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436200412()JournalofFudanUniversity(NaturalScience)Vol.43No.6Dec.2004:0427-7104(2004)06-1056-05X倪伟明,黄路标,王荫榆,刘国元,张伯生,任大明(,,200433):.,AS1.2984.PCR2,-pNZ8037,,.pNZ8037,.MRS,.:;;;pNZ8037;;;:Q782:A,,,[1].[2].:[3].,Nisin[4].,,.,.,,[1].,EfV583[5],.2,,GFP,GFP,Nisin.,.11.1AS1.2984AS1.2986,.1.2pNZ8037(-,,NisinPnisA),pSG1164(GFP)NIZOFoodResearch.TPromega.NisinPWY7130-gfp,.1.3TaKaRa();IPTGX-galPromega;dNTP;NisinSigma;.X:2004-03-25:(1978)),,;.1.4(a)MRS:[4]:10g10g5gK2HPO42g2g-801mLNaAc5gMgSO40.5gMnSO40.25g20g,1L,pH6.2~6.6,830min.100mL1.6g.(b)MRS[4]:100mLMRS20g.(c)MRS:MRS.(d)LB:10gNaCl10g5g,1L,pH7.4.100mL1.6g.(e)PEB2(PEB)[4]:0.5mol/L0.5mmol/LMgCl21mmol/L,pH7.4.1.51.5.1AS1.2984,AS1.2984EfV583,,EfV583[5]AS1.2984.BDGPNeuralNetworkPromoterPredictionEfV583(GAP)(LDH),,300bp.1.5.2pLDH-GFP-pNZ8037pGAP-GFP-pNZ8037AS1.2984,PCRGAPLDH.GAP(+)5c-GCAGATCTATGGTTATGCTCAA-3c(-)5c-CCCTGCAGAACTGTCATTAAA-3c;LDH(+)5c-CGAGATCTCTGAAGCGACACA-3c(-)5c-CGCTGCAGTACTTTCATCGTTC-3c.PCR(50LL):10@buffer5LL;dNTP(2.5mmol/L)1LL;(10Lmol/L)1LL,1LL;Taq(1U/LL)1LL;DNA(50ng/LL)1LL;ddH2O40LL.:94e5min,94e30s,53e30s,72e45s,30;72e10min.PCR(8g/L),GelExtraction,T-VectorProtocolPCRT-Vector,CaCl2,T-pGAPT-pLDH.T-pGAPT-pLDHpNZ8037BglIIPstI,,pLDHpGAPPnisApNZ8037,pLDHpGAPPnisApNZ8037,,pLDH-pNZ8037pGAP-pNZ8037.pLDH-pNZ8037pGAP-pNZ8037pSG1164PstIXbaI,,,,pLDH-GFP-pNZ8037pGAP-GFP-pNZ8037.,GFPpLDHpGAP,pLDHpGAP.1.5.3pLDH-GFP-pNZ8037pGAP-GFP-pNZ8037AS1.2984pLDH-GFP-pNZ8037pGAP-GFP-pNZ8037,[4]AS1.2984.2LL40LL,(0.2cm),5min.Bio-Rad,1.8kV,25LF,2008.,400LLMRS,1.5mLeppendorf37e2h,100LL5Lg/mLMRS,36h,.1.5.410576:MRS,pH7.40.1mol/LTris-Cl,,LeicaTCSNT,NisinPWY7130-gfp,pLDHpGAP.MRS.22.1BDGPNeuralNetworkPromoterPredictionEfV583GAPLDH400bp,:¹GAP0.80,,400bp40.80.,1.00,0.80.GAP400bp,300bp.49941.00ttctttgatgaagaaggaaaagtggtctataaaattccacGaatt891340.97gaattggccttcaattaaaaaacttacaagaaattccctaTgtcg2352801.00gagattttaaaaggggtaaccctttaaaataaaaaattttTgatt2683130.99aaatttttgattttcataaggaggaaatctttaatgacagTtaaaºLDH0.80,,400bp70.80.,300bp.36810.97atttttggaagttcatagctacagctaccgataactagttGaaaa681130.97aactagttgaaaacggttcggctgtttttcattattgcttGcata991440.88ttattgcttgcataatacactgttgtcgttgtcattttgcCtcct1241690.86tcgttgtcattttgcctccttatctcctctattttcctacTctta1932380.93cttatttaaattgttaataaagactattacagattggtatAacca2012460.99aattgttaataaagactattacagattggtataaccacacCtaaa2482930.87gttcgtgatataattcactaagaaagaaatattacagaaaGaaga2.2AS1.2984DNA,GAPLDHPCR,T-Vector,GFPpNZ8037,pLDH-GFP-pNZ8037pGAP-GFP-pNZ8037.,.1.2.3AS1.2984LeicaTCSNTpGAP-GFP-pNZ8037pLDH-GFP-pNZ8037AS1.2984GFP-pNZ8037AS1.2984NisinPWY7130-gfpAS1.2984,pGAP-GFP-pNZ8037pLDH-GFP-pNZ8037.NisinPWY7130-gfp,pGAP-GFP-pNZ8037pLDH-GFP-pNZ8037,.2..,,MRS,GFP,GFP1058()43.3.1A.pLDH-GFP-pNZ8037;B.pGAP-GFP-pNZ8037Fig.1Mapofconstructedplasmids2Fig.2ExpressionofGFPA.AS1.2984;B.GFP-pNZ8037AS1.2984;C.NisinPWY7130-gfpAS1.2984;D.pGAP-GFP-pNZ8037;E.pLDH-GFP-pNZ80373Fig.3RegulationofthepromotersA.,pH6,pGAP-GFP-pNZ8037GFP;B.,pH11,pGAP-GFP-pNZ8037GFP;C.,pH6,pLDH-GFP-pNZ8037GFP;D.,pH11,pLDH-GFP-pNZ8037GFP3,..,;.2[3],.10596:[4],.,,.,,,.GFP,,.MRS,,GFP,MRS,,MRSMRS.,GFP,,.,,GFP,,.H+OH-,,GFP.,,.:[1].[M].:,2002.[2].[J].中国乳业,2001,6:21-24.[3]RenDM,WangYY,WangZL,etal.CompleteDNAsequenceandanalysisoftwocrypticplasmidsisolatedfromLactobacillusplantarum[J].Plasmid,2003,50:70-73.[4],,,.[J].生物工程学报,2002,18(4):516-520.[5]PaulsenI,BanerjeiL,MyersG,etal.RoleofMobileDNAintheEvolutionofVancomycin-ResistantEntero-coccusfacalis[J].Science,2003,299:2071-2074.ConstructionoftheExpressionPlasmidinLABNIWe-iming,HUANGLu-biao,WANGYin-yu,LIUGuo-yuan,ZHANGBo-sheng,RENDa-ming(StateKeyLaboratoryofGeneticsEngineering,InstituteofGenetics,SchoolofLifeSciences,FudanUniversity,Shanghai200433,China)Abstract:Enterococcusfacalisisonekindoflacticacidbacteria(LAB).Byusingbioinformatics,constitutivepromot-ersofL-lactatedehydrogenaseandglyceraldehyde3-phosphatedehydrogenasewerepredictedfromthegenomeofEn-terococcusfacalisAS1.2984.Afterdesigning5cand3cprimers,thesequencesofthesetwopromoterswereobtainedbyPCR.ThenexpressionplasmidspGAP-GFP-pNZ8037andpLDH-GFP-pNZ8037wereconstructedwithreportingpro-tein-GFPjustunderthecontrolofthesetwopromotersintheshuttleplasmid-pNZ8037,andtheyweretransformedin-toEnterococcusfacalisbyelectricpulse.TheexpressionofGFPisconfirmedbyconfocusmicroscope.TheexpressionofGFPisdifferentattheexistenceofsomereagentssuchaslacticacid,HClandNaOH.Soitisassumedthatacidicandalkalinematerialcanregulatethesetwopromoters.TheseresultsshowthatmorepowerfulpromoterscanbefoundintheLAB.sgenomebybioinformaticsintheshorttime.Keywords:LAB;Enterococcusfacalis;promoter;pNZ8037;GFP;confocusmicroscope;orallytakenvaccine1060()43