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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 construct a eukaryotic expression plasmid containing B domain-deleted human FⅧ (BDDhFⅧ) with deletion of B region and transfected into HepG2 cells to stably express human factor Ⅷ. METHODS: BDDhFVIII gene fragment was inserted into pcDNA4 / v5-his empty vector to construct a recombinant eukaryotic expression plasmid. After correct sequencing, HepG2 cells were transformed into HepG2 cells and purified by Ni-NTA. The expression of coagulation factor Ⅷ in HepG2 cells was detected by Western blot , Continuous culture to obtain stable expression of BDDhF Ⅷ protein cell lines. Results: The recombinant plasmid pcDNA4 / v5-his-BDDhFⅧ was successfully constructed by restriction enzyme digestion and sequencing. After transfected into HepG2 cells, Western blot confirmed that human factor Ⅷ could be correctly expressed in HepG2 cells. Conclusion: The stable cell line of human factor Ⅷ is successfully constructed, and the target protein can be expressed in HepG2 cells.