摘要
目的:构建葡激酶(Staphylokinase,SAK)基因的原核表达质粒,在大肠杆菌中表达SAK蛋白质,纯化并初步鉴定其生物学活性。方法:基于生物信息学方法分析优化基因序列,使用基因合成技术合成SAK基因,转化感受态大肠杆菌B834菌株,使在其中表达,利用离子交换柱,分子筛(Superdex 75)等进行分离纯化,应用纤维蛋白平板溶圈法测定评价其生物活性。结果:SAK在大肠杆菌中以可溶上清的方式高效表达,利用离子交换柱和分子筛等纯化效果良好,纯度达95%,体外实验显示其纤溶活性与尿激酶标准品相当。结论:应用全基因合成经过优化的基因可在大肠杆菌系统中高效上清表达具有较高纤溶活性的SAK。
Objective：To construct the expression vector of human staphylokinase（SAK） gene,express SAK protein in E.coli,purify recombinant SAK protein and to verify its bioactivity.Methods：On the basis of bioinformatics,gene sequence was analyzed and optimized,SAK plasmid was constructed by gene synthesize methods and the protein was expressed by B834 strain.The recombinant protein was purified by ion exchange coloum（DEAE） and size exclusion chromatography（Superdex 75）.Soluble fibrin plate method was used to characterize its fibrinolytic activity.Results：The recombinant SAK was highly expressed in soluble supernatant by E coli system.The recombinant protein with 95% purity was obtained by using of DEAE and Superdex 75.Experiments in vitro revealed that the fibrinolytic activity of SAK was approximately equal to that of urokinase.Conclusion：The recombinant SAK with high fibrinolytic activity can be highly expressed in E coli system by utilizing sequence optimizing and gene synthesize.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2012年第3期232-235,共4页
Journal of Chongqing Medical University
基金
重庆市科委资助项目(编号:2009BB5406)
关键词
葡激酶
表达纯化
体外活性
staphylokinase
expression and purification
activity in vitro
作者简介
杨可（1986-），女，硕士，研究方向：冠脉溶栓治疗。
通信作者：周建中，男，教授，Email：zhou_jianzhong27@yahoo．com．cn。
