中国医学科学院学报

高级检索
中国医学科学院学报

中国医学科学院学报 ›› 2008, Vol. 30 ›› Issue (6): 690-695.doi: 10.3881/j.issn.1000-503X.2008.06.014

• 论著 • 上一篇    下一篇

抗肿瘤坏死因子相关凋亡诱导配体受体死亡受体5嵌合抗体的构建及其真核表达

陈凤1,2;郭雅彬1;刘士廉1;郑德先1;刘彦信1   

  1. 1中国医学科学院北京协和医学院基础医学研究所医学分子生物学国家重点实验室,北京 100005 2北京大学临床肿瘤学院北京市肿瘤防治研究所北京肿瘤分子生物学实验室,北京 100142
  • 收稿日期:2008-04-17 修回日期:1900-01-01 出版日期:2008-12-30 发布日期:2008-12-30
  • 通讯作者: 刘彦信

Construction and Expression of Anti-tumor Necrosis Factor Related Apoptosis-inducing Ligand Receptor Death Receptor 5 Chimeric Antibody in Eukaryotic Cells

CHEN Feng1,2;GUO Ya-bin1;LIU Shi-lian1;ZHENG De-xian1;LIU Yan-xin1   

  1. 1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China 2Laboratory of Molecular Oncology, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing 100142, China
  • Received:2008-04-17 Revised:1900-01-01 Online:2008-12-30 Published:2008-12-30
  • Contact: LIU Yan-xin

摘要: 摘要:目的 构建以人肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体死亡受体5(DR5)为抗原的鼠/人重组嵌合抗体。方法 采用基因工程技术,扩增和克隆抗DR5嵌合抗体的轻、重链表达载体,共转染二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞(CHO-dhfr-),筛选稳定分泌表达抗DR5嵌合抗体的细胞株。采用ELISA和Western bloting法鉴定抗体的人源性和抗体-抗原结合活性,四唑盐/吩嗪硫酸甲酯比色法检测抗体的生物学活性。结果 获得了稳定表达和分泌抗人DR5的嵌合抗体(anti-DR5 mV-hH)的重组细胞;与人DR5蛋白具有高的特异性结合活性;能使体外培养的人髓性白血病Jurkat细胞和人结肠癌HCT116细胞的存活率分别下降到73.15%和77.30%。结论 抗DR5嵌合抗体anti-DR5 mV-hH具有抗肿瘤活性,为发展人源化抗体药物的研究奠定了基础。

关键词: 抗死亡受体5嵌合抗体, 中国仓鼠卵巢细胞, 人IgG轻链, 人IgG重链

Abstract: ABSTRACT:Objective To construct the human/mouse chimeric antibody of a functional anti-death receptor 5 (DR5) antibody. Methods The viable region of light chain (VL) and viable region of heavy chain (VH) genes of anti-DR5 antibody were amplified and cloned into the light- and heavy-chain expression vectors respectively, then the recombinant plasmids were co-transfected into dihydrofolate reductase- Chinese hamster ovary cell (CHO-dhfr-) for expression. The positive clone was screened by the two selective genes (neo and dhfr). The humanization and specificity of chimeric antibody was identified by ELISA and Western bloting, and the tumoricidal activity of the expressed chimeric antibody was detected by tetrazolium salt phenazine methosulfate assay. Results The expression vectors stably expressed chimeric antibody in CHO-dhfr-. In the cell supernatant of the F4’clone, the human IgG heavy constant region and light constant region were identified. Moreover, the secreted chimeric antibody retained the binding capacity to the antigen (DR5) and decreased the cell viability of Jurkat and HCT116 cells to 73.15% and 77.30% in vitro respectively. Conclusion The human/mouse anti-DR5 chimeric antibody has been successfully expressed in eukaryotic cells and shows tumoricidal activity, which establishes a foundation for the future research of humanized antibody medicine.

Key words: anti-deatd receptor 5 cdimeric antibody, cdinese damster ovary cell, duman icc licdt cdain, duman icc deavy cdain