鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备

doi:10.11843/j.issn.0366-6964.2023.06.036

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (6): 2596-2604.doi: 10.11843/j.issn.0366-6964.2023.06.036

鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备

刘青^1,2, 吴少鹏^1,2, 石彬^1,2, 邵红霞^1,2,3, 钱琨^1,2,3, 叶建强^1,2,3,4, 秦爱建^1,2,3,4*

1. 扬州大学兽医学院教育部禽类预防医学重点实验室, 扬州 225009;
2. 扬州大学兽医学院江苏省动物预防医学重点实验室, 扬州 225009;
3. 扬州大学兽医学院江苏高校动物重要疫病与人畜共患病防控协同控制中心, 扬州 225009;
4. 扬州大学教育部农业与农产品安全国际合作联合实验室, 扬州 225009

收稿日期:2022-07-04 出版日期:2023-06-23 发布日期:2023-06-16
通讯作者: 秦爱建,主要从事预防兽医学研究,E-mail:aijian@yzu.edu.cn
作者简介:刘青(1996-),女,山东潍坊人,硕士生,主要从事预防兽医学研究,E-mail:1358401919@qq.com
基金资助:
国家自然科学基金(31972717)

Expression of Chicken CTLA-4 Protein in Insect Cells and the Preparation of Its Monoclonal Antibody

LIU Qing^1,2, WU Shaopeng^1,2, SHI Bin^1,2, SHAO Hongxia^1,2,3, QIAN Kun^1,2,3, YE Jianqiang^1,2,3,4, QIN Aijian^1,2,3,4*

1. Ministry of Education Key Laboratory for Avian Preventive Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. Jiangsu Province Key Laboratory of Preventive Veterinary Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
3. Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
4. Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China

Received:2022-07-04 Online:2023-06-23 Published:2023-06-16

摘要/Abstract

摘要： 旨在制备鸡细胞毒性T淋巴细胞相关抗原4(CTLA-4)的特异性单克隆抗体,为家禽免疫检查点和疫苗研发提供有益制剂。利用杆状病毒表达系统体外表达鸡的CTLA-4蛋白,以其为免疫原免疫8周龄Balb/c小鼠,经B淋巴细胞融合技术制备、筛选出针对鸡CTLA-4蛋白的单克隆抗体。通过间接免疫荧光、蛋白免疫印迹、流式细胞术等方法分析抗CTLA-4单克隆抗体的生物学特性。结果显示:构建了在Sf9昆虫细胞中表达CTLA-4蛋白的重组杆状病毒,成功筛选出3株能稳定分泌抗鸡CTLA-4蛋白的单克隆抗体,分别命名为:mAb-CTLA4-3D7、mAb-CTLA4-5A4、mAb-CTLA4-6A12。亚类鉴定表明,mAb-CTLA4-3D7为IgG2a,Lambda链;mAb-CTLA4-5A4为IgG3,Kappa链;mAb-CTLA4-6A12为IgG2a,Kappa链。3株单克隆抗体均能与转染真核表达质粒pCAGGS-CTLA-4-Flag的DF-1细胞和感染重组杆状病毒rBac-CTLA-4的Sf9昆虫细胞反应。单克隆抗体mAb-CTLA4-3D7与鸡的PBMC有较好的结合活性,且能与CD3⁺T淋巴细胞结合,结合率约为16%。本研究首次成功制备了抗鸡CTLA-4蛋白的单克隆抗体,并分析了其反应活性及生物学特性,为免疫检查点在禽病的发病机制、信号通路、疫苗研发等研究领域提供了材料。

关键词: 鸡CTLA-4, 杆状病毒, 单克隆抗体, 生物学特性

Abstract: The aim of this study was to prepare monoclonal antibody specific to chicken CTLA-4, and provide useful tools for research on the poultry immune checkpoint and vaccine development. Baculovirus expression system was used to express chicken CTLA-4 protein in vitro. The monoclonal antibodies against chicken CTLA-4 protein were prepared by fusion between SP2/0 cells and spleen cells from the 8-week-old Balb/c mice immunized with the expressed chicken CTLA-4 protein. The charcterization of monoclonal antibodies were analyzed by indirect immunofluorescence, Western blot and flow cytometry. The recombinant baculovirus expressing CTLA-4 protein in Sf9 insect cells was constructed. Three monoclonal antibodies against chicken CTLA-4 protein were successfully obtained, named as mAb-CTLA4-3D7, mAb-CTLA4-5A4 and mAb-CTLA4-6A12. The subclass of mAb-CTLA4-3D7 was IgG2a and type Lambda; The subclass of mAb-CTLA4-5A4 was IgG3 and type Kappa; The subclass of mAb-CTLA4-6A12 was IgG2a and type Kappa. All three monoclonal antibodies could react with DF-1 cells transfected with pCAGGS-CTLA-4-Flag and Sf9 insect cells infected with rBac-CTLA-4. Among them, mAb-CTLA4-3D7 had a good binding activity with chicken PBMC and could react to CD3⁺T lymphocytes, with the binding rate of about 16%. In this study, monoclonal antibodies against chicken CTLA-4 protein were successfully developed, and their reactivity and biological characteristics were analyzed, which provided a reference for immune checkpoints in the pathogenesis, signaling pathways, vaccine development and other research fields of avian diseases.

Key words: chicken CTLA-4, baculovirus, monoclonal antibody, biological characteristics

中图分类号:

S852.4

刘青, 吴少鹏, 石彬, 邵红霞, 钱琨, 叶建强, 秦爱建. 鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备[J]. 畜牧兽医学报, 2023, 54(6): 2596-2604.

LIU Qing, WU Shaopeng, SHI Bin, SHAO Hongxia, QIAN Kun, YE Jianqiang, QIN Aijian. Expression of Chicken CTLA-4 Protein in Insect Cells and the Preparation of Its Monoclonal Antibody[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2596-2604.

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鸡CTLA-4蛋白在昆虫细胞中的表达及其单克隆抗体的制备

Expression of Chicken CTLA-4 Protein in Insect Cells and the Preparation of Its Monoclonal Antibody

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