摘要
利用体外特定点突变技术,将原核生物核糖体结合位点及十碱基间隔子(spacer)引科兔肝经胞色素b_5 cDNA的5′端,并构建了一个兔红血球b_5cDNA克隆,重组入表达载体pKK223-3质粒中,在大肠杆菌中成功地表达了兔肝和红血球两种形式的b_5蛋白。产量分别为每毫升培养液1.5μg和4.5μg。
By using site-directed mutagenesis, rabbit liver cytochrome b5 mRNA was modified for the in vitro expression of both liver and erythrocyte cytochrome b5s.A prokaryotic ribosome binding site 10 base pair from the start codcn ATG and a premature stop codon at amino acid position 99 were introduced.Using guinea pig antibody against rabbit liver cytochrome b5 we monitored the synthesis or both form of b5s on western blotting after inducing the expression vectors with IPTG The inducib-le level were 1.5********ug(liver)and 4.5*******ug(erythrocyte)b5/mL of culture.
关键词
细胞色素B5
特定位点突变
基因表达
Cytochrome b5
Site-directed mutagenesis
in vitro gene expression
