ALuciferaseReporterMinigenomeSystemfor.docx
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ALuciferaseReporterMinigenomeSystemfor.docx
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ALuciferaseReporterMinigenomeSystemfor
ALuciferaseReporterMinigenomeSystemfor-…
ALUCIFERASEREPORTERMINIGENOMESYSTEMFOR
QUANTIFYINGRESPIRATORYSYNCYTIALVIRUSREPLICATION1111MelanieJ.Aston,MichaelH.Chi,MonicaK.Deterding,MatthewM.Huckabee,22MartinL.Moore,andR.StokesPeebles,Jr.12DepartmentofBiomedicalEngineering,VanderbiltUniversityandDepartmentofMedicine,VanderbiltUniversity
SchoolofMedicine,Nashville,TN
RespiratorySyncytialVirusistheleadingcauseofrespiratorytractinfectionininfantsintheUnitedStatesandworldwide.ThereiscurrentlynovaccineavailabletotreatRSV.ThecurrentmethodtodetermineRSVtiterinthelaboratoryistheviralplaqueassay,alabor,materials,andtimeintensiveprocedure.Thereisaneedforahighthroughput,inexpensive,andhighlysensitivemethodtoquantifyinfectiousRSV.WeengineeredanRSVminigenomecontainingaluciferasereporterforhigh-throughputquantificationofRSVreplication.TheminigenomeisundercontroloftheCMVpromoterforconstitutiveexpression.HEp-2cellsweretransfected,andstably-transfectedcelllineswereselected.Luciferasebioluminescencewasmeasured48hoursafterRSVinfectionina96-wellplate.LuciferaseactivitywasRSV-specificanddose-dependent.
Introduction
RespiratorySyncytialVirusisaparamyxovirusthatconsistsoftengenesina15kbpnegativesensegenome.Figure1showsaschematicoftheRSVgenomeanditslifecyclewithinacell.Thegenesinpink(N,P,
M2,andL)arenecessaryforthetranscription
andreplicationofthevirus.Thevirallifecycle
withinthecellproceedsinseveralsteps.First
thevirusbindstothetargetcellviaan
unknownreceptor.Thegenomeunfoldsand
Figure1:
RSVgenomeandintracellularlifecyclehostcellmachinerytranscribestheviral
genes.Thesegenesaretranscribedindividuallyalongagradient.ThemRNA’sarethen
translatedbythehostcellmachineryintoprotein.Atsomepoint,whentheproteinsbuildupinthecellthenegativesensegenomeisreplicatedintoapositivesenseantigenome,whichservesasatemplateforreplicationofmanycopiesoftheviralgenome.Thesenewviralgenomesarethenpackagedintonewvirusparticles.
RSVistheleadingcauseofbronchiolitisandpneumoniaininfantsunderoneyearofage
(1).CurrentestimatesplacetheannualinfectionandmortalityfiguresforRSVat64millionand800,000respectfully.Itistheleadingcauseofhospitalization,respiratoryfailure,mechanicalventilation,andviraldeathininfantsintheUnitedStatesand
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worldwide
(1).TherearecurrentlynovaccinesordrugsavailabletotreatRSV.ThebiggestchallengewithcreatingaviableRSVvaccineisthebottleneckinlaboratoryresearch.Thisbottleneckarisesfromtheinefficienciesindeterminingviraltiter.
ThecurrentmethodtodetermineRSVtiterinthelaboratoryistheviralplaqueassay.Theplaqueassayisalabor,materials,andtimeintensiveprocedure.Plaqueassayscantakeupto7daystocomplete.Figure2showsaflowchartfortheplaqueassay.Inaddition,theplaqueassayisnothighlysensitive.Tocountaplaquetheresearchermust“eyeball”theplateand
manuallycounttheplaquesor
“holes”formedbydeadcells.This
procedureintroducessubjectivity
tothemeasurementandasa
resultismorepronetohuman
error.
Clearlythereisaneedfora
highthroughput,inexpensive,and
Figure2:
FlowChartfortheViralPlaqueAssay(majordelayshighlysensitivemethodtoareinred)
quantifyinfectiousRSV.We
proposeanovelluciferasebasedminigenomereportersystem.
AteamattheNationalInstituteofHealthledbyDr.PeterCollinssuccessfullycreatedaminigenomesystemthatexpressesareportergeneinresponsetoRSVinfection(6).ThissysteminvolvedanRSVminigenomeunderthecontroloftheT7promoter.TheCollinsgroupfoundthattheN,P,M2-1andLproteinsweresufficientandnecessaryforexpressionoftheminigenomeandreportergene.WechosetoadoptthissystembecausereportergeneactivitywasRSVspecificanddependent.
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Methods
Thisplasmidreporterwasconstructedfromfourseparatesequences.Theyare:
apcDNA3.1vector,aleadersequence,acopyoftheluciferasegeneandatrailersequence.Theywerefabricatedorisolatedindividuallyandfinallyligatedtogether.In
silcodesignwasconductedusingVectorNTIcloningsoftware.WenamedourplasmidpRSVlucM5.
ThevectorbackboneselectedwaspcDNA3.1(+),a5428basepairplasmid
purchasedfromInvitrogen.Thisplasmidwaschosenforthepresenceofunique
restrictionsites,theCMVpromoter,
andthegeneticinandampicillin
resistancegenestoallowforselection.
Second,pGEM-lucwasselectedas
theluciferasegenesource.Figure3.(A)DiagramoftheTrailersequence,(B)Diagramofthe
LeadersequenceTheleaderandtrailerregionsof
theplasmidweredesignedbasedontheconstructcreatedbytheCollinsgroup(6).Withintheleaderisa44nucleotide(nt)RSVleaderregion,a9ntgenestartsignalofRSVnonstructuralprotein1(NS1),anda32ntnontranslatedregionoftheNS1gene(Figure3B).Thetrailersequenceincludes12ntofthenontranslatedregionoftheRSVlargepolymerase(L)gene,the12ntLgeneendsignal,andthe155ntRSVtrailerregion(Figure3A).Thesequencesweredesignedwiththeproperrestrictionsites
necessaryforfutureligation.
Afterindividualdesigninsilcothe
sequenceswerecompiledtocreate
thefinalplasmidconstruct(Figure4).
Theleaderandtrailersequences
werefabricatedbyIntegratedDNA
Technologies.Thetrailersequence
wasorderedasa191basepair
elementinaminigeneplasmid.
FabricationoftheleadersequenceFigure4:
DiagramofpRSVlucM5provedtobemoredifficult.The
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lengthoftheleadersequenceandthepresenceofahairpinintheribozymemadeitimpossibletofabricateasaminigeneplasmid.Togetaroundthisproblemweconstructedtheleaderfromfourseparateoligonucleotidefragments.Thisrequiredadditionalworkbeforetheleader
couldbeligated.
Thefouroligonucleotideshad
tobeannealedtogether
(matchingoneendofthedouble
helixtotheother)creatingtwo
leaderportionsreferredtoas
LeaderA(madeupofoligos1&
2)andLeaderB(madeupof
oligos3&4)(SeeFigure5).
InitialligationsofLeaderAFigure5:
Diagramofthefabricationprocess
andLeaderBproved
unsuccessful.Thiswasduetothelackof5'phosphorylationofthesequences.T4polynucleotidekinasepurchasedfromNEBwasusedtophosphorylateLeaderAandLeaderBandtheligationwasattemptedagain.TheresultsoftheligationofLeaderAandLeaderBfollowingphosphorylationprovedinconclusive.
Thetrailersequencehadtobeisolatedfromthepurchasedminigeneplasmid.ItwasdesignedwiththeHindIIIandXhoIrestrictionsites.Usingtherespectiverestrictionenzymes,thetrailerwascutoutoftheminigeneplasmid.Thetrailerdigestwasrunthrougha2%agarosegelusinganelectrophoresismachinesetat100Vfor2hoursand45minutestoseparatethetrailerfromtheremainderoftheplasmid.Thetrailersequencewasthenextractedfromthegelandsavedforfutureuse.
TheluciferasegenewasisolatedfromthepGEMlucvectorinasimilarfashion.pGEM-lucwasfirstcutwithrestrictionenzymeXhoIfollowedbyBamHI,resultingintwoseparatepiecesofthevector,theluciferasegene(1716basepairs),andtheremainderoftheplasmid(3215basepairs).Gelpurificationwasconductedinordertoisolatetheluciferasegene.A0.7%agarosegelwaspreparedandtheluciferasedigestwasrunat
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80Vfor1hour,andthen100Vforanadditionalhour.Theluciferasegenewasthenextractedfromthegelandstoredforlateruse.
pcDNA3.1wasalsocutinordertoallowforfutureligationwiththeothersequences.ThevectorwascutwithHindIII,andthenlaterwithNotI.Theenzymecutscreatedtwofragmentsofthevector,one5360basepairslong(functional),theother68basepairslong(nonfunctional).Another0.7%agarosegelwasprepared,loadedwiththedigest,andrunat100Vforanhourandahalf.Thefunctional5360bpsequencewasextractedfromthegelandstoredforlaterligation.Duetoinconclusiveresultsfrom
theLeaderAandLeaderBligationwedecidedtoconductonelargeligationofLeaderA,LeaderB,luciferase,Trailer,andpcDNA3.1.
ThefourbasicpartsofRSVlucM5(pcDNA3.1,luciferase,leader,andtrailer)
weredesignedwithendssuchthatwhentheywerecutwiththeappropriaterestrictionenzymes,theycouldonlyassembleinaparticularway.However,itwaspossiblethataseriesofself-dimerizationscouldoccurthatwouldresultinotherviablealternativestopRSVlucM5.VectorNTIprovidedfourviablealternateconfigurations.Theywere10720bp,11090bp,11188bp,and21440bpinlength.Fromthedesignweknowthatourplasmidisonly7495basepairsinlength.InordertoscreenforthecorrectplasmidanadditionaldigestwithrestrictionenzymeSphIwasconducted.
Bacteriawerefirsttransformedwiththeplasmid.Thisbacteriawasgrownonampicillinplates–thiswasdonetoweedoutanybacteriathatdidnotincorporateourplasmid.Wewerethenableto
screenforthecorrectplasmidfrom
allcoloniesthatgrew.Aninitial24
colonieswereselectedforthefirst
roundofscreening.
TheSphIdigestcutthe
DNAfromthe24selectedcolonies.
A0.7%agarosegelwasagain
preparedandall24cutsamples
wereloaded.BasedontheinsilcoFigure6:
Gelimageofthetotalplasmiddigestwithbandsindicateddesignadigestofourplasmidwith
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SphI,wouldresultinfivesegmentsofthefollowinglengths4908bp,1146bp,7
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