欢迎访问《现代生物医学进展》编辑部！

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。

英文摘要:

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.

查看全文查看/发表评论下载PDF阅读器

关闭