CAJ | 학술논문

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 31．／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 31．／myc‐His‐SIRT7，经 Two‐step PCR获得突变体pcDNA 31．／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。
목적：구건SIRT7기인을선화활성결실적정점돌변진핵표체재체。방법용RT‐PCR법종293T세포중확증SIRT7기인，병극륭도진핵재체 pcDNA 31．／myc‐His（－）A상。채용 Two‐step PCR정점돌변기술，구건SIRT7기인적 H187Y돌변질립，경측서학증정점돌변성공，재장SIRT7급돌변재체전염293T세포，Western blot검측융합단백표체。결과극륭출SIRT7기인，구건료진핵재체pcDNA 31．／myc‐His‐SIRT7，경 Two‐step PCR획득돌변체pcDNA 31．／myc‐His‐SIRT7H187Y。DNA측서결과표명，편마187위안기산적560～562위감기유CAC돌변위TAC ，기타감기균무돌변。SIRT7화 H187Y돌변재체전염293T 세포후，가검측출myc‐SIRT7융합단백표체。결론성공구건료SIRT7이급 H187Y돌변적진핵표체재체。
Objective To construct eukaryotic expression vectors for SIRT 7 gene with acetylation activity deletion induced by site directed mutagenesis .Methods SIRT7 gene was amplified by RT‐PCR from 293T cells 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 H187Y . The point mutation was verified by DNA sequencing . The mammalian 293T cell 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 293T cells transfected with the mutant vectors . Conclusion The eukaryotic expression vectors for SIRT 7 and its mutants H187Y were successfully constructed .