heavy chains belong to IgG1 and 4 MAbs heavy chains belong to IgG2a. All light chains were κ. The results of immunohistochemistry showed that 5 MAbs had positive reactivity with the ASFV in lung, tonsil, lymph node of pigs infected with ASFV. The monoclonal antibodies against p62 protein obtained from the study laid a foundation for the establishment of immunological detection method of African swine fever virus and could provide important biomaterials for research on the structure and function of p62 protein.

Key words: African swine fever, p62 protein, monoclonal antibody, immunohistochemistry

中图分类号:

S852.659.1

白晨雨, 王同燕, 赵少若, 王衡, 郝丽影, 李雪锋, 白露露, 邓跃, 王孟月, 邓均华, 李向东, 张桂红, 田克恭. 非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用[J]. 畜牧兽医学报, 2020, 51(5): 1074-1082.

BAI Chenyu, WANG Tongyan, ZHAO Shaoruo, WANG Heng, HAO Liying, LI Xuefeng, BAI Lulu, DENG Yue, WANG Mengyue, DENG Junhua, LI Xiangdong, ZHANG Guihong, TIAN Kegong. Preparation of Monoclonal Antibodies against Recombinant p62 Protein of African Swine Fever Virus and Its Preliminary Application[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1074-1082.

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非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用

Preparation of Monoclonal Antibodies against Recombinant p62 Protein of African Swine Fever Virus and Its Preliminary Application

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