计算机在生物学中的作用期末试题

计算机在生物学中的作用期末试题

揣惴也賞99寸"LgaoozDin潞¥go齋6000sasiH舁册专M逐淋外范娠iH逐外鲁东大学生命科学学学院2011—2012学年第1学期《计算机技术在生物学中的应用》课程论文课程号：2512290-200论文题目:1、请以E.coliheat-labileenterotoxinBsubunit基因在转基因植物中高效表达为研究题材，查询相关基因序列，相关中外文文献、结合已学知识，以其中1篇中文文章的部分内容为素材，运用Endnote,以planta期刊为模板，书写论文。2、鼓励个性发展，也可不局限上述研究题材。论文要求：（对论文题目、内容、行文、字数等作出判分规定。）1、基因序列查询（5分），中外文文献查询（10分）。2、论文目录自动生成（10分），参考文献中外文（10分），两图片合理放在一起（15分）、论文引用（10分）、论文结构合理（15分）、正确运用多种生物软件（20分）。3、统一上交以本课程论文模板为标准的论文。打印的论文除上交一份纸质论文外,还需以学号+姓名为标题向qiushen弊han£001@126・com发电子版。（5分）教师评语:教师签字：年月日正文\nOralVaccinationofBaculovirus-ExpressedVP2DisplaysEnhancedProtectionagainstWhiteSpotSyndromeVirusinPenaeusmonodonSyedMusthaqS1,JimmyKwang1,2*AnimalHealthBiotechnology,TemasekLifesciencesLaboratory,NationalUniversityofSingapore,Singapore,Singapore,2DepartmentofMicrobiology,FacultyofMedicine,NationalUniversityofSingapore,Singapore,SingaporeAbstractWhiteSpotSyndromeVirus(WSSV)isaninfectiouspathogenofshrimpandotherCrustaceans,andneithereffectivevaccinesnoradequatetreatmentsarecurrentlyavailable・WSSVisanenvelopeddsDNAvirus,andoneofitsmajorenvelopeproteins,VP28,playsapivotalroleinWSSVinfection.InanattempttodevelopavaccineagainstWSSV,weinsertedtheVP28geneintoabaculovirusvectortailoredtoexpressVP28onthebaculovirussurfaceundertheWSSVie1promoter(Bac-VP28).TheBac-VP28incorporatedabundantquantity(65.3mg/ml)ofVP28・ShrimpweretreatedbyoralandimmersionvaccinationwitheitherBac-VP28orwild-typebaculovirus(Bac-wt).ThetreatmentwasfollowedbychallengewithWSSVafter3and15days・Bac-VP28vaccinatedshrimpshowedsignificantlyhighersurvivalrates(oral:81.7%and76.7%;immersion:75%and68.4%)thanBac-wtornon-treatedshrimp(100%mortality).ToverifytheprotectiveeffectsofBac-VP28,weexaminedinvivoexpressionofVP28byimmunohistochemistryandquantifiedtheWSSVcopynumberbyqPCR.Inadditiontothat,wequantifiedtheexpressionlevelsshrimpgenesLGBPandSTATbyreal-timeRT-PCRfromthesamplesobtainedfromBac-VP28vaccinatedshrimpatdifferentdurationofvaccineregime.OurfindingsindicatethatoralvaccinationofshrimpwithBac-VP28isanattractivepreventativemeasureagainstWSSVinfectionthatcanbeusedinthefield・Citation:SSM,KwangJ(2011)OralVaccinationofBaculovirus-ExpressedVP28DisplaysEnhancedProtectionagainstWhiteSpotSyndromeVirusinPenaeusmonodon.PLoSONE6(11):e26428.doi:10.1371/journal.pone.0026428Editor:PierreBoudinot,INRA,FranceReceivedJuly29,2011;AcceptedSeptember26,2011;PublishedNovember1,2011Copyright:_2011S：Kwang.ThisisanoperraccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense：whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited・Funding:Theauthorshavenosupportorfundingtoreport・\nCompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.•E-mail:kwana@UI.ora.saIntroductionWithahigheconomicoutput一USS9.0billionannually—shrimpcultureplaysamajorroleinglobalaquaculture[1]・Thesuccessfulproductionofshrimpishamperedbyseveralviraldiseases,particularlyWhiteSpotSyndromeVirus(WSSV).Ithasbeenoneofthemostthreateninginfectiouspathogenstotheshrimpcultureindustryoverthepasttwodecades・WSSV-relatedcumulativemortalitytypicallyreaches100%within2to5daysoftheonsetofclinicalsigns.Thevirushasawidehostrangethatincludcsfreshwaterprawns,lobsters,freshwatercrabsandscvcralspccicsofmarinecrabs[2,3,4,5]・WSSVbelongstothegenusWhispovirus(www.ncbi.nih.gov/ICTVdb/Ictv/index.htm)undertheviralfamilyNimaviridae・I【isarod-shaped,envelopeddsDNAtail-likeappendagesatoneend・SequencingofthefullgenomeofWSSVrevealedthatithas184ORFs,buttheroleofmanyofitsviralproteinsisstillunknown・Asofthisstudy,39structuralproteinshavebeenidentifiedinWSSV,including22envelopeproteins[6].TheWSSVgenomecontainsatfiveknownmajorstructuralproteins:VP2&VP19,VP26,VP24andVP15.StudicsonWSSviralproteinshavedemonstratedthatVP28andVP19areassociatedwiththevirionenvelope・VP26actsasategumentproteinlinkingthetwonucleocapsid-associatedproteinsVP24andVP15totheenvelope⑺.Novelviralcontrolstrategies,includingvaccination,arenecessaryduetothecontinuedhighgrowthofshrimpcultivationandthebroadhostrangeofWSSV.Invertebrateslackatrueadaptiveimmuneresponseandrelysolelyonaninnateimmuncrcsponsc.sountilrecently,vaccinationofshrimpagainstWSSVwasnotconsideredaviablestrategy.However,Venegasetal.[8]demonstratedtheexistenceofquasi-immuneresponseinPenaeusjaponicusthathadsurvivedpreviousexposuretoWSSV.Inanotherstudy,shrimpbecameresistanttoWSSVabout3weeksafterinfectionandhemolymphfromresistantshrimpcontainedvirusneutralizingfactors[9].TheseresultsstimulatedreseiirchintothepossibilityofvaccinatingshrimpagainstWSSV.Withinafewyears,numerouspublishedstudiesexploreddifferentWSSVvaccinationstrategicstoprotectshrimp,includinginactivatcdWSSVvaccines110,11subunitrecombinantvaccines!12,13,14,15],oralrecombinantvaccines[16,17],DNAvaccineslnmostWSSVvaccines,VP28hasbeenamajortarget,astheenvelopestructuralproteinsofWSSVplayapivotalroleininitialviralinfection.Theenvelopeproteinsareoftennecessaryforviralbudding,entryandassembly.VP28isamajorenvelopeproteinofWSSVandisinvolvedinthesystemicinfectionofshrimp123,24J.DuringWSSVinfection,VP28interactswithhostcellularprotcinssuchasPmRab7[25],heatshockcognateprotein70[26]andthesignaltransducerandactivatoroftranscription(STAT)[27]tobringabouttheviralinfection.PreviousreportshavedemonstratedthatVP28-basedrecombinantvaccinesprovideprotectionandincreasethesurvivalrateofchallengedshrimpduringWSSVexperimentswhencomparedwithunvaccinatedshrimp[10,15,16,17].However,theprotectionofferedbytheseVP28derivedrecombinantvaccinesisnothigh,andtheprotectiveresponseisdrasticallylowat10dayspost-challcngc.1(maybcpossiblctoimprovevaccineefficacybyexpressingtherecombinantVP28proteininabaculoviruseukaryoticexpressionsystem.Inthepresentstudy,weinsertedVP28geneintobaculoviralvectorunderthecontroloftheWSSVielpromoterandexpressedVP28gene(Bac・VP28)onbaculovirussurface・Ourquantitativewesternblottingtechniqueshowed,abundantquantityofVP28wasexpressedonBac-VP2&Next,wedemonstrateforthefirsttimeanefficaciousimmunisationofWSSVimmcrsion-challcngcdshrimpbyadministeringBac-VP28viaoralandimmersionroutcs.ThcprotectiveresponsegeneratedinBac-VP28immunizedshrimpagainstWSSVwasmeasuredbyquantificationofWSSV\nviralcopynumbersbyquantitativereal-timePCR(qPCR)andinvivoexpressionofVP28byimmunehistochemistry(IHC).ToauthenticatevaccineefficacywequantifiedshrimpLipopolysaccharideandb-l,3-glucanbindingprotein(LGBP)andSTATgcncsexpressionprofilesbyreal-timeRT-PCRfromthesamplcsobtaincdfromvaccinatedshrimpatdifferentdurationofvaccineregime・Hence,Bac-VP28isanattractivepreventativemeasureforshrirnpcultureagainstWSSVinfectionthatcanbeusedinthefieldapplicableapproach・MaterialsandMethodsCollectionandmaintenanceofexperimentalanimalsShrimp,Penaeusmonodon(10-12gbodyweight),wereimportedfromMalaysiaandmaintainedin1000-1fiberglasstankswithairliftbiologicalfiltersatanambienttemperatureof27-30uCwithsalinilybetween20and25ppt.Naturalseawaterwasusedinalltheexperiments.ItwaspumpedfromtheadjacentseatoSingaporeandallowedtosedimenttoremovethesandandothersuspendedparticles.Theseawaterwasthenchlorinatedbytreatingwithsodiumhypochloriteattheconcentrationof25ppmandthcndcchlorinatedbyvigorousaeration,beforebeingpassedthroughasandfilterandusedfortheexperiments・Theanimalswerefedwithartificialpelletfeed(BTAfeed,Malaysia).TemperatureandpHwererecorded,salinitywasmeasuredwithasalinometer(Aquafauna,Japan)anddissolvedoxygenwasestimatedbytheWinklermethod.Theanimalswerekeptintanksfor5daysandacclimatizedpriortotheexperiments・Fromtheexperimentalanimals,5fromagroupof30wererandomlyselectedandscrccncdforthepresenceofWSSVbypolymerasechainrcaction(PCR)usingtheprimersdesignedbyTakahashietal.[281andonlyhealthyshrimpwereusedfortheexperimentsPreparationofWSSVviralinoculumThelaboratoryWSSVstockwasusedinthisstudyanditwaspropagatedbyinjectingintohealthyshrimp.Thehemolymphwasdrawndirectlyfromtheheartsofmoribundshrimpusingstcrilcsyringcsfollowedbycentrifugation(30006gfor20minat4uC).Thesupernatantfluidwasthenre-centrifuged(80006gfor30minat4uC)andthefinalsupernatantfluidwasfilteredthrougha0.4mmfilter.Thefiltratewasthenstoredat280uCforexperimentalstudies.GenerationofrecombinantbaculovirusFortheconstructionofVP28geneintopFASTBacHTA(lifetechnologies,USA)baculovirustransfervectorcontainspolyhedrinpromoterincludingHistag,ATGandmultiplecloningsiteswasreplacedwithWSSVielpromoterwithRsrIIandHindlllrestrictionenzymes・TheielpromoterwasamplifiedfromWSSVDNAusingtheprimersWSSVielF-59-CCTACGTATCAATTTTATGTGGCTAATGGAGA-39andWSSVie1R-59-CGCGTCGACCTTGAGTGGAGAGAGAGCTAGTTATAA-39theninsertedThefulllengthORFofVP28genewasamplifiedfromWSSVgenomeusingtheprimersBac-VP28F59-CGCCGGTCCGATGGATCTTTCTTTCACTCTTTC-39andBac-VP28R59-CCGAAGCTTTTACTCGGTCTCAGTGCCAG39withRsrIIandHindlllrestrictionenzymesandclonedintomodifiedpFASTBacHTAvector.ForthegenerationofrecombinantbaculoviruscstheconstructswcrcintegratedintothebaculovirusgenomewithinDH10BacTM(lifetechnologies,USA)throughsite-specifictranspositionaccordingtotheprotocolofBac-to-Bacsystem(lifetechnologies).Therecombinant\nbaculoviruseswithVP28genewasnamedasBac-VP28andwithoutVP28genewasknownasBac-wt.FurtherrecombinantbaculoviruswerepropagatedinSF-900IISFM(lifctcchnologics)at27uCandinfectingSf9-ccllsasdescribedbySyedMusthaqetal.[291.ImmunofIuorescenceassaytodetecttheexpressionofVP28ininsectcellsTodetecttheImmunofluorescencesignals,Sf9cellsweregrownin24wellplatesandinfectedwithBae-VP28andBac-wtbaculovirusataMOIof0.5.After48hrspostinfection,thecelIswerefixedwithparaformaldehydefor20minandblockedwith1%gelatinfor30minatroomtemperature・Thefixedcellswerethenincubatedwithanti-mousePrVP28(prokaryoticexpressedrecombinantVP28)polyclonalantibodypreparedbySyedMusthaqetal.[29]atadilutionof1:100forIhrat37uC.FITC-conjugatedrabbitanti-mouse(DakoCytomation,Dcnmark)atadilutionof1:100wassubsequentlyincubatedwiththecellsforihr.Thefluorescencesignalwasdetectedwithaninvertedfluorescencemicroscope(Olympus,UK)andtheimageswerecapturedbyadigitalimagingsystem(Nikon,USA).WesternblotanalysisofVP28ForWesternblotanalysis,theBac-VP28infectedcellsorsupernatantweremixedwithLacmmlisamplebufferandSDSPAGEwascarriedoutasdescribedbyLaemmli[30].VP28frompurifiedWSSVvirionsservedasareferencewhileBac-wtwasincludedasanegativecontrol.ThegelwastransferredtonitrocellulosemembraneandWesternblotwasperformedbythemethodofTalbotetal.[31]・Theanti-mousePrVP28polyclonalantibodiesatadilutionof1:2000wereusedasprimaryantibodyandrabbitanti-mouseIgG(DakoCytomation,Dcnmark)atadilutionof1:1000wereusedassecondaryantibodytodeteetthebaculovirusexpressionofVP28・QuantitativewesternblottingOdysseyInfraredImager(LI-COR,Biotechnology)wasusedtocalculatetheamountofVP28presentonrecombinantbaculovirus.His-tagfusionpartnerwasusedasastandard.Theanti-mouseVP28polyclonalantibodiesatthedilutionsof1:2000andadilutionof1:10.000ofdonkeyanti-mouseIRdye800CWIgG(LICOR,Biotechnology)wasusedasprimaryandsecondaryantibodyrespectively.ThemembranewasdevelopedandbandintensitieswereanalysedbyOdysseyApplicationsoftwareversionl.2・TheamountofVP28displayedonBac-VP28wascalculatedbycomparingwithPrVP28asstandardcontrol.ResultsGenerationofrecombinantbaculovirusanditsconfirmationTheWSSVgenomeencodingenvelopeproteinVP28wasselectedforthegenerationofrecombinantbaculoviruswithanimmediateearlypromoter1(iel)derivedfromtheWSSVgenomeandnamedasBac・VP28(Fig・la).Theaboveconstructwithoutcalculation,theamountofVP28presentsinBac-VP28wasmeasuredontheaverageof65.3mg/ml(10s\npfu)obtainedfromthreeindependentbatchesofbaculovirusculture(Fig.ld)・VP28geneistermedasBac-wt(Fig・la)・TherecombinantbaculovirusesaregeneratedinSf9-IIcellsandfollowedthemethoddescribedbyBac-tBacexpressionsystem(lifetechnologies,USA).TheexpressionofVP28wasanalyzedbyImmunoflourescenceassayandresultsindicatedthatbaculoviruscounterpartVP28expressedininsectcellsandmaintainsitsstructuralandantigcnicconformity(Fig.lb).Next,wccomparedthemolccularmassofVP28presentinthebaculovirusinfectedcelllysateorcompletevirionandpurifiedWSSVvirionsbythewesternblotanalysis,whichdetectedabandbothwiththesimilarsizeof28kDa(Fig.lc)withanti-mousePrVP28polyclonalantibody.However,neitherfluorescentsignalnorspecificproductwasdetectedinBac-wtderivedfrominsectcellsorlysate\n0.20.40.€04rVP28proteinstandardspg/ml(ii)BM-VP28Samples(Hi)Figure1・Schematicstructureofrecombinantbaculovirusconstruct,analysistheexpressionofVP28onbaculovirussystemusinganti-mouseVP28polyclonalantibodiesandQuantificationofVP28expressedfromBac-VP28・(a)TheVP28genewascioneddownstreamofWSSVie1promoterandupstreamofTsv40ofpFasBacHTvectorandnamedasBac-VP28andaboveconstructwithoutVP28genenamedasBac-wt.(b)ImmunofluoresceneeassayofexpressionofVP28inSf9cells・ThecellswereinfectedwithBac-VP28andBac-wtatMOIof0.5andat48hrspostinfectionthecellsfixedandanalysed.(c)WesternblotanalysisofbaculovirusexpressedVP28comparedwithWSSVvirions.Lane1-supernatantofbaculovirusexpressedVP28;Lane2-completeBac-VP28virions;Lane3-completeBac-wtvirions;Lane4-purifiedwildtypeWSSVvirionsfromWSSVinfectedshrimptissue・(d)(i)・quantitativewesternblotanalysisofVP28expressedinbaculovirussystemandcomparedwithdifferentconcentrationofPrVP28expressedinbacterialsystemasastandard・Lane1toLane6・purifiedPrVP28proteinfrom0.01mg,0.05mg,0.1mg,0.25mg,0.5mgand1mg;LaneBac-VP28-1mgtotalprotein;LaneBac-wt-1mgoftotalprotein.ThenitrocellulosemembranewasseannedandanalysedbyOdysseyInfraredImager.(ii)-rVP28proteinstandardcurve;(iii)-theamountofVP28proteinpresentsinBac-VP28culture.doi:10.1371/journal.pone.0026428.g001AcknowledgmentsTheauthorsarethankfultoMr.AnbuKumarKaruppannanformanuscripteditingandMr.RamaiahRajkapoorinanimalmaintenance・AuthorContributionsConceivedanddesignedtheexperiments:SMSJK.Performedtheexperiments:SMS.Analyzedthedata:SMSJK.Contributedreagents/materials/analysistools:SMSJK.Wrotethepaper:SMS・References1.FAOyearbook2008.Available:(http://www.fao.0rg/docrep/013/i1890t/i1890t.pdf).Accessed2011July8.\n1.SahulHameedAS,YoganandhanK,SathishS,MuruganV,RasheedM,etal.(2001)Experimentalpathogenicityofwhitespotsyndromevirus(WSSV)intwofreshwatercrabs(PartelphusahydrodomousandP.pulvinata)・Aquaculture201:179-186.2.SahulHameedAS,MurthiBLM,RasheedM,SathishS,YoganandhanK,etal.(2002)AninvestigationofArtemiaasapossiblevectorforwhitespotsyndromevirus(WSSV)transmissiontoPenaeusindicus・Aquaculture204:1-10.3.SahulHameedAS,BalasubramanianG,SyedMusthaqS,YoganandhanK(2003)ExperimentalinfectionoftwentyspeciesofIndianmarinecrabswithwhitespotsyndromevirus(WSSV).DisAquaOrg57:157-161.4.SyedMusthaqS,SudhakaranR,BalasubramanianG,SahulHameedAS(2006b)Experimentaltransmissionandtissuetropismofwhitespotsyndrome(WSSV)virusintwospeciesoflobsters,PanulirushomarusandPanulirusornatus.JInvertPathol93:75-80.5.TsaiJM,WangHC,LeuJH,HsiaoHH,WangAH,etal.(2004)GenomicandProteomicAnalysisofThirty-NineStructuralProteinsofShrimpWhiteSpotSyndromeVirus.JVirol78:11360-11370.6.TsaiJM,WangHC,LeuJH,WangAHJ,ZhuangY,etal.(2006)IdentificationoftheNucelocapsid,TegumentedandEnvelopeproteinsofthe\nShrimpWhiteSpotSyndromeVirusVirion.JVirol80:3021-3029.1.VenegasCA,NonakaL,MushiakeK,NishizawaT,MurogaK,etal.(2000)Quasi-immuneresponseofPenaeusjaponicustopenaeidrodshapedDNAvirus(PRDV).DisAquatOrgan42:83—9.2.WuJL,NishiokaT,MoriK,NishizawaT,MurogaK(2002)Atime-coursestudyontheresistanceofPenaeusjaponicusinducedbyartificialinfectionwithwhitespotsyndromevirus・FishShellfishImmunol13:391-403.\nDNAWSSVielF-59-CCTACGTATCAATTTTATGTGGCTAATGGAGA-39andWSSVieIR-59-CGCGTCGACCTTGAGTGGAGAGAGAGCTAGTTATAA-39Bac-VP28F59-CGCCGGTCCGATGGATCTTTCTTTCACTCTTTC-39andBac-VP28R59-CCGAAGCTTTTACTCGGTCTCAGTGCCAG39图片