抗KOD聚合酶单克隆抗体的制备.pdf

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1、亿学与生物互程2010，Vo1．27No．3固Chemistry&BioengineeringPreparationofMonoclonalAntibodiesAgainstKODDNAPolymeraseYUXiao-lan，LIYu，WANGFei，ZHONGXing，CHENLiang，MALi—xin(CollegeofLifeScience，HubeiUniversity，Wuhan430062，China)Abstract：Monoclonalantibody(McAb)wasadvancedinhomogeneityandspe

2、cificity．McAbagainstKODDNApolymerasecanbindwithpolymeraseandthebindingofMcAbwithpolymerasecaninhibittheactivityoftheKODDNApolymeraseatroomtemperatureandimprovethespecificityofPCRremarkably．Twohybrido—malinesproducingMcAbagainstKODpolymerasehavebeenestablished．BALB／cmicewasi

3、mmunizedwithK0DpolymeraseexpressedinE．coli，thestimulatedspleenocyteswereconfusedwithSP2／0mousemyelomasinHAT-selectiveculturetoproducehybridomas．TwohybridomalinesspecifictoKODpolymerasewereselect—ed，designated1F5，3G6，expressedantibodiesofsubclassesIgG1andIgG2arespectively．Th

4、enumberofchro—mosomeofthetwohybridomalineswas85～90and83～86respectively．TheELSAtitersofcellsupernatantandascitesof1F5and3G6were1：2。，1：2。and1：2u，1：2nrespectively．Byindirectimmunofluorescenceassay,thetwoMcAbreactedwithcellstransfectedwithpcK0Danddidnotreactwithcontrolcells．Thi

5、sre—sultfoundedabaseforestablishinghigherspecificityPCRpathway．Keywords：KODDNApolymerase；monoclonalantibody(McAb)；ELISAassay；preparationInrecentrapiddevelopmentofmolecularbiolo—ityduringthefirstdenaturingstepofPCR．RecentlygY。polymerasechainreaction(PCR)，whichuseshotstartPCR

6、usingantiDNApolymerasemono—thethermostableDNApolymerase，isoneoftheclonalantibodies(McAb)hasbeenreported，themostimportantcontributionstoproteinandgeneticMcAbsincubatedwithTaqDNApolymeraseandresearchbecauseitenablesamplificationofaspecificaddedtoPCRtubesatambienttemperature，y

7、ieldDNAfragmentinvitro[．Sofar，morethan50specificDNAfragmentsuponamplificationwhenu—DNApolymerasegeneshavebeenclonedandse—singhighnumbersoftemperaturecyclesandaveryquencedfromvariousorganisms[’引．Intheconv'en—lowcopynumberoftargetDNAin8complexDNAtionalmethodofPCR，DNApolymeras

8、esisolatedbackgroundE，．fromthermophilicbacteriaandhyperthermophilicar—Inthisstudy，