小鼠干扰素γ受体β基因启动子荧光报告载体构建及转录活性分析
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摘要
目的克隆小鼠干扰素γ受体β(Ifngr2)基因启动子,并应用双荧光素酶报告系统对其进行功能分析。方法首先应用欧洲启动子在线数据库(EPD)获得小鼠Ifngr2基因的启动子区域,包含转录起始位点在内的-1344～+48的DNA序列。然后以小鼠MEF细胞基因组DNA为模板,利用PCR扩增调取该序列,并进一步插入pGL4.19-luc2CP荧光素酶报告载体。通过PCR扩增获得启动子系列截短体并分别插入pGL4.19-luc2CP载体,共获得包含6个不同长度启动子序列的荧光素酶报告载体。进一步通过双荧光报告实验检测不同截短体对Ifngr2基因转录活性影响,确定其影响转录活性的关键区域。通过点突变探究潜在转录因子对Ifngr2基因转录的影响。结果成功构建小鼠Ifngr2基因启动子荧光素酶报告载体,获得有转录活性的启动子报告基因载体。启动子系列截短分析显示,-132～-97和-1059～-727包含启动子重要元件。生物信息学分析发现,在-132～-97区域内存在潜在的NF-κB结合位点,进而通过点突变实验得到验证。结论通过构建小鼠Ifngr2基因启动子报告载体并对Ifngr2启动子进行功能分析,发现小鼠Ifngr2基因启动子中的转录因子NF-κB结合区域,提示NF-κB在Ifngr2基因表达调控中起重要作用。
OBJECTIVE To functionally analyze the promoter using dual luciferases reporter assay system by cloning the promoter region of mouse interferon-γ receptor 2(lfngr2) gene. METHODS The5′ sequence(-1344-+48) containing the transcription start site of the mouse Ifngr2 gene was acquired from the Eukaryotic Promoter Database, and the gene fragment was cloned into the luciferase reporter vector. Series of 5′ deleted truncated variants were further constructed to obtain six luciferase reporter vectors with different lengths of promoters. The transcriptional activity of different Ifngr2 promoter truncations was determined by the dual luciferase analysis. In addition, site-directed mutation was applied to explore the effect of NF-κB on mouse Ifngr2 transcription. RESULTS The constructed mouse Ifngr2 promoter luciferase reporter vector was confirmed by gene sequencing and alignment. Promoter deletion analysis showed that the-132--97 and-1059--727 regions were important for mouse Ifngr2 gene transcription. Site-directed mutation indicated that the NF-κB binding region was essential for the transcription of Ifngr2 gene. CONCLUSION Mouse Ifngr2 gene promoter luciferase vectors are successfully constructed. Functional analysis of the mouse Ifngr2 gene promoter reveals an NF-κB binding site,which is essential for transcription of Ifngr2 gene.
OBJECTIVE To functionally analyze the promoter using dual luciferases reporter assay system by cloning the promoter region of mouse interferon-γ receptor 2(lfngr2) gene. METHODS The5′ sequence(-1344-+48) containing the transcription start site of the mouse Ifngr2 gene was acquired from the Eukaryotic Promoter Database, and the gene fragment was cloned into the luciferase reporter vector. Series of 5′ deleted truncated variants were further constructed to obtain six luciferase reporter vectors with different lengths of promoters. The transcriptional activity of different Ifngr2 promoter truncations was determined by the dual luciferase analysis. In addition, site-directed mutation was applied to explore the effect of NF-κB on mouse Ifngr2 transcription. RESULTS The constructed mouse Ifngr2 promoter luciferase reporter vector was confirmed by gene sequencing and alignment. Promoter deletion analysis showed that the-132--97 and-1059--727 regions were important for mouse Ifngr2 gene transcription. Site-directed mutation indicated that the NF-κB binding region was essential for the transcription of Ifngr2 gene. CONCLUSION Mouse Ifngr2 gene promoter luciferase vectors are successfully constructed. Functional analysis of the mouse Ifngr2 gene promoter reveals an NF-κB binding site,which is essential for transcription of Ifngr2 gene.
引文
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[24]Huang T,Cao YX,Zhang ZJ,Zhou XL,Wei HJ,Wu ZH.Construction and determination of the activity of luciferase reporter vector of human DNMT1gene promoter[J].J Wannan Med Coll(皖南医学院学报)2018,37(1):1-3.
[2]Dunn GP,Koebel CM,Schreiber RD.Interferons,immunity and cancer immunoediting[J].Nat Rev Immunol,2006,6(11):836-848.
[3]Borden EC,Sen GC,Uze G,Silverman RH,Ransohoff RM,Foster GR,et al.Interferons at age 50:past,current and future impact on biomedicine[J].Nat Rev Drug Discov,2007,6(12):975-990.
[4]Schneider WM,Chevillotte MD,Rice CM.Interferonstimulated genes:a complex web of host defenses[J].Annu Rev Immunol,2014,32:513-545.
[5]Lasfar A,Cook JR,Cohen Solal KA,Reuhl K,Kotenko SV,Langer JA,et al.Critical role of the endogenous interferon ligand-receptors in typeⅠand typeⅡinterferons response[J].Immunology,2014,142(3):442-552.
[6]de Weerd NA,Nguyen T.The interferons and their receptors-distribution and regulation[J].Immunol Cell Biol,2012,90(5):483-491.
[7]Pestka S,Kotenko SV,Muthukumaran G,Izotova LS,Cook JR,Garotta G.The interferon gamma(IFN-gamma)receptor:a paradigm for the multichain cytokine receptor[J].Cytokine Growth Factor Rev,1997,8(3):189-206.
[8]Griggs ND,Jarpe MA,Pace JL,Russell SW,Johnson HM.The N-terminus and C-terminus of IFN-gamma are binding domains for cloned soluble IFN-gamma receptor[J].J Immunol,1992,149(2):517-520.
[9]Ahmed CM,Johnson HM.IFN-gamma and its receptor subunit IFNGR1 are recruited to the IFN-gammaactivated sequence element at the promoter site of IFN-gamma-activated genes:evidence of transactivational activity in IFNGR1[J].J Immunol,2006,177(1):315-321.
[10]Kudinov Y,Wiseman CL,Kharazi AI.Phorbol myristate acetate and Bryostatin 1 rescue IFN-gamma inducibility of MHC classⅡmolecules in LS1034 colorectal carcinoma cel line[J/OL].Cancer Cell Int,2003,3:4(2003-03-25).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC153529/
[11]Kemp K,Bruunsgaard H.Identification of IFN-gammaproducing CD4+T cells following PMA stimulation[J].J Interferon Cytokine Res,2001,21(7):503-506.
[12]Schroder K,Hertzog PJ,Ravasi T,Hume DA.Interferon-gamma:an overview of signals,mechanisms and functions[J].J Leukoc Biol,2004,75(2):163-189.
[13]Dustin ML,Rothlein R,Bhan AK,Dinarello CA,Springer TA.Induction by IL-1 and interferon-gamma:tissue distribution,biochemistry,and function of a natural adherence molecule(ICAM-1)[J].J Immunol,1986,137(1):245-254.
[14]Rawlings JS,Rosler KM,Harrison DA.The JAK/STAT signaling pathway[J].J Cell Sci,2004,117(Pt 8):1281-1283.
[15]Qin Z,Blankenstein T.CD4+T cel-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by nonhematopoietic cells[J].Immunity,2000,12(6):677-686.
[16]Lugo-Vilarino G,Maldonado-Lopez R,Possemato R,Penaranda C,Glimcher LH.T-bet is required for optimal production of IFN-gamma and antigen-specific T cell activation by dendritic cells[J].Proc Natl Acad Sci USA,2003,100(13):7749-7754.
[17]Zhou F.Molecular mechanisms of IFN-gamma to up-regulate MHC classⅠantigen processing and presentation[J].Int Rev Immunol,2009,28(3-4):239-260.
[18]Gao J,Shi LZ,Zhao H,Chen J,Xiong L,He Q,et al.Loss of IFN-γpathway genes in tumor cel s as a mechanism of resistance to anti-CTLA-4 therapy[J].Cell,2016,167(2):397-404.e9.
[19]Zaretsky JM,Garcia-Diaz A,Shin DS,Escuin-Ordinas H,Hugo W,Hu-Lieskovan S,et al.Mutations associated with acquired resistance to PD-1 blockade in melanoma[J].N Engl J Med,2016,375(9):819-829.
[20]Benci JL,Xu B,Qiu Y,Wu TJ,Dada H,TwymanSaint Victor C,et al.Tumor interferon signaling regulates a multigenic resistance program to immune checkpoint blockade[J].Cell,2016,167(6):1540-1554.e12.
[21]Rosenzweig SD,Schwartz OM,Brown MR,Leto TL,Holland SM.Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness[J].J Immunol,2004,173(6):3991-3999.
[22]Hu QY,Wang Q.Molecular mechanism of calpain in regulating macrophage polarization mediated by NF-κB/STAT3 signaling pathways[J].Fudan Univ J Med Sci[复旦学报(医学版)],2018,45(3):297-304.
[23]Xiong JT,Li XS,Yang AN,Yang SH,Deng M,Wang L,et al.Effect of homocysteine on activity of human FABP4 promoter in macrophages[J].China J Mod Med(中国现代医学杂志),2018,28(29):25-30.
[24]Huang T,Cao YX,Zhang ZJ,Zhou XL,Wei HJ,Wu ZH.Construction and determination of the activity of luciferase reporter vector of human DNMT1gene promoter[J].J Wannan Med Coll(皖南医学院学报)2018,37(1):1-3.