含人LINGO-1慢病毒干扰载体的构建与鉴定  

Construction and Identification of Lentiviral Interference Vector That Including Human LINGO-1

作　　者：索磊[1] 杨印祥[2] 栾佐[1,2] 

机构地区：[1]南方医科大学第三临床医学院,广东省广州市510515 [2]中国人民解放军海军总医院儿科

出　　处：《中国全科医学》2016年第24期2962-2966,共5页Chinese General Practice

基　　金：国家自然科学基金面上项目(81471486)

摘　　要：目的体外构建含人LINGO-1慢病毒干扰载体,并测定其对靶基因LINGO-1的干扰效应。方法2015年3—11月,根据Gen Bank数据库中报道的人LINGO-1基因序列,设计合成针对人LINGO-1短发夹RNA(LINGO-1 shRNA)干扰序列,并将其克隆至重组质粒载体,测序验证后在293T细胞内包装慢病毒并测定病毒滴度。将慢病毒载体分为目标基因干扰载体组(实验组)和错配序列干扰载体组(对照组),并转染至人胶质细胞瘤细胞U251,荧光显微镜下观察其感染效率,并采用免疫荧光染色、实时荧光定量聚合酶链式反应(q PCR)法检测LINGO-1 mRNA表达水平,采用Western blotting法检测LINGO-1表达水平。结果成功构建两组慢病毒干扰载体,经过包装分别得到滴度为2×108TU/ml和4×108TU/ml的病毒原液。病毒载体感染人胶质细胞瘤细胞U251后,实验组和对照组载体转染效率分别为96.6%、95.2%。免疫荧光染色显示,人胶质细胞瘤细胞U251高表达LINGO-1,经慢病毒载体转染后,实验组免疫荧光呈现减弱现象。实验组LINGO-1 mRNA表达水平(0.09±0.01)较对照组的(1.00±0.00)降低(t=12.87,P<0.01),实验组LINGO-1 mRNA相对表达抑制率为91.0%。实验组LINGO-1表达水平(0.28±0.02)较对照组的(1.00±0.00)降低(t=-8.13,P<0.01)。结论本研究成功构建了含人LINGO-1shRNA干扰载体,并证实了干扰载体对靶基因有较好的干扰效果,为研究LINGO-1在轴突再生中的作用奠定了基础。Objective To construct the lentiviral interference vector that including human LINGO - 1 in vitro and evaluate its interference effects on the target gene LINGO - 1. Methods From March to November in 2015,interference sequences that targeted at human LINGO - 1 interfered by short hairpin RNA（LINGO - 1 shRNA）were designed and synthesized according to the LINGO - 1 gene sequence reported in GenBank database,LINGO - 1 shRNA was cloned into recombinant plasmid vectors. They were assembled in 293T cells after sequence verification. The test divided into interference vector group of target gene（experimental group）and interference vector group of mismatch sequence（control group）,the lentiviral vectors were transfected into U251 cells of human glioma, and its efficiency of infection was observed by fluorescence microscopy. Immunofluorescent staining and real - time fluorescent quantitative polymerase chain reaction（qPCR）method were adopted to detect the expression level of LINGO - 1 mRNA. The expression level of LINGO - 1 was detected by Western blotting method. Results Two groups of lentiviral interference vectors were successfully constructed,and the virus stock solutions with a titer of 2 ＆#215; 108 TU/ ml and 4 ＆#215; 108 TU/ ml were obtained respectively after package. After U251 cells of human glioma were transfected with virus vectors,the transfection efficiency of vectors in experimental group and control group were 96. 6% and 95. 2% respectively. Immunofluorescent staining presented that LINGO - 1 was highly expressed in U251 cells of human glioma, and immunofluorescence in experimental group weakened after the transfection of lentiviral vectors. The expression level of LINGO- 1 mRNA in experimental group（0. 09 ± 0. 01）was significantly decreased compared with that in control group（1. 00 ± 0. 00） （t = 12. 87,P ﹤ 0. 01）,and the interference rate of LINGO - 1 mRNA relative expression in experimental group was 91. 0% . Compared with control group（1. 00 ± 0. 00）,the LINGO - 1

关 键 词：慢病毒属 RNA干扰 LINGO-1 

分 类 号：R349.65[医药卫生—基础医学]