汉坦病毒GM04-38株包膜糖蛋白上融合肽的初步确定

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1、山东大学硕士学位论文DEFININGTHEFUSIONPEPTIDEoNTHEGLYCoPROTEINOFHANTAVIRUSGM04．38STRAINMajor：EpidemiologyandHealthStatisticsGraduateStudent：CAOHaixiaAdvisor：WANGZhiyu,ProfessorAbstractObjectivePriorcomputationalmethodsshowedthattheglycoproteinG2ofHantaviruswasitsfusionprotein,whichWascla

2、ssIIfusionprotein,andthattheinternalhighlyconserveddomain(aa763"--785)maybewascriticalformembranefusionandWasnamedfusionpeptide．Inaddition,therewereexperimentalresultsdemonstratingthefusionpeptidewaslocated011glycoproteinG2butnotG1．Findingoutthat也erewasasimilarconserveddomaini

3、nHantavirusGM04-38strain,weinvestigatedtheroleofthisdomaintoestimatewhetheritWasitsfusionpeptidepreliminarily,SOastoprovideelementsforelucidatingthefusionmechanismofHantavirusanddevelopingeffectivevaccinesanddrugsforHantavirus．MethodsPrimersweredesignedaccordingtothesequenceof

4、MsegmenteDNA-byPrimer5．RecombinantPCRWasusedforconstructingtenclonemutants，inwhichtherewasonlyoneaminoacidmutated．ThenweamplifiedtheirglycoproteinG2codingregionbyPCRdigestedtheproducts晰nlKmIandXhoI．ThesamedigestionWasmadeforthevectorpCAGGS／MCS．Consequently，theG2codingregionand

5、thevectorwereligatedandtransformedintoE．coliDH5a．Afterbeingscreenedbyampicillinresistant,theexpressingmutantswereconstructed．Afterbeingconfirmedbyenzymedigestionandsequenceanalysis，thewildOlBglandGmG2／oroneoftheexpressingmutantswereCO-transfectedintoVetoE6cells．IFAwasperformed

6、toobserveexpressionefficiencies．Aftertreated晰tllacidicMEM，the7山东大学硕士学位论文cellswerefixedandstainedbyGiemsaandobservedundermicroscopetoexaminecellfusionactivities．Results1．Tenclonemutantswereconstructed，inwhichthereWaSonlyoneaminoacidmutated．Accordingtotheaminoacidsubstituted，the

7、ywerenamedC765A,P767D，P768D，C770A，P771D，G772D，G774D，G776D，C777AandC780A．TheyweredigestedwithKpnIandHindllI，thefragmentswere1．6kband2．7kbrespectively．Theresultswereconfirmedbysequenceanalysis．2．Tenexpressingmutantswereconstructed，inwhichtherewasonlyoneaminoacidmutated．Theyweren

8、amedGmG2-C765A,GmG2一P767D，GmG2一P768D，GmG2一C770A，GmG2一P771D，Gr