重组变异体二氢叶酸还原酶和绿色荧光蛋白腺相关病毒载体的构建及外源

,1,1(,1,510515)(mDHFR)(GFP)(AAV),NIH3T3,NIH3T3mDHFRGFP,PCR,T,AAV,NIH3T3,PCR:PCRNIH3T3DNAmDHFRGFPcDNA,25%,MTTMTX:AAVmDHFRGFPNIH3T3,MTX,mDHFRAAV;;;;Q782;R730141ConstructionofAdeno2AssociatedVirusVectorCarriedMutatedDihydrofolateReductaseandGreenFluorescentProteinandItsExpressioninNIH3T3CellsLILI2Bo,FENGRu1,ZHOUShu2Yun1(DepartmentofOncologyand1DepartmentHematology,NanfangHospital,TheFirstMilitaryMedicalUniversity,Guangzhou,510515)AbstractTheaimofthisstudywastoconstructrecombinantmDHFR2GFP/AAVvectorcontainingmutateddihydrofolateredutase(mDHFR)andgreenfluorescentprotein(GFP)fusiongenesanditsexpressioninNIH3T3cells,toinvestigatetheresistanceofthecellstomethotrexate.AmplifiedcDNAofmDHFRandGFPsegmentedfromtheirplasmidseparatelywerelinkedbyPCRwiththeaminoaceticacidlinker.ThefusiongenewasinsertedintoTvector,andafterenzymecuttingthefusiongenefragmentwasinsertedintoAAVvector,thenpackagingthevectorintorecombinedAAVandinfectedNIH3T3cells.ExpressionofgenefusionwasobservedbyPCR,fluorescentmicroscopyandflowcytometry.mDHFRandGFPcDNAwerefoundinNIH3T3genomicDNA,theGFPexpressionratewasabout25%,andresistanceofthetransferedcellstoMTXwasincreasedmarkedly.TheresultsshowedthatAAVvectorcantransfermDHFRandGFPfusiongeneintoNIH3T3cellsandincreaseresistancetoMTXingenemodifiedcells.ThisdataprovidedabasisforapplicationofmDHFRandAAVvectoringenetherapy.Keywordsadeno2associatedvirus;mutateddihydrofolatereductase;greenfluorescentprotein;vectorconstruction;geneexpression,,,,,,:;,;,(mutateddihydrofolatereductaseL22Y/F31R,mDHFR)2231DHFR,,(GFP),Ca2+,,,[1],AAV/mDHFR2GFP:39670826::[phone].:[phone].E2mail:[e-mail]212JournalofExperimentalHematology2002;10(3):212-217©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.NIH3T3,PCRMTTAAVpWAV,(Ad8),(Ad5)293()pWAVAAV(ITR),repcap,ITRCMV(Nhe,Xho,PstBamH)polyAMSCV2DHFR(L)(StJudeBrianSorrentino),pCMX2GFPNIH3T3DH5NikonEclipseE600Nikon,(BioRad)DNA(Promega),DMEM(Gibco),,Lipofection(Gibco),,Polybrene,MTT,DMSO(Sigma),PCR10buffer,4dNTP(10mmol/L),MgCl2(25mmol/L),DNATaq(2U/l)()DNAMarker(),(Promega)DHFR,1mDHFR17,mDHFR,mDHFRGFP4515,2mDHFR1731331GFP17,4GFP171:5ATGGTTGGTTCGCTAAA3;2:5CCGAC2CCACCACCGCCCGAGCCACCGCCACCATCATTC2TTCTCATATA3;3:5GGGCGGTGGT2GGGTCGGGTGGCGGCGGATCTATGGTGAGCA2AGGGCGA3;4:5TTACTTGTACAGC2TCGT3:5GGTGGCG2GTGGCTCGGGCGGTGGTGGGTCGGGTGGCGG2CGGATCT3;5AGATCCGCCGCCACCGACCC2ACCACCGCCCGAGCCACCGCCACC3PCR12MSCV2DHFR(L)mDHFR,34pCMX2GFPGFP;mDHFRGFPPCRmDHFR2GFP,14mDHFR2GFPmDHFR2GFPmDHFR2GFP(1326bp),T,,T,BamHXhoT,BamHXhomDHFR2GFP,(1);BamHXhopWAV;XhoBamHmDHFR2GFP(1326bp)pWAV,rAAV/mDHFR2GFP(2)Figure1ProductionofmDHFRandGFPfusiongenebyPCR312mDHFRGFP©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Figure2ConstructionofthehuammDHFRandGFPcDNAexpressionvectordirectedbyCMVpromoterNIH3T3,5%DMEM37,5%CO2,70%,DMEM1,LipofectinDNALipofectin5l(2mg/ml),DNA4g,24,481104/96,(150nmol/L),2,6MTT15,72,DNADNANIH3T3LipofectinAD8293,12AD5293,48-72,293,3-5,,,NIH3T3,72,DNAPCRDNA,PCR:945,Taq0.3l:9440,4840,7260,30,7210PCR[1]200l,889g,490nm,475nm(40),(40),AAV/mDHFR2GFP2PCRmDHFR2GFP,3,pWAVAAV/mDHFR2GFPFigure3PCRamplifiedproductofmDHFRandGFPfusiongene(DG).Land1,3and6aresamesample.M:markerAAV/mDHFR2GFP6.8kbXhoBamH,1.3kb,BamHXbaI,1.0kb,4mDHFR2GFPAAV,mDHFR2GFPcDNAAAV/mDHFR2GFPNIH3T3PCRNIH3T3mDHFRGFPcDNA,564bp720bp,,5AAV/mDHFR2GFPNIH3T348,,21,6,MTTSPSS,412JExpHematol2002;10(3)'1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Figure4RestrictionanalysisofrecombinantDNA.Lane1and2:byBamHandXhorestrictionenzyme.Lane3:byBamHandXbarestrictionenzyme.M:markerFigure5PCRanalysisofmDHFRandGFPcDNAinNIH3T3cellsinfectedwithrecombinantAAV/mDHFR2GFPvirus.Lane1:GFP.Lane2:mDHFR.M:markerOD,t,P0.01,OD()TableCellviabilityoftransgenicanduntransgenicNIH3T3cellsassayedbyMTTmethodGroupNumbersofsamplesODvalueTransgeniccell100.4540.0213Untransgeniccell100.3780.0123t=3.827,P0.00520%,48(6,7)Figure6TransgenicNIH3T3cellsunderphasecontrastfluroscencemicroscop(40)Figure7TransferredNIH3T3cellsunderfluroscencemicro2scope(40)8AAV/mDHFR2GFPNIH3T3,48-7222.5%,,,,9610%,DNARNA,,,,DNA,[2]AAV512mDHFRGFP©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Figure8ThetransfectionrateofrecombinantAAV/mDHFR2GFPvirusbyflowcytometry.A:selectionofnormalsizeNIH3T3cellstoremovecellscrap.B:control.C:thetransfectionrateof24hoursis8.5%.D:therateof48hoursis22.5%.E:therateof72hoursis18.2%.F:therateof96hoursis10.8%,DNA,4675bp,322repcap,rep,cap[3]AAV(ad8)(ad5)(293)[4]AAV(repcap),AAVITR,ITR,DNA(4.6kb),120%,AAVMDR1,rAAV/MDR1AAV,,[5],mDHFRGFP,AAVGFPAequoreaVictoria,1,,,GFP[6],AAV4:Nhe,Xho,Pst,BamH[7],MSCV2DHFR(L)pCMX2GFP,PCRmDHFRGFP,,145,15,,,612JExpHematol2002;10(3)'1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.(DHFR),22kDDHFR,,DHFR,DNARNA,DHFR(MTX)MTX,,MTXDHFR,,DHFRFlasshove[8]S31DHFRCD34+,DHFRMTX2.5mDHFR(L22Y/F31R)2231DHFR,,,AAV/mDHFR2GFP,NIH3T3,mDHFR2GFP,mDHFRMTX,,MTX,1LarrickJW,BalintRF,YouvanDC.Greenfluorescentprotein:un2tappedpotentialinimmunotechnology.Immunotechnology,1995;1:83-862CrystalRG.Transferofgenestohumans:earlylessonsan