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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。
Objective To construct RNA interference (RNAi) lentiviral vector of human THAP11 gene and prepare high titer virus particles to knock down THAP11 expression in K562 cells and cord blood CD34 + cells. Methods The third generation lentiviral packaging system was used to clone the DNA sequence containing specific interference THAP11 into shuttle plasmid pSicoR to construct siTHAP11-pSicoR plasmid. In liposome-mediated, siTHAP11-pSicoR and packaging plasmid pLP1, pLP2, pLP / VSVG co-transfected HEK293T cells to obtain high-titer lentivirus particles. K562 cells were infected to detect endogenous THAP11 knockdown effect. Infection of human CD34 + cells, GFP-positive cells were sorted by flow cytometry, and the knockdown effect of THAP11 in primary cells was examined. Results RNAi lentiviral vectors siTHAP11-1 and siTHAP11-2 targeting at two sites of THAP11 were successfully constructed and lentiviral particles were obtained. The titer of virus was up to 1.8 × 108TU / ml. Stable strains were established after infection with K562 cells, and THAP11 protein and mRNA levels were down-regulated. The efficiency of infection of CD34 + cells was over 30%, siT-HAP11-1 down-regulated about 80% of THAP11 mRNA, and siTHAP11-2 down-regulated about 85%. Conclusions RNAi lentiviral vector of THAP11 was successfully constructed. The encapsulated virus particles can effectively knock down the expression of endogenous THAP11 in cell lines and primary cells, which lays the foundation for further studies on the effect of THAP11 on CD34 + cells.