摘要
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。
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 μg/ml by specific ELISA techniques. The biological activity of recombinant C1-INH was assayed by using the inhibition of C1 estrolytic activity.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
1997年第1期41-47,共7页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金!39270620
关键词
补体1抑制物
基因表达
双顺反子
CHO细胞
C1-inhibitor
gene expression
dicistronic
expression vector
CHO cells
