大鼠Sirt1基因原核表达质粒的构建及融合蛋白表达

目的构建大鼠Sirt1基因重组原核表达质粒pET41-Sirt1,表达其重组蛋白,为进一步研究Sirt1在哺乳动物细胞内的作用奠定基础。方法根据GenBank中大鼠Sirt1基因序列设计引物,用RT-PCR扩增得到含BamHI/XhoI酶切位点的Sirt1片段,克隆至pGEM-Teasy载体后亚克隆至原核表达载体pET41,测序鉴定后,转化宿主菌进行表达、蛋白纯化,SDS-PAGE鉴定。结果从大鼠脑的mRNA中扩增出特异Sirt1基因片段长506bp,经酶切鉴定及DNA序列测定,证实pET41-Sirt1重组质粒构建正确,表达融合蛋白分子量约为48kD。结论成功构建重组原核表达质粒pET41-Sirt1,并能表达融合蛋白,其分子量大小与预期一致。

Construction of prokaryotic expression plasmid with rat Sirt 1 gene and fusion protein expression

Objective To construct a recombinant expression plasmid with rat Sirt1 cDNA and to obtain the fusion protein.Methods A fragment of rat Sirt1 cDNA containing BamHI/XhoI were amplified by RT-PCR.The fragment was cloned into pGEM-T easy vector and subcloned into pET41 vector.Subsequently,E.coli BL21 cells were transformed by the recombinant plasmid.The fusion protein,which was induced by IPTG was purified and was analyzed by SDS-PAGE.Results A fragment of 506 bp of rau Sirt1 cDNA was amplified by RT-PCR.The construction of the recombinant plasmid pET41-Sirt1 was confirmed by sequencing.The fusion protein of 48 kD was expressed and purified.Conclusions The recombinant plasmid has been successfully constructed.The fusion protein of rat Sirt1 can be expressed in E.coli BL21 cells with the expected molecular weight.