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小鼠CHD5基因siRNA重组腺病毒载体的构建及鉴定
引用本文:陈妮,左国伟,潘玥,赖国旗,杨根岭,向延秀,韩建红,黄云.小鼠CHD5基因siRNA重组腺病毒载体的构建及鉴定[J].西南农业大学学报,2011,33(8).
作者姓名:陈妮  左国伟  潘玥  赖国旗  杨根岭  向延秀  韩建红  黄云
作者单位:重庆医科大学实验动物中心;重庆医科大学医学检验系;重庆医科大学感染性疾病分子生物学教育部重点实验室;重庆医科大学附属第一医院分子肿瘤及表观遗传学实验室;泸州医学院实验动物科;泸州市畜牧局;
基金项目:国家自然科学基金资助项目(81072148/H1622)
摘    要:目的:构建小鼠染色质区解旋酶DNA结合蛋白5(CHD5)基因干扰RNA(small interfering RNA,siRNA)重组腺病毒载体并在NIH3T3细胞中验证其干扰作用.方法:人工合成靶向CHD5的siRNA干扰序列,用分子克隆的方法插入到穿梭载体pSES-HUS上,并与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,得到pAdeasy-SES-HUS-CHD5siRNA重组质粒,在HEK293细胞中包装成重组腺病毒Adr-simCHD5,然后感染NIH3T3细胞,用RT-PCR和Western Blot方法检测其对CHD5mRNA和蛋白表达的干扰效果.结果:得到高滴度Adr-CHD5siRNA重组腺病毒,RT-PCR和Western Blot结果表明该重组腺病毒能有效地降低CHD5基因的表达.结论:成功构建Adr-CHD5siRNA重组腺病毒载体,并在NIH3T3细胞上实现CHD5基因表达抑制,为进一步研究CHD5基因功能奠定了基础.

关 键 词:染色质区解旋酶DNA结合蛋白5(CHD5)  RNA干扰  腺病毒载体  

Construction and Identification of a siRNA Recombinant Adenovirus Vector for Mouse Chromodomain Helicase DNA Binding Domain 5 Gene
CHEN Ni,ZUO Guo-wei,PAN Yue,LAI Guo-qi,YANG Gen-ling,XIANG Ting-xiu,HAN Jian-hong,HUANG Yun.Construction and Identification of a siRNA Recombinant Adenovirus Vector for Mouse Chromodomain Helicase DNA Binding Domain 5 Gene[J].Journal of Southwest Agricultural University,2011,33(8).
Authors:CHEN Ni  ZUO Guo-wei  PAN Yue  LAI Guo-qi    YANG Gen-ling  XIANG Ting-xiu  HAN Jian-hong  HUANG Yun
Institution:CHEN Ni1,ZUO Guo-wei2,PAN Yue1,LAI Guo-qi1,3,YANG Gen-ling1,XIANG Ting-xiu4,HAN Jian-hong5,HUANG Yun6 1.Center of Experimental Animals,Chongqing Medical University,Chongqing 400016,China,2.Department of Laboratory Medicine,3.Key Laboratory of Infectious Disease Molecular Biology of Ministry Education,4.Molecular Oncology and Epigenetics Laboratory of the First Affiliated Hospital,Chongqing Medical Unive...
Abstract:Objective: To construct a recombinant adenovirus carrying a short interfering RNA(siRNA) targeting CHD5d(chromodomain helicase DNA binding domain 5) gene and indentify its inhibitive effect in NIH3T3 cells.Methods: The siRNA sequences targeting CHD5 gene were synthesized and cloned into the shuttle plasmid pSES-HUS and then homogenously recombined with the adenovirus backbone plasmid pAdeasy-1 in E.coli BJ5183 to generate the recombinant adenoviral plasmid pAdeasy-SES-HUS-CHD5siRNA.pAdeasy-SES-HUS-CHD5siRNA...
Keywords:chromodomain helicase DNA binding domain 5  RNA interference  adenovirus vector  
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