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为实现h EGFR全长抗体(anti-h EGFR)轻、重链共表达以及建立快速、高效筛选方法并获得共表达细胞株。采用分子克隆技术获得重链和轻链基因,将之连入peedual真核表达载体;经GS-Neo加压及流式细胞术筛选获得稳定表达细胞株;通过protein A亲和层析技术纯化出目标抗体;应用cck-8法、Annexin V/PI法以及q PCR技术检测anti-h EGFR对A431细胞增殖、凋亡影响。SDS-PAGE分析结果显示,纯化后抗体纯度为95.0%、分子质量约为150 ku。cck-8试验中不同浓度anti-h EGFR对A431细胞增殖均有极显著抑制作用,且与商品化Cetuximab效果相近;anti-h EGFR与Cetuximab均可诱导A431细胞产生凋亡,作用效果相近,均达极显著。全长抗体轻、重链的共表达,省去分别表达的重组过程,保证抗体天然结构;GS-Neo双加压结合流式筛选,实现阳性细胞株快速、高效分离;建立省时、高效抗体共表达平台,为抗体类药物真核构建及筛选提供新思路。
To achieve light and heavy chain co-expression of h EGFR full-length antibody (anti-h EGFR), a rapid and efficient screening method was established and co-expressed cell lines were obtained. The heavy chain and light chain genes were obtained by molecular cloning technology and linked into peedual eukaryotic expression vector. Stably expressing cell lines were obtained by GS-Neo pressurization and flow cytometry screening, and purified by protein A affinity chromatography Target antibody. The effects of anti-h EGFR on the proliferation and apoptosis of A431 cells were detected by cck-8 assay, Annexin V / PI assay and q PCR. SDS-PAGE analysis showed that the purity of purified antibody was 95.0% and the molecular weight was about 150 ku. Different concentrations of anti-h EGFR in cck-8 test significantly inhibited the proliferation of A431 cells and showed similar effects with commercial Cetuximab. Both anti-h EGFR and Cetuximab induced apoptosis in A431 cells with similar effects Extremely significant. The total length of the antibody light and heavy chain co-expression, eliminating the recombinant expression of the respective expression, to ensure that the natural structure of antibodies; GS-Neo dual pressurized flow cytometry screening to achieve rapid and efficient isolation of positive cell lines; the establishment of time-saving and efficient antibody Co-expression platform for the eukaryotic antibody drug construction and screening to provide new ideas.