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目的利用Flp/FRT位点特异性重组技术,在Flp-In-CHO细胞中表达重组人凝血因子Ⅶ(rhFⅦ),并进行鉴定。方法用限制性内切酶EcoRⅠ和EcoRⅤ分别双酶切质粒pT-FⅦ与表达载体pcDNA5/FRT/TO,将回收的目的基因片段和载体片段连接,构建重组表达质粒pcDNA5/FRT/TO-FⅦ,并与辅助质粒pOG44共转染Flp-In-CHO细胞,经400μg/ml潮霉素B压力筛选,获得抗性细胞株。采用PCR、RT-PCR、Western blot和PT等方法对筛选出的细胞株分别进行基因组水平、mRNA水平、表达水平和蛋白活性的鉴定。结果重组表达质粒pcDNA5/FRT/TO-FⅦ经PCR、双酶切及测序鉴定证明构建正确;筛选出9株抗性细胞;外源基因FⅦ定点整合到Flp-In-CHO细胞基因组中并得到表达;重组蛋白表现出与血浆FⅦ相一致的促凝活性。结论利用位点特异性表达系统Flp/FRT,在Flp-In-CHO细胞中成功表达了hFⅦ,为其制备和纯化奠定了基础。
Objective To express recombinant human coagulation factor Ⅶ (rhFⅦ) in Flp-In-CHO cells using Flp / FRT site-specific recombination technology. Methods Plasmid pT-FⅦ and expression vector pcDNA5 / FRT / TO were digested with restriction endonucleases EcoRⅠ and EcoRⅤ respectively to construct a recombinant plasmid pcDNA5 / FRT / TO-FⅦ. The recombinant plasmid pcDNA5 / Flp-In-CHO cells were cotransfected with the helper plasmid pOG44 and then screened with 400 μg / ml hygromycin B to obtain resistant cell lines. The selected cell lines were identified by PCR, RT-PCR, Western blot and PT methods respectively. The results of genotyping, mRNA expression, expression and protein activity were identified. Results The recombinant plasmid pcDNA5 / FRT / TO-FⅦ was confirmed by PCR, restriction endonuclease digestion and DNA sequencing. 9 resistant cells were screened out and the exogenous gene FⅦ was integrated into the genome of Flp-In-CHO cells and expressed ; Recombinant protein showed procoagulant activity consistent with plasma FVII. Conclusion The hFⅦ was successfully expressed in Flp-In-CHO cells by using the site-specific expression system Flp / FRT, which laid the foundation for its preparation and purification.