Anti-hEGFR轻、重链共表达及高表达细胞株快速筛选

Co-expression of anti-hEGFR light and heavy chain and rapid screening of cell lines

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。

The aim of this study was to co-express h EGFR full length antibody（anti-h EGFR） light and heavy chains, also to establish a rapid screening method and obtain co-expressing cell lines. fulllength heavy chain and light chain were connected to peedual eukaryotic expression vector, which cloned by molecular cloning techniques. stably expressing cell lines were screened by GS-Neo pressure combining flow cytometry; the target antibody was purified by protein A affinity chromatography; To validate the influence for cell proliferation and apoptosis of anti-h EGFR, cck-8 assay,Annexin V/PI method and q PCR were applicated to A431 cell. SDS-PAGE showed that molecular weight of anti-h EGFR was about 150 ku and relative purity was 95.0%. In cck-8 assay, the proliferation of A431 cells were significantly inhibited, and anti-h EGFR had similar effects with Cetuximab. anti-h EGFR had the same effect with Cetuximab in terms of pro-apoptotic of A431 cells. Co-expression could eliminate the refolding process and ensure the activity; The method of GS-Neo pressure combining flow cytometry could achieve a fast and efficient separation of positive cell lines; In this study, the province and efficient co-expression platform was established, which could offer new ideas for the production of antibody drugs.