重组人凝血因子Ⅶ在Flp-In-CHO细胞中的表达及鉴定  

Expression of Recombinant Human Coagulation Factor Ⅶ in Flp-In-CHO Cells and Identification of Expressed Product

作　　者：王卓[1,2] 吕茂民[1] 冯晶晶[1] 赵媛媛[1] 章金刚[1] 

机构地区：[1]军事医学科学院野战输血研究所血液生物制品研究室,北京100850 [2]解放军第457医院,武汉430012

出　　处：《中国生物制品学杂志》2010年第3期248-251,255,共5页Chinese Journal of Biologicals

摘　　要：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 by using Flp/FRT site specific recombination technique and identify the expressed product.Methods Plasmid pT-FⅦand expression vector pcDNA5/FRT/TO were digested with EcoRⅠand EcoRⅤseparately,and the recovered target gene fragment and vector were linked.The constructed recombinant plasmid pcDNA5/FRT/TO-FⅦ were co-transfected with accessory plasmid pOG44 to Flp-In-CHO cells,from which positive clones were obtained by pressure screening with 400μg/ml hygromycin B and identified for genome,mRNA,expression and protein activity by PCR,RT-PCR,Western blot and PT respectively.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pcDNA5/FRT/TO-FⅦwas constructed correctly.Nine hygromycin B-resistant clones were screened.FⅦgene was integrated to the genome of Flp-In-CHO cells and expressed.The expressed product showed the same coagulation activity as that of plasma FⅦ.Conclusion hFⅦwas successfully expressed in Flp-In-CHO cells by using site specific expression system Flp/FRT,which laid a foundation of preparation and purification of hFⅦ.

关 键 词：人凝血因子Ⅶ 位点特异性重组 Flp—In—CHO细胞 

分 类 号：R818.052.4[医药卫生—放射医学] Q786[医药卫生—临床医学]