肝素黄杆菌2-O-sulfatase基因的克隆与表达  

Cloning and Expression of Flavobacterium heparinum 2-O-sulfatase

作　　者：余永莉[1] 陈敬华 郑化[1] 

机构地区：[1]武汉理工大学化学工程学院,湖北武汉430070 [2]江南大学医药学院,江苏无锡214122

出　　处：《安徽农业科学》2011年第12期7286-7288,共3页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金项目(50873080)

摘　　要：[目的]研究肝素黄杆菌2-O-sulfatase基因的克隆与表达。[方法]以肝素黄肝菌为模板,通过PCR从肝素黄杆菌基因组DNA中扩增2-O-sulfatase的基因,测序正确后插入到表达质粒PET-28a(+)上构建表达载体,重组质粒转化E.coil BL21(DE3)进行蛋白质表达。[结果]成功地将PCR扩增得到的测序正确的2-O-sulfatase基因构建入PET-28a(+)载体上,重组质粒转入E.coil BL21(DE3)后,表达产物经SDS-PAGE凝胶电泳鉴定得到目的蛋白。[结论]克隆并成功表达了黄杆菌2-O-sulfatase基因,为分析肝素多糖分子及其降解产物结构奠定了基础。[Objective] To clone and express 2-O-sulfatase gene from Flavobacterium heparinum.[Method] The 2-O-sulfatase gene was amplified with PCR from the genomic DNA of Flavobacterium heparinum,and it was linked into expression plasmid pET-28a after sequencing,then the recombined plasmid was expressed in E.coli BL21（DE3）.[Result] The recombined pET-28a（＋）with the PCR product of 2-O-sulfatase which was confirmed by DNA sequencing was successfully constructed.The expression product of the recombined plasmid pET-28a in E.coli BL21（DE3） was tested by SDS-PAGE electrophoresis.[Conclusion] The 2-O-sulfatase gene from Flavobacterium heparinum has been cloned and expressed successfully.These findings provide a framework that enables the use of 2-O-sulfatase as a tool for the determination of GAG and its degradation products＇ fine structure.

关 键 词：肝素黄杆菌 2-O-sulfatase 克隆 蛋白表达 纯化 

分 类 号：Q786[生物学—分子生物学]