AKT2基因短发夹结构RNA慢病毒载体的构建及鉴定

目的：构建丝/苏氨酸蛋白激酶2（AKT2）基因RNA干扰（RNAi）慢病毒载体。方法：利用公用网站按照RNAi序列设计原则,设计RNAi靶点序列并合成靶序列的Oligo DNA,退火形成双链DNA,与经MluI和ClaI进行酶切后的PLVTHM载体连接产生shRNA慢病毒载体。应用shRNA慢病毒载体转染293T细胞及U87细胞,测定病毒滴度,流式细胞仪测定U87细胞的转染效率,PCR及Western blot鉴定AKT2基因在U87细胞中的下调作用。结果：成功构建了shRNA-AKT2慢病毒载体,经测序与设计合成的靶向链完全一致。荧光显微镜下观察293T细胞感染效率大于90%,病毒滴度为3.59×107TU/ml;流式细胞仪测定对AKT2细胞的转染效率为86.93%。PCR测定shRNA载体感染U87细胞后AKT2的干扰效率为68%。Western Blot结果显示该慢病毒载体对AKT2的表达有较为显著的敲减作用。结论：成功构建了人胶质瘤细胞株AKT2基因RNAi慢病毒载体,为后续的体内外功能学试验创造了条件。

Construction and Identification of Lentiviral Vector-Mediated Short Hairpin RNA of AKT2 Gene

Objective： To construct a lentiviral vector-mediated RNA interference（RNAi）of AKT2 gene.Methods：In accordance with RNAi sequence design principles in the public Web site,RNAi target sequences were designed,then the target sequences of Oligo DNA were synthesized and annealed to double-stranded DNA,which were connected with PLVTHM-GFP vector digested by MluI and ClaI.Short hairpin RNA lentiviral vectors were constructed.293T cells and U87 cells were transfected by shRNA lentiviral vector,and virus titer was determined.Transfection efficiency U87 cell was determined by flow cytometry.The expression of the AKT2 gene in U87 cells was identified by PCR and Western blot.Results： The lentiviral vector of AKT2-shRNA-oligonucleotide chain was inserted correctly;Infection efficiency of 293T cells observed under fluorescence microscope was more than 90%,the virus titer was 3.59 × 107 TU/ml;Transfection efficiency was 86.93%.AKT2 interference rate was 68%.shRNA lentiviral vectors inhibited the expression of AKT2.Conclusion： A lentiviral vector of AKT2-gene RNAi was constructed successfully by the genetic engineering technology,and it provides a condition forfurther research in vitro and vivo.