摘要
目的:构建抗人肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体2(死亡受体5,DR5)的人-鼠嵌合抗体表达载体,获得稳定表达该嵌合抗体的细胞株,并分析嵌合抗体的抗肿瘤活性。方法:采用DNA重组技术,扩增抗人DR5的鼠源单克隆抗体(mAb)AD5-10的重链(HC)、轻链(LC)可变区基因片段,并将其分别插入含有人IgG重链、轻链恒定区基因的真核表达载体RpCI-neo,以重、轻链表达质粒共转染中国仓鼠卵巢细胞(CHO),筛选稳定表达抗人DR5嵌合抗体(hmAD5-10)的重组细胞。采用Western blot和间接ELISA检测嵌合抗体的表达量及其与抗原DR5的结合活性。采用MTS比色法检测嵌合抗体的生物学活性。并对重组细胞株进行无血清培养驯化。结果:获得了2株稳定表达嵌合抗体的重组细胞株CHO-A5和CHO-B11,抗体的表达水平分别为(0.36±0.11)mg/L和(0.16±0.01)mg/L,嵌合抗体与DR5有较好的结合活性,对体外培养的人T淋巴细胞白血病细胞SVT35有显著的杀伤作用。结论:在真核细胞中表达了具有生物学活性的抗DR5的人-鼠嵌合抗体,为其应用于肿瘤治疗研究奠定了基础。
AIM：To establish an human-mouse chimeric antibody against tumor necrosis factor-related apoptosis-inducing ligand（TRAIL） receptor 2（death receptor 5,DR5） in an eukaryotic cell line and analyse its tumoricidal activity.METHODS： The cDNAs encoding for the variable regions of heavy chain（VH） and light chain（VL） of AD5-10 were amplified by PCR and inserted into the human IgG heavy and light chain containing expression vector RpCI-neo,respectively.The recombinant plasmids were co-transfected into HEK293 and/or CHO cells.The production of anti-DR5 human-mouse chimeric antibody（hmAD5-10） and the antibody affinity for DR5 were identified by ELISA and Western blot assay.The tumoricidal activity of hmAD5-10 was demonstrated by MTS assay.The stable expression cells were selected and cultured in serum-free medium.RESULTS： Two stable CHO cells CHO-A5 and CHO-B11 with the chimeric antibody hmAD5-10 expression were established,in which the production of hmAD5-10 were reached at（0.36±0.11） mg/L and（0.16±0.01） mg/L,respectively.The hmAD5-10 secreted from the cells can well bind with DR5 and kill the cultured leukemia SVT35 cells by apoptosis remarkably.CONCLUSION： The human-mouse chimeric antibody hmAD5-10 was successfully expressed in the eukaryotic cells and resulted tumor cell death by apoptosis.This study lays a fundamental basis for the potential application of the recombinant chimeric antibody in cancer therapy.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2011年第4期415-418,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30623009
30972684)
国家重大基础研究发展计划(973)资助项目(2007CB507404)
关键词
肿瘤坏死因子相关凋亡诱导配体受体2
嵌合抗体
中国仓鼠卵巢细胞
tumor necrosis factor-related apoptosis-inducing ligand receptor 2
chimeric antibody
chinese hamster ovary cells
