摘要
Background Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. It is converted to factor VIIa that plays an important role in the coagulation cascade. The aim of this study was isolating and cloning the genes of human factor VII and hepsin and subsequent co-transfection of the constructs to Chinese hamster ovary (CHO) cell line to obtain rFVIIa.
Methods Factor VII and hepsin cDNAs were isolated from HepG2 cell line and cloned into pcDNA3·1 (+) vector. The constructs were co-trasfected to CHO cell line. A cell line that permanently expressed recombinant factor VII (rFVII) and hepsin was established. The expression of rFVII was confirmed by reverse transcriptase–polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot analysis. Biological activity of rFVII was evaluated by prothrombin time assay.
Results The results showed that the genes of FVII and hepsin were successfully cloned and expressed. Stable CHO cells co-transfected with pcNDA3·1-FVII and pcNDA3·1-hepsin expressed FVII and hepsin mRNA, but there was no expression in the CHO cells transfected with insert free pcDNA3·1. FVIIa protein was secreted to medium of CHO cells co-transfected with pcNDA3·1-FVII and pcNDA3·1-hepsin. The expected band of rFVII was detected in Western blot analysis. A three- to fourfold decrease in clotting time was observed when human FVII-depleted plasma was used in combination with human thromboplastin in the presence of rFVII, confirming the biological activity of rFVII.
Conclusion As we are aware, this is the first report of establishing a cell line expressing FVIIa using genetic engineering methods.
摘要译文
背景因子VII（FVII）是一种血浆糖蛋白，参与凝血过程，导致产生纤维蛋白。它被转换成因子Ⅶa播放在凝血级联中起重要作用。本研究的目的是分离和克隆人因子VII和hepsin和随后的共转染的构建体，以中国仓鼠卵巢（CHO）细胞系的基因来获得的rFVIIa。方法因子VII和hepsin的cDNA从HepG2细胞中分离，并克隆入载体pcDNA3·1（）载体中。所述构建体共同trasfected到CHO细胞系。永久地表达的重组因子VII（rFVII）和hepsin的细胞系正式成立。 rFVII的表达通过逆转录酶 - 聚合酶链反应证实，酶联免疫吸附试验和Western blot分析。 rFVII的生物活性，通过凝血酶原时间测定法进行评估。结果结果表明，FVII和hepsin的基因成功地克隆和表达。稳定CHO细胞共转染pcNDA3·1-FⅦ和pcNDA3·1- hepsin表达的FVII和hepsin的mRNA，但没有表达在CHO细胞转染了插入免费的pcDNA3·1。FVIIa的蛋白被分泌到CHO细胞共转染pcNDA3·1-FⅦ和pcNDA3·1- hepsin的介质。在Western印迹分析法检测rFVII的预期条带。的三轮中凝血时间四倍下降时观察到人FVII的血浆被用于与人凝血活酶组合在rFVII的存在下，确认rFVII的生物活性。结论正如我们所知，这是建立利用基因工程方法表达的FVIIa的细胞系的第一份报告。
R. Halabian[1]; M. H. Roudkenar[1]; N. S. Esmaeili[1]; N. Masroori[1]; A. M. Roushandeh[2] and A. J. Najafabadi[3];. Establishment of a cell line expressing recombinant factor VII and its subsequent conversion to active form FVIIa through hepsin by genetic engineering method[J]. Vox Sanguinis, 2009,96(4): 309-315