重组抗HER2人源化单克隆抗体的瞬时表达及其抗肿瘤活性

发布时间：2017-12-28 16:28

本文关键词：重组抗HER2人源化单克隆抗体的瞬时表达及其抗肿瘤活性　出处：《中国医药工业杂志》2016年07期 　论文类型：期刊论文

【摘要】：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%。
[Abstract]:Transient expression of recombinant human anti HEK293F HER2 monoclonal antibody (RH HER2-m Ab) was used to optimize transfection conditions, and initially identify the anti-tumor activity of the purified antibody. RH HER2-m Ab heavy and light chain expression vectors (P CMV-HC and P CMV-LC) were constructed respectively. Polyethylenimine (PEI) was used as a transfection reagent to co transfect heavy and light chain plasmids into HEK293F cells. The antibody was purified by Protein A affinity chromatography, and the antitumor effect in vitro was detected by WST-8 method. After optimization, the optimal conditions for transient expression of RH HER2-m Ab in HEK293F cells were: cell inoculation density 4 * 106 cells/ml, DNA concentration 2 g/106 cells, DNA PEI PEI 1: 2, heavy chain light chain 1: 1, antibody expression up to 73 mg/L, antibody purity greater than 98%. The inhibition rate of RH HER2-m Ab on breast cancer BT-474 cells with high expression of HER2 was (72.3 + 2)%, and the inhibition rate on SK-BR-3 cells expressing HER2 was about (32.1 + 1.2)%, but it had no significant inhibition on MCF-7 cells expressing low HER2. The apoptosis test showed that, compared with the control group, the apoptosis rate of RH HER2-m Ab to BT-474 cells was about 25%, and that of SK-BR-3 cells was nearly 15%.
【作者单位】：暨南大学生命科学技术学院广东省生物工程药物重点实验室基因工程药物国家工程研究中心;
【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)
【分类号】：R96
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