目的克隆小鼠的SIrT5基因,及制备SIrT5多克隆抗体。方法提取小鼠肝细胞总rnA进行rT-PCr,PCr法扩增SIrT5基因,将此基因克隆至PET-28A载体,转化E.COlI bl21(dE3),经异丙-β-d-硫代吡喃半乳糖苷(IPTg)诱导表达后,利用nI 2+-nTA亲和层析柱纯化;纯化的目的蛋白免疫bAl/C小鼠后,收获血清并鉴定。结果成功构建了SIrT5原核表达载体,并能在宿主菌E.COlI bl21(dE3)中高效表达;利用纯化的蛋白制备了理想的多克隆抗体。结论利用分子克隆技术,获得了高纯度的SIrT5蛋白并制备了多克隆抗体,为进一步研究奠定了基础。Objective To construct prokaryotic expression vector of SIRT5and prepare its polyclonal antibody.Methods Total RNA isolated from mice liver cell was RT-PCR.Sirt5was amplified by RT-PCR and cloned into pET-28aplasmid,and the recombinant plasmid was transformed into E.coli BL21(DE3).After induction by IPTG,the fusion protein of SIRT5was produced,the protein was purified by Ni 2+-NTA afinity chromatography.Bal/c mice were immuned with the purified protein of SIRT5,and mice serum was collected for identification.Results SIRT5was successfully constructed and highly expressed in E.coli BL21(DE3).The purified protein was used to prepare polyclonal antibody.Conclusion Using moleccular cloning techonlogy,purified SIRT5protein can be obtained and prepared its polyclonal antibody,which may provide an foundation for further investigation
