英文文献汇报教学文案

英文文献汇报Avianleukosisviruses(ALVs),whichbelongtothefamilyRetroviridae,areclassifiedintoA,B,C,D,E,andJsubgroups[1,2].TheA,BandJsubgroupsofALVarethemostcommonALVsincommercialpoultry,whereassubgroupsCandDhaverarelybeenreported.SubgroupEisaubiquitousendogenousleukosisvirusoflowpathogenicity[3].ALVpredominantlycauseslymphocyticleukemia,myeloidleukosisandothersarcomasandcanalsoleadtoimmunosuppressioneffects,suchastheabnormaldevelopmentofimmuneorgans,growthretardation,anddecreasedimmuneresponses.[4,5].ALVsubgroupJ(ALV-J)wasfirstisolatedintheUKfromwhitemeat-typechickensin1988[2].InChina,ALV-Jwasfirstreportedin1999.Sincethen,ALV-Jisolateshavebeendetectedinlayerchickens,broilerbreedersandlocalchickensthroughoutmostregionsofChinaandhavecausedvarioustumorsinchickens[6–8].Inrecentyears,ALV-Jhasbecomewidespreadandhasresultedinsevereeconomiclossestothepoultryindustry[6,8].1.IntroductionEffectivevaccinesagainstALVarenotavailable.ThecontrolofALVinfectionoccursprimarilybyestablishingexogenousALV-freepoultryflocksbyadoptingeradicationasthestrategyofchoice[9].BecauseofsubstantialantigenicandgeneticvariationamongALVisolatesandhighlevelsofverticalandhorizontaltransmission,eradicationhasbeendifficult[10,11].Thus,effectivemethodsfortheaccuratedetectionofALVantigensinchickensarecriticalforthecontrolofALVinfections.ThegenomeofALVconsistsof5’-LTR-UTR-gag-polenv-UTR-LTR-3’.Thegag,polandenvgenesaretheviralstructuralgenes[11].TheP27protein,whichisencodedbythegaggene,isthecapsidproteinandactsasagroup-specificantigen.TheALVP27geneishighlyconservedandexhibits96%sequenceidentityamongtheexogenoussubgroups(A,B,C,DandJ).Inaddition,theP27proteincontentaccountsformorethan30%oftheviralprotein,andP27hasmanyviralantigensitesthatareeasytodetect.Therefore,theP27proteinisthefirstchoiceforpreparingantibodiesfordetection.Epitopesofproteinsareclassifiedaseithercontinuousordiscontinuousdependingonwhethertheaminoacidsincludedintheepitopearecontinuousinthepeptidechainornot[12–14].Continuousepitopessometimesdonotrepresenttheentireantigenicepitopeintheviralproteinbutinsteadonlyashortcross-reactiveportionofalarger,discontinuousepitope[12,13].B-cellepitopesareregionsthatarerecognizedbythebindingsitesorparatopesofantibodymoleculeswhentheyarepresentintheirfreeforminserumorasmembrane-boundB-cellreceptors[12].CorrectidentificationofB-cellepitopeswithinanantigenicproteinmayopenthedoorforthedesignofmoleculesthatmimicpotentiallyprotectiveepitopesandcouldbeusedtoraisespecificAbsorbeusedasprophylacticortherapeuticvaccines[15–17].IdentificationofB-cellepitopescouldpromoteprotectiveimmunityinthecontextofemergingandre-emerginginfectiousdiseasesandpotentialbioterroristthreats[15].TheaimofthisstudywastogenerateaP27-specificantibodyagainstALVusingpurifiedrecombinantP27proteinastheimmunogenandsubsequentlytoidentifytheB-cellepitoperecognizedbytheantibody.TheinformationdescribedinthisstudywillfacilitatethedevelopmentofALVdiagnostictoolsandwillfurtherourunderstandingoftheantigenicstructureoftheP27protein.2.Materialsandmethods重组质粒的构建构建重叠片段短肽免疫小鼠制备单抗IFA&Westernblot检测单抗特异性鉴定最小抗原表位基序分析表位保守性3.ResultsFig.1CharacterizationoftherecombinantP27proteinandmAb3A9.(A)ExpressionandpurificationoftherecombinantP27protein.M,molecularmarker(kDa);1,recombinantP27proteinbeforeIPTGinduction;2,recombinantP27proteinafterIPTGinduction;3,purifiedrecombinantP27protein.(B)RecognitionofthepurifiedALV-JP27proteinbymAb3A9inwesternblotanalysis.M,proteinmarker(kDa);1,purifiedrecombinantP27protein;2,negativecontrol(prokaryoticexpressionvectorpET-30a).(C)IFAanalysisofmAb3A9usingdifferentviralstrains.mAb3A9onDF1cellsinfectedwith(A)RAV-1;(B)RAV-2;(C)HPRS103;(D)NX0101;(E)ADOL-Hc1;(F)ADOL-7501;(G)HuB09JY03;(H)JL08CH3-1;(I)LN08SY10;(J)SD09DP04;(K)HLJ09SH01;(L)negativecontrolFig.2IdentificationoftheminimallinearepitoperecognizedbymAb3A9.(A)Strategyfortheidentificationoftheepitope.Thirteenoverlappingpeptideswereexpressedandsubjectedtowesternblotanalysis.Theredpeptidesaretheimmunodominantregions,andtheblueonesarethenon-immunodominantregions.(B)WesternblotanalysisofthereactivityoftheGSTfusionsusingmAb3A9.GSTfusionscoveringaminoacids181to197weredesignedandexpressed.GSTwasencodedbytheprokaryoticexpressionvectorpGEX6p-1,whichservedasanegativecontrol.M,molecularmarker(kDa);1,GST-S;2,GST-DCFRQKS;3,GST-IIDCFRQKS;4,GSTVIIDCFRQKS;5,GST-PVIIDCFRQKS;6,GST-APVIIDCFRQKS;7,GSTRAPVIIDCFRQKS;8,GST-ARAPVIIDCFRQKS;9,GST.(C)Aminoacidsequence,positionintheP27proteinandreactionwith3A9foreachoftheGSTfusions.*representsthereactionbetweentheGSTfusionsandmAb3A9;?indicatesthat3A9couldidentifytheGSTfusion;–indicatesthat3A9couldnotidentifytheGSTfusionFig.3ELISAanalysisandaminoacidsequencealignmentanalysisoftheidentifiedminimallinearepitope.(A)ELISAanalysisforreactivityoftheidentifiedepitopewithmouseanti-ALV-JP27sera.Threeexpressedproteins(GST,theminimallinearepitope,andtherecombinantpET-30a-p27protein)wereusedtocoattheELISAplates.(B)ELISAanalysisforreactivityoftheidentifiedepitopewithchickenanti-ALVsera.Errorbarsreflecttheresultsofthreeexperimentalrepeats.(C)AminoacidalignmentanalysisoftheidentifiedepitopeindifferentALV-A,ALV-BandALV-Jstrains.Residuesthatmatcharerepresentedby‘‘’’4.DiscussionSincetheemergenceofALV,infectionwiththisvirushasbecomewidelyprevalentandhasbeenidentifiedasthecauseofvarioustumorsinmanychickenspecies,includingmeat-typechickens,layerchickens,andlocalbreederflocks[6–8,25].AlthoughwesterncountrieshavesuccessfullyeradicatedALVfrombreedingchickens,thevirushasexpandedrobustlyinChinasince2008[26].AneffectivevaccineagainstALVisnotavailable.Earlydetectionanderadicationofvirus-infectedbirdstoreducethespreadofinfectiontootherbirdsareveryimportantforthecontrolofALVinfection.Thus,effectivemethodsfortherapidandaccuratedetectionofALVinfectionareessentialforeffectivecontrolofALVspread.TraditionalmethodsusedtodiagnoseALVinfection,suchasreversetranscriptionPCR,virusisolation,andIFA,areslowandmightyieldinconclusiveresults[27–29].Incontrast,aspecificmAbcouldbeappliedtodevelopasensitive,rapid,andreliablemethodtodetectvirusinfection.Inthisstudy,we