实时荧光定量PCR构建奶牛生殖系统PrP基因标准品质粒和标

PCRPrP121111(1./,100094;2.,510642):20060707:(30571399;30500371);(20050019031):,,E2mail:[e-mail];,,,,,E2mail:[e-mail],,RT2PCRPrPEcoR302bp,r2=0.999,y=-0.29x+9.48,PrP,PrPPrP;RT2PCR;PCR;;;BSES85519+910074333(2007)02000104AConstructionofcowreproductivesystemPrPgenestandardplasmidandcurveusingreal2timeRT2PCRZhangTaixiang1,NingZhangyong2,ZhaoDeming1,ZhouXiangmei1,YangJianmin1,YiXiaomin1(1.NationalLaboratoryofAnimalTransmissibleSpongiformEncephalopathies,CollegeofVeterinaryMedicine,ChinaAgriculturalUniversity,Beijing100094,China;2.CollegeofVeterinaryMedicine,SouthChinaAgriculturalUniversity,Guangzhou510642,China)AbstractThepurposeofthisstudywastoelucidatethenormalPrPgeneexpressionanditsnormalphysiologicalfunc2tions,confirmtheroleofthereproductivesysteminthematernitytransmissionrouteoftheBSEandestablishagoodfoundationforfurthermolecularstudiesonBSEspread.HerewereporttheconstructionofcowreproductivesystemPrPgenestandardplasmidandcurveusingreal2timeRT2PCR.TheresultoftheEcoRtotheplasmidincludingthetargetgeneshowedtheproductwas302bp,whichwasaccordingtotheexpectedresult.Theconstructedstandardcurvehadarelativecoefficientr2=01999andtheregressingequation:y=-0129x+9148suggesteditwassuccessful.Theconstructedcalibrationcurvewasusedtoquantifytheunknownsamplesofcow.KeywordsPrP;RT2PCR;real2timequantitativePCR;standardcurve;cowreproductivesystem;BSE,[14](PrPc)(PrPsc),[5]PrP,,PrPmRNA,,PrP[3],,[46]PrP,PrPPrPcPrPsc,PrPsc2007,12(2):14JournalofChinaAgriculturalUniversity©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[7]PCR(real2timequantitative,PCR),[8]PrP,PCRPrP,PrP1111:;:E.coliDH5TRIZOLRNA(Gibco)dNTPs(TaKaRa)TaqDNA(TaKaRa)M2MLV(Promega)DEPC(Promega)RNASEOUTRibonuclease(Promega)SYBRGreenPCRKit(FINNZYMES)EcoR(TaKaRa)()PGEM2TEasyVectorSystems(Promega)E.Z.N.AGelExtractionKit(Promega)PCR(PTC2150,MJResearch)PCR(OPTICON2,MJResearch)112RNA011%(,)DEPC,011%DEPC372h,DEPC,50100mg1mLRNA2Solv(OmegaBio2tek)Eppendorf,;1mLRNA2Solv012mL(4),10min,12000g20min;80%Eppendorf,500L(4),10min,12000g10min,;1mL75%()(4),7500g5min;,1020min,3050LDEPCRNA,-86[1011]113RNART2PCRPrPmRNA(GenBankAY247262),,PrimerPrimer510,(PE3900DNA):PrP,:5GCTACATGCTGGG2AAGTG3,:5TTGGTAATAAGCCTGGGAT3,302bp012mL:primer(T18)1L,10mmol/LdNTP1L,RNA(015g/L)5L,DEPC7L655min,:5buffer4L,RNA1L,M2MLV1L20L,37,1h;70,15min,cDNA-20PCR,25L,:10buffer215LdNTPs2LPrP(20mol/L)015LExTaq012LcDNA2L1713L:945min;9430s5130s7240s,36;728min114E.Z.N.AGelExtractionKitPCRPGEM2T2EasyVectorSystemsPGEM2T2easy,10L,:PGEM2T2EasyVector(50ng)5LPCR115LT4DNA1L115L,4200LDH5,,Amp-LB1h,Amp+X2GalIPTGLB,37442200712©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(6595,01211s)Opticonanalysissoftware(version2101)2211RNARNAA260/A280118210,RNA212EcoR,pGEM2T2EasyEcoR,,302bp(1)DNA,DNAPubmedblast100%,M:MarkerDL22000;1,2EcoR;3,4PCR1PrPPCRFig.1AgarosegelelectrophoresisofPrPrecombinantT2easy2vectorsfollowingdigestionandproductofPCRusingtheplasmidastemplate213108102,,,102;,,r2=01999,:y=-0129x+9148(2)2PrPmRNART2PCRFig.2Exampleofreal2timePCRforquantificationofPrPmRNA21465950121,,,(2(c))1%()302bp,3RT2PCRmRNA,[10]PCRPCR,PCRPCR,PCR,[9,1112]PCRmRNART2PCR,,32:PCRPrP©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[11]Ct,,DNAPCR,,Ct,Ct,,Ct,,10Ct01999,,[1]PrusinerSB.Novelproteinaceousinfectiousparticlescausescrapie[J].Science,1982,216:136144[2]StahlN,BorcheltDR,HsaioK,etal.Scrapieprionproteincontainsaphosphatidylinositolglycolipid[J].Cell,1987,51:229240[3]PrusinerSB.Prions[J].ProcNatlAcadSciUSA,1998,95:1336383[4]LiemannS,GlockshuberR.Transmissiblespongiformencephalopathies[J].BiochemBiophysResCommun,1998,250:187190[5]Bruce,ME,ChreeA,McConnellI,etal.Transmissionofbovinespongiformencephalopathyandscrapietomice:strainvariationandthespeciesbarrier[J].PhilosTransRSocLondBBiolSci,1994,343,405411[6]SkrettingG,EspenesA,UlvundMJ,etal.cDNArep2resentationaldifferenceanalysisofilealPeyerspatchesinlambsafteroralinoculationwithscrapie[J].BiochemicalandBiophysicalResearchCommunications,2004,316:272279[7]SafarJ,RollerPR,GajdusekDC,etalConformationaltransitions,dissociationandunfoldingofscrapieamyloid(prion)protein[J].JBiolChem,1993,268:2027620284[8]WalkerNJ.Real2timeandquantitativePCR:applica2tionstomechanism2basedtoxicology[J].JBiochemMolToxicol,2001,15(3):121127[9]WhelanJA,RussellNB,WhelanMA.AmethodfortheabsolutequantificationofcDNAusingreal2timePCR[J].JournalofImmunologicalMethods,2003,278:261269[10]TichopadA,PfafflMW,DidierA.Tissue2specificex2pressionpatternofbovinepriongene:quantificationus2ingreal2timeRT2PCR[J].MolecularandCellularProbes,2003,17:510[11]NingZhanyong,ZhaoDeming,LiuHongxiang,etal.Quantificationofpriongeneexpressioninbrainandpe2ripheralorgansofgoldenhamsterbyreal2timeRT2PCR[J].AnimalBiotechnology,2005,16:111