利用双顺反子表达质粒在CHO细胞中有效表达人补体1抑制物

利用基因工程手段获取编码人补体1抑制物（C1-inhibitor，C1-INH）的基因片段，将其插入双顺反子表达载体pED中，构建成功可在CHO细胞中有效表达人C1-INH的表达质粒。采用Lipofectin法将其转染二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞（CHO-dhfr-），获得有效表达人C1-INH的CHO-dhfr 细胞克隆。经Northern印迹、免疫及Western印迹等试验检测证实，重组的细胞克隆可以表达人C1-INH，特异性ELISA检测其表达水平在0.4μg／ml左右。重组C1-INH活性检测采用酯酶裂解抑制法。

Efficient Expression of Human C1-inhibitor in CHO Cells by Using a Dicistronic Expression Vector

The full-length cDNA fragment encoding human C1-INH was obtained by gene recombination techniques and a stable expression plasmid was constructed by inserting the human C1-INH cDNA into an efficient dicistronic expression vector pED. This plasmid was transfected into DHFR-deficient Chinese Hamster Ovary cells (CHO-dhfr-) by Lipofectin method, and the positive CHO-dhfr+ cell clones which stably express C1-INH was generated. Expression of C1-INH mRNA was detected by Northern blot. Expression of C1-INH protein was confirmed by immunoprecipitation and Western blot analysis. The level of expressed C1-INH was estimated at 0.4 microgram/ml by specific ELISA techniques. The biological activity of recombinant C1-INH was assayed by using the inhibition of C1 estrolytic activity.