豬γ干擾素cDNA的克隆與表達及其生物學活性的研究.pdf

1、中國農業(yè)大學博士學位論文豬γ干擾素cDNA的克隆與表達及其生物學活性的研究姓名：吳文學申請學位級別：博士專業(yè)：預防獸醫(yī)學指導教師：蔣金書夏春2002.5.1AbstractPorcineinterferongamma(PoIFNf)isanimportantcytokine，canexertantiviralactivityandanumberofimmunomodulatoryeffectssuchasenhancementofTce

2、ilmediatedcytotoxicity，Bcelldifferentiation，MHCIandMHCIIexpression，andmacrophageactivationInordertoleadtheresearchontherecombinantPolFNy，apairofprimersweredesignedaccordingtothePolFN—YgenereposedbyRoger。AnewsubtypePolFN／

3、eDNAwasclonedfromConAstimulatedPorcinespleencellstotalRNAbyRTPCR，andanucleotidesubstitutionwasfoundat462position111ePolFNy2genecodingformatureproteinof150aminoacideswithsphIandHindllIrestrictionsitewassubcloned，digestedw

4、ithSphIandHindlII，andligatedwiththeexpressionvectorpQE30ThepositiverecombinantvectorpQE30／PolFNy2identifiedbyPCRandEnzymewasUansformedintoEcoilJMl09andSGl3009(pREP4)，andindueed、vitllIPTGSDSPAGEandWestemblottingconfirmedt

5、hatconfluentproteinof6xhisrecombinantPorcineinterferongamma(6HisrPolFNf2)wasexpressedefficientlyininclusionbodiesiIIEcoilwiththeyieldabove30％AtierpurificationwithNiNTAagarosethepurityof6HisrPoIFN72w∞greaterthan90％6HisrPo

6、lFNy2soluiblizedinadenaturedsolutioncontaining8mol／lurearefoldedbydialysishaveantiviralactivityof810316X104units／mgAneweucaryoticexpressionsystemofrecombinantPoIFN7wasalsoconstructedsothatthedenaturalizationandrefoldingp

7、rocessisavoidedThetargetgenecodingPolFN72matureproteinwassubclonedintoexpressionvectortransformedintoPichiapastoris，TheHisMut5phenotypetransformantswerescreened，fermentedinflasksandinducedby1％methan01Theexpressedproducti

8、nculturesolutionandsedimentationhaveantivimlactivityonVSVandimmunologicalactivityprovedbyindixectedimmunofluorescenceantibodyandDotblottingassay。TherecombinantPolFNyexpressedbyEcoliSGl3009andPichiapastorisalsoshowinhibit