细菌内同源重组法构建pAd-EGFP-hSDF-1

pAd-EGFP-hSDF-11,2,1,1,1,1,1,1(1;2,442000)[]:-1(hSDF-1):5’EcoRV/XhoIhSDF-1cDNA,(PCR)pORF-hSDF-1hSDF-1cDNA,pGEM-TEasy,pGEM-T-hSDF-1,EcoRV/XhoI279bp,8.8kbpShuttle-IRES-hrGFP-2,DH5,,pShuttle-EGFP-hSDF-1EcoRV/XhoIpShuttle-EGFP-hSDF-1PmeI,pAdeasy-1BJ5183,pAd-EGFP-hSDF-1;pAd-EGFP-hSDF-1PacPCR:pShuttle-EGFP-hSDF-1pAdeasy-1BJ5183,23kb4.5kb,PCR279bp:hSDF-1cDNA,hSDF-1hSDF-1[],;T;-1;;[]Q813.2[]A[]1006-9674(2006)03-0193-05ConstructionofRecombinantAdenovirusPlasmidContaininghSDF-1cDNAByHomologousRecombinationinBacteriaTANGJun-ming1,2,HUANGYong-zhang1,GUOLing-yun1,PANGuo-dong1,KONGXia1,YANGJian-ye1,WANGJia-ning1.(1InstituteofClinicalMedicine,RenminHospital;2DepartmentofPhysiology,YunyangMedicalCol2lege,Shiyan,Hubei442000,China)Abstract:ObjectiveToconstructrecombinantadenovirusplasmidcontaininghSDF-1cDNAbyusinganovelandhighefficientmethodofhomologousrecombinationinbacteria.MethodshSDF-1cDNAprimersweresynthesizedwithforwardprimerandre2verseprimercontainingEcoRVandXhoI,respectively.hSDF-1cDNAwasamplifiedbypolymerasechainreaction(PCR),andsubclonedintopGEM-TEasyvectortoconstructpGEM-T-hSDF-1.Thefragmentof279bphSDF-1cDNArecoveredfromEcoRV/XhoI-digestedpGEM-T-hSDF-1wassubclonedintopShuttle-IRES-hrGFP-2.TherecombinantplasmidwasnamedafterpShuttle-EGFP-hSDF-1andidentifiedwithEcoRVandXhoIdigestion.AdenovirusgenomicDNAplasmidofpAdeasy-1wastransformedintoBJ5183bacteriaandultracompletentBJ5183containingpAdeasy-1wasprepared,pShuttle-EGFP-hSDF-1waslinealizedwithPmeIandtransformedintoultracompletentBJ5183containingpAdeasy-1,theidentificationofrecombinantadenovira1plasmidpAd-EGFP-hSDF-1wasperformedwithdigestionwithPacIandPCR.ResultsThereweretwobands,4.5kbandlargerthan23kbwhenpAd-EGFP-hSDF-1wasdigestedwithPacIandelectrophoresis.Thereap2peared279bphSDF-1cDNAfragmentwhenPCRwasperformedwithpAd-EGFP-hSDF-1astemplate.ConclusionTherecombinantadenoviralplasmidcarryinghSDF-1cDNAwassuccessfullyconstructedwithhomologousrecombinationinbacteria.ThisstudyprovidesabasisforthepreparationofrecombinantadenovirusexpressinghSDF-1andforexploringthemigrationmechanismofbonemarrow-derivedstemcellsintoinjuredtissue.Keywords:Homlogousrecombination;pGEM-TEasyvector;hSDF-1;Adenovirus;EGFP[](2005ABA079),(JX2B68),(Q200524003,B200624006)[](1974-),,,,,[](1967-),,,,,:-1(stromalderivedfactor-1,SDF-1)CXC:SDF-1SDF-1,CXC4(CXCchemokinereceptor4,CXCR4)7G[1]1997,AiutiSDF-1CD34+,CD34+SDF-1391,pAd-EGFP-hSDF-1©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[2],SDF-1CXCR4[1,3],SDF-1,,SDF-1[4];,G-CSF,G-CSFSDF-1/CXCR4[5]CD34+/SDF-1CXCR4[1.4],SDF-1CXCR4///[6]/,,,hSDF-1,hSDF-1hSDF-1cDNA,hSDF-111.11.1.1:pORF-hSDF-1Invivogen;pAdeasy-1pShuttle-IRES-hrGFP-2BJ5183XL10-goldStratagene;pGEM-TEasyvectorPromega;DH51.1.2:EcoRVXhoIPacIPmeINewEnglandBiolabs;DNAHindmarker200bpDNAladderdNTPTaqT4DNA;hSDF-1cDNA::5’GATATCATGAACGCCAAGGTCGTGGTC3’,:5’CTCGAGTTACTTGTTTAAAGCTTTCTC3’,5OD,20mmol/L1.1.3:(UNIVERSAL32R,)PCR(Biometra,)1.21.2.1hSDF-1cDNA:pORF-hSDF-1,20mmol/L5l,pfuDNA1l,10buffer5l,10dNTP5l,50l:945min,9430s,5530s,7230s,30,7210min279bphSDF-1cDNA1.2.2T:[7,8],279bpPCR,10PCRbuff2er5l,10MgCl25l,2mmol/LdATP2l,Taq2l,50l,7230minAhSDF-1cDNA(281bp),10T4DNAbuffer5l,pGEM-TEasyvector0.5l,T4DNA1l,10l,3hDH5,(100g/ml)LB12h,,(100g/ml)TB12h,EcoRVXhoIpGEM-T-hSDF-11.2.3pShuttle-EGFP-hSDF-1:pShuttle-IRES-HrGFP-2pGEM-hSDF-1EcoRV/XhoI,,8.8kb279bphSDF-1cDNA,T4DH5,,,DNA1.2.4pAd-EGFP-hSDF-1:1.0gpAdeasy-1BJ5183,pAdeasy-1pAdeasy-1BJ5183,pAdeasy-1BJ5183pShuttle-EGFP-hSDF-1Pmel24h,DNA,(C1AP)5030minpShuttle-EGFP-hSDF-10.7%,pShuttle-EGFP-hSDF-1,pAdeasy-1BJ5183pAdeasy-1,pShuttle-EGFP-hSDF-1,491(JYMC)20066,25(3):193©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.:pAd-EG2FP-hSDF-11lPCR,hSDF-1cDNAPCRPCR22.1hSDF-1cDNApORF-hSDF-1,hSDF-1PCR,2%279bp(1)1hSDF-1cDNAFig1TheidentificationofhSDF-1cDNAPCRamplificationproductbyagarosegelelectrophoresis1.200bpDNAladder2.PCRproductofhSDF-1cDNA(279bp)2.2ThSDF-1cDNATaqA,T4DNApGEM-Teasyvector,DH5,,,EcoRVXhoI,2%279bp(2)pGEM-T-hSDF-1,:pGEM-T-hSDF-1hS2DF-1cDNApGEM-T-easyvector,hSDF-1In2vivogen(3)hSDF-1:ATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCTTTAAACAAG2pGEM-T-hSDF-1Fig2pGEM-T-hSDF-1wasidentifiedwithEcoRVandXhoIdigestion1.200bpDNAladder2.pGEM-T-hSDF-1wasdigestedwithEcoRVandXhoI(279bp,3kb)2.3pShuttle-EGFP-hSDF-1hSDF-1cDNApGEM-T-hSDF-1EcoRVXhoI,pShuttle-IRES-HrGFP-2,pShuttle-EGFP-SDF-1EcoRVXhoI8.8bp279bp,pShuttle-IRES-HrGFP-24pShuttle-EGFP-hSDF-1591,pAd-EGFP-hSDF-1©1994-2007ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(279bp,8.8kb)2.4(pAd-EGFP-hSDF-1)pShuttle-EGFP-hS