摘要
目的 :构建人源抗_HAV全分子抗体的CHO细胞表达体系。方法 :将抗_HAV抗体轻链全分子 (信号肽与VL_CL)基因克隆至表达载体pCI_neo中 ;将重链信号肽、重链 (γ)VH_CH1 和Fc基因进行重组形成重链全分子基因 ,再将其克隆到真核表达载体pCdhfr1中。将轻、重链全分子表达载体共转染CHO dhfr_细胞 ,用G_4 18和甲氨蝶呤(MTX)筛选细胞克隆 ,ELISA法检测细胞培养上清中的抗_HAV活性 ,增加MTX浓度进行加压筛选。用蛋白L亲和层析柱从培养上清中纯化重组抗体。结果 :构建的真核表达载体可实现抗_HAV全分子抗体在CHO细胞中的分泌表达 ,纯化的重组抗体在还原SDS_PAGE中表现为相对分子质量约 2 5 0 0 0、5 0 0 0 0两条带 ;Western印迹表明 ,重组抗体全分子和重链分子均可和羊抗人Fc抗体发生特异性免疫反应。结论 :抗_HAV全分子抗体在CHO细胞中表达 。
Objective:To establish the CHO expression system for the whole human anti_HAV antibody.Methods:The light chain (V L_C L) of anti_HAV and its signal sequence were linked, then cloned into a mammalian expression vector, pCI_neo. The heavy chain signal sequence, the variable region, the first constant region (V H_C H1 ) and Fc fragment sequence were ligated to form a full length heavy chain ORF, which was then cloned into another mammalian expression vector, pCdhfr1. CHO/ dhfr _ cells were cotransfected with the light and heavy chain expression vectors, and cell clones expressing human anti_HAV antibodies were selected by G418 and methotrexate (MTX). The recombinant human antibodies were purified with protein L affinity chromatography from the cell culture medium.Results:ELISA revealed that anti_HAV antibodies were expressed in culture medium. In reducing SDS_PAGE, the recombinant IgG exhibited two bands of approximately 50?000 and 25?000, respectively. Western_blot demonstrated that both the whole IgG without reductant and the heavy chain with reductant reacted with goat anti_human Fc antiserum.Conclusions:Whole human anti_HAV antibody was expressed in CHO cells.The result provides the basis for genetically engineered production of antibody wtih complete function.
出处
《军事医学科学院院刊》
CSCD
北大核心
2002年第3期176-178,共3页
Bulletin of the Academy of Military Medical Sciences
基金
国家"8 63"高技术基金 (Z18_0 1)资助
关键词
基因工程抗体
甲型肝炎病毒
哺乳动物细胞
真核表达
genetically engineered antibody
hepatitis A virus
mammalian cell
eukaryotic expression
