SIRT2去乙酰化酶活性位点突变体的构建及活性鉴定  

Plasmid construction and deacetylase activity detection of SIRT2 enzyme mutants

作　　者：朱蕊 武幸濡 邸杰 许雅淇 谭克[1] 樊玉梅[1] ZHU Rui;WU Xing-ru;DI Jie;XU Ya-qi;TAN Ke;FAN Yu-mei(College of Life Science,Hebei Normal University,Shijiazhuang 050024,China)

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024

出　　处：《生物技术》2023年第2期135-142,共8页Biotechnology

基　　金：河北省自然科学基金项目(C2020205003);河北省大学生创新创业训练计划一般项目(S202110094022)。

摘　　要：[目的]构建人沉默信息调节因子2(silent information regulator 2,SIRT2)的去乙酰化酶活性位点突变体,并验证其去乙酰化酶活性。[方法]设计合成人SIRT2的酶活性关键位点(Q167、N168和H187)突变基因PCR引物。利用重叠延伸PCR技术,获得hSIRT2突变基因。利用分子克隆技术构建hSIRT2突变体。利用Western Blot技术检测hSIRT2突变体的表达及其去乙酰化酶活性。[结果]成功获得了hSIRT2的168、187、167/168双位点和167/168/187三位点突变基因,片段大小为1190 bp。所构建的突变基因重组质粒经DNA测序验证,显示成功插入。细胞内检测到清晰明确的突变体的表达信号。与野生型SIRT2相比,突变体的去乙酰化酶活性具有不同程度的改变。[结论]成功构建了hSIRT2的4种不同的去乙酰化酶活性位点突变体,其去乙酰化酶活性有不同程度降低。[Objective]To construct the eukaryotic expression vectors of human silence information regulator 2(hSIRT2)enzyme activity mutant genes and verify their deacetylase activity.[Method]The specific primers were designed based on the hSIRT2 gene specific deacetylase activity sites(Q167,N168 and H187).The human SIRT2 gene mutants at 168,187,dual locus 167/168 and three locus 167/168/187 were obtained by overlap extension PCR.hSIRT2 mutants were constructed by molecular cloning.Western Blot was used to detect the expression and deacetylase activity of hSIRT2 mutants.[Result]The mutated genes of dual locus 168,187,167/168 and 167/168/187 for hSIRT2 were obtained successfully,and the fragment size was 1190 bp.DNA sequencing showed that the sequences of the mutated genes were correct.Clear protein signals of the mutants were detected in the cells.hSIRT2 mutants showed different deacetylase activity compared with wild-type SIRT2.[Conclusion]Present study shows that four hSIRT2 mutants with different deacetylase activity are successfully constructed.

关 键 词：SIRT2 去乙酰化酶活性 重叠延伸PCR 分子克隆 

分 类 号：Q785[生物学—分子生物学]