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ID:39133652
大小:3.19 MB
页数:57页
时间:2019-06-25
《汉坦病毒GM04-38株包膜糖蛋白上融合肽的初步确定》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
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
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