TNF-α诱导的NF-κB慢病毒报告基因系统的构建

Construction of TNF-α-Induced NF-κB Lentiviral Report⁃er Gene System

目的:构建一种肿瘤坏死因子α(TNF-α)诱导的NF-κB慢病毒报告基因系统。方法:以NF-κB信号通路为基础,设计并构建含有NF-κB响应元件和mCherry报告基因序列的表达载体,并进一步建立受NF-κB调控表达mCherry的细胞株,最后使用TNF-α进行刺激验证。结果:质粒经过双酶切鉴定,得到约1500 bp的目的片段,符合预计大小且测序分析正确,表明质粒构建成功;在TNF-α刺激实验中,NF-κB信号通路的刺激物TNF-α作用于构建的NF-κB调控表达mCherry荧光蛋白的细胞株后出现特异性荧光反应,TNF-α诱导组的细胞有较强的mCherry表达,阳性率约为90%,而未加TNF-α组细胞mCherry阳性表达率约为10%。结论:构建了受NF-κB调控稳定表达mCherry的慢病毒报告基因系统,可用于对NF-κB活化效果的检测及筛选,具有普适性的应用价值。

Objective:To construct a tumor necrosis factorα(TNF-α)-induced NF-κB lentiviral reporter gene system.Methods:Based on the NF-κB signaling pathway,we designed and constructed a vector containing NF-κB response elements and mCherry reporter gene sequence.Then,monoclonal RAW264.7 cell strain expressing mCherry reporter gene under control of the NF-κB response element was established and verified with TNF-αstimulation.Results:The inserted sequences were verified by restriction enzyme digestion and sequencing.The target fragment of about 1500 bp was obtained,which was in line with the expected size and the sequencing analysis was correct,indicating that the plasmid was constructed successfully.TNF-α,as an NF-κB activator,acted on the constructed RAW264.7 cell strain and led to the mCherry expression.The mCherry positive expression rate was about 90%,while without TNF-α,the rate was about 10%.Conclusion:TNF-α-induced NF-κB lentiviral reporter gene system was successfully constructed.The constructed reporter system in this study could be applied to detecting and screening the NF-κB transcriptional activity.