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

作　　者：李德山[1] 刘春香[1] 姜媛媛 刘铭瑶[1] 张瑛杰[1] 袁清艳 郭笑辰 任桂萍[1] 吴强[1] 李婷婷[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]哈药集团技术中心,哈尔滨150025

出　　处：《东北农业大学学报》2015年第10期56-62,共7页Journal of Northeast Agricultural University

基　　金：国家自然基金东北农业大学生物学理科基地科研训练及科研能力提高项目(J1210069/J0116);黑龙江省应用技术研究与开发计划项目(GC13C104)

摘　　要：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.

关 键 词：anti-hEGFR 共表达载体 GS-Neo筛选 流式细胞术 抗肿瘤 

分 类 号：Q816[生物学—生物工程]