凝血因子VII高表达细胞的高通量筛选方法

High-Throughput Method for Screening Recombinant Coagulation Factor VII Expressing Cells

DOI：10.3969/j.issn.1673-1689.2018.07.008

中文关键词: 高通量 中国仓鼠卵巢细胞 流式细胞仪 重组蛋白

英文关键词: high-throughput,CHO,flow cytometry,recombinant protein

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中文摘要:

针对哺乳动物细胞表达克隆的耗时较长的筛选过程，作者通过流式细胞仪对细胞进行快速筛选和纯度检测。利用流式细胞仪对电转细胞进行分选，利用点杂交和western blot方法筛选高表达克隆。流式细胞仪分选后的细胞，经点杂交和WB检测后获得的CHO-rFVII-1细胞，表达rFVII细胞的比例为99.9 %，同时与传统筛选方法获得的CHO-rFVII克隆比较后发现，两株细胞具有相同的rFVII产量。作者基于流式细胞仪建立了快速筛选高表达重组药物蛋白克隆的筛选的高通量方法和纯度检测的方法，为其他在哺乳动物细胞中表达重组蛋白的筛选提供参考。

英文摘要:

To overcome the time-consuming screening process，mammalian cells expressing recombinant therapeutic protein were selected by flow cytometry and method of cell purity analysis was established. Transfected CHO cells were sorted by flow cytometer and high expression sub-clone was screened by dot blot and western blot. CHO cells after transfecting with rFVII expression vector were divided by flow cytometry to 96 micro-plates and recombinant coagulation factor VII expression was analyzed by dot blot and western blot. High rFVII expression clone，CHO-rFVII-1，was obtained and the purity of CHO-rFVII-1 was 99.9 %. Moreover，CHO-rFVII-1 had similar viable cell density and rFVII expression compared to CHO-rFVII，which was obtained by traditional screening method. Screening high expression clones by flow cytometry was a rapid and high-throughput method and will be beneficial to screen high recombinant therapeutic protein expression clone in other mammalian cells.

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