人可溶性CD14及其信號轉(zhuǎn)導(dǎo)缺失突變體的克隆表達(dá)與鑒定.pdf

1、軍事醫(yī)學(xué)科學(xué)院碩士學(xué)位論文人可溶性CD14及其信號轉(zhuǎn)導(dǎo)缺失突變體的克隆表達(dá)與鑒定姓名：王威申請學(xué)位級別：碩士專業(yè)：微生物學(xué)指導(dǎo)教師：蔭俊2000.6.1英文摘要AbstractHumansolubleCDl4eDNAfragmentwasamplifiedfromthetotalRNAextractedfomtheU937cellsbyusingRTPCRtechnologyThefragmentwasclonedintopGEMTve

2、ctorandthensequencedTheresultshowedthatthesequenceoftheinsertedfragmentwasinlinewiththepublishedsequenceofthenativesCDl4completelyThesCDl4fragmentwasnextsubclonedintoeukaryoticexpressionvectorpClneoTherecombinantvectorth

3、enwasintroducedintoCOS7cellsSDSPAGEdemonstratedCOS7cellshadexpresseda53kDaproteinWestemblotandELISAshowedtheproteincouldreactwithantisCDl4antibodiesspecitificallyTheexpressedproteinwaspurifiedbyimmunoaffinitychromatograp

4、hyonacolumntowhichCDl4mAb3C10wascoupledtoSepharose4BSDSPIAGEshowedthepuritywasabove95％ThepurifiedproteinmanifestedtheexpressionIevelIntheconditionedmediumoftheCOS7jsabout58扯g／m1TheabilityofsCDl4toa艉cltheLPSdependentTNF—q

5、secretioninU937cellswasobservedwhichshowedtheeukaryoticexpressedsCDl4hassomebiologicalactivityofthenativeproteinNext，sitedirectedmutagenesisbydoublePCRwasusedtointroducealaninesubstitutiontotheNterminal813and97一101aminoa

6、cidswhicharetheputativesignaltransductiondomainsofsCDl4ThreePCRfragments，sCDl4(97101)A152aa，sCDl4(s131A152a。andsCDl4(s13)A348aawereobtained，thenclonedintopGEMTvectorrespectivelyandsequencedTheresultsshowedalanineswereint

7、roducedinto813and9710IaminoacidsregionoftheNterminalofsCDl4sCDl4(s13)A152aathenwascolonedintoprokaryoticexpressionvectorpET28a，andrecombinantvector、vastransferedtoEcoliBL21fDE3)InducingwithIPTGfor25hours，SDSPAGEshowedane

8、wproteinband，whichwasafusionproteinandwaslargerthanthenonfusioncounterpartThensCDl4(97101)A152n，sCDl4(813)A152nandsCDl4(813)6348fragmentsweresubclonedintopCIneoandthe1asttwecombinantvectorswereintroducedintoCOS7cellsThec

9、elllysatewasanalyzedbySDS—PAGEandWestemblotTheresultsshowedpClneo／sCDl4(813】A152transfectedCOS7cellsexpresseda26KDaProtein，whichcouldreactwithantisCDl4antibodiesspecitificallywhilethiswasnotthecasewiththepCIneo／sCDl4(s13

10、)A348aatraadfectedCOS7cellsInthisstudysCDl4andasignaltransductiondomainmutatedsCDl4wereclonedandexpressed，sCDl4waspurifiedanditsbiologicalactivityWasroughlyobservedAllthesewillbeusefulforfurtherstudyofthestructureandbiol