酪氨酸激酶受体B基因RNA干扰慢病毒载体的构建与鉴定

Construction and identification of a lentiviral vector mediating RNA interference of tyrosine kinase B gene

目的构建有效抑制酪氨酸激酶受体B（TrkB）基因的RNA干扰慢病毒表达载体。方法根据大鼠TrkB基因的不同部位设计4对短发卡RNA（short hairpin RNA，shRNA）寡核苷酸片段，克隆到慢病毒干扎载体pXZRNAi1．0中，构建shRNA慢病毒表达载体pXZRNAi-shTrkB-1，2，3，4。获得较高滴度的慢病毒颗粒后感染大鼠神经干细胞，应用实时荧光PCR和Westernblot方法检测对TrkB的干扰效果。结果酶切鉴定和测序结果提示慢病毒载体的寡核苷酸链插入正确。包装浓缩慢病毒颗粒的滴度为8．6×105cfu／ml，对神经干细胞感染效率可达80％。与未感染组相比，转染pXZRNAi-shTrkB-3和4的神经干细胞中TrkBmRNA表达水平分别为（66．7±5．5）％和（76．8±4．9）％，TrkB蛋白的表达水平分别为（68．5±4．3）％和（78．2±5．1）％，均差异有统计学意义（p〈0．05）。结论成功构建可高效沉默TrkB基因的pXZRNAi-shTrkB慢病毒载体，为研究TrkB在神经干细胞中的作用提供了有力工具。

Objective To construct a tyrosine kinase B （TrkB） targeted RNA interference （RNAi） lenti- viral vector. Methods Four oligonucleotides targeting rat TrkB gene were synthesized and cloned into lentiviral vector pXZRNAi 1.0 to construct recombinant lentiviral vectors pXZRNAi-shTrkB-1,2,3,4. Neural stem cells prepared from rat hippocampus were infected with these high-titer viruses. Real-time PCR was employed to detect the TrkB mRNA expression and western blot was used to assess the gene silencing efficacy of these recombinants. Results Enzyme digestion and DNA sequencing results demonstrated that these shRNAs were correctly inserted into lentiviral vectors and the four recombinants were constructed successfully with the titer of 8.6 x 105cfu/ml. The infection efficiency of the letivirus on neural stem ceils reached 80%. Compared with the uninfection group, the expression levels of TrkB mRNA in neural stem cells decreased significantly after transfected with pXZRNAi- shTrkB-3 and 4 （ （ 66.7 ± 5.5 ） % and（ 76.8 ± 4.9 ） % respectively, P 〈 0.05 ） ; and the protein expression levels were also significantly decreased （（68. 5 ± 4. 3）% and （78. 2 ± 5. 1）% respectively, P 〈 0. 05）. Conclusion The lentiviral vectors for TrkB have been successfully constructed with high yield of lentivirus, which provides versatile method for assessing gene function in neural stem cells.