靶向大鼠HIF-1α的RNAi慢病毒载体的构建及鉴定  

Construction and identification of HIF-1α targeted RNAi lentiviral vector

作　　者：陈蕾[1] 曾天芳[1] 周东[1] 李耀华[1] 

机构地区：[1]四川大学华西医院神经内科,四川成都610041

出　　处：《西部医学》2012年第6期1050-1053,共4页Medical Journal of West China

基　　金：国家自然科学基金资助项目(编号:30900470/C090301)

摘　　要：目的构建靶向HIF-1α的RNAi慢病毒载体,为进一步观察其对难治性癫痫大鼠模型脑内HIF1α基因过度表达的干扰作用奠定基础。方法根据GenBank报道的大鼠HIF-1α基因序列,选择4个靶序列,设计并合成4对含编码短发夹RNA(shRNA)序列的寡核苷酸,双链退火后,插入经限制性内切酶Age I和EcoR I处理所得的线性化慢病毒载体pGCSIL-GFP(表达绿色荧光蛋白,GFP)的U6启动子下游,合成4种重组病毒pGCSIL-GFP-shRNA1,2,3,4,经酶切和测序鉴定构建正确后,分别与Lipofectamine 2000共转染293T细胞包装,收集富含慢病毒颗粒的细胞上清液浓缩后得到高滴度的慢病毒液,孔稀释法测定病毒滴度。采用同样方法构建阴性对照重组慢病毒。结果酶切和测序分析证实成功构建了靶向大鼠HIF-1α的慢病毒表达载体4对,滴度为(5～7)×108 TU/ml。结论本实验成功构建了针对大鼠HIF-1α基因的RNAi慢病毒载体,为实现在细胞和动物模型以慢病毒为载体的HIF-1α靶向治疗提供了良好的实验基础。Objective To construct a HIF-1α targeted RNA interference （RNAi） lentiviral vector and establish a basis for further observation of its interference effection of HIF-1α expression on the rat model of refractory epilepsy. Methods Four target sequences for shRNA expression were selected and designed according to HIF-1α sequence of rat. Annealed oligos were inserted into the downstream of pGCSIL-GFP（linearizated by restriction enzymes Age I and EcoR D U6 promoter to construct recombinant virus pGCSIL-GFP-shRNA1,2,3,4. After these virus were confirmed as right by enzyme digestion and sequencing respectively, were co-transfected with Lipofectamine 2000 into 293T cells, packaging, collection of lentiviral particles rich in concentrated supernatant and condensed to high-titer virus solution,then tested ti- ters. Using the same method to build a negative control recombinant lentivirus. Results DNA sequencing and enzyme di- gestion results demonstrated that four HIF-1α targeted RNA interference （RNAi） Lentiviral Vector were successfully constructed with titer of 5-7 × 10^8 TU/mL. Conclusion The recombinant lentiviral vectors target to rat HIF-1α are suc- cessfully constructed and packaged, which provides a good experimental basis for further study in rat model gene therapy.

关 键 词：RNA干扰 HIF-1Α 慢病毒 

分 类 号：R456[医药卫生—治疗学]