凝血因子Ⅶ基因表达载体的构建及其在BHK细胞中的表达

Construction of Coagulant Factor Ⅶ Gene Expression Vector and Expression in BHK Cell Line

凝血因子Ⅶ是凝血块形成的起始关键分子之一,它对外源性凝血途径的启动具有重要的作用。为表达具有促凝活性的重组人凝血因子Ⅶ,通过PCR的方法从质粒pUC-FⅦ中扩增人凝血因子ⅦcDNA,并将其定向克隆入真核表达载体pIRESneo中,构建重组表达载体pIRES-FⅦ,测序正确后用脂质体介导的方法转染BHK-21细胞,经G418加压筛选、细胞有限稀释等方法获得克隆细胞株,收集无皿清培养上清,进行SDS-PAGE、Western blot和活性鉴定。结果成功构建了重组表达载体pIRES-FⅦ,实现了其在BHK-21细胞中的表达,且表达产物具有促凝活性。重组人凝血因子Ⅶ在哺乳动物细胞中的成功表达,为整体止血剂的研究奠定了基础。

In order to achieve the expression of recombinant human coagulant factor Ⅶ (rhF Ⅶ) in mammalian cell lines and to study its biological activity. The hF Ⅶ cDNA was amplified using PCR method, and the expression vector was constructed by subcloning method. After identification by DNA sequencing and obtaining a correct recombinant vector, the recombinant expression vector pIRES-F Ⅶ was transfected into BHK-21 cell line by means of lipofectin procedure. The resultant transfected cells were selected by G418.The positive cell clones were cultured and the culture supernatants were collected for identification by SDS-PAGE, Western blot and bioassays. The results showed that the bioactive rhF Ⅶ was expressed in BHK-21 cell line, and laid a foundation for further research.