Preparation and Identification of Neutralizing Monoclonal Antibodies against Classical Swine Fever Virus

doi:10.15933/j.cnki.1004-3268.2019.12.017

Journal of Henan Agricultural Sciences ›› 2019, Vol. 48 ›› Issue (12): 114-120.DOI: 10.15933/j.cnki.1004-3268.2019.12.017

• Animal Husbandry and Veterinary Medicine • Previous Articles Next Articles

Preparation and Identification of Neutralizing Monoclonal Antibodies against Classical Swine Fever Virus

LIU Yunchao¹,CHEN Yumei²,FENG Lili³,WANG Lei¹,WANG Jucai¹,LU Dongfeng⁴,ZHANG Gaiping¹

(1.Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences/Key Laboratory of Animal Immunology,Ministry of Agriculture,Zhengzhou 450002,China; 2. School of Life Sciences,Zhengzhou University,Zhengzhou 450001,China; 3.Institute of Agricultural Economics and Information,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China; 4.Henan Huahuaniu Industrial Cooparation,Zhengzhou 450006,China)

Received:2019-08-10 Published:2019-12-15 Online:2019-12-15

抗猪瘟病毒中和性单克隆抗体的制备与鉴定

刘运超¹，陈玉梅²，冯丽丽³，汪磊¹，王聚财¹，陆东峰⁴，张改平¹

（1.河南省农业科学院动物免疫学重点实验室/农业部动物免疫学重点开放实验室，河南郑州 450002；2.郑州大学生命科学学院，河南郑州 450001； 3.河南省农业科学院农业经济与信息研究所，河南郑州 450002； 4.河南花花牛实业总公司，河南郑州 450006）

通讯作者: 张改平（1960-），男，河南内黄人，研究员，中国工程院院士，博士，主要从事动物免疫学研究。E-mail：zhanggaiping2003@[KG-*5]163.com
作者简介:刘运超（1982-），男，河南周口人，助理研究员，博士，主要从事动物免疫学研究。E-mail：yunchaoliu2012@163.com
基金资助:
国家重点研发计划项目（2016YFD0500704；2017YFD0501103）

Abstract

Abstract: In order to obtain neutralizing monoclonal antibodies against classical swine fever virus(CSFV),BABL/c mice were immunized with purified CSFV and CSFV E2 protein from baculovirus expression system, respectively.Monoclonal antibodies against CSFV were prepared by hybridoma cell fusion.Immunoperoxidase monolayer assay(IPMA) was established for screening and identification of monoclonal antibodies.After subcloning and screening,four monoclonal hybridoma cell lines with stable secretion of anti-CSFV monoclonal antibody, including 5D1, 8H7, 9A1 and 13B2, were successfully obtained.Four monoclonal antibodies light chain were identified to belong to kappa type,of which 5D1,8H7,13B2 were IgG1,and 9A1 was IgG2c.The ascites titers of four monoclonal antibodies detected by indirect ELISA ranged from 2.56×105 to 1.02×10⁶.The titers of IPMA were 6.4×10⁴,1.28×10⁵,2.56×10⁵ and 1.28×10⁵,respectively.The results of SDS-PAGE and Western blot showed that all four monoclonal antibodies could react specifically with CSFV E2 protein which was obtained from prokaryotic expression system and baculovirus expression system. IPMA detection showed that all four monoclonal antibodies could react specifically with CSFV.There was no cross-reaction with BVDV,PCV2 and PRRSV.Viral neutralization test confirmed that 13B2 had neutralization activity,and the neutralization titer was 1.28× 10⁴.In conclusion, four hybridoma cell lines secreting specific anti-CSFV antibodies were successfully screened in this study, among which monoclonal antibody 13B2 had the activity of neutralizing CSFV infection.

Key words: Classical swine fever virus, E2 protein, Neutralizing monoclonal antibodies, Antibody neutralization titer, Immunoperoxidase monolayer assay

摘要： 为了获得具有中和活性的抗猪瘟病毒（CSFV）单克隆抗体，分别将CSFV和杆状病毒表达系统表达纯化的CSFV E2 蛋白与佐剂混合后免疫BABL/c小鼠，经杂交瘤细胞融合制备抗CSFV单克隆抗体，经过多轮亚克隆和免疫过氧化物酶单层试验（IPMA）筛选，成功获得5D1、8H7、9A1和13B2共4株能够稳定分泌抗 CSFV单克隆抗体的杂交瘤细胞株。4株单克隆抗体轻链均属于κ型，其中5D1、8H7、13B2为IgG1亚型，9A1为IgG2c亚型。间接ELISA检测结果显示，4株单克隆抗体的腹水效价在2.56×10⁵~1.02×10⁶；IPMA效价分别为6.40×10⁴、1.28×10⁵、2.56×10⁵、1.28×10⁵；SDS-PAGE和Western blot检测结果显示，4株单克隆抗体均能与经原核表达系统及杆状病毒表达系统获得的CSFV E2蛋白发生特异性反应；IPMA检测结果显示，4株单克隆抗体均能与CSFV发生特异性反应，而与牛流行性腹泻病毒（BVDV）、猪繁殖与呼吸综合征病毒（PRRSV）和猪圆环病毒（PCV2）均无交叉反应。病毒中和试验证实，单克隆抗体13B2具有中和活性，其中和效价为1.28×10⁴。综上，成功筛选出4株能够分泌抗CSFV特异性抗体的杂交瘤细胞株，其中单克隆抗体13B2具有中和CSFV感染活性。

关键词: 猪瘟病毒, E2蛋白, 中和性单克隆抗体, 抗体中和效价, 免疫过氧化物酶单层细胞试验

CLC Number:

S855.3

LIU Yunchao, CHEN Yumei, FENG Lili, WANG Lei, WANG Jucai, LU Dongfeng, ZHANG Gaiping. Preparation and Identification of Neutralizing Monoclonal Antibodies against Classical Swine Fever Virus[J]. Journal of Henan Agricultural Sciences, 2019, 48(12): 114-120.

刘运超, 陈玉梅, 冯丽丽, 汪磊, 王聚财, 陆东峰, 张改平. 抗猪瘟病毒中和性单克隆抗体的制备与鉴定[J]. 河南农业科学, 2019, 48(12): 114-120.

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Preparation and Identification of Neutralizing Monoclonal Antibodies against Classical Swine Fever Virus

抗猪瘟病毒中和性单克隆抗体的制备与鉴定

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