黄杆菌肝素酶Ⅱ的克隆与表达

Cloning and Expression of Flavobacteriun heparinum HeparinaseⅡ

目的克隆并表达黄杆菌肝素酶Ⅱ(HepⅡ)的基因。方法通过PCR从黄杆菌基因组DNA中扩增出黄杆菌HepⅡ的基因,经验证后插入到表达质粒pET-28a上构建表达载体,重组质粒转化E.coli 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HepⅡ基因构建入pET-28a载体上,重组质粒转入E.coli BL21(DE3)后表达产物经SDS-PAGE凝胶电泳鉴定得到目的蛋白,测活显示重组HepⅡ具有活性。结论克隆并成功表达了黄杆菌HepⅡ基因。

Objective To clone and express the heparinase Ⅱ gene from Flavobacterium heparinum. Methods The heparinase Ⅱgene 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）. Results The recombined pET-28a with the PCR product of heparinase Ⅱ 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. The enzyme activity of the recombined heparinase Ⅱ was verified. Conclusion The heparinase Ⅱ gene from Flavobacterium heparinum has been cloned and expressed successfully.