人白介素-8 shRNA慢病毒载体的构建和鉴定

Construction and identification of shRNA lentiviral vector targeting human interleukin-8 gene

目的:构建携载人白介素-8(IL-8)shRNA的慢病毒并鉴定其对MDA-MB-231细胞该基因的沉默效率。方法:设计两个IL-8基因特异性siRNA靶点,与pMagic7.1慢病毒载体质粒重组,经PCR鉴定并测序,通过293T细胞实现慢病毒包装,DNA定量试剂盒分别检测两种慢病毒滴度。将这两种慢病毒分别以最佳感染复数感染MDA-MB-231细胞,通过Real-time PCR检测IL-8沉默效率。结果:PCR鉴定阳性的shRNA重组质粒测序正确,包装后两种病毒滴度分别为1.93×109 IU/mL和1.89×109IU/mL;分别以MOI=40感染MDA-MB-231细胞后,IL-8沉默效率分别为80%和90%。结论:成功构建IL-8 shRNA慢病毒并达到较高的沉默效率,为下一步研究沉默靶基因后细胞生物学功能变化提供实验基础。

Objective： This study aims to target human interleukin-8 gene by constructing shRNA lentiviral vectors and detect its silencing effect in MDA-MB-231 cells. Methods： Two specific siRNA sequences targeting human IL-8 gene were designed and recombined with pMagic7.1 lentiviral vector plasmids. The recombined plasmids were identified by PCR and then sequenced. The lentivirus particles were packaged using 293T cells,the titer of which were detected by quantitative DNA technique. Then MDA-MB-231 cells were transfected with the optimized MOI. The silencing effect on IL-8 was detected by real-time PCR. Results： The sequencing of shRNA plasmids which been identified positive by PCR were correctly constructed. The titer of the packaged virus was 1.93 × 10^9 IU/mL and 1.89 x× 10^9 IU/mL separately. After infecting the MDA-MB-231 ceils with MQI=40, the silencing efficiency were 80% and 90% respectively. Conelusion：The recombinant lentiviral shRNA has been constructed successfully, with a highly silence efficiency, which ensure the reliability for the further study on cell functional assay.