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。
In order 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.
作者
刘青
吴少鹏
石彬
邵红霞
钱琨
叶建强
秦爱建
LIU Qing;WU Shaopeng;SHI Bin;SHAO Hongxia;QIAN Kun;YE Jianqiang;QIN Aijian(Ministry of Education Key Laboratory for Avian Preventive Medicine,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Province Key Laboratory of Preventive Veterinary Medicine,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Joint International Research Laboratory of Agriculture and Agri-Product Safety,the Ministry of Education of China,Yangzhou University,Yangzhou 225009,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2023年第6期2596-2604,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31972717)。
