摘要
目的获得稳定高表达人GPC3的SK-Hep-1细胞系。方法构建人GPC3真核表达载体pc DNA3.1-GPC3,通过电穿孔的方法转染至SK-Hep-1细胞,利用G418进行细胞筛选,获得稳定高表达GPC3的细胞系。再利用Western blot和流式细胞术进行鉴定,分析稳定转染细胞表面GPC3蛋白的表达情况。结果成功构建的真核表达质粒pc DNA3.1-GPC3,检测发现SK-Hep-1细胞G418最佳筛选浓度为700μg/m L,利用该浓度G418筛选转染SK-Hep-1细胞,获得了细胞表面能够稳定高表达人GPC3蛋白的SK-Hep-1细胞系。结论建立高表达人GPC3蛋白的SK-Hep-1稳定细胞系,为研究靶向GPC3肝癌抗体提供了物质基础。
Objective To establish a stably expressing human GPC3 SK-Hep-1 cell line.Methods The eukaryotic expression vector pc DNA3.1-GPC3 was constructed and transfected into SK-Hep-1 cells by electroporation.The G418 optimum screening concentration for SK-Hep-1 cells was 700μg/m L.Transfected cells with the target gene were screened with G418 for 20 days.Analysis of GPC3 expression in the stable cell line was performed by Western blot and flow cytometry.Results Western blot result showed that the GPC3 expressing level of SK-Hep-1/GPC3 was markedly higher than that of HepG2.Flow cytometry demonstrated GPC3 protein on the surface of SK-Hep-1/GPC3 cells.Conclusions SK-Hep-1/GPC3 cells that stably expressed GPC3 protein were successfully established.These result provide a solid foundation for further research on GPC3 therapeutic antibodies.
作者
何琦琳
郎巧利
余琳
黄楠
杨希
葛良鹏
HE Qilin;LANG Qiaoli;YU Lin;HUANG Nan;YANG Xi;GE Liangpeng(Chongqing Academy of Animal Sciences Bioengineering institute,Chongqing 402460,China)
出处
《中国比较医学杂志》
CAS
北大核心
2020年第2期59-63,共5页
Chinese Journal of Comparative Medicine
基金
重庆市农发资金资助项目(17406)
重庆市科研院所绩效激励引导专项(cqjxjl201709)
国家自然科学基金(5167070727).
