黄杆菌肝素酶I基因的克隆与表达

Cloning and Expression of Flavobacterium heparinum Heparinase I Gene

目的克隆并表达黄杆菌肝素酶I(HepI)基因。方法通过PCR从黄杆菌基因组DNA中扩增黄杆菌HepI基因,验证后插入到表达质粒pET-22b中构建表达载体,重组质粒转化大肠杆菌BL21(DE3)进行蛋白质表达,并用Azure A法测定酶活性。结果PCR扩增得到序列正确的HepI基因,并将该基因成功插入到pET-22b表达载体中。重组质粒转入E.coli BL21(DE3)后表达的蛋白质经SDS-PAGE分析,与目的蛋白质的相对分子质量基本一致,其活性约为50 U/LA600。结论克隆并成功表达了黄杆菌HepI基因。

Objective To clone and express the heparinase Ⅰgene from Flavobacterium heparinum. Methods The heparinase Ⅰ gene was amplified with PCR from the genomic DNA of Flavobacterium heparinum, and it was linked into the expression plasmid pET-22b after sequencing, then, the recombinant plasmid was expressed in E.coli BL21 （DE3） and the enzyme activity of recombinant heparinase Ⅰ was verified by Azure A assay. Results The recombi- nant pET-22b with the PCR product of heparinase I which was confirmed by DNA sequencing was successfully constructed. The expression product of recombinant plasmid pET-22b in E.coli BL21 （DE3） was tested by SDSPAGE electrophoresis. The enzyme activity of recombinant heparinase Ⅰ was 50 U/LA600. Conclusion The heparinase Ⅰ gene from Flavobacterium heparinum can be cloned and expressed successfully.