豬瘟病毒HL-LY地方株E2基因在畢赤酵母中的表達(dá)及高效表達(dá)條件的探索.pdf

1、東北農(nóng)業(yè)大學(xué)碩士學(xué)位論文豬瘟病毒HLLY地方株E2基因在畢赤酵母中的表達(dá)及高效表達(dá)條件的探索姓名：滿朝來申請學(xué)位級別：碩士專業(yè)：預(yù)防獸醫(yī)學(xué)指導(dǎo)教師：李一經(jīng)2003.5.1。，一。。，；—一!一。?！?dāng)型喹篁篁鶩鯊鎏圣一。。。。。一，。。，。Expression程囂2GenefromClassicalSwineFeverVirusHLLYIsolateinPichiapastorisandExploringforItsExpressio

2、nConditionsAbstractClassicalSwineFever(CSF)isoneofmajorcontagiousdiseasesinpigbreedingindustryInordertogainmassivediagnosticantigenCSFV，andlargescaleofCSFVspecificmajorprotectiveantigenprotein(E2envolopeglycoproteimineuc

3、aryoticcells，whicharepreparedintosubunitvaccineforeffectiveproventionagainstCSFE2genefromCSFVHLLYisolatewasclonedandexpressedinPichiaPastorisexpressionsystem，moreovefconditionswereexplorerdforexpressingtheE2envelopeglyco

4、proteinTheresultsmshowedasfollowsAccordingtoCSFV’sE2genesequencepublishedinGenBankandthesequenceofPPAstorisexpressionvectorpPIC9’sMultipleCloningSite(MCS)，apairofprimersweredesignedwithOlignandPrimer50softwarasE2genewasa

5、mplifiedbyRT—PCRfromHLLYisolate，TheproductofRTPCRwasclonedtopMDl8Tvector，andidentifiedb～restrictionendonuclease，PCRandsequencing，whichwasnamedasTE2秘IeE2gene’ssequencewasanalyzedbyhomologycomparingwithpublishedE2geneofCSF

6、VAIfonstrain，Bresciastrain，SHIM[ENstrain，Cstrain，CVLZstrain，GStTstrainandGS。LXstrainThehomologyofE2genecompletesequenceshares9920％。9544％，965I％，95，53％，8912％，7866％and78，23％innucleotiderespectivelyandshares9893％，95。98％，97。3

7、2％，95。71％，8954％，9316％and8342％identitiesinaminoacidsequencerespectivelyThemajorantigenicregionofthesequencewasalsoanalyzedbyhomologycomparing，whichshares9890％，9505％，9670％，9615％，9542％，8242％，and8315％nnucleotiderespectivelya

8、ndshares98。35％，9505％，97。25％，9560％，9505％，87，36％and88。46％identitiesinaminoacidsequencerespectivelyTheresultshowsthatE2geneishigherconservative，butthereexistCSFVE2genemutationstrainsinChinaaccordingtoitslowerhomologieswithC

9、VLZ，GS【TandGSL1’strainswhichwereisolatedfromthedifierentregionsinChinaE2genewascutwithrestrictionendonucleasefromtherecombinationplasmidTE2，andsubclonedintotheexpressionvectorpPIC9’sMCS，whichwasidcmtifiedbyenzymedigestio

10、nartdPCRamplificationTherecombinationexpressionVeCtOr，pPIC9。B2，waslinearizedbvSalIandelectroporatedintoEPastorisGSll5RecombinantEPAstorisGSll5designatedGSl15pPIC9E2，wasidentifiedbyPCRandtheproductofthePeRwasanalyzedbvseq

11、uencingbeforeitsexpressionfoCSFV￡2proteinExpressionofCSPVE2fusionproteinwasfollowedinRecombinantEPastorisGS115一pPlC9E2withtheinductionof1％methanolat30“C，shakingat200rpmAstrongproteinbandofmolecularmassestimation5054kuwit