Objective: To construct the HDAC2 deletion and point mutants with deacetylation inactivation using double-tube method mutant construction technology and detect SUMO E3 ligase activity of these mutants for investigating on the impact of HDAC2 SUMO E3 ligase on deactylayion function. Methods: Primers were designed for HDAC2 100-322 amino acid lack mutant and 142 amino acid point H→A mutant. HDAC2 pcDNA3.1 eukaryotic expression plasmid was used as the template. Corresponding single DNA in double-tube was amplified by heat efficiency Fusion enzyme. Mutant eukaryotic expression vectors were constructed by the following steps, including annealing, template enzyme cutting, purification of ethanol, screening with resistance flat and sequencing appraisal. The expression of mutants in cells were detected by western blotting and the sumolation E3 ligase activity was measured by LUC report gene. Results: Fragment lack mutant pcDNA3.1/HDAC2 DEL(100-322AA) and point mutant pcDNA3.1/HDAC2(142 H→A) were constructed successfully. The western blotting of C-myc detection showed that these mutants expressed in cells. LUC report gene results showed that the mutants still have the E3 ligase activity. Conclusion: The E3 sumolation ligase activity of HDAC2 had no effect on deacetylation enzyme function. C-terminus of the HDAC2 has the function of E3 ligase activity.%目的:针对人源HDAC2外显子拼接功能区(CDS)基因,运用双管法突变体构建技术,构建HDAC2去乙酰化酶功能失活的片段缺失与关键位点突变体,检测突变体的SUMO化修饰E3连接酶功能活性,探究此HDAC2新功能是否受到其固有的去乙酰化酶功能的影响.方法:设计HDAC2 100-322位氨基酸密码子缺失的片段突变体引物与142位H→A点突变体引物,利用HDAC2的pcDNA3.1真核表达质粒为模板,通过耐热Fusion酶PCR反应双管扩增相应的单链DNA,并经退火、模板酶切、乙醇纯化、感受态转化、抗性平板筛选、测序鉴定获得两种HDAC2去乙酰化酶功能失活的pcDNA3.1真核表达质粒.免疫印迹检测突变体质粒在细胞中的表达,荧光素酶(LUC)报告基因实验检测其SUMO化修饰E3连接酶功能活性.结果:成功构建HDAC2去乙酰化功能失活的片段缺失突变体pcDNA3.1/HDAC2 (DEL 100-322AA)与关键位点突变体pcDNA3.1/HDAC2(142 H→A).C-myc标签的免疫印迹检测显示两种突变体在细胞中可稳定表达.将突变体转染进入三种细胞,LUC报告基因分析结果显示突变体仍具有E3连接酶功能活性.结论:HDAC2的E3连接酶功能不依赖于其去乙酰化酶活性,并且E3连接酶功能结构域位于其序列C端.
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