Preparation and Identification of Monoclonal Antibodies against African Swine Fever Virus p17 Protein

doi:10.15933/j.cnki.1004-3268.2020.12.020

Journal of Henan Agricultural Sciences ›› 2020, Vol. 49 ›› Issue (12): 137-143.DOI: 10.15933/j.cnki.1004-3268.2020.12.020

Special Issue: 非洲猪瘟专题

• Animal Husbandry and Veterinary Medicine • Previous Articles Next Articles

Preparation and Identification of Monoclonal Antibodies against African Swine Fever Virus p17 Protein

BAI Jingjing¹,SONG Huanhuan²,BAI Chenyu¹,HAO Liying¹,YAN Shijun²,DU Mengmeng²,LI Xiangdong²,DENG Junhua¹,TIAN Kegong^1,2

(1.Luoyang Putai Bio-Tech Co.,Ltd.,Luoyang 471000,China; 2.National Research Center for Veterinary Medicine,Luoyang 471000,China)

Received:2020-05-21 Published:2020-12-15 Online:2020-12-15

非洲猪瘟病毒p17蛋白单克隆抗体的制备与鉴定

白晶晶¹，宋欢欢²，白晨雨¹，郝丽影¹，颜世君²，杜萌萌²，李向东²，邓均华¹，田克恭^1,2

（1.洛阳普泰生物技术有限公司，河南洛阳 471000；2.国家兽用药品工程技术研究中心，河南洛阳 471000）

通讯作者: 邓均华（1978-），女，四川阆中人，兽医师，硕士，主要从事动物疫病诊断与防控技术研究。E-mail:dengbetter88@163.com
作者简介:白晶晶（1987-），女，河南巩义人，助理工程师，本科，主要从事兽用诊断试剂研究。E-mail:15138755156@163.com宋欢欢为同等贡献作者
基金资助:
郑洛新国家自主创新示范区创新引领型产业集群项目（181200211700）；洛阳市重大科技专项；国家“万人计划”青年拔尖人才项目

Abstract

Abstract: In order to develop ASFV immunodiagnostic reagents,the purified recombinant ASFV p17 protein that expressed in baculovirus was utilized for immunization of BALB / c mice,and then the positive hybridoma which produced after fusion of spleen cells of the mice with high serum antibody titers to myeloma cells,was conducted by indirect ELISA. A total of 16 hybridomas that secreted specific MAbs against ASFV p17 were obtained,and the titers of ascites in all strains were between 1∶2.560×10⁶and 1∶1.024 ×10⁷.MAbs isotype assay showed that heavy chain of 6 MAbs(6H6,4D1,7E8,3D1,2F4 and 2B4) were IgG1,and the rest of 10 MAbs(7H12,10H6,10F3,6E11,4B3,5F7,7H9,6C4,2F1 and 4H7) were IgG2a,while the light chain type of all the MAbs was κ.The results of IFA showed that 8 MAbs could react with ASFV.In summary,MAbs against ASFV p17 protein was successfully prepared.

Key words: African swine fever virus(ASFV), p17 protein, Baculovirus expression system, Monoclonal antibody, Indirect immunofluorescence assay

摘要： 为研发非洲猪瘟病毒（ASFV）的免疫学诊断试剂，利用杆状病毒表达系统表达了重组ASFV p17蛋白并纯化，然后将其免疫BALB/c小鼠，之后将高血清抗体效价小鼠的脾细胞融合骨髓瘤细胞，通过间接ELISA方法筛选阳性杂交瘤细胞，筛选到16株能稳定分泌抗p17蛋白特异性单克隆抗体的杂交瘤细胞株，其腹水的效价均介于1∶2.560×10⁶～1∶1.024×10⁷。抗体亚类鉴定结果显示，6株单克隆抗体（6H6、4D1、7E8、3D1、2F4和2B4）的重链亚类均为IgG1，其余10株单克隆抗体（7H12、10H6、10F3、6E11、4B3、5F7、7H9、6C4、2F1和4H7）的重链亚类均为IgG2a，所有单克隆抗体的轻链亚类均为κ链；IFA鉴定结果表明，共有8株单克隆抗体可特异性识别ASFV。综上，成功制备了ASFV p17蛋白单克隆抗体。

关键词: 非洲猪瘟病毒, p17蛋白, 杆状病毒表达系统, 单克隆抗体, 间接免疫荧光试验

CLC Number:

S852.6

BAI Jingjing, SONG Huanhuan, BAI Chenyu, HAO Liying, YAN Shijun, DU Mengmeng, LI Xiangdong, DENG Junhua, TIAN Kegong. Preparation and Identification of Monoclonal Antibodies against African Swine Fever Virus p17 Protein[J]. Journal of Henan Agricultural Sciences, 2020, 49(12): 137-143.

白晶晶, 宋欢欢, 白晨雨, 郝丽影, 颜世君, 杜萌萌, 李向东, 邓均华, 田克恭. 非洲猪瘟病毒p17蛋白单克隆抗体的制备与鉴定[J]. 河南农业科学, 2020, 49(12): 137-143.

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Preparation and Identification of Monoclonal Antibodies against African Swine Fever Virus p17 Protein

非洲猪瘟病毒p17蛋白单克隆抗体的制备与鉴定

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