人凋亡相关因子基因RNA干扰慢病毒载体的构建与包装

目的:构建人凋亡相关因子(Fas)基因短发夹RNA慢病毒载体,实现沉默人脐带间充质干细胞Fas基因的表达.方法:根据Fas基因mRNA序列,设计4组靶向Fas基因的短发夹RNA序列,合成、退火形成双链DNA片段,与经过DNA内切酶BamHⅠ、EcoRⅠ双酶切后的LV3载体连接转入感受态细胞,对长出的克隆进行菌落PCR鉴定,并对阳性克隆进行测序及比对分析.通过转染293T细胞对慢病毒进行包装和滴度测定.结果:经酶切及基因测序鉴定证明慢病毒载体构建成功,通过荧光显微镜观察证明病毒转入293T细胞,感染效率 > 90%,并获得高滴度的慢病毒载体,病毒滴度为3×108TU/ml.结论:成功构建人Fas基因RNA干扰慢病毒载体,为下一步转染脐带间充质干细胞提供理论参考.

Construction and packaging of the recombination lentivirus vector with RNA interference gene targeting human Fas

Objective:To construct the recombination lentivirus vector with short hairpin RNA gene targeting human factor associated suicide(Fas), and silence the Fas gene expression in umbilical cord mesenchymal stem cells.Methods:According to Fas gene mRNA sequence, four groups of the short hairpin RNA gene sequence targeting Fas were designed, synthesised and annealed to form double-stranded DNA fragments, which was inserted the LV3 vector digested by BamHⅠ and EcoRⅠ enzyme, then it was transfected into the competent cells.The positive clones were identified by PCR, and analyzed by sequencing.The packaging and titer of lentivirus were determined after transfecting 293T cells.Results:Lentiviral vector was constructed successfully by the identification of restriction enzyme digestion and gene sequencing.The lentivirus vector was transfected into 293 T cells using the fluorescence microscope observation, the infection efficiency of which was more than 90%.The high titer of Lentiviral vector was obtained, which was 3×108 TU/ml.Conclusions:The recombination lentivirus vector with interference gene targeting human Fas is successfully constructed, which can provide the basis of transfecting umbilical cord mesenchymal stem cells.