重庆市畜牧科学院,重庆市医用动物资源开发与利用工程技术研究中心,重庆 402460
R-33
Chongqing Academy of Animal Sciences Bioengineering institute, Chongqing 402460, China
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 pcDNA3. 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 pcDNA3. 1-GPC3,检测发现 SK-Hep-1 细胞 G418 最佳筛选浓度为 700 μg / mL,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 pcDNA3. 1-GPC3 was constructed and transfected into SK-Hep-1 cells by electroporation. The G418 optimum screening concentration for SK-Hep-1 cells was 700 μg / mL. 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.
何琦琳,郎巧利,余 琳,黄 楠,杨 希,葛良鹏.稳定高表达人 GPC3 的 SK-Hep-1 细胞系的构建[J].中国比较医学杂志,2020,30(2):59~63.
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