人凝血因子Ⅶ在CHO/DG44细胞中的表达

The expression of human coagulation factor Ⅶ in CHO/DG44 cell

PCR法扩增FⅦ基因,构建重组表达质粒FⅦ-pOptiVEC,酶切、测序鉴定.脂质体法将FⅦ-pOptiVEC质粒转入CHO/DG44细胞,并用氨甲喋呤(MTX)进行分级加压筛选,获得高表达重组FⅦ蛋白的阳性细胞克隆.亲和层析法纯化FⅦ,并通过Western-blot检测蛋白表达情况.采用FⅦ促凝活性检测试剂盒(凝固法)检测FⅦ的凝血活性.结果表明筛选出的细胞克隆能稳定表达目的蛋白,MTX加压使其表达量明显升高.促凝活性检测结果证明获得的FⅦ蛋白具有凝血活性.

FⅦ gene was amplified by PCR and inserted into pOptiVEC vector. Then the recombinant plasmid was transfected into CHO/DG44 cells after being identified by enzyme digestion and sequencing. The obtained positive clones were further screened at different concentrations of MTX. FⅦ protein was purified by affinity chromatography and confirmed by Western-blot and PT. The Results showed that the recombinant plasmid was construct correctly and the positive cell clones could steadily express F Ⅶ which had the coagulation activity.