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:(30471617);863(20024032):100730(),();();();():yongzhelipumch@yahoo1com1cn:,,1964,1987,,,gp210p62LBRgp210p62LBR,,,(ELISA),gp210p62LBR(PBC),BL21M15;Ni2NAT,2(SDS2PAGE)(WB);ELISA,6068(PBC)6060gp210p62LBR,gp210p62LBR,,;SDS2PAGE690006200027000,WBgp210p62LBRELISA,PBC,gp210p62LBR3618%3019%519%;,gp210117%,3PBC3,(P0105P0101)gp210p62LBR,ELISAgp210p62LBR,PBC,PBC,;;Cloningandexpressionofnuclearenvelopeproteingp210p62LBRautoantigenegeneaswellasclinicalapplicationofthecorrelativeantibodiesindiagnosisofprimarybiliarycirrhosisLIYong2zhe3,LIULei,SUNQing2guo,ZENGChang2qian,ZHAOZhen2guo,TONGDa2wei,ZHANGShu2lan,GAOYang,YUMeng2xue,ZHULi2ping13DepartmentofClinicalLaboratory,PekingUnionMedicalCollegeHospital,PUMC&CAMS,Beijing100730,ChinaEmail:yongzhelipumch@yahoo1com1cnAbstractObjectiveTocloneandconstructthehumanplasmidcontaininggp210p62LBRofhumanperipheralbloodmononuclearcell,thenpurifyandidentifytheimmunoreactivityoftherecombinantprotein,andestablishtheenzymelinkedimmunosorbentassay(ELISA)todetectgp210p62LBRautoantigenecorrelativeantibodiesindiagnosisofprimarybiliarycirrhosis1MethodsTheconstructedplasmidwastransformedintoEcoliBL21M151ThisfusionproteinwaspurifiedbyNATchromatographyanditsimmunoreactivitywas02112005112811ChinJLabMed,November2005,Vol28,No111identifiedbySDS2PAGEandWesternblot1Theenzymelinkedimmunosorbentassay(ELISA)withthefusionproteinwasestablishedtodetectthegp210p62LBRautoantigenecorrelativeantibodiesinserumofpatientswithPBC(68)patientswithviralhepatitis(60)patientswithCTD(60))andnormalcontrols(60)1ResultsThesequenceofgp210p62LBRautoantigenegenewasthesameasthesequencereportedontheliteratures1Thefusionproteinwerefound69kD62kD27kDspecialstriponSDS2PAGEgel1Westernblotanalysisshowedthatthefusionproteinhadthesameimmunoreactivityashumangp210p62LBRautoantigene1WhenanalyzingtheserumofpatientswithPBCbyELISA,theresultindicatedthepositiverateofanti2gp210,anti2p62andanti2LBRantibodieswere3618%,3119%,517%respectively1Butalltheantibodieswerenegativeinothercontrolsexceptanti2gp210,whichshowed117%positiveinpatientwithCTD1ThepositivitiesofallthethreeautoantibodiesweresignificantdifferencesbetweenPBCanddiseasecontrolsornormalcontrols1ConclusionThehumanplasmidcontaininggp210p62LBRissuccessfullyclonedintoEcoliBL21M151TheresultsofELISAshowitsgoodantigenicityandspecificity1ItalsoprovidestestreferenceforclinicaldiagnosisofPBCforitsgoodantigenicity1KeywordsLivercirrhosis,biliary;Autoantgen;Nuclearenvelopeproteins(PBC),(AMA)PBC[1](ANA)PBC50%,[()][2],gp210p62LBR3PBC[3]AMAPBC[4]gp210p62LBR,gp210p62LBR,PBC11:PBC68,9,59,5914(3077)PBC:;AMA5:;;;IgM;Schirmer,212PBCPBC68PBC60,,38,2260,,20,20,206021:PET230aE1coliBL21,E1coliDH5aM15(pREP4),pQE30Qiagen31:T4DNATaqDNA[(HindBgSalI),TaKaRa],Olig(dt)(Promega),PlatinumTaqDNApolymeraseNi2NAT(Invitrogen),IgG2HRP(),DNA(),RNA(Qiagen),IPTG(ANA)(AMA)M2(AMA2M2)()gp210p62LBRIMTECWernerSch;Bler,HRPIgG(Sigma),PE2400DNA(ABI),TransBlotSD(Bio2Rad)41RNA:,RNARNA,cDNA,gp210p62LBR,(PCR)gp210:5HindIIIBglII:52GAAGATCTGCTCAACCCTGAAATAGAAGCA23;:52CCCAAGCTTGGTGGGAGGCATAGGCTGGGCT23PCR:953min,9550s,601min,72150s,25,PlatinumTaqDNApolymerase014l,9550s,601min,72150s,20,7210minp62:(Primer1):52CGTCGACGAATGAGCGGGTTTAATTTTG23(SalI);12112005112811ChinJLabMed,November2005,Vol28,No111(Primer2):32CAAGCTTCTAGGGACCTGCGGGCCCCAG25(HindIII)PCR:945min,9430s,602min,7245s,30,725min,LBR:(Primer1):52CGGATCCATGCCAAGTAGGAAATTTGC23(BamHI);(Primer2):32CAAGCTTCTCCAAGTCCTTTGCCCGGATG25(HindIII)PCR4112%,51:(3),PCR(gp210:HindIIIBglII;p62:SalIHindIII;LBR:BamHIHindIII),,PET30a2gp210pQE302p62pQE302LBR,,E1coliBL21(gp210)E1coliDH5a(p62LBR)PCR,DNA61:gp210:,IPTG,IPTG1mmol/L,225h,45000r/min15min20ml(50mmol/LNaPO4,015mol/LNaCl,pH810),,440000r/min20min,Ni2NATp62:pQE302p62M15[pREP4],Amp(100g/ml)Kan(25g/ml),DNA,110LBAmp+Kan,250r/minA(600)016018,1mol/LIPTG1mmol/L,170r/min4h,10000r/min,100ml5mlbuffer1,,buffer,1mg/ml,30min,5min,37,1,,410000r/min30min,50%Ni+2NTAsephrose160l,41h,LBR:71(WB):10%2(SDS2PAGE),TransblotSD,,3%BSA2PBS4011%Tween2PBS,gp210p62LBR,,IgG/HRP(1500)3%0134%2128.(ELISA):4g/ml,,4TPBS4,l0%,3730min1100100l,372h,4,18000IgG2HRP100l,371h,5,TMB/H2O2100l,3710min450nmA,A+29.:SPSS1110,21.:PET30a2gp210:1%,PBR322/BstNI1:PBR322/BstNI;2:RT2PCR1gp210,1819bp(1)HindIIIBglII,PET2302(2)pQE302p62:p62cDNAPCR,1600bp,p62cDNApQE30pQE302p62,3,2,PCR1600bppQE302LBR:LBRcDNA22112005112811ChinJLabMed,November2005,Vol28,No111PCR,1:PET30a2gp210/HindIIIBglII;2:DNA/HindIII;3:PET230agp210/HindIII2PET30a2gp210650bp,LBRcDNApQE30pQE302LBR,12,PCR650bp3,,gp210p62LBR100%,,,32.:PET30a2gp210:SDS2PAGE,1:;2:;3:;4:;5:gp2103gp210SDS2PAGE69000,10%(3),90%pQE302p62:IPTGpQE302p6210%SDS2PAGER2250,,65000(p6262000,3000)pQE302p6265000pQE302LBR:IPTGpQE302LBR10%SDS2PAGE,R2250,,27000(LBRM:;1:;2:1h;3:2h;4LBRSDS2PAGE25000,2000)pQE302LBR27000(4)31:WB,gp210p62LBR690006200027000,WBgp210p62LBR(5)M:;1:;2:5p62WESTERNSDS2PAGE41:ELISA,68PBC,gp210p62LBR3618%3019%519%;,gp210117%,3PBC3,(P0105P0101)11gp210p62LBRELISA(%)gp210p62LBRPBC682536182130194519600010001000106011170000106000100010001032112005112811ChinJLabMed,November200