金黄色葡萄球菌IsdD基因的克隆与表达

金黄色葡萄球菌是一种重要的人畜致病菌,IsdD属于金黄色葡萄球菌Isd系统中的重要成员。本研究根据GenBank报道的金黄色葡萄球菌IsdD基因序列设计一对特异性引物,以金黄色葡萄球菌基因组DNA为模板扩增出目的片段IsdD,将其用NcoⅠ和XhoⅠ双酶切后连接到载体pET32a(+)上,构建重组质粒pET32a(+)-IsdD。序列分析结果显示,目的基因序列与网上的IsdD序列相比核苷酸序列和氨基酸序列同源性均在98%以上。将鉴定正确的重组质粒转化到大肠杆菌E.coli BL21中并成功诱导表达出大小为61.3 ku的蛋白,这为下一步研究IsdD蛋白的结构和功能奠定了良好基础。

Cloning and Expression of lsdD gene from Staphylococcus aureus

Staphylococcus aureus causes a wide spectrum of human and animal diseases,including skin and soft-tissue infections and infectious endocarditis. IsdD gene is one of the important members of iron-regulated surface determinant (Isd) system from S.aureus. According to the sequence of IsdD gene published in GenBank,a pair of specific primers was designed and synthesized.Using the total genome DNA from S.aureus Wood46 strain as template,IsdD coding sequences were amplified by PCR method. The PCR product which had been digested by NcoⅠ and XhoⅠ restriction endonucleases was inserted into pET32a(+) vector subsequently. The recombinant plasmids,designated as pET32a(+)-IsdD,were transformed into E.coli BL21. The sequence analysis results showed the homologies of both nucleoside sequence of target gene and its deduced amino acid sequence to compare with standard sequence of IsdD could reach above 98%. SDS-PAGE analysis results indicated the recombinant IsdD protein of 61.3 ku was successfully expressed by E.coli BL21 induced with IPTG. The results obtained in this report provide important basis for further study on structure and function of the IsdD protein.