【摘要】 目的构建携带livin基因短片段发卡RNA(livin shRNA)的慢病毒载体。方法针对已经筛选确定的干扰人livin基因的有效靶序列,设计、合成靶序列的寡聚脱氧核苷酸DNA序列(Oligo DNA),退火形成双链DNA,与经Hpa I和Xho I酶切后的携带U6启动子和绿色荧光蛋白的pGCL-GFP载体连接产生短片段发卡RNA慢病毒载体,PCR筛选阳性克隆,测序鉴定。结果PCR鉴定与DNA测序证实合成的含livin shRNA慢病毒载体寡核苷酸链插入正确。结论成功构建人livin shRNA慢病毒载体。更多还原

【Abstract】 Objective To construct a lentiviral vector for RNA interference of human livin gene.Methods The effective target sequence of livin gene was confirmed in our previous study.The Oligo DNA containing both sense and antisense chains of the target sequences was designed,synthesized,reannealed to double-stranded DNA and cloned into the pGCL-GFP vector,which contained U6 promoter and green fluorescent protein(GFP).A lentiviral vector which expressed short hairpin RNA(shRNA)was then constructed and was identified by PCR and DNA sequencing.Results PCR identification and DNA sequencing demonstrated that the oligonucleotide were correctly inserted into the lentiviral vector.Conclusion The lentiviral-mediated shRNA vector for human livin gene is constructed successfully.更多还原