当前位置:首页 > 商业/管理/HR > 经营企划 > 大肠杆菌和铜绿假单胞菌中蛋白表达监测系统的构建
i大肠杆菌和铜绿假单胞菌中蛋白表达监测系统的构建中文摘要20世纪90年代,“人类基因组计划”的推进和完成,标志着“后基因组时代”的到来,“功能基因组学”中蛋白质组学的研究提上了未来生命科学研究的日程。目前蛋白质研究的主要方法是通过2-DE配合质谱对蛋白质的分离鉴定。虽然对蛋白质的研究技术在不断地改进,但是目前的生物化学技术已不能满足研究蛋白质组所需要的高灵敏度、高通量以及可规模化的要求,对于蛋白质组学的研究期待着新型的技术方法的突破。本课题以大肠杆菌和铜绿假单胞菌为研究对象,采用了将带有RBS的目的基因克隆至缺失自身RBS的报道基因luxABCDE上游,构建二者共用RBS的预期的蛋白表达监测系统;报道基因的表达量取决于克隆基因的转录及翻译水平,利用发光值读取仪检测CPS值衡量目的基因蛋白质的表达量。对该监测系统进行验证,并将两种菌的全基因组克隆至各自相应的监测系统中,建立反映蛋白质表达情况的荧光值数据库。在大肠杆菌中,将重组载体pUCNot-lux中得到的报道基因luxABCDE克隆至蛋白表达载体pPROEXHTa、pPROEXHTb、pPROEXHTc,初步构建得到大肠杆菌的蛋白表达监测系统,命名为pPROEXHTa-lux、pPROEXHTb-lux、pPROEXHTc-lux。报道基因luxABCDE在载体pPROEXHTc-lux中翻译时能够正确阅读;并在IPTG梯度浓度条件下,测定含有载体pPROEXHTc-lux的细菌发光量,由结果可知克隆到蛋白表达载体中的缺失自身RBS的报道基因luxABCDE可以使用载体上游的trc启动子及其RBS,并且该报道基因的蛋白表达受到trc启动子调节因素的调节。测定预期的蛋白表达载体pPROEXHTc-lux中的报道基因luxABCDE两侧的序列,结果表明载体pPROEXHTc与报道基因luxABCDE连接处的碱基序列与已知碱基序列相同。在铜绿假单胞菌中,将从已构建的大肠杆菌蛋白表达监测载体酶切后得到的luxABCDE(a)、luxABCDE(b)、luxABCDE(c)克隆至去除原有报道基因luxCDABE的pMS402剩余载体中,初步构建铜绿假单胞菌蛋白表达监测系统,命名为pMS402-luxABCDE(a)、pMS402-luxABCDE(b)、pMS402-luxABCDE(c)。在大肠杆菌和铜绿假单胞菌中构建的蛋白表达监测方法可以实现高通量、高灵敏度、规模化以及对低丰度蛋白表达的监测;该监测方法不仅可用于蛋白质组学的基础理论性研究,而且可用于攻克重大疾病和开发生物医药等领域的实践应用性研究。关键词:大肠杆菌,铜绿假单胞菌,蛋白表达监测系统iiConstructionofProteinExpressionMonitoringSysteminEscherichiacoliK12andPseudomonasaeruginosaPAO1AbstractGenomicshasbeenfocusedonbyresearchersduringthe20thcentury.Thenproteomicswasbeingthemainstudyingdirectioninthepost-genomiceraaftertheaccomplishmentofthe“HumanGenomeProject”.Variousmethodslike2-DEtechniquewithMassSpectrometrywereusuallyusedforisolatingandidentifyingproteins.However,thosetechniquesstillcouldn’tmeettheneedsofproteomedevelopment.Therefore,exploringahighsensitiveandhigh-throughputandlarge-scaleresearchmethodisimperactiveandbeingexpected.Inthisstudy,Escherichiacoli(E.coli)andPseudomonasaeruginosaPAO1wereselectedashostcellsforconstructingaproteinexpressionmonitoringsystem,respectively,usingthereportergenewithoutitsself-RBS.SincethereportergenecouldsharetheRBSoftheclonedgene,thetranscriptionalandtranslationallevelsoftheclonedgeneweredependedontheintensityofluminescencesothattheexpressionoftheclonedgenecouldbedeterminedbydetectingtheCPS.Toverifythemonitoringsystemconstructedaboveandfurthertoestablishthefluorescencedatabaseforthecorrespondingproteinofthewhole-gemoneofE.coliandPAO1weregoingtobestudiedinthefollowingresearch.TheproteinexpressionmonitoringvectorswereconstructedinE.coliandbeennamedpPROEXHTa-luxandpPROEXHTb-luxandpPROEXHTc-lux,repectively.pPROEXHTc-luxwastheappreciatevectorfortheexpressionofluxABCDEincorrectreadingframe.TheresultofmeasuringtheintensityofluminescenceinE.colicontainingpPROEXHTc-luxwithIPTGshowedthetrcpromotercouldtranscriptthereportergeneluxABCDE,andtheexpressionoftheluxABCDEalsocouldbeinducedbytheregulatingfactoroftrcpromoter.Meanwhile,theregionaroundthestartcodonofluxABCDEgeneofpPROEXHTc-luxwassequencedtoindicatethatthesequencingregionofpPROEXHTc-luxwasmatchedwithexpected.Besides,theproteinexpressionmonitoringvectorswereconstructedinPAO1whichnamedpMS402-luxABCDE(a),pMS402-luxABCDE(b),pMS402-luxABCDE(c)aswell.TheproteinexpressionmonitoringsystemsestablishedinE.coliandPAO1inthisstudywerenewtechniqueswhichcouldachievehigh-throughputandhighsensitiveandlarge-scaledetection.Thisnewmethodcouldbeusedfornotonlyproteomicstheorystudy,butalsothepracticalfieldslikebiomedicalinvestigation.Keywords:E.coli,Pseudomonasaeruginosa,Monitoringsystemofproteinexpressioniii缩写对比表PA铜绿假单胞菌E.coliDH10B大肠埃希氏菌DH10B菌株RBS核糖体结合位点CPS每秒钟化学发光MCS多克隆位点PCR聚合酶链式反应DNA脱氧核苷三磷酸Min分钟Amp氨苄青霉素Tc四环素Gm庆大霉素Kan卡那霉素Tmp甲氧苄氨嘧啶EDTA乙二胺四乙酸lacZΒ-半乳糖苷酶基因Ori复制起始位点SDS十二烷基硫酸钠TETE缓冲液HEPESN-2-羟乙基哌嗪N-2-乙磺酸RNAaseRNA酶DMSO二甲基亚砜iv目录中文摘要.....................................................................................................................................iAbstract.....................................................................................................................................ii缩写对比表...............................................................................................................................iii图表目录..................................................................................................................................vii第一部分引言........................................................................................................................11.1蛋白质组学研究的背景..............................................................................................11.2蛋白质组学的研究方法..............................................................................................31.2.1蛋白质的分离技术............................................................................................31.2.1.1二维SDS-聚丙烯酰胺凝胶电泳技术.............................................................31.2.1.2色谱技术.................................................................................................................41.2.1.3一维电泳(色谱)-质谱技术...........................................................................51.2.2蛋白质的鉴定技术——生物质谱技术............................................................51.2.2.1基质辅助激光解吸电离质谱(MALDI-MS)......................
本文标题:大肠杆菌和铜绿假单胞菌中蛋白表达监测系统的构建
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