NSPc1-siRNA重组慢病毒的构建及其对U87细胞NSPc1基因的沉默效应

发布时间：2019-02-11 16:22

【摘要】：【目的】构建包装NSPc1-si RNA慢病毒颗粒,并在U87胶质瘤细胞中鉴定其感染及基因沉默效果。【方法】根据Gen Bank中基因信息,采用干扰序列设计软件设计靶点,制备合成GV118-si RNA目的质粒,转化感受态细胞,对于长出的克隆应用菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和对比分析,重组病毒质粒与另外2种辅助包装载体质粒通过Lipofectamine TM2 000共转染293T细胞,培养48 h后,收集细胞培养上清液,将病毒浓缩后在293T细胞中测定病毒滴度;并检测病毒颗粒在目的细胞U87胶质瘤细胞中的感染效率,实时荧光定量PCR(RT-PCR)和Western blotting方法检测NSPc1 si RNA慢病毒对U87中NSPc1的干扰作用。【结果】成功构建NSPc1 si RNA慢病毒载体GV118-si RNA,重组病毒滴度为4×108TU/ml;用该病毒体外感染U87胶质瘤细胞,当感染复数(MOI)为10时,感染效率大于90%;RT-PCR和Western blotting方法检测NSPc1基因沉默的效率分别为66.4%、60.0%。【结论】成功构建了NSPc1 si RNA慢病毒载体GV118-si RNA,该重组病毒包装后在体外感染U87胶质瘤细胞的效率较高,且具有显著的基因沉默效果。
[Abstract]:[objective] to construct the packaged NSPc1-si RNA lentivirus particles and identify the effect of infection and gene silencing in U87 glioma cells. [methods] according to the gene information in Gen Bank, the target was designed by interference sequence design software. The target plasmid of GV118-si RNA was prepared and transformed into receptive cells. The clones were identified by colony PCR, and the positive clones identified by PCR were sequenced and compared. The recombinant virus plasmid and the other two kinds of auxiliary package vector plasmids were co-transfected into 293T cells by Lipofectamine TM2 000. After 48 hours of culture, the supernatants of cell culture were collected and the virus titers were measured in 293T cells after the virus was concentrated. The infection efficiency of virus particles in U87 glioma cells was detected. Real-time fluorescence quantitative PCR (RT-PCR) and Western blotting were used to detect the interference of NSPc1 si RNA lentivirus to NSPc1 in U87. [results] the recombinant NSPc1 si RNA lentivirus vector GV118-si RNA, was successfully constructed with a titer of 4 脳 108 TU / ml. U87 glioma cells were infected with the virus in vitro. When the complex (MOI) was 10:00, the infection efficiency was greater than 90%. The detection efficiency of NSPc1 gene silencing by RT-PCR and Western blotting was 66.4% and 60.0% respectively. [conclusion] NSPc1 si RNA lentivirus vector GV118-si RNA, was successfully constructed. The recombinant virus can infect U87 glioma cells in vitro with high efficiency and remarkable gene silencing effect.
【作者单位】：武警后勤学院生物化学与分子生物学教研室;武警后勤学院人体解剖与组织胚胎学教研室;武警后勤学院附属医院脑科医院神经内科;
【基金】：国家自然科学基金青年项目(81201757) 天津市自然科学基金青年项目(13JCQNJC09600) 武警后勤部项目(WJHQ2012-13)
【分类号】：R346

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