人干扰素λ1基因启动子荧光素酶报告基因质粒的构建及其转录活性分析

Construction and transcriptional activity of recombinant plasmid containing human interferon λ1 gene promoter luciferase reporter gene

目的构建人干扰素λ1(interferonλ1,IFNλ1)基因启动子荧光素酶报告基因载体,并探讨仙台病毒及质粒IRF3-5D和HA-IRF7对其转录活性的影响。方法提取BEAS-2B细胞基因组DNA,以其为模板,PCR扩增IFNλ1基因启动子,插入荧光素酶报告基因载体pGL3-enhancer,构建IFNλ1基因启动子荧光素酶报告基因质粒IFNL1-luc,将其转染293T细胞,同时用质粒IFNβ-luc和pGL3-enhancer转染作为对照。转染24 h后感染仙台病毒,于感染前和感染后4、12、18和24 h收集细胞,进行双荧光素酶活性检测;用质粒IRF3-5D或HA-IRF7与质粒IFNL1-luc共转染,36 h后收集细胞,进行双荧光素酶活性检测。结果 IFNλ1基因启动子荧光素酶报告基因质粒IFNL1-luc经双酶切及测序鉴定,证明构建正确;分别转染质粒IFNL1-luc和IFNβ-luc的细胞感染仙台病毒后,荧光素酶活性倍数均随时间延长呈明显上升趋势,至18 h达到峰值(分别为1 000倍和2 300倍),转染质粒pGL3-enhancer的细胞感染仙台病毒后,不同时间点收集的细胞,荧光素酶活性倍数一直处于较低水平;质粒HA-IRF7或IRF3-5D对IFNL1-luc的活化倍数明显高于pGL3-enhancer(P<0.05)。结论成功构建了IFNλ1基因启动子荧光素酶报告基因质粒IFNL1-luc,仙台病毒及质粒IRF3-5D和HA-IRF7均能激活IFNL1-luc的转录,为进一步研究调控IFNλ1表达的信号转导通路奠定了基础。

Objective To construct the recombinant plasmid containing human interferon （IFN）λ 1 promoter recombined luciferase reporter gene, and investigate the effect of plasmids IRF3-5D and HA-IRF7 on its transcriptional activity, Methods The promoter of human IFNkl gene was amplified by PCR using the extracted genomic DNA of BEAS-2B cells as a template, and inserted into the luciferase reporter vector pGL3-enhancer. The constructed recombinant plasmid IFNLI-luc was transfected to 293T cells, using plasmid IFN-β-luc and pGL3-enhancer as controls. The ceils were infected with Sendal virus 24 h after transfection. The cells were collected before and 4, 12, 18 and 24 h after infection, and determined for luciferase activity. The ceils were co-transfected with plasmids IFNLI-luc and IRF3-5D or HA-IRFT, and collected 36 h later for determination of luciferase activity. Results Both restriction analysis and sequencing proved that recombinant plasmid IFNLI-luc was constructed correctly. The folds of luciferase activation of cells transfected with plasmids IFNL1-luc and IFNβ-luc increased significantly and reached peak values of 1 000 and 2 300 18 h after infection with Sendal virus. However, the folds of luciferase activation of cells transfected with pGL3-enhancer were maintained at low levels at various time points after infection with Sendal virus. The activation folds of plasmids HA- IRF7 or IRF3-5D to IFNL1-luc were significantly higher than that of pGL3-enhancer （P 〈 0. 05 ）. Conclusion The recombinant plasmid IFNL1-lue containing human interferon （IFN） λ1 promoter recombined luciferase reporter gene was constructed successfully. Sendal virus as well as plasmids IRF3-5D and HA-IRF7 activated the transcription of IFNL1-luc, which laid a foundation of further study on the signaling pathway regulating IFNλI expression.