NF-κ Bp65基因siRNA腺病毒载体的构建及其对EOMA细胞的影响

Construction of An Adenovirus Vector Encoding NF-κ Bp65 siRNA and Its Expression in EOMA Cells

目的构建含NF-κ Bp65基因siRNA腺病毒载体,并研究其对小鼠血管瘤细胞EOMA中NF-κ Bp65基因表达的影响。方法首先构建含有NF-κ Bp65 shRNA的人H1启动子真核表达载体pSuperH1/shp65,利用NotⅠ与SalⅠ双酶切将人H1载体启动子和NF-κ Bp65 shRNA序列亚克隆至经相同酶切的腺病毒穿梭质粒pShuttle中,形成转移质粒pShuttle-H1/shp65,然后转化含有pAdeasy的大肠杆菌BJ5183中进行同源重组,提取含目的基因的腺病毒重组体质粒,经PacⅠ酶切线性化后用脂质体转染293细胞,包装成重组腺病毒Ad-shp65,扩增、纯化,并对病毒滴度进行检测。利用得到的重组腺病毒感染小鼠血管瘤细胞EOMA,采用RT-PCR和荧光定量PCR检测感染前后NF-κ Bp65表达水平的变化,观察Ad-shp65对EOMA中NF-κ Bp65表达的影响。结果成功构建NF-κB p65 siRNA腺病毒载体,该载体可明显抑制EOMA细胞中NF-κ Bp65的表达。结论构建的Ad-shp65腺病毒能有效抑制NF-κ Bp65基因在小鼠血管瘤细胞中的表达,为研究异种移植延迟性排斥反应提供了新的方法。

Objective To construct an adenovirus vector for efficient delivery of NF-κBp65 siRNA and to investigate its effect on EOMA cells. Methods After digestion with NotⅠand SalⅠ,the H1 promoter and NF-κ Bp65 shRNA were cloned into the shuttle vector. Once constructed,the shuttle vector is linearized with Pme Ⅰ and transformed into BJ5183 cells pretransformed with the pAdEasy plasmid. Transformants are selected for kanamy-cin resistance,and recombinants are subsequently identified by restriction digestion. Purifie...