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利用HEK293F细胞瞬时表达重组抗HER2人源化单克隆抗体(rh HER2-m Ab),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rh HER2-m Ab重、轻链表达载体(p CMV-HC和p CMV-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rh HER2-m Ab的最佳条件为:细胞接种密度4×106 cells/ml,DNA浓度2.0?g/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%。rh HER2-m Ab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,rh HER2-m Ab对BT-474细胞的凋亡率约25%,对SK-BR-3细胞则近15%。
Recombinant anti-HER2 humanized monoclonal antibody (rh HER2-m Ab) was transiently expressed in HEK293F cells and the transfection conditions were optimized. The anti-tumor activity of the purified antibody was initially identified. The heavy and light chain expression vectors (p CMV-HC and p CMV-LC) of rh HER2-m Ab were constructed respectively, and the heavy and light chain plasmids were co-transfected into HEK293F cells using polyethylenimine (PEI) as transfection reagent Express. The protein was purified by Protein A affinity chromatography and its antitumor activity was tested by WST-8 method in vitro. After optimization, the optimal conditions for transient expression of rh HER2-m Ab in HEK293F cells were: cell seeding density 4 × 106 cells / ml, DNA concentration 2.0 μg / 106 cells, DNA: PEI 1: 2, heavy chain: light The chain was 1: 1, the antibody expression reached 73.0 mg / L and the antibody purity was more than 98%. The inhibitory rate of rh HER2-m Ab to BT-474 cells expressing HER2 was (72.3 ± 2.0)% and that of SK-BR-3 cells expressing HER2 was (32.1 ± 1.2)% No significant inhibitory effect on breast cancer MCF-7 cells with low expression of HER2. The result of the apoptosis test showed that rh HER2-m Ab had a apoptosis rate of about 25% in BT-474 cells and 15% in SK-BR-3 cells relative to the control group.