Description:

Baculovirusesareinsectviruses,predominantlyinfectinginsectlarvaeofthe orderLepidoptera(butterfliesandmoths).Abaculovirusexpressionvectoris arecombinantbaculovirusthathasbeengeneticallymodifiedtocontaina foreigngeneofinterest,whichcanthenbeexpressedininsectcellsunder controlofabaculovirusgenepromoter.Themostcommonlyusedbaculovirus forforeigngeneexpressionisAutographacalifornicanucleopolyhedrovirus (AcMNPV).AcMNPVhasacircular,double-stranded,super-coiledDNA genome(133894bp;Accession:NC_001623),packagedinarod-shaped nucleocapsid.Thenucleocapsidcanbeextendedlengthwaysandthusthe DNAgenomecanaccommodatequitelargeinsertionsofforeignDNA.The AcMNPVgenomeformsthebasisoftheflashBACDNAprovidedinthiskit. AcMNPVhasabi-phasiclifecycleresultinginthe productionoftwovirus phenotypes:buddedvirus(BV)andocclusion-derivedvirus(ODV).BVs containsingle,rod-shapednucleocapsidsenclosedbyanenvelope(Figure1) containingamembrane-fusionprotein(GP64).GP64isacquiredwhenthe nucleocapsidsbudthroughthehostcellplasmamembrane.TheBVformof thevirusis1000-foldmoreinfectiousforculturedinsectcells,comparedto theODVphenotype,andisresponsIBLeforcell-to-celltransmissionintheearly stagesofinfection.ItistheBVformofthevirusthatdeliverstheforeigngene intothehostinsectcell. Inthelatestagesofinfectionlargenumbersofocclusionbodies(OB)or polyhedraareformed.Theseconsistofmultiplerodshapednucleocapsids enclosedbyanenvelope,acquireddenovointhenucleus,andembedded withinthepara-crystallinematrixoftheOB/polyhedra.Themajorcomponent oftheOBmatrixispolyhedrin,aproteinthatisproducedbythepowerful transcriptionalactivityofthepolyhedringene(polh)promoter.

OBsprotectthevirusandallowthemtosurvivebetweenhosts,withinthe environment.Mostbaculovirusexpressionvectorsdonotproducepolyhedra (seebelowfordetails),justtheBVformofthevirus.Thisisausefulsafety featurebecauserecombinantviruscannotpersistintheenvironmentinthe absenceofpolyhedra. 

Thebaculoviruspolyhedringeneisnon-essentialforvirusreplicationininsect cellsandthishasledtothedevelopmentofthewidely-usedbaculovirus expressionvectorsystem,firstdescribedbySmith etal.3.Thecoding sequencesofthepolyhedringenearereplacedbythoseofaforeigngene,to producearecombinantbaculovirusinwhichthepowerfulpolyhedrinpromoter drivesexpressionoftheforeigngene.Hencerecombinantbaculovirusesare sometimesreferredtoaspolyhedrin/polyhedra-negativeviruses. Expressionofforeigngenesininsectcellsusingrecombinantbaculoviruses hasbecomeoneofthemostwidelyusedexpressionsystems,andisoftenthe firstchoiceeukaryoticsystem. 

Thebaculovirusexpressionsystemhasseveraladvantagesoverbacterial systems: 

Safetouse. 

Canaccommodatelargeormultiplegenes

Usesavarietyofpromotersforearlyand/orlategeneexpression 

Usesveryefficientgenepromoters 

Proteinsproducedarealmostalwaysfunctional 

Proteinsareprocessed:signalpeptidecleavage,nucleartargeting, membranetargeting,secretion,phosphorylation,glycosylation,acylation 

However,itisnotwithoutitsdisadvantagesandtheseliemainlyinthelabourintensiveandtechnicallydemandingstepsneededtoproducerecombinant viruses.Thefollowingoutlinesthedevelopmentofthebaculovirusexpression systemandthefine-tuningthathasbeenusedtoimprovethesystemoverthe years. Generally,thebaculovirusgenomeisconsideredtoolargeinwhichtoinsert theforeigngenedirectly.Insteadtheforeigngeneisclonedintoatransfer vector,whichcontainssequencesthatflankthepolyhedringeneinthevirus genome.Thevirusgenomeandthetransfervectorareintroducedintothe hostinsectcellandhomologousrecombination,betweentheflanking sequencescommontobothDNAmolecules,effectsinsertionoftheforeign geneintothevirusgenome,resultinginarecombinantvirusgenome.The genomethenreplicatestoproducerecombinantvirus(BVphenotypeonly,as thepolyhedringeneisnolongerfunctional),whichcanbeharvestedfromthe culturemedium. Inmostbaculovirusexpressionsystemsavailablethatusehomologous recombinationtotransfertheforeigngeneintothevirusgenome,amixtureof recombinantandoriginalparentalvirusisproducedaftertheinitialroundof replication.Beforeusingthevirusasanexpressionvector,therecombinant virushastobeseparatedfromtheparentalvirus.TrADItionallythishasbeen achievedbyplaque-assayorplaque-purification.Thisprocessislabourintensive,technicallydemandingandtime-consuming. Manydevelopmentshaveattemptedtoimprovethemethodsbywhich recombinantandparentalvirusmaybeseparated.Thefrequencyof recombinationusingthissystemislow(<1%)andrecombinantvirusplaques canbeobscuredbyparentalvirusplaques.Thisproblemwaspartially addressedbytheinsertionofthe EscherichiacolilacZgeneintothevirus genome,inadditiontothegeneofinterest.Therecombinantvirusplaques couldthenbestainedbluebytheadditionofX-gal(5-bromo-4-chloro-3-indolyl β-D-galactopranoside)againstabackgroundofcolourlessparentplaques.

However,thisdidnotimprovethelowrecombinationefficiencyandresultedin thecontaminationofrecombinantproteinwithβ-galactosidase. Theefficiencywithwhichrecombinantviruscouldberecoveredwasimproved bytheadditionofauniquerestrictionenzymesite(Bsu36I)atthepolyhedrin locus(AcRP6-SC).Linearizationofthevirusgenomepriortohomologous recombinationreducedtheinfectivityofthevirus DNAbutincreasedthe proportionofrecombinantvirusrecoveredto30%.Homologousrecombination betweenthetransfervectorandthelinearDNAre-circularisedthevirus genome,restoringinfectivityandtheproductionofvirusparticles. LacZwas thenintroducedatthepolyhedringenelocus,replacingthepolyhedrincoding region,producingAcRP23.lacZ.ABsu36IrestrictionsitewithinlacZallowed formoreefficientrestrictionofthelinearDNApriortohomologous recombinationandthepresenceof lacZallowedtheselectionofcolourless recombinantvirusplaquesagainstabackgroundofblueparentalvirus plaquesinthepresenceofX-gal11. Greaterthan90%recoveryofrecombinantvirusplaqueswasachievedby furthermodificationstoproduceBacPAK612.BacPAK6containsthe E.colilacZgeneinsertedatthepolyhedringenelocusandBsu36Irestrictionenzyme sitesintwoflankinggenesoneithersideof lacZ.Digestionwith Bsu36I removesthe lacZgeneandafragmentofanessentialgene(ORF1629)10producinglinearvirusDNA(BacPAK6)thatisunabletoreplicatewithininsect cells.Co-transfectionofinsectcellswithBacPAK6DNAandatransfervector containingthegeneofinterest,underthecontrol ofthepolyhedringene promoter,restoresORF1629andre-circularisesthe virusDNAbyallelic replacement.TherecombinantbaculovirusDNAisthenabletoreplicatein insectcellsandinthelatephaseofinfection,virionsareassembledand recombinantbaculovirusesareproduced.However,Bsu36Idigestionisnever 100%efficientandthefinalviruspopulationwillalwayscontainamixtureof recombinantandparentalvirusthatrequirespurificationbyplaque-assay. Despitethefine-tuningandoptimisationofthesystem,anumberofstepsare stillrequiredtoproduceandisolaterecombinantvirus.Hencecomparedto bacterialexpressionsystems,ithasnotbeenamenabletohigh throughputorautomatedsystems. 

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Name	baculoCOMPLETEproteinexpressionkit
Concentration	LotSpecific
StABIlity	6months
Storage	Part4°C,Part-20°C,PartLiq.N2
IntendedUse	ResearchUseOnly