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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。
OBJECTIVE: To construct a human-mouse chimeric antibody expression vector against human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (death receptor 5, DR5), and to obtain a cell strain stably expressing the chimeric antibody Anti-tumor activity of chimeric antibodies was analyzed. Methods: The heavy chain (HC) and light chain (LC) variable region genes of murine monoclonal antibody (mAb) AD5-10 against human DR5 were amplified by DNA recombination technique and inserted into human IgG Heavy chain, light chain constant region gene of eukaryotic expression vector RpCI-neo, heavy and light chain expression plasmid cotransfection of Chinese hamster ovary cells (CHO), screening stable expression of anti-human DR5 chimeric antibody (hmAD5-10) Recombinant cells. The expression of chimeric antibody and its binding activity with antigen DR5 were detected by Western blot and indirect ELISA. MTS colorimetric assay was used to detect the biological activity of chimeric antibody. Recombinant cell lines were cultured in serum-free medium. Results: Two recombinant CHO-A5 and CHO-B11 cell lines stably expressing chimeric antibody were obtained. The antibody expression levels were (0.36 ± 0.11) mg / L and (0.16 ± 0.01) mg / Has a good binding activity with DR5, and has a significant killing effect on SVT35 cultured in vitro in human T lymphocyte leukemia cells. CONCLUSION: The biological activity of the human-mouse chimeric antibody against DR5 is expressed in eukaryotic cells, which lays the foundation for its application in the treatment of cancer.