利用HEK293F 细胞瞬时表达重组抗HER2 人源化单克隆抗体(rhHER2-mAb)，优化转染条件，并初步鉴定纯化后的抗体抗肿瘤活性。分别构建rhHER2-mAb 重、轻链表达载体(pCMV-HC 和pCMV-LC)，采用聚乙烯亚胺(PEI) 为转染试剂，共转染重、轻链质粒到HEK293F 细胞中进行表达。采用Protein A 亲和色谱纯化抗体，以WST-8 法检测其体外抗肿瘤效果。经优化后，HEK293F 细胞瞬时表达rhHER2-mAb 的最佳条件为：细胞接种密度4×106 cells/ml，DNA 浓度2.0 μg/106 cells，DNA ∶ PEI 为1 ∶ 2，重链∶轻链为1 ∶ 1，抗体表达量可达73.0 mg/L，抗体纯度大于98％。rhHER2-mAb 对高表达HER2 的乳腺癌BT-474 细胞抑制率为(72.3±2.0)％，对中表达HER2 的乳腺癌SK-BR-3 细胞抑制率约(32.1±1.2)％，但对低表达HER2 的乳腺癌MCF-7 细胞无显著抑制作用。细胞凋亡试验结果表明，相对于对照组，rhHER2-mAb 对BT-474 细胞的凋亡率约25％，对SK-BR-3 细胞则近15％。
Abstract
Recombinant humanized anti-HER2 monoclonal antibody (rhHER2-mAb) was expressed through transient transfection into HEK293F cells. The transfection conditions were optimized, and the antitumor activity of the purified antibodies was analyzed. The expression vectors, pCMV-HC and pCMV-LC, which contained heavy and light chains of rhHER2-mAb were constructed respectively, and co-transfected into HEK293F cells using polyethylenimine (PEI) as the tranfectant. The recombinant protein was purified by Protein A affinity chromatography. Its antitumor activity was determined by WST-8 method. Under the optimal transfection conditions as the cell density of 4×106 cells/ml, DNA concentration of 2.0 μg/106 cells, DNA∶PEI of 1∶2, and heavy chain∶light chain of 1∶1, the highest titer of
rhHER2-mAb reached 73.0 mg/L, with a purity over 98％. The inhibitory rates of rhHER2-mAb on the BT-474 cells with HER2 overexpression was up to (72.3±2.0)％, and on the SK-BR-3 cells with moderate HER2 expression was (32.1± 1.2)％, but it showed no inhibitory activity on the MCF-7 cells with low HER2 expression. In addition, the rhHER2-mAb could induce the BT-474 cells apoptosis up to 25％ compared with the control group, while it was only about 15％ to the SK-BR-3 cells.
关键词
重组抗HER2 人源化单克隆抗体 /
瞬时基因表达 /
HEK293F 细胞 /
抗肿瘤活性
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Key words
recombinant anti-HER2 humanized monoclonal antibody /
transient gene expression /
HEK293F cell /
antitumor activity
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脚注
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基金
国家“ 十二五” “ 重大新药创制” 科技重大专项(2012ZX09202-301-001)、广东省战略性新兴产业核心技术攻关项目(2012A080800008)、广东省重大科技专项(2012A080202014)
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