重组人凝血因子Ⅷ表达质粒的构建及其在HepG2细胞中的表达  

Construction and expression of the recombinant plasmid containing BDDhFⅧ in HepG2 cells

作　　者：赵倩[1] 解金辉[2] 李双玉[2] 董磊[2] 种靖慧[2] 闫莉娜[2] 刘运德[1] 袁玉华[2] 

机构地区：[1]天津医科大学医学检验学院,天津300203 [2]天津市血液中心,天津300110

出　　处：《中国应用生理学杂志》2012年第3期259-262,共4页Chinese Journal of Applied Physiology

基　　金：天津市卫生局科技基金资助项目(09KY33)

摘　　要：目的:构建含有B区缺失型(△760aa-1639aa)人凝血因子Ⅷ(B domain-deleted human FⅧ,BDDhFⅧ)的真核表达质粒,转染HepG2细胞使其稳定表达人凝血因子Ⅷ。方法:将BDDhFVIII基因片段插入pcDNA4/v5-his空载体中构建重组真核表达质粒,测序正确后电转入HepG2细胞,经Ni-NTA纯化,利用Western blot检测凝血因子Ⅷ在HepG2细胞中的表达,持续培养获得稳定表达BDDhFⅧ蛋白的细胞株。结果:经限制性酶切和测序鉴定均证实重组真核表达质粒pcDNA4/v5-his-BDDhFⅧ成功构建,在转染HepG2细胞后,Western blot检测证实人凝血因子Ⅷ可以在HepG2细胞中正确表达。结论:成功构建了人凝血因子Ⅷ的稳定细胞株,并能在HepG2细胞表达目的蛋白。Objective： To get stable cell line expressing B domain-deleted human FVIII （BDDhFVIII） by constructing the eukaryotic ex- pression plasmid. Methods： Eukaryotic expression plasmid containing BDDhFVIII was constructed and transfected into HepG2 cells via elec- troporation. The expression and purification of the target protein was detected by Western blot. Results： Results of enzyme digestion and se- quence analysis demonstrated that the gene of BDDhFVIII was correctly inserted into the eukaryotic expression vector pcDNA4/vS-his. Western blot confirmed the successful expression of BDDhFVIII at the protein levels in HepG2 cells. Conclusion： The constructed eukaryotic expression vector was able to generate high level expression of human FVIII in HepG2 cells, thus could construct human blood coagulation FVIII stable cell line successfully.

关 键 词：B区缺失型人凝血因子VIII 重组真核表达质粒 重组凝血因子VIII 血友病A 

分 类 号：Q782[生物学—分子生物学]