核干细胞因子RNA干扰慢病毒载体的构建及鉴定  

Construction and identification of a lentiviral vector for RNA interference of nucleostemin

作　　者：孙晓莉[1] Abdallah Dlykan 贾宇[1] 魏园玉 刘帅[1] 岳保红[1] 

机构地区：[1]郑州大学第一附属医院检验科,450052

出　　处：《中华临床医师杂志（电子版）》2013年第11期104-107,共4页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学基金(81271911);河南省医学科技攻关重点项目(201002006)

摘　　要：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 expression vector for RNA interference （RNAi）of nucleostemin（NS） and detect its interference efficiency in three leukemia cell lines （HL-60,NB4 and K562）.Methods Effective RNAi target sequence was designed and screened towards NS gene sequence.Single-stranded DNA oligo containing RNAi sequence,Loop circle,Age I/EcoR I enzyme cutting site,and termination signal sequence was synthesized and annealed to double-stranded DNA,which was subsequently connected to the Age I/EcoR I-digested GV248 lentiviral vector with GFP and puromycin resistance marker to form a recombinant lentiviral vector.Then it was transformed into competent E.coli cells,and the positive clones were confirmed by colony PCR and sequencing.The recombinant vector and two lentivims packing plasmids （pHelperl.0 and pHelper2.0） were cotransfected into 293T cells to obtain packaged lentivirus particles.Viral titer was then determined.Subsequently,the recombinant lentivirus were transfected into three human leukemia cell lines （HL-60,NB4 and K562）,then the transfection efficiency were observed under inverted fluorescence microscope,and the inhibition rates were detected by real-time PCR.Results DNA sequencing showed that RNAi sequence was inserted into the GV248 vector and the recombinant lentiviral vector was constructed successfully.The recombinant lentivims harvested from 293T cells had a titer of 4 × 108 TU/ml.Observation under inverted fluorescence microscope showed the lentiviral transfection efficiency was higher than 80％ in all the three cell lines.Real-time PCR showed NS mRNA expression level of experimental group was significantly lower than negative control group（P ＜ 0.05）.The interference efficiency in HL-60,NB4 and K562 were 52.3％,80.5％,62.3％,respectively.Conclusion The lentiviral vector for RNAi of NS has been successfully constructed with high titer,and it could inhibit NS mRNA expression effectively in three human leukemia cell lines HL-60,NB4 and K562,which laid the foun

关 键 词：RNA干扰 核干细胞因子 慢病毒载体 白血病细胞 

分 类 号：R733.7[医药卫生—肿瘤]