人MUC┐1全长cDNA基因真核表达载体构建及在COS┐7细胞中的表达.doc

MUC1cDNACOS7 :MUC-1;COS-7; :MUC-1cDNACOS-7.MUC-1pGEM-3zf-DNA.pcDNA3.1+pcDNA3.1+-MUC-1.COS-7MUC-1.MUC-1cDNAMUC-1COS-7.MUC-1cDNACOS-7. Keywards:MUC-1gene;COS-7cell;electroporation;eu-karyoticexpression Abstract:AIMToconstructaneukaryoticexpressionvec-torcontainingthecodingregionofhumanfulllengthMUC1geneandtodetectitsexpressioninCOS-7cells.METHODSTheMUC1genewasligatedintothesitesofHindIIIandSalIofcloningvectorpGEM-3zf.AfteridentificationthegenewassubclonedintoeukaryoticexpressionvectorpcDNA3.1+.TherecombinantpcDNA3.1+-MUC-1wastrans-fectedintoCOS-7cellsbyelectroporation.Im-munoflourescenceandFCMwereusedtodetecttheMUC1geneexpressioninCOS-7cells.RESULTSRestrictionanal-ysisandDNAsequencingshowedthattherecombinantplas-midcontainedthecodingregionofhumanfulllengthMUC1gene.TransfectionexperimentverifiedthatMUC1genecouldbeexpressedinCOS-7cells.CONCLUSIONTheeu-karyoticexpressionvectorcontainingtheMUC1genewassuccesfullyconstructedandexpressedwhichisabasisforthestudyforDNAvaccine. 0 MUC-1PEMpolymorphicepithelialmucin1-3polymorphism2variablenumberoftandemre-peatsVNTRsVNTR60GC.VNTRs20125.MUC-1.4-7MUC-1.;MUC-1MUC-1MHCMHCTCTLsCTLsMUC-18.MUC-1.MUC-1MUC-1cDNACOS-7. 1 1.1 1.1.1E.coliDH5pGEM-3zf-MUC-1pVax-MUC-132tandemrepeatspcDNA3.1+ImperialCancerResearchFundTaylor-Papadimitriou.COS-7. 1.1.2HindSalXhoGibco;T4DNATaKaRa;.E.Z.N.ARplasmidMiniprepKitOmega;NucleoTrapRGelExtractionKitClontech;DMEMGibco;MUC-1mAbAntibodyDiagnostica;IgG-FITC;. 1.2 1.2.1MUC-19.pVax-MUC-1pGEM-3zf-HindXhoHindSalSalXholNucleoTrapRGelExtractionKit3.5kbpMUC-1DNApGEM-3zf-DNA3116.2LDH5DH5100mg・L-1LB37.16hHindXba. 1.2.2DNAE.Z.N.ARplasmidMiniprepKitpGEM-3zf-MUC-1T7promoterSP6.DNA. 1.2.3pGEM-3zf-MUC-1MUC-1HindXhopcD-NA3.1+.DH510.pcDNA3.1+-MUC-1. 1.2.4COS-7100mL・L-1FCSDMEM100mL75%10g・L-12g・L-1EDTA2000r・min-110min1PBS21010・L-1. 1.2.5COS-710pcDNA3.1+-MUC-1pcDNA3.1+OmegaE.Z.N.ARplasmidMiniprepKitDNA.DNA1g・L-1.40g0.8mL0.4cm10min50F450V25010min100mL・L-1FCSDMEM3cm3750mL・L-1CO2. 1.2.6MUC-154h2g・L-1EDTACOS-71PBS0.51.01010・L-1;40LMUC-1mAb10L5mL50L120430min22mL/1000r・min-15min;50LIg-FITC430min22mL/1000r・min-15min;100L. 1.2.7MUC-1COS-7. 2 2.1MUC13.5kbFig1MUC-1cDNApGEM-3zf-MUC-1. 1 2.2MUC1pGEM-3zf-MUC-1DNA13MUC-1cDNA. 2.3pcDNA3.1+MUC1pcDNA3.1+-MUC-1DNAHindXholMUC-1pcDNA3.1+Fig2. 2.4MUC1COS7pcDNA3.1+pcDNA3.1+-MUC-1COS-7pcDNA3.1+-MUC-1COS-7Fig3. 2.5MUC-1COS-7Fig4. 24 3 MUC-1Mucin3240630nmCTL1112.DNAimsc.MUC-1.MUC-113-15. pGEM-3zf-MUC-1MUC-1pcDNA3.1+-MUC-1MUC-1.COS-7MUC-1MUC-1COS-7.MUC-1COS-7MUC-1MUC-1. : 1MollerHSerttasNPaulsenHBurchellJMTaylor-Pa-padimitriouJBerndM.NMR-baseddeterminationofthebind-ingepitopeandconformationalanalysisofMUC-1glycopeptidesandpeptidesboundtothebreastcancer-selectivemonoclonalantibodySM3J.EurJBiochem2002;2695:1444-1455. 2MilesDWTaylor-PapadimitriouJ.TherapeuticaspectsofpolymorphicepithelialmucininadenocarcinomaJ.PharmacolTher1999;821:97-106. 3GendlerSJLancasterCATaylor-PapadimitriouJDuhigTPeatNBurchellJPembertonLLalaniEI-NWilsonD.MolecularCloningandExpressionofHumanTumor-associatedPolymorphicEpithelialMucinJ.JBiolChem1990;26525:15286-15293. 4RahnJJDabbaghLPasdarMHughJC.TheimportanceofMUC1cellularlocalizationinpatientswithbreastcarcinoma:Animmunohistologicstudyof71patientsandreviewofthelit-eratureJ.Cancer2001;9111:1973-1982. 5vonMensdorff-PouillySSnijdewintFGVerstraetenAAVer-heijenRHKenemansP.HumanMUC1mucin:AmultifacetedglycoproteinJ.IntJBiolMarkers2000;154:343-356. 6CuiDXWangLYanXJSuCZWangJB.Alterationofex-pressionofnm23-H1andmuc1andtheirclinicalsignificanceJ.Di-siJunyiDaxueXuebaoJFourthMilMedUniv1999;209:823-826. 7LalaniEI-NBerdichevskyFBoshellMShearerMWilsonDStaussHGendlerSJTaylor-PapadimitriouJ.ExpressionofthegenecodingforahumanmucininmousemammarytumorcellcanaffecttheirtumorigenicityJ.JBiolChem1991;26623:15420-15426. 8MukherjeePGinardiARTinderTLSternerCJGendlerSJ.MUC1-specificcytotoxicTlymphocyteseradicatetumorswhenadoptivelytransferredinvivoJ.ClinCancerRes2001;73Suppl:848s-855s. 9DavisLKuehlMBatteyJ.Basicmethodsinmolecularbiology2ndeditionM.NorwalkConnecticut:AppletonLange1993:237-245612-624. 10FongZHHuangpuYM.MedicalmolecularbiologyM.Wuhan:WuhanPress1998:265-267. 11SnijdewintFGvonMensdorff-PouillySKaruntu-WanamartaAHVerstraetenAALivingstonPOHilgersJKenemansP.Antibody-dependentcell-mediatedcytotoxicitycanbeinducedbyMUC1peptidevaccinationofbreastcancerpatientsJ.IntJCancer2001;931:97-106. 12GrahamRABurchellJMBeverleyPTaylor-PapadimitriouJ.IntramuscularimmunisationwithMUC1cDNAcanprotectC57micechallengedwithMUC1-expressingsyngeneicmousetumourcellsJ.IntJCancer1996;655:664-670. 13JohnenHKulbeHPecherG.Long-termtumorgrowthsup-pressioninmiceimmunizedwithnakedDNAofthehumantu-morantigenmucinMUC1J.CancerImmunolImmunother2001;507:356-360. 14SoaresMMMehtaVFinnOJ.Threedifferentvaccinesbasedonthe140-aminoacidMUC1peptidewithseventandemlyre-peatedtumor-specificepitopeselicitdistinctimmuneeffectormechanismsinwild-typevers