CD3E蛋白真核表达及全人源CD3单克隆抗体的制备  

Eukaryotic expression of CD3E protein and generation of human CD3 monocl-onal antibody

作　　者：杨希 郎巧利 黄楠 余琳 吴梦(指导)[1] 葛良鹏[1] YANG Xi;LANG Qiao-Li;HUANG Nan;YU Lin;WU Meng;GE Liang-Peng(Chongqing Academy of Animal Sciences,Chongqing Research Center for the Development and Utilization of Medical Animal Resources,Chongqing 402460,China)

机构地区：[1]重庆市畜牧科学院,重庆市医用动物资源的开发与利用工程技术研究中心,重庆402460

出　　处：《中国免疫学杂志》2020年第8期971-976,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金(5167070727);重庆市科研院所绩效激励引导专项(cstc2018jxjl0043);重庆市农发资金资助项目(17406)资助。

摘　　要：目的:高效真核表达和纯化获得CD3E蛋白,制备全人源CD3抗体。方法:构建CD3E链胞外区真核表达质粒,利用293F悬浮细胞表达系统表达并纯化获得高纯度蛋白。选取人源化抗体转基因小鼠CAMouse-HG76进行免疫,通过细胞融合的方法获得杂交瘤细胞,再利用ELISA法和流式细胞分析法筛选CD3特异性单克隆抗体。结果:成功构建重组表达载体pcDNA3.4-CD3E-mFc,真核细胞表达并纯化获得纯度为85%的融合蛋白。免疫小鼠后效价高达1∶102400。杂交瘤细胞筛选获得3株CD3特异性单克隆抗体。结论:本研究成功获得了CD3E融合蛋白和CD3单克隆抗体,为后续建立ELISA检测方法、全人源治疗性抗体药物及相关研究提供基础。Objective:To obtain CD3E protein by eukaryotic expression system,and to prepare all-human CD3 antibody.Methods:The extracellular region of CD3E chain was inserted into eukaryotic expression plasmid.The high-purity protein was obtained by the expression system of 293F suspension cells.Humanized antibody transgenic mice(CAMouse-HG76)were selected for immunization.Hybrid tumor cells were obtained by cell fusion,and the CD3-specific monoclonal antibody was screened by ELISA and flow cytometry.Results:The recombinant expression vector pcDNA3.4-CD3E-mFc was successfully constructed,and the fusion protein with a purity of 85%was generated by eukaryotic expression and purification.The titer of immunized mice was around 1∶102400.Three strains of CD3-specific monoclonal antibody were generated from hybridoma cells.Conclusion:CD3E fusion protein and CD3 monoclonal antibody were successfully obtained in this study,which loads a good foundation for the development of CD3 ELISA detection methods,all-human therapeutic antibody drugs and other related studies.

关 键 词：人CD3E 293F悬浮细胞 真核表达 单克隆抗体 

分 类 号：R392.12[医药卫生—免疫学]