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目的:利用双荧光素酶报告系统探讨活化T细胞核因子(NFAT)能否调控常见组成性启动子CMV,SV40和TK,为不同条件下选择合适双荧光报告系统内参提供依据。方法:用限制性内切酶BglⅡ和HindⅢ分别从质粒pCDNA3.1和pRL-TK中切下CMV和TK启动子,克隆至pGL3-basic载体中,构建成pCMV-Luc和pTK-Luc载体。将构建pCMV-Luc和pTK-Luc以及商品化的pGL3-control(SV40启动子驱动),分别与SV40(pBIND)和TK(pRL-TK)两种启动子驱动的两种内参质粒共转染入HEK293细胞;观察过表达组成性活化NFAT后相对荧光素酶活性读数的改变。结果:成功构建了pCMV-Luc和pTK-Luc质粒,荧光素酶活性检测发现,常见组成性启动子SV40启动子对过表达组成性活化的NFAT存在一定的反应。结论:T细胞活化过程中重要的转录因子NFAT能够调控SV40启动子活性;表明常见组成性启动子SV40并非真正、绝对的组成性不变。因此,在荧光素酶报告系统内参选择时需要充分考虑该问题,本研究为合理选择内参质粒提供了一个可行策略。
OBJECTIVE: To investigate whether NFAT regulated the common constitutive promoters CMV, SV40 and TK by using dual luciferase reporter system, and provide the basis for selecting appropriate double fluorescent reporter system internal control under different conditions. Methods: The CMV and TK promoters were excised from plasmids pCDNA3.1 and pRL-TK respectively by restriction endonucleases BglII and HindⅢ and cloned into pGL3-basic vector to construct pCMV-Luc and pTK-Luc vectors. Construction of pCMV-Luc and pTK-Luc and commercial pGL3-control (SV40 promoter driven) were co-transfected with two internal control plasmids driven by two promoters of SV40 (pBIND) and TK (pRL-TK) HEK293 cells; changes in relative luciferase activity readings observed after overexpression of constitutively activated NFAT. Results: The pCMV-Luc and pTK-Luc plasmids were successfully constructed. The luciferase activity assay showed that SV40 promoter, a common constitutive promoter, had a certain response to constitutively activated NFAT over-expression. CONCLUSION: NFAT, an important transcription factor during T cell activation, regulates the activity of SV40 promoter. It shows that SV40, a common constitutive promoter, is not truly and absolutely constitutively constitutive. Therefore, this problem needs to be fully considered in the internal selection of luciferase reporter system. This study provides a feasible strategy for the rational selection of the reference plasmid.