SIRT7基因乙酰化活性缺失突变真核表达载体的构建

The construction of eukaryotic expression vectors for SIRT7 gene with acetylation activity deletion

目的构建SIRT7基因乙酰化活性缺失的定点突变真核表达载体。方法用RT-PCR法从293T细胞中扩增SIRT7基因,并克隆到真核载体pcDNA 3.1/myc-His（-）A上。采用Two-step PCR定点突变技术,构建SIRT7基因的H187Y突变质粒,经测序确证定点突变成功,再将SIRT7及突变载体转染293T细胞,Western blot检测融合蛋白表达。结果克隆出SIRT7基因,构建了真核载体pcDNA 3.1/myc-His-SIRT7,经Two-step PCR获得突变体pcDNA 3.1/myc-His-SIRT7H187Y。DNA测序结果表明,编码187位氨基酸的560-562位碱基由CAC突变为TAC,其他碱基均无突变。SIRT7和H187Y突变载体转染293T细胞后,可检测出myc-SIRT7融合蛋白表达。结论成功构建了SIRT7以及H187Y突变的真核表达载体。

Objective To construct eukaryotic expression vectors for SIRT7 gene with acetylation activity deletion induced by site directed mutagenesis.Methods SIRT7 gene was amplified by RT-PCR from 293 Tcells and cloned into the eukaryotic vector pcDNA 3.1/myc-His（-）A.Site directed mutagenesis method based on two-step PCR was performed to construct dominant negative（DN）mutants H187 Y.The point mutation was verified by DNA sequencing.The mammalian 293 Tcell was transfected with SIRT7 and the mutants.The expression of the fused proteins was determined by Western blot.Results SIRT7 gene was cloned and the eukaryotic vector pcDNA 3.1/myc-His-SIRT7 was constructed.The mutants were obtained by two-step PCR.DNA sequencing showed that CAC at 560-562 bps sites,which encoded the 187 amino acids,was changed to TAC.No mutation was found in other base pair sites of the recombinant plasmids.The fusion protein myc-SIRT7 was detectable by Western blot in 293 T cells transfected with the mutant vectors.Conclusion The eukaryotic expression vectors for SIRT7 and its mutantsH187 Y were successfully constructed.