猪SIRT1基因的克隆、表达及多克隆抗体制备

Cloning,expression and preparation of polyclonal antibody of porcine SIRT1 gene

根据GenBank中猪SIRT1mRNA序列设计引物,使用杜大长商品猪大脑RNA经RT-PCR扩增得到猪SIRT1基因全长表达序列,通过Ω-PCR将SIRT1蛋白末端抗原表位序列插入载体pET-28a(+),在大肠杆菌中表达并纯化得到45ku大小的蛋白,以其免疫新西兰大白兔并制备出抗体滴度达212的多克隆抗体。结果表明,成功制备出可以特异性识别猪SIRT1蛋白的抗体。

Based on the porcine SIRT1 mRNA sequence from GenBank,primers of porcine SIRT1 gene were designed and coding region of SIRT1 gene was amplified.The epitope sequences located on the rear section of SIRT1 protein were then inserted into pET-28a(+)byΩ-PCR.The SIRT1 protein was expressed in E.coli,purified and its polyclonal antibody was produced by immuning rabbit,the protein weight is about 45ku and the antibody titers is about 212.The results showed that antibody is capable of detecting the porcine SIRT1 protein specifically and provided a new tool for studies of the cellular path way involving in porcine SIRT1.