猫IL-1β阻断单克隆抗体的筛选与猫源化改造
Felinized development of blocking McAbs against cat IL-1β
王定一 1王阅 1张晨曦 1李秋燕 1周登元 1司有辉 1曹胜波 1叶静1
作者信息
- 1. 华中农业大学动物医学院,湖北武汉 430070
- 折叠
摘要
为给猫的炎症性疾病治疗提供具有与猫白细胞介素-1β(IL-1β)高亲和力的单克隆抗体,将表达后纯化的重组猫IL-1β蛋白接种小鼠后制备单克隆抗体,经验证单抗对猫IL-1β蛋白的阻断效果并分析其靶向位点后,将小鼠单克隆抗体的轻/重链可变区基因片段连接到表达猫抗体的真核质粒中,转染真核细胞后检测猫源化改造抗体效果.结果表明,成功扩增了猫IL-1β基因,并成功构建了原核表达质粒pET-30a-IL-1β并表达猫IL-1β重组蛋白;在使用猫IL-1β重组蛋白免疫小鼠以及细胞融合、杂交瘤细胞筛选后,成功得到2株分别能够与猫IL-1β重组蛋白第二、第三结构域反应的小鼠源单克隆抗体C4C、H10E;将单克隆抗体C4C、H10E猫源化基因改造后的重组抗体仍具有阻断效果.本试验研究结果为开发针对猫IL-1β的阻断单克隆抗体药物奠定了基础,也为猫炎症性疾病的治疗提供了一种新的方案.
Abstract
In order to develop monoclonal antibody(McAb)with binding cat interleukin-1β(IL-1β)to block cat inflammatory disease as a new drug product,the blocking mouse McAb to cat IL-1β protein was de-tected,and the action site of the antibody was analyzed.Then the light/heavy chain variable region genes of the blocking McAb were amplified and linked to the constant region of cat antibody gene to construct eu-karyotic plasmid transfection to detect the effect of cat source modified antibody.The results showed that the cat IL-1β gene was successfully amplified,and the prokaryotic expression plasmid pET-30a-IL-1β was successfully constructed,and the recombinant cat IL-1β protein was expressed.After immunizing mice with recombinant cat IL-1β protein,cell fusion and hybridoma cell screening,two murine monoclonal antibodies C4C and H10E which can react with the second and third domain of cat IL-1β recombinant protein were success-fully obtained.The recombinant antibody genetically modified by monoclonal antibodies C4C and H10E still had blocking effect.The results of this study laid a foundation for the development of blocking monoclonal antibody drugs against IL-1β in cats,and also provided a new scheme for the treatment of inflammatory diseases in cats.
关键词
白介素1β/单克隆抗体/阻断效果/猫源化抗体改造Key words
interleukin-1β/monoclonal antibody/blocking effect/cat source modification引用本文复制引用
基金项目
湖北省自然科学基金(2019CFA010)
出版年
2024
国家科技期刊平台
NSTL
万方数据