定点突变内皮抑素Zn2+的结合位点及突变基因的克隆表达

从人胚肝组织中提取总RNA， 以逆转录聚合酶链式反应（RT-PCR）法获得人内皮抑素编码序列， 采用定点突变技术将His2和His4双突变为Leu2和Val4. 将突变基因cDNA插入含有T7启动子的质粒pET-28b中构建表达质粒pMendo， 转化大肠杆菌BL21(DE3)， 筛选表达菌株BL21-Mute， 表达菌株经IPTG诱导后以包涵体方式产生大量内皮抑素突变蛋白. SDS-PAGE分析表明， 表达的重组蛋白占菌株可溶性蛋白质的30%. 复性、 纯化的内皮抑素突变蛋白纯度达到98%， 失去抑制人脐静脉内皮细胞增殖的活性.

Site Directed Mutation on Zn2 + Binding Site of Endostatin and Cloning, Expression of Mutant Gene

Human endostatin cDNA was cloned from total RNA of human embryo liver tissue by RT-PCR. (Endostatin) DNA sequence encoded His2 and His4 were double changed to Leu2 and Val4 using site directed mutation technique. The mutant endostatin cDNA was inserted into the pET28b containing promoter T7. The recombinant plasmid transformed the E.coli BL21(DE3). Recombinant human endostatin was highly (expressed) as inclusion body when the expression strain BL21-Mute was induced with IPTG. The result of (SDS-PAGE) analysis reveals that recombinant mutant endostatin accounts for up to 30% of soluble protein in E.coli. The mutant endostatin was purified and refolded farther, its purity was up to 98%. Recombinant (mutant) endostatin was inactive in inhibiting endothelial cell proliferation.