贵州医科大学学报

2015, v.40;No.178(07) 683-687

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HDAC2基因RNA干扰载体的构建
Construction and Identification of HDAC2 Gene RNA Interference Vector

刘燕青;黄健;黄韻祝;娄方方;孔维莹;
LIU Yanqing;HUANG Jian;HUANG Yunzhu;LOU Fangfang;KONG Weiying;Department of Medical Laboratory,Guizhou Medical University;Department of Biochemistry,Affiliated Hospital of Guizhou Medical University;

摘要(Abstract):

目的:构建组蛋白去乙酰化酶2(HDAC2)基因RNA慢病毒干扰质粒载体。方法:对比HDAC2基因序列,设计并合成短发夹RNA(shRNA)干扰靶点序列,克隆入经Age I与EcoRI双酶切后的RNA干扰载体p LKO.1-puro,构建p LKO.1-HDAC2-shRNA重组质粒,采用酶切法和测序法验证重组质粒。结果:成功退火合成3对短发夹RNA序列并将其克隆入p LKO.1-puro载体中,构建重组质粒p LKO.1-HDAC2-shRNA1、p LKO.1-HDAC2-shRNA2及p LKO.1-HDAC2-shRNA3,经酶切鉴定和测序验证重组质粒载体与预期靶序列相同。结论:成功构建HDAC2-shRNA慢病毒干扰载体,为进一步研究HDAC2的功能及应用提供有效工具。
Objective: To construct a lentivirus vector for RNA interference of HDAC2 gene. Methods: The shRNA interference sequences targeting human HDAC2 gene were designed,synthesized,and then ligated with the linearized p LKO. 1-puro vector which had been digested with the Age I and EcoRI,then the p LKO. 1-HDAC2-shRNA recombinant lentiviral vectors were constructed and confirmed by enzyme digestion and DNA sequencing. Results: 3 pairs of synthesized short hairpin RNA sequences were successfully annealed and cloned into p LKO. 1-puro vector to construct recombinant plasmid p LKO. 1-HDAC2-shRNA1,p LKO. 1-HDAC2-shRNA2,p LKO. 1-HDAC2-shRNA3. Enzyme digestion and DNA sequencing confirmed that the sequence in the recombinant plasmid vector was the same as the target sequence,which meaned three shRNA recmbinant lentiviral plasmid vectors were successfully constructed. Conclusion: Three shRNA recmbinant lentiviral plasmid vectors are successfully constructed and provide an effective tool for further study of function and clinical application of HDAC2 gene.

关键词(KeyWords): 组蛋白去乙酰化酶2;RNA干扰;慢病毒载体;质粒;DNA,重组
histone deacetylase 2;RNA interference;lentivirus vector;plasmid;DNA,recommbinant

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基金项目(Foundation): 贵州省联合基金[黔科合LH字(2014)7114]

作者(Author): 刘燕青;黄健;黄韻祝;娄方方;孔维莹;
LIU Yanqing;HUANG Jian;HUANG Yunzhu;LOU Fangfang;KONG Weiying;Department of Medical Laboratory,Guizhou Medical University;Department of Biochemistry,Affiliated Hospital of Guizhou Medical University;

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