通过启动子同源重组研究人凝血因子的组成型表达

(C)305SCIENCEINCHINA(SeriesC)200010***(,200433)B,HeLahFIX.p921,,GCVG418,PCR,hCMVhFIXhCMV,.hFIXHeLa.,,,.B(hFIX),[1].,.cDNA,,[2].,,hFIX,hFIX,,.,hFIXDNA5CMVhFIX,hFIXHeLa,,.hFIX,ELISA.1,T4-DNANewEnglandBiolabs;Promega;pSSC-9;pA1pA2B.George;HeLaATCC;1999-08-03,2000-03-21*(:39880019)“863”(:863-Z20-02-01)(:98QMA1409)**(E-mail:[e-mail])536(C)30BioRadGenePulserElectroporating0.4cm;3A6Yoshioka;PCR:hFIXDNA5,5-GAGCAAACACATTCAAAAGC-3,pSSC-9neo,5-TAATATGCGAAGTGGACCTG-3;RT-PCR:2,5-CTTGTCATGAAAACGCCAA-3,3,5-AACATACTG-CTTGGAAAATT-3.(1)[3].(2):HeLaDMEM+10,100U/mL,100μg/mL;37,5CO2.G418200μg/mL,GCV500ng/mL.(3)DNA(PCR):[4]DNA,PCR94,30s,58,45s,68,2min,35.,1.0%,80V1h.(4)RT-PCR:RNA[5],PCR:90,30s,45,30s,70,2min,30.(5)(ELISA)[6][7].22.1p9212.1.1p921p921pSSC-9,.hFIXhFIX(ACCACTTT)+1.pA2PBS,hFIXDNA-7000-2200.pSSC-9hFIXDNA5-3460-2250,p92,1,:NhepA2,1800bp,XbapSSC-9,T41800bpp92.2.1.2p921pA1PBS,hFIX11125700,NheCla,p921.pCGCMV1100bpBamHNhe,p92BamHCla,,p921.2.2p921HeLap921Sfi,400V,100μF,600ΩHeLa.neoPCR.PCRHeLahFIXDNA5-flanking,2,PCR1900bp.p921HeLa,PCR1900bp(3),hFIXneohCMV.:5372.3hCMV2.3.1RT-PCRHeLa/p9215#HeLa/p921RNA,RT-PCR,PCRhFIX23.,RT-PCR183bp(4),hFIX1p921538(C)30hCMV,mRNA.2.3.2ELISAHeLa/p921HeLa1106,ELISA24h(5).HeLa0.01ng/(10624h).HeLa/p9212.0ng/(10624h),11.2ng/(10624h).FIX,85,FIXcDNA.3,23PCRλ/HindMarker;2;3pA2TDNA;4HeLaDNA;51#DNA;65#DNA;78#DNA4hFIX2,3RT-PCR1;2HeLa;35#;4PBSIXDNA;5PBR322/MspMarker:539,,.,.,.pSSC-9,,neoHSV-TK.HSV-TK,HSV-TK,.+1,hFIXDNA5-4060-2250+12+5753,7500bp.-219+15hFIX.-219-202DBP,NF1-99-76,ARP-36-22,HNF4-27-15,COUP-Ear3-15-36.,HNF4hFIX,ARPCOUP-Ear3hFIX.5,-1900-1600(silencer).-2250+12.hCMV,,cDNA,,.,hFIXhCMV.,hCMV,hFIX,1ATG34bp.,.HeLa,.,.HeLahFIX,.HeLa.,hCMVhFIX,,.ELISAFIXγ-,3A6.,,..,,,,Leyden.,.,,..,,.1DiscipioRG,DavieEW.CharacterizationofproteinS,agamma-carboxyglutamicacidcontainingproteinfrombovineand5HeLa/p921hFIXFIX3540(C)30humanplasma.Biochemistry,1979,18(5):8999042,,,.B.,B,1993,23(1):53603SambrookJ,FristonEF,ManiatisR.MolecularCloning:ALaboratoryMannual.NewYork:ColdSpringHarborLaboratoryPress,19894,,.DNA.,1990,35(8):6356285DuanJB,CaiX,ZhouLM,etal.Single-stepmethodoftotalRNAislationbysodiumdodecylsulfate/phenolextractionfromculturedcells.AnalyticalBiochemistry,1997,251:2912926,,..,1989,34(12):9329347,,.cDNAB.,B,1992,(5):611618