生物发光技术及其应用

Confidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.Promega Founded 1978 Over 50 distributors in Europe, Asia, Africa and the Americas 1,400+ employees worldwide ISO 13485 certified Manufacturing in US, Korea and Shanghai Over 3,000 products for life science research and applied science including drug discovery Promega BioSystems Sunnyvale, Inc., CAPromega BioSciences, San Luis Obispo, CAPromega Headquarters and Manufacturing Madison, WisconsinPromega BioSystems, Seoul, KoreaShanghai Promega, ChinaConfidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.Promega Provides Outstanding Life Science Research ToolsSystems BiologyProteomicsGenomicsCellomicsDNARNAProteinCellTissue Cellular Analysis Protein Protein Interaction Drug Screening Research Tools Little Animal Imaging Cell-Free Protein Expression DNA , RNA Purification and Analysis Epigenetics Research Tool©2010, Promega Corporation. Confidential and Proprietary. Not for Further Disclosure.段文娟 Senior Field Application Specialist wenjuan.duanpromega.com萤光素酶报告基因技术及其应用Confidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.Content-目录目录1.报告基因技术简介报告基因技术简介2.萤光素酶报告基因技术应用3.萤光素酶报告基因技术检测法Confidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.5Reporter Gene 报告基因的产生报告基因的产生PromoterExon 12345Enhancertranscription start siteEnhancerReporter GenePromoter优势优势: 报告基因通常是容易检测的蛋白 报告基因可以通过生物发光、荧光、比色或放射的方法进行检测 采用酶类做为报告基因，具有放大信号的作用Reporter 问题问题:下游蛋白难以检测、方法繁琐Confidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.Absorbance 物质的结构不同吸光波长也不同 物质对光的吸收与其浓度成正比 灵敏度有限灵敏度有限Four Methods For Assay 四种检测方法学四种检测方法学AbsorbanceAbsorbanceRadiationRadiationFluorescenceFluorescenceBioluminescenceBioluminescenceFluorescence Excitation from a photonBioluminescence Excitation from a chemical ReactionExcitation （photon）Emission （photon）Excitation （chemical）Emission （photon）Confidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.萤光素酶反应原理萤光素酶反应原理LIGHTFirefly Luciferase: Common Firefly Photinus pyralis Wavelenghty:550-570nmRenilla Lucierase: Renilla reniformis Sea Pansy Wavelenghty:480nmLIGHTConfidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.什么是理想的报告基因？没有内源性表达易于定量高灵敏度线性范围宽反应迅速ReporterNon- RadioactiveEnzymaticBackgroundSensitivityActive FormHalf-lifeFirefly LuciferaseYY+Monomer3 hoursRenilla LuciferaseYY+Monomer5 hoursCAT(氯霉素乙酰 转移酶)NY+Trimer50 hoursb-Galactosidase (-半乳糖苷酶)YY+/+Tetramer20 hours其他：GUS (葡萄糖醛酸酶)、SEAP (分泌型碱性磷酸酶)、GFP (绿色荧光蛋白)Luciferase is the REPORTER of choice!Confidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.Reporter Assay Concept 报告基因检测大体流程报告基因检测大体流程transcriptionREPORTERASSAYTRANSFECT REPORTER CONSTRUCTVector ConstructVector ConstructTreatment 适用范围广: 总RNA 、细胞裂解物以及纯 化的RNA都可用于miRNA的定量检测需要仪器设备northern杂交杂交检测miRNA在组织细胞中的表达水平 结合RNA marker，经凝胶电泳检测miRNA 的分子大小对样品的需求量较高：需要微克级样品才 能避免假阴性原位杂交原位杂交检测不同细胞系单个细胞中的miRNA表达 不经分类和分离的情况下，比较不同细胞系 中miRNA的表达水平操作复杂基因芯片基因芯片高通量的检测基因表达谱需要昂贵的仪器设备； 半定量 重复性较差 不能同时优化所有待测miRNA的杂交环境， 因而不能区分高度类似的miRNAConfidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.20Promega miRNAs /siRNAs 靶标研究工具靶标研究工具载体特点载体特点：1.插入片段可以是目的基因的3-UTR或一段 siRNA/shRNA/miRNA的结合序列2.目的片段插入萤光素酶基因3端终止密码子后， 与萤光素酶基因共同转录，产生一个mRNA， 但目的片段不翻译3.不生成融合蛋白，不用担心过表达功能蛋白 影响细胞状态，可用于研究对细胞有毒性的 蛋白4.蛋白水平直接验证，siRNA/ shRNA/ miRNA有 效时检测不到萤光；无效时检测到萤光应用应用：1.鉴鉴定定miRNA 靶标位点靶标位点2.研究细胞内源性研究细胞内源性miRNA活性活性3.高通量筛选有效的高通量筛选有效的siRNAsPromega miRNA/siRNA 靶标载体靶标载体Translation stop coden萤光素酶萤光素酶靶序列靶序列53mRNAmiRNA/siRNA/shRNA发光发光发光发光Inhibition of translation or Cleavage of mRNAConfidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.21多克隆位点多克隆位点主报告基因主报告基因PGK启动子启动子新一代新一代miRNA/siRNA靶标载体靶标载体Cat.#载体载体骨架骨架主报告主报告基因基因内参基因内参基因 靶标靶标/内参启动子内参启动子 大小大小应用应用SizeE1330pmirGLOpGL4luc2hRluc-neo fusionPGK/SV40 7.35kb siRNA 适用范围广: 总RNA 、细胞裂解物以及纯 化的RNA都可用于miRNA的定量检测需要仪器设备northern杂交杂交检测miRNA在组织细胞中的表达水平 结合RNA marker，经凝胶电泳检测miRNA 的分子大小对样品的需求量较高：需要微克级样品才 能避免假阴性原位杂交原位杂交检测不同细胞系单个细胞中的miRNA表达 不经分类和分离的情况下，比较不同细胞系 中miRNA的表达水平操作复杂基因芯片基因芯片高通量的检测基因表达谱需要昂贵的仪器设备； 半定量 重复性较差 不能同时优化所有待测miRNA的杂交环境， 因而不能区分高度类似的miRNAConfidential and Proprietary. Not for Further Disclosure.Confidential and Proprietary. Not for Further Disclosure.报告基因应用报告基因应用Experimental firefly luciferase constructControl Renilla luciferase constructexpression level varies little with treatmentPROMOTER DISSECTIONSIGNALING PATHWAYNUCLEAR RECEPTORSPROTEIN INTERACTIONSPOST-TRANSCRIPTIONMIRNA CONTROLexpression level varies with treatmentLITTLEANIMAL IMAGINGENZYMEACTIVITYConfidential and Pro