人THAP11基因RNAi慢病毒表达系统的建立

目的构建人THAP11基因RNA干扰（RNAi）慢病毒载体,制备高滴度病毒颗粒,敲低K562细胞和脐带血CD34＋细胞中THAP11表达。方法采用第三代慢病毒包装系统,将含有特异性干涉THAP11的DNA序列克隆入穿梭质粒pSicoR中,构建siTHAP11-pSicoR质粒。在脂质体介导下,siTHAP11-pSicoR与包装质粒pLP1,pLP2,pLP/VSVG共转染HEK293T细胞,获得高滴度慢病毒颗粒。感染K562细胞,检测内源THAP11敲低效果。感染人CD34＋细胞,流式分选GFP阳性细胞,检测原代细胞中THAP11的敲低效果。结果成功构建针对THAP11两个位点的RNAi慢病毒载体siTHAP11-1和siTHAP11-2并获得慢病毒颗粒,病毒滴度检测可达1.8×108TU/ml。感染K562细胞后建立稳定株,THAP11的蛋白水平和mRNA水平均有下调。感染CD34＋细胞效率达30%以上,siT-HAP11-1可下调THAP11mRNA约80%,siTHAP11-2约下调85%。结论成功构建THAP11的RNAi慢病毒载体,包装后的病毒颗粒可有效敲低细胞株及原代细胞中内源性THAP11的表达,为后续研究THAP11对CD34＋细胞的影响奠定基础。

Construction of lentiviral vectors containing THAPll RNAi and establishment of expression systems

Objective To prepare the lentivirus granules interfering human THAP11 gene and knock down the expres- sion of endogenous THAP11 in K562 cells and primary cord blood CD34 ＋ cells. Methods DNA segments carrying the specific THAP11 interfering sequences were cloned into the plasmid pSicoR to generate siTHAP11-pSicoR vectors. The siT- HAP11-pSicoR plasmids and three packing plasmids pLP1, pLP2 and pLP/VSVG were transfected into 293T cells to pro- duce lentivirus granules interfering THAP11. The siTHAP11-K562 cell line was established after infecting the lentivirus granules interfering THAP11 and the interference effect was evaluated by Western blot. Primary cord blood CD34＋ cells were infected with lentivirus granules and the expression of THAP11 was investigated by real-time PCR. Results Two lentivirus vectors carrying the interfering sequence against THAP11 were successfully generated and the titer of the lentivir- us was 1.8 × 10^8 TU/ml. In K562 cells, THAPll protein and mRNA levels were knocked down by the two siRNA lentivir- us vectors. In CD34＋ cells infected with siTHAP11-1 and siTHAP11-2, THAP11 mRNA levels were downregulated by 80% and 85%, respectively. Conclusion Human THAP1 ! siRNA lentiviral granules have been successfully generated.