小鼠CHD5基因siRNA重组腺病毒载体的构建及鉴定

Construction and Identification of a siRNA Recombinant Adenovirus Vector for Mouse Chromodomain Helicase DNA Binding Domain 5 Gene

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.

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 was then transfected into HEK293 cells for packing of Adr-CHD5siRNA virus.The expression of CHD5 was determined by RT-PCR and western blot after infection of Adr-simCHD5 in NIH3T3 cells for evaluating the inhibitive effect.Results： The Adr-CHD5siRNA obtained was shown to have a high titer and reduce the expression of CHD5 determined by RT-PCR and western blot after infection.Conclusion： The recombinant adenovirus Adr-CHD5siRNA was successfully constructed and expressed in NIH3T3 cells,which could provide a basis for further research on the function and mechanism of CHD5.