qRT-PCRwasusedtoexaminetheexpressionpatternsofBjvipinthedifferenttissuesofB.japonicum.TotalRNAswereextractedwithTrizol(Invitrogen)fromthedifferenttissuesincludingthegill,hepaticcecum,hind-gut,testis,ovary,notochordandmuscle.AfterdigestionwithRNase-freeDNase(Promega)toeliminatethegenomiccontamination,cDNAsweresynthesizedandusedastemplateforreversetranscriptionsystemusingoligod(T)primer.ThePCRprimersP6eFandP6eRspeci?cofBjvipandtheprimersP7eFandP7eRspeci?cofb-actingene(Table1)weredesignedusingtheprimerpremier5.0program.Thereactionmixture(?nalvolume20ml)wasconsistedof10mlofSYBRPremixExTaq?(TliRNaseHPlus),0.4mlofROXReferenceDyeII,0.5mloftemplateand200nMofeachsenseandantisenseprimers.Theb-actingenewaschosenasthereferenceforinternalstandardization.qRT-PCRex-perimentswereconductedintriplicate.Theampli?cationwasperformedonABI7500real-timePCRsystem(AppliedBiosystems)at95??Cfor15s,followedby40cyclesof95??Cfor5s,60??Cfor15s,and72??Cfor30s.Meltingcurveanalysisofampli?cationproductswasperformedattheendofeachPCRtocon?rmthatonlyonePCRproductwasampli?edanddetected.TheexpressionlevelsofBjviprelativetothatofb-actingenewerecalculatedbythecomparativeCTmethod(2àDDCT)(LivakandSchmittgen,2001).

TotesttheeffectoftheviralmimicpolyI:C,asyntheticdouble-strandedRNA(FengandRise,2011;Horietal.,2012;Riseetal.,2008),onBjvipexpression,theadultamphioxus(B.japonicum)wererandomlydividedintotwogroups(eachconsistingof100individuals,andmaintainedatroomtemperature.Amphioxusingroupone(control)wereculturedinsterilizedseawater,whilethe

animalsingrouptwowererearedinsterilizedseawatercontaining50mg/lofpolyI:C.Atotalof10amphioxuswassampledfromeachgroupat2,8,12hafterchallengewithpolyI:C,andthedifferenttissuesincludingthehepaticcecum,muscle,gillandhind-gutweredissectedoutoftheanimalsaseptically.TotalRNAextraction,cDNAsynthesisandqRT-PCRwereperformedasdescribedabove.2.3.Eukaryoticexpressionvectorconstruction

ThecompletecodingregionofBjvipwasampli?edbyPCRusingtheprimerP8eFandP8eR(Table1),andthePCRproductwasdigestedwithEcoRIandXhoIandsubclonedintotheeukaryoticexpressionvectorpcDNA3.1/V5-HisAvector(Invitrogen,Carlsbad,CA,USA)previouslycutwiththesamerestrictionenzymestoconstructpcDNA3.1/Bjvipvector.Subsequently,thegreen?uores-centprotein(GFP)genewasampli?edbyPCRusingtheprimersP9eFandP9eR(Table1),andthePCRproductwasdigestedwithKpnIandBamHIandligatedintothepcDNA3.1/Bjvipvector(whichwascutwiththesamerestrictionenzymes)upstream,toconstructtherecombinanteukaryoticexpressionvector,pcDNA3.1/GFP/Bjvip,whichwasthenveri?edbysequencing.2.4.Cellcultureandtransfection

ThecontinuouscelllineFG-9307establishedfromthegillsofParalichthysolivaceus(Tongetal.,1997)wereculturedat22??Cinminimalessentialmedium(MEM),supplementedwith10%bovinecalfserum(BCS),100U/mlpenicillin,and100mg/mlstreptomycin.Lymphocystisdiseasevirus(LCDV)wasextractedaspreviouslydescribedbyChengetal.(2006)fromtheLCDV-infected?oundersofWeihai?shfarm,Shandongprovince.TheviruswasdilutedwithsterilizedPBS,andtheviraltitersweremeasuredvia50%tissuecultureinfectivedose(TCID50)assayaccordingtothemethoddescribedbyReedandMuench(1938).Fortransfection,theFGcellsweredistributedinto6-wellcultureplates(105cells/ml)inOpti-MEMmedium(Gibco,InvitrogenCorp.,Carlsbad,CA)withoutan-tibiotics.ThetransfectionofthecellswithpcDNA3.1/GFP/BjvipandpcDNA3.1/GFP(control)wereperformedwithLipofectamine?2000reagent(Invitrogen,Carlsbad,CA,USA)accordingtothein-structionsofthemanufacturer.At48hposttransfection,thecellswereexaminedbyLeica?uorescencemicroscopy(Germany).2.5.ViralinfectionofFGcellsandassayforantiviralactivityofBjVipinvitro

TheFGcellstransfectedwithpcDNA3.1/GFP/BjvipandpcDNA3.1/GFPweresimilarlytreatedwith500mlofLCDVsus-pension(2.7?104TCID50/ml).Afterincubationat16??Cfor2h,1.5mlofMEMwith2%BCSwasaddedtoeachwell.Thecellswerethenincubatedat16??Cfor24,48and72h,respectively,andhar-vestedbyscraping.GenomicDNAswereextractedfromthecellswithaDNApuri?cationkit(CWBIO,China)accordingtomanu-facturerprotocol,andwereusedastemplates.qRT-PCRwasemployedtoanalyzetherelativequantityofLCDVineachsample,usingapairofprimersP10eFandP10eR(Table1)speci?cofLCDVdesignedaccordingtothesequenceofmcpgene(GenBankno.EF059991.1)ofLCDV-cn.TheprimersP11eFandP11eRspeci?cof?ounderb-actingenewereusedasthereferencefornormalization.2.6.Prokaryoticexpressionvectorconstruction

ThecDNAregionencodingmaturepeptideofBjvipwasampli-?edbyPCRasaboveusingtheprimersP12eFandP12eR(Table1).ThePCRproductwasdigestedwithNdeIandEcoRI,andsub-clonedintotheplasmidexpressionvectorpET28a(Novagen,Darmstadt,

296M.Leietal./DevelopmentalandComparativeImmunology53(2015)293e302

Germany)previouslycutwiththesamerestrictionenzymes.Therecombinantplasmidwasveri?edbysequencing,andnamedpET28a/Bjvip.

2.7.Expression,puri?cationandrefoldingofrecombinantBjVip(rBjVip)

ThecellsofE.coliBL21(DE)weretransformedwiththeplasmidpET28a/Bjvipandculturedover-nightinLBbrothcontainingkanamycin(50mg/ml).Theculturewasdiluted1:100withLBbrothandsubjectedtofurtherincubationat37??Cfor3h.TheexpressionofBjVipwasinducedbyaddingisopropyld-thiogalactoside(IPTG)tothecultureata?nalconcentrationof0.1mM.TheinclusionbodieswerepreparedbythemethodofLiuandZhang(2009),andanalyzedona12%SDS-PAGEgel.TherecombinantproteinrBjVipwaspuri?edbychromatographyonaNi-NTAresincolumn(Novagen,Darmstadt,Germany),andrefoldedasdescribedbyXuandZhang(2012).TherefoldedrBjVipwasrunona12%SDS-PAGEgelandimmunostainedwiththemouseanti-His-taganti-bodyastheprimaryantibody(LiuandZhang,2009).Proteincon-centrationsweredeterminedbythemethodofBradford(1976)withBCAproteinassaykit(CWBIO,China),usingbovineserumalbuminasastandard.

2.8.Shrimpculture,viruschallengeandassayforantiviralactivityofrBjVipinvivo

TotestifrBjViphasanyantiviralactivity,healthywhiteshrimps(Litopenaeusvannamei)withaveragebodyweightofabout15e20gwerepurchasedfromalocalshrimpfarm,andacclimatedforoneweekbeforeexperimentbycultureat25??Cin500ltankswithaeratedcirculatingseawaterandfeedingwithcommercialdiettwiceaday.Eightoutof100shrimpsweretestedforthepresenceofWSSV(Pilotexperiments),andtheresultsshowedthattheywereallwhitespotsyndromevirus(WSSV)free(Datanotshown).Thustheywererandomlydividedintothreegroupsforthefollowingexperiments.Theshrimpsingroupone(Blankcontrol)wereinjectedwith50mlofTris-NaCl(pH9.5)atthethirdabdominalsegment,theshrimpsingrouptwo(Control)injectedwith30mlofWSSV(2?105virions/ml)plus20mlofTris-NaCl(pH9.5),andtheshrimpsingroupthreeinjectedwith30mlofWSSV(2?105virions/ml)plus20mlof1mg/mlrBjVipTris-NaCl.ThemixtureofWSSVwithrBjVipwaspre-incubatedat25??Cfor2hbeforeinjection.

Fiveshrimpsweresampledat2,8,12,24and48hafterinjec-tion,andthehepatopancreasandmusclesweredissectedoutoftheshrimps.ThegenomicDNAswereextractedfromthehepatopan-creasandmusclesusingthemarineanimalDNAkit(CWBIO,China)followingthemanufacturer'sinstructionsandwereusedastem-plates.qRT-PCRwasconductedforthedeterminationofrelativequantityofWSSV.ApairofprimersP13eFandP13eR(Table1)speci?cofWSSVweredesignedaccordingtothesequenceofvp28gene(GenBank:DQ681069.1).TheprimersP14eFandP14eRspe-ci?cofL.vannameib-actingenewereusedasthereference.

2.9.Statisticalanalysis

Alltheexperimentswereperformedintriplicateandrepeatedatleastthreetimes.Dataweresubjectedtostatisticalevaluationwithone-wayanalysisofvariance(ANOVA),andthedifferenceatp<0.05wasconsideredsigni?cant.Alldatawereexpressedasmean±S.E.M.

3.Results

3.1.Sequence,characteristicsandphylogeneticsofBjvip

AcDNAfragmentof559bpwasobtainedbyPCRusingtheprimersP1eFandP1eR.Basedonthispartialsequence,twofrag-mentsof530bpand835bpwereclonedbymeansof50-RACEand30-RACE.BjvipcDNA(GenBanknumber:KP979737)wasobtainedbyassemblingtheoverlappingcDNAfragments,whichis1540bpinlengthandcontainsanopenreadingframe(ORF)of1074bp,a50-untranslatedregion(UTR)of71bpanda30-UTRof395bp(SupplementaryS1).TheORFencodesadeducedproteinof357aminoacidswithacalculatedmolecularmassofabout40.4kDaandanisoelectricpoint(pI)of8.5.TheanalysisbySMARTprogramidenti?edaconserveddomainofelongatorprotein3(Elp3)/radicalSAMsuperfamilyinBjVip,whichcontainsCxxxCxxCintheformof79

CNYKCGFC86typicalofviperinhomologues.Sequencealignmentindicatedthatthededucedproteinshares60.7%e87.8%overallsequenceidentitywiththeviperinhomologuesofhuman,chim-panzee,mouse,platypus,chicken,zebra?nch,anolelizard,frog,zebra?sh,tilapia,elephantsharkandFloridaamphioxus(SupplementaryS2).AnalysisbySignalPrevealedthepresenceofasignalpeptideof23aminoacidsintheN-terminus.AllthesedatashowedthatthecDNAcodesfortheviperinhomologueofB.japonicum,BjVip.Thiswasfurthersupportedbythe3DmodelingofBjVip,consistingof9a-helixand9b-sheet,whichiscloselysimilartothe3Dstructureofhumanandzebra?shviperinhomo-logues(Fig.1A).