NRG1-siRNA慢病毒载体的构建

Construction of neuregulin 1-small interference RNA lentivirus vector

目的 构建神经调节蛋白1（NRG1）-小干扰RNA （siRNA）慢病毒载体.方法 将NRG1干扰序列构建入线性化的慢病毒载体GV123上,得到NRG1-siRNA慢病毒载体.将重组病毒质粒包装和浓缩,并在293T细胞中测定病毒滴度.然后转染体外培养的星形胶质细胞,72 h时分别采用RT-PCR法和Western blot法检测NRG1 mRNA及其蛋白表达水平.结果 成功构建NRG1-RNAi慢病毒载体GV123-siRNA,病毒滴度为8＆#215;108 TU/ml,该病毒转染星形胶质细胞后,NRG1 mRNA及其蛋白表达均下调.结论 成功构建了NRG1-siRNA慢病毒载体.

Objective To construct the neuregulin 1 （NRG1）-small interference RNA （siRNA） lentivirus vector.Methods NRG1-siRNA lentivirus vector was constructed by transfecting lentivirus vector GV123 with interfering sequences for NRG1 gene.The recombinant plasmid was packed and concentrated.The titer of lentivirus vector was measured in 293T cells.Then the astrocytes isolated and cultured in vitro were transfected with the lentivirus.NRG1 mRNA and protein expression was determined using RT-PCR and Western blot,respectively,72 h later.Results The NRG1-siRNA lentivirus vector GV123-siRNA was constructed successfully,and the titer of lentivirus vector was 8 ＆#215; 108 TU/mL.The expression of NRG1 mRNA and protein was significantly down-regulated in the astrocytes after being transfected with the lentivirus vector.Conclusion The NRG1-siRNA lentivirus vector is constructed successfully.