抗人CD4嵌合抗体基因真核表达质粒的构建及其瞬时表达  

Construction and Transient Expression of Eukaryotic Expression Vector for Chimeric Antibody Gene against Human CD4

作　　者：胡迪超[1] 张爱华[1] 潘勇兵[1] 端义坤[1] 孙可芳[1] 张银川[1] 杨晓明[1] 

机构地区：[1]武汉生物制品研究所,430060

出　　处：《中国生物制品学杂志》2011年第9期997-1002,共6页Chinese Journal of Biologicals

摘　　要：目的构建抗人CD4嵌合抗体基因真核表达质粒,并在哺乳动物细胞中进行瞬时表达。方法采用RLM-RACE法克隆WuT4抗体可变区和信号肽序列,并采用基因拼接法构建嵌合抗体基因真核表达质粒。采用脂质体法瞬时转染HEK293T、CHO-DHFR-和COS-7 3种哺乳动物细胞,ELISA法检测转染细胞上清中嵌合抗体的浓度,流式细胞术、Western blot和Dotblot分析嵌合抗体的抗原结合活性,并对表达的嵌合抗体进行免疫荧光分析。结果成功克隆了WuT4抗体可变区和信号肽序列;抗人CD4嵌合抗体共表达质粒pOptiVEC-T4H和pcDNA3.3-T4L构建正确;表达的嵌合抗体保留了亲本抗体的抗原结合力,并能特异性识别人CD4分子;免疫荧光分析表明,表达的嵌合抗体含人抗体的重、轻链恒定区。结论 已成功构建了抗人CD4嵌合抗体基因真核表达质粒,并能在哺乳动物细胞中瞬时表达,为其进一步研究奠定了基础。Objective To construct a eukaryotic expression vector for chimeric antibody gene against human CD4 and transiently express in mammalian cells.Methods The gene sequences encoding variable region and signal peptide of WuT4 antibody were cloned by RLM-RACE method,based on which a eukaryotic expression vector for chimeric antibody was constructed by gene splicing method and transiently transfected to HEK 293T,CHO-DHFR-and COS-7 cells in mediation of liposome.The chimeric antibody in supernatant of transfected cells was determined for concentration by ELISA,for antigen binding activity by flow cytometry,Western blot and Dot blot,and identified by IFA.Results The gene sequences of variable region and signal peptide of WuT4 antibody were successfully cloned.The co-expression vectors pOptiVEC-T4H and pcDNA3.3-T4L for chimeric antibody against human CD4 were constructed correctly.The expressed chimeric antibody retained the antigen binding activity of its parental antibody,and recognized human CD4 molecules specifically.IFA showed that the expressed chimeric antibody contained the constant regions of both heavy and light chains of human antibody.Conclusion The eukaryotic expression vector for chimeric antibody gene against human CD4 was successfully constructed and transiently expressed in mammalian cells,which laid a foundation of further study.

关 键 词：抗原 CD4 RLM-RACE 基因拼接 嵌合抗体 瞬时表达 

分 类 号：Q782[生物学—分子生物学]