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

[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.