Anti-hEGFR轻、重链共表达及高表达细胞株快速筛选

发布时间：2017-12-27 11:00

本文关键词：Anti-hEGFR轻、重链共表达及高表达细胞株快速筛选　出处：《东北农业大学学报》2015年10期 　论文类型：期刊论文

【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。
[Abstract]:In order to realize the light and heavy chain co expression of the full length antibody (anti-H EGFR) of H EGFR and to establish a rapid and efficient screening method, the co expression of cell lines was obtained. Get the heavy chain and light chain gene by molecular cloning technology, will be connected to the peedual eukaryotic expression vector; after GS-Neo compression and stable cell lines were obtained by flow cytometry screening; protein A affinity chromatography purified target antibody; using CCK-8 method, Annexin V/PI method and Q method for detection of PCR anti-H EGFR on the proliferation and apoptosis of A431 cells. The results of SDS-PAGE analysis showed that the purity of the purified antibody was 95% and the molecular weight was about 150 Ku. In the CCK-8 test, different concentrations of anti-H EGFR significantly inhibited the proliferation of A431 cells, and the effect was similar to that of commercialized Cetuximab. Anti-H EGFR and Cetuximab could induce A431 cells to produce apoptosis, and the effect was similar. Co expression of full-length antibody light and heavy chain, recombinant expression respectively to eliminate the process of natural structure, ensure the antibody; GS-Neo double compression combined with flow cytometry screening positive cell lines, to achieve fast and efficient separation; the establishment of time saving and efficient platform for co expression of antibodies, antibody drugs eukaryotic construction and provide new ideas for screening.
【作者单位】：东北农业大学生命科学学院;哈药集团技术中心;
【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)
【分类号】：R392;Q78
【正文快照】： Co-expression of anti-h EGFR light and heavy chain and rapidscreening of cell lines/LI Deshan1,LIU Chunxiang1,JIANG Yuanyuan2,LIU Mingyao1,ZHANG Yingjie1,YUAN Qingyan1,GUO Xiaochen1,REN Guiping1,WU Qiang1,LI Tingting1(1.School of Life Sciences,Northeast

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