草鱼脂多糖结合蛋白的分离纯化（英文）

草鱼脂多糖结合蛋白的分离纯化（英文） 草鱼脂多糖结合蛋白的分离纯化(英文) 虿北基业学报2010,l9(1):611 ActaAgriculturaeBoreali—occidentalisSinica IsolationandPurificationofLipOp0lysaccharide—bindingProtein (LBP)fromGrassCarp,Ctenopharyngodonidellus HUANGTeng,SUJianguoandDONGJie (CollegeofAnimalScienceandTechnology.NorthwestA&FUniversity.ShaanxiKeyLaboratoryof MolecularBiologyforAgriculture,YanglingShaanxi712100,China) Abstract:Lipopo1ysaccharidebindingprotein(LBP)isasolubleacutephaseproteinthatbindstobac— terialLPStoelicitimmuneresponsesbypresentingtheLPStoimportantcellsurfacepatternrecogni—n tionreceptors,involvedintheacute—phaseimmunologicresponsetogram—negativebacterialinfec — tions.Inpresentstudy,freshwholebloodwascollectedfromgrasscarp(Ce,zo户口rg’odonidellus) challengedwithAeromonashydrophilafor24h.Theplasmawasobtainedbycentrifuge.Integrated activitytestofLBPandammoniumsulphateprecipitation,CMSephadexC一 50cation—exchangechro— matographyandDEAESephadexA25anion—exchangechromatography,Jipopolysaccharidebinding proteins(LBPs)wereisolatedandpurified.TheresultsindicatedthatthepurifiedIBPshadrelatively highaffinitytoFluoresceinisothiocyanatelabeledlipopolysaccharide(FITC—LPS).SDS—PAGEsta ined withCoomassieBrilliantBlue,visualizedthattherewerethreedistinctbandswithmolecularweightof 68kDa,53kDaand48kDarespectively.Thisresearchdescribedasimpleandmaneuverablemethod forisolationandpurificationofLBPsandservedthefurtherstudyofLBPfunctions. Keywords:Lipopolysaccharidebindingprotein;Grasscarp;Isolation;Purification CICnumber:S965.1l2Documentcode:AArticleID.】0041389(2O】0)0l一0006—06 草鱼脂多糖结合蛋白的分离纯化 黄腾,苏建国,董捷 (西北农林科技大学动物科技学院,陕西省农业分子生物学重点实验室,陕西杨凌712100) 摘要:脂多糖结合蛋白(1ipopolysaccharidebindingproteins,LBP)是机体对革兰氏阴性菌感染 产生的可溶 性急性期蛋白,结合并提呈LPS给细胞面的模式识别I,C—IPS)有较强的结合能力.在 SDS-PAGE电泳后,考马斯亮蓝染色可见3条明显的 带,分子量大约为68,53和48kDa.同时探索一条简便分离纯化脂多糖结合蛋白的方法,为进 一步研究I.BP 的功能奠定基础. 关键词:脂多糖结合蛋白;草鱼;分离;纯化 Receiveddate:2009一O7一O7Returneddate:2009O8,29 Foundationitems:NationalNaturalScienceFoundationofChina(30871917),ProgramforNewCentur yExcellentTalentsinUni versity(NCET一080466),andtheProjectsponsoredbySRFforROCS,SEM(14110104). Biography:HuangTeng(1985 一),male,borninGuangxi,masterstudent,majorinimmunologyofhydrobiology. *Corresp0ndingauthor:SuJianguo.Email:su.jianguo@gmail.corn HUANGTeng,el”:IsolationandPurificationofLipopolysaccharide—binding tI.BP)fromGrassCarp,Cten()phnryngOa()nidellus Likeothervertebrates,teleostimmunesys— ternisconventionallvdividedintotheinnateand theadaptiveimmunesystem.However,thea daptiveimmunesysteminfishisconsidered primitiveandinferiorincomparisonwiththatin mammalianspecies,whichaccountsforlesssen sitiveimmunememoryresponsetopathogenin vasion卜.Thustheinnateimmunitvasthe firstlineofhostdefenceplaysapivotalrolein non——selfrecognitionandinductionoftheadap—— tiveimmunity. Asolubleglycoproteinsecretedintheacute phase,termdaslipopolysaccharidebindingpro tein(IBP),canspecificallyrecognizeandbind lip.polysaccharide(LPS),anecessarycompo nentofGram—negativebacteria.Itservesascat— alystforactivatingTolllikereceptor4(TLR4) signallingpathwayandeventuallyinducesthein— fectedcelltoreleasepro—inflammatorycytokines (TNF—a,II一1BandII6etc.)thataccelerate tissue1esion.. LBPwasfirstpurifiedfromrabbitserum jj ,afterwardsotherLBPswereconfirmedin human,mouse,ratandbovinerespctivelyE. Todate,IBP/BPI(bactericidalpermeability—in— creasingprotein)geneshavebeenclonedand characterizedinsomefishspecies【….Howev— er,theevolutionattyconservedstructuresbe— tweenLBPandBPI1cadtomassivechallengesin differentiatingthem.Moreover,Itremainsun— clearandcontroversialwhetherfishpossesthe capacityofrecognizingIPSornot.Priortothe latestidentifiedTIR4homotogofzebrafishthat doesnotrecognizeLPS,ithasbeenestablished thatfishareresistantIothetoxiceffectofLPS . Arecentstudysuggestedthatfish,inparti cularrainbowtroutOm’orhynchusrnykiss3]and AtlanticcodGadusmorhuaI.[,maylackan importantcomponentoftheLPSrecognitionre ceptorcomplex.Inthissense,itcanbeinferred thatthefishattenuatedsensitivitytoLPSisat— tributedtothelOSSoffunctionalIBP.Onthe otherhand,thefindingsfromtrout,codand largeyellowcroakerPseudosciaenacroceawere supportiveofaspeculationthatthefishLBP/ BPIhomologiscomparabletothemammalian BPI[1516].Theabsenceofthefunctiona1LBP purifiedundernormalorinfectedconditioninvi— vo,alongwiththecomplexityofpost—transla— tionalmodificationamongdisparatespecies, triggersmoreinterestinsettlingthesecontro— vers1es. Aeromonashydrophila,aGram—negative bacteriumisthemajorsuspectthatcauseshigh mortalityandconsiderableeconomiclossesofthe culturedgrasscarpinChinaEl7].Inthepresent study,weisolatedgrasscarpIBP(gcLBP)and identifieditsbiologicalfunctions.Ourresults suggestedthatgcLBPisresponsivetoA.hy— drophilastimulationandbindstoIPS.These findingsindicatethatLBPsofvertebratesare functionallyconservedininflammation—inducible properties.Expectedly.IBPblockagecouldbea promisingtherapeuticsagainstgrasscarpsepsis andenhancefishviabilityunderunfavorablecul— tureconditions. 1Materialsandmethods 1.1Experimentalfishandbacterialinfection Healthyadultgrasscarps(bodyweight800 ?100g)werepurchasedfromthelocalmarket. Afterdisinfection,theyweretemporarilyraised for1dintanksat2OC.0.5mLof1.0×10 CFU/mL(OD6o0—0.643)Aeromonashydrophi— lawasinjectedperkilogrambodyweightintrap— eritoneally.Theiniectedindividualsweremain— tainedinaeratedaquariaat2OCfor24h. 1.2Preliminaryextractionofplasmaprotein Approximate100mLofbloodwascollected fromcaudalveinoftheinjectedfish,addinglmg ofanticoagulantEDTANa2forlmIofblood. Unlessotherwisementioned,allprocesseswere manipulatedat4?.Themixturewascentri— fugedat3000r/minforl5mintocollectsuper— natants.Saturatedammoniumsulphatesolution wasinfusedintotheobtainedplasmatoaresul— tingsaturationof50.Centrifugationwascar— riedoutat4500r/minfor30min,andsedi— mentsweredissolvedin50mmol/LPB ? 8?西北农业学报l9卷 (Na2HPO4一NaH2POdbuffer,pH7.3)anddia— lyzedagainstthesamebufferfor24h.Afterde— salting,theharvestedextractswerecentrifuged at3000r/minfor15minandthesupernatants werekeptat,20?. 1.3Determinationofproteincontent ReferredtoBradford[.thismodifiedas— saywasperformedinaccordancewithfollowing protocols:(1)Dissolve100mgCoomassieBril一 1iantBlueG一250in50mL95%ethanol,add1OO mL85%(w/v)phosphoricacid.Diluteto1liter whenthedyehascompletelydissolved,andfil, terthroughWhatmanNo.1filterpaperjustbe, foreuse;(2)Prepareastandardcurveofabsor, banceversusarangeof5to100micrograms BSA(BovineSerumAlbumin)in100”L.Add 5mLdyereagentandincubate5rain.(3)Meas, uretheabsorbanceat595nm.Calculateconcen, trationsoftheamountprotein. I.4Proteinisolationandpurification Plasmaextractionwasfractionatedwith30 mIofCMSephadexC一50(Pharmacia,Uppsala, Sweden)equilibratedwith40mmol/LNaC1in 50mmol/Lphosphatebuffer(pH7.3)contai— ning2mmol/LEDTA(phosphate—EDTA). 3mLofsamplewas1oadedinthecolumn.and thecolumnwaswashedwithphosphate—EDTA untiltheA280oftheeluatewaslessthan0.02 absorbanceunit.ProteinabsorbedtotheSepha— dexwaselutedwithgradientsolutionof220 mmol/L,35Ommol/1,500mmol/Land1mol/ LNaCIinphosphate—EDTA.Collectionoffrac— tionstobetestedforLBPactivityweredialyzed against50mmol/Iphosphatebuffer(pH7.3) andconcentratedbyusingAmiconUltra——4cen—- trifugalfilterdevice.Alltheflow—throughfrac— tionswerekeptformeasuringLBPactivityand furtherpurification. FractionscontainingLBPactivityweread— sorbedonaDEAESephadexA一25column (Pharmacia,Uppsala,Sweden)equilibratedin 25mmol/LTris—Clbuffer(PH8.3).Themate— rialwaselutedbynormalliquidchromatogra phy.Beforeloadingsample,proteinswereelu tedwithalinear solutionmixed0 gradientammoniumsulphate and330mmol/L.Fractions containingLBPactivitywerecollected,dialyzed againstphosphatebuffer(pH7.3),andloaded onSephadexG一50chromatographytodesalt. 1.5DetectionofLBPactivity AIIthecollectedfractionsweredilutedto 50/~g/mLwithcoatingbuffer(1.5g/LNa2CO3, 2.9g/LNaHCO3,pH9.6).Thetestwascar— riedoutin96,wellplatewherefractionsand1 BSAasblankwerecoated.Threewellsforeach treatmentwerefilledwith200/,Lofthecoating bufferaheadofbeingincubatedat4?over— night.Washedofftheunboundsamples,then filledthewellswithPBS—Tween(NaC18.0g, KH2PO0.2g,Na2HPO4?12H2O2.9g,KCl 0.2g,Tween一200.5mL,in1000mL,pH 7.4),washingtwice.Blocknon—specificbinding byadding200uLof1BSA/PBSfor2hat 37?.50uLof2×10一g/LFITC—LPS(Fluo— resceinisothiocyanatelabeled1ipopolysaccha— ride)wasaddedtoeachwellandwasincubated for4hat37?,washingtheplate6timesasa— hove.Theresultsofthisassaywereobservedby fluorescentmicroscopy. 1.6Identificationoftargetprotein SDS—PAGEwascarriedoutaccordingto Laemmli,with59/5stackinggeland12 separatingge1.Stainedwith0.05Coomassie BrilliantBlueR250,theseparatinggelwasre- peatedlywashedwith0.5mol/INaC1solution at65?toeliminatebackgrounduntilthepro— teinbandswasclearlyvisualized. 2Results 2.1Variationsinproteincontents AsshowninFig.1.theoriginalconcentra— tionofplasmaproteinintheinfectedfishwas 450/zg/mL;cation,exchangeremoveda】arge portionofneutralproteins,andthustherecov— eryofproteinsfeltto90/,g/mL.Thefinalfrac— tioncontaininggcIBPsfromanion—exchangede— creasedto27#g/mL. 1期HUANGTeng,et口f:IsolationandPurificationofIipopolysaccharide—bindingProtein (LBP)fromGrassCarp,Cf已”.户nrg0oidellus TheinfectedFractionBFractionB3 Fig.1Variationsofproteincontentsinthe processofLBPisolation 2.2BiochemicalpropertiesofthepurifiedLBPs FractionsAandBwerecollectedduringCM SephadexC一50chromatography.ByFITC—LPS assay,itwasfoundthatfractionBwasableto bindLPS,whereasnoremarkablef1uorescence ineitherfractionAortheblankwasobserved (Fig.2). ThefractionBwassubjectedtoDEAE SephadexA一25chromatography.SeenfromFig. 3,sevenfractionsfromB1toB7wereobtained; FITC—LPSassayindicatedfractionB3hada strongeraffinitytoLPS.B3waswashedoutun— dertheconditionofpH8.3,itcouldbejudged thattheisoelectricpointofgrLBPsshouldbeal— kaline.WiththecondensationofgcLBPs,fluo— rescentintensityandspotsincreasedaccordingly (Fig.4).InfractionB3,therewerethreedis— tinctbandswithmolecularweightof68kDa,53 kDaand48kDarespectively(Fig.5). Fig.2ElutioncurveofgrasscarpplasmathroughCM SephadexC-50exchangechromatographycolumn O.02 0 0l234567 Time/h Fig.3ElutioncurveofgrasscarpplasmathroughDEAE SephadexA一25exchangechromatographycolumn 3Discussion 3.1gcLBPisafunctionallyconservedprotein andbindstoLPS Multiplegenesinvolvedinnaturalhostde— lensesystemarenotonlydiscoveredinverte— bratesbutalsoininvertebrates.itcanberea— sonedthatinnateimmunitymusthaveformedin theprimaryphylogenyofmulti——cellularorgan—— isms[z0].LBP,anacute—phasereactantfoundin humanandotheranimalserum,isasingle—chain glycoproteinof50,60kDa,featuringwithin, tensecoherencetolipidA(acomponentofLPS orendotoxin)..LBPproteinsequencesare highlyconservativefrommolluskstomammals throughblastpwithhumanLBPsequence(Ac— cessionNo.NP004130),however,thenucleo— tidesequencesvaryalotbetweenmammalsand fishes.Inhuman,theknownmembersofaram— ilyofLPS-bindingproteinsincludeBPI,LBP, andCETP(cholestero1estertransferprotein)in asizerangefrom50to60kDa[233.Ithasbeen confirmedthatrainbowtroutLBP/BPI一1and LBP/BPI一2withapredictedsizeof5lkDa formedaclusterwithmammalLBPandBPI[. Inaccordancewiththeirnoticeableaffinityto LPS,thethreepurifiedproteinswegainedhere, withtheirsizeof68,53and48kDarespective— ly,couldbebettercategorizedintoafamilyof LPS—bindingproteinsexistingingrasscarp.As acomplementarypieceofevidencetocharacter— izethegcLBP,wehaveclonedthefull—length gcLBPcDNAandcalculatedthemolecular ??% OOO00O0 一gI10?—uII对qJ0? OOOOOOOOOOO如柏如加:2. _【\TI一\_口_口0u口一o.I(_昌d0?00?1Io?D《 1期HUANGTeng,etal:IsolationandPurificationofIipopo1ysaccharide—bindingProtein (LBP)fromGrassCarp,C,0户 rgo0idellus ducedingrasscarpandfunctionsimilarlyto thoseofthemammals.Inthefuture,moreef— fortswillbeinvestedtoverifytheaminoacid constitutionofthesethreepurifiedproteinsand clarifytheinteractionswiththedownstream moleculesthatareresponsibleforLBP—mediated bacterialdiseases. RefeFences: r1]PilarAP.Fishimmunityandparasiteinfections:fromin nateimmunitytoimmunoprophylacticprospects[J].Vet erinaryImmunolandImmunopathol,2008,l26:171】98. [2]张艳秋,詹勇,许梓荣.鱼类免疫机制及其影响因子EJ] 水产养殖,2005,269(3):15. 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