目的构建核干细胞因子基因RNA干扰(RNA interference,RNAi)慢病毒表达载体并对其在白血病细胞株中的干扰效果进行鉴定,为后期研究奠定基础。方法针对核干细胞因子基因的序列设计RNAi有效靶点,合成含RNA干扰序列、Loop环、AgeⅠ和EcoRⅠ酶切位点,以及终止信号的单链DNA oligo,经退火形成双链DNA,与双酶切线性化的带有GFP荧光标记和嘌呤霉素抗性标记的GV248慢病毒空载体进行连接产生重组慢病毒载体,转化感受态细菌,挑取重组阳性转化子进行菌落PCR反应,并对PCR阳性克隆进行测序。将重组慢病毒载体及其两种辅助包装原件载体质粒pHelper1.0和pHelper2.0共转染293T细胞进行病毒的包装,收集、浓缩病毒液后测定其滴度,并转染HL-60、NB4以及K562等三种人白血病细胞株,倒置荧光显微镜下观察其转染效率,real-time PCR检测核干细胞因子基因的敲减效率。结果经测序证实正确构建出了核干细胞因子基因的RNAi重组慢病毒载体,包装、浓缩后其滴度为4×108TU/ml,荧光显微镜下显示其能有效转染进入HL-60、NB4及K562等白血病细胞株中,转染效率均在80%以上;real-time PCR显示转染后核干细胞因子基因mRNA表达水平较阴性对照组显著下降(P<0.05),在HL-60、NB4和K562细胞中核干细胞因子基因的抑制效率分别为52.3%、80.5%、62.3%。结论成功构建出了核干细胞因子基因的RNAi慢病毒载体,其能有效干扰人白血病细胞株HL-60、NB4及K562中的核干细胞因子基因表达。 Objective To construct a lentiviral RNA interference expression vector of nucleostemin gene and identify its interference effect in leukemia cell lines, which will lay the foundation for later research. Methods According to the sequence of nucleostemin cytokines, RNAi targeting sites were designed and RNA interference sequences, Loop loop, AgeⅠ and EcoRⅠ restriction sites, and single-stranded DNA oligo of the termination signal were synthesized. Double-stranded DNA was annealed to form double- The linearized GV248 lentiviral empty vector with the GFP fluorescent marker and the puromycin resistance marker was ligated to generate a recombinant lentiviral vector and transformed into competent bacteria. The recombinant positive transformants were picked for colony PCR reaction and PCR positive Clones were sequenced. The 293T cells were co-transfected with the recombinant lentiviral vector and its two auxiliary packaging vector plasmids pHelper1.0 and pHelper2.0 to collect the virus and collect the virus solution. The titer of the virus was determined and transfected into HL-60, NB4 And K562 and other three human leukemia cell lines were observed under inverted fluorescence microscope transfection efficiency, real-time PCR detection of nuclear stem cell factor gene knockdown efficiency. Results RNAi recombinant lentiviral vector with nucleostemin gene was successfully constructed and sequenced. The titer was 4 × 108TU / ml after being packaged and was transfected into HL-60, NB4 and K562 cells under fluorescence microscope The transfection efficiency was above 80% in leukemia cell lines. Real-time PCR showed that the mRNA expression levels of nuclear factor were significantly decreased in HL-60, NB4 and K562 cells after transfection compared with the negative control group (P <0.05) The inhibitory rates of nuclear stem cell factor genes in the cells were 52.3%, 80.5% and 62.3%, respectively. Conclusion RNAi lentiviral vector of nucleostemin gene was successfully constructed, which can effectively interfere with the gene expression of nucleostemin in human leukemia cell lines HL-60, NB4 and K562.