全 文 ：*This work was supported by grants from The Scientific
Research-FlandersofBelgium(G.0113.01),GuangdongNaturalScience
Foundation, China(04010899), MinistryofEducationofChina
(2004-6),ShaoguanScientificandResearchProgram,China(2004-02),
theOpenProjectoftheStateKeyLaboratoryofBiocontrolatSun
Yet-senUniversity(2004-2006) andChinaPostdoctoralScience
Foundation(2005-2006).
**Corespondingauthor.
Tel:86-755-26036381,Fax:86-755-26036740
E-mail:wfwang1@yahoo.com.cn
Received:June17,2006 Accepted:July28,2006
PreparationofMonospecificPolyclonalAntibodies
AgainstGlechomahederaceaAgglutinin*
WANGWei-Fang1,3)**,VANDAMMEEls2),HEChao-Zu1)
(1)LifeScienceDivision,GraduateSchoolatShenzhen,TsinghuaUniversity,Shenzhen518055,China;
2)DepartmentofMolecularBiotechnology,FacultyofBioscienceEngineering,GhentUniversity,Gent9000,Belgium;
3)DepartmentofBioscienceEngineering,FacultyofYingdongBioscienceEngineering,ShaoguanUniversity,Shaoguan512005,China)
Abstract Glechomahederaceaagglutinin(Gleheda)isanovelglycosylatedlectinisolatedfromtheleavesofG.hederacea.Likeother
glycosylatedproteins,thedetectionofGlehedabyimmunologicalmethodsisoftenhamperedbythecross-reactivityofthepolyclonal
antibodieswithunrelatedglycoproteins.Henceaprotocoltopurifymonospecificpolyclonalantibodiesfromacrudeantiserumraised
againstGlehedawasdeveloped.Afterselectiveammoniumsulfateprecipitationandsuccessiveafinitychromatographyoncolumnsof
Sepharose4BwithimmobilizedGlehedaandRobiniapseudoacaciaagglutinin(RPA),respectively,ion-exchangechromatographyona
columnofQFastFlowwasusedforfurtherpurification.Thespecificityoftheantibodyfractionsfromeachstepwastestedbydouble
immunodifusionassayandanalyzedbyWesternblot.Resultsrevealedthatafinitychromatographyoftheimmunoglobulinfractionon
theimmobilizedGlehedaantigenyieldedanantibodypreparationthatstilcross-reactedwithmanyproteinsinleafextracts.Depletion
ofnonspecificcross-reactingantibodiesdirectedagainsttheglycanpartoftheglycoproteinbyafinitychromatographyonimmobilized
RPAremovedmostbutnotalnonspecificalyreactingantibodies.Onlyuponfurtherpurificationbyionexchangechromatographyan
IgGfractionofmonospecificantibodiesthatreactedexclusivelywithGlehedacouldbeobtainedandaccordinglywassuitablefor
immunodetectionstudies.Thisantibodypurificationprocedurepromisessimplicityandeficiency.Inaddition,thismethoddoesnot
requireexpensivefacilities.
Keywords monospecificpolyclonalantibodies,antibodypurification,Glechomahederaceaagglutinin(Gleheda)
生物化学与生物物理进展 ProgressinBiochemistryandBiophysics,2006,33(11):1113~1119
www.pibb.ac.cn
Leavesofgroundivy(Glechomahederacea),
whichisatypicalrepresentativeoftheplantfamily
Lamiaceaeexpresshighconcentrationsofalectin
caledG.hederaceaagglutinin(Gleheda).Biochemical
analysesindicated thatGlehedaisatetrameric
glycoproteinconsistingof26kuand28kusubunits,
whichcontainoneandtwoglycan(s),respectively.
Molecularcloningandmolecularmodelingofthe
deducedsequencedemonstratedthatGlehedashares
highsequencesimilaritywiththelegumelectinsand
exhibitsthesameoveralfoldandthree-dimensional
structureastheclassicallegumelectins,indicatingthat
Glehedabelongstothesamelectinfamilyasthe
legumelectins[1].Insectfeedingbioassaysrevealedthat
Gleheda dramaticaly afects larvalgrowth and
reproductionofbothcaterpilarsandbeetles[2,3] but
exhibitsnocytotoxicitytowardshumanandanimal
cels[2,4].
TheidentificationofaLamiaceaelectinwith
insecticidalactivityraisesgreatinterestwithrespectto
astudyoftheinsecticidalactivityandmodeofaction
ofGlehedaaswelaspossibleapplicationsforthe
generation of transgenic plants with enhanced
resistanceagainstsomepestinsects.Todevelophighly
specific sensitive detection/quantification methods
生物化学与生物物理进展 Prog.Biochem.Biophys. 2006;33(11)
basedonimmunologicaltechniques(likeWesternblot
analysisandimmunocytochemistry) apolyclonal
antiserumwasraisedinarabbitagainsthighlypurified
Gleheda.However,preliminaryexperimentsindicated
thatevenafterpartialpurificationtheantiserumstil
cross-reactedextensivelywithnumerousunrelated
proteins,andhencewasnotsuitedforaspecific
detectionand/orquantificationofthelectin.
Thisreportdescribesthedevelopmentofa
protocol for the purification of monospecific
polyclonalantibodiesfromacrudeantiserumraised
againstGleheda.Usingacombinationofselective
ammonium sulfateprecipitation, doubleafinity
chromatography on immobilized Gleheda and
immobilizedRobiniapseudoacaciaagglutinin(RPA),
respectively,andion-exchangechromatographyan
antibodyfractionwasobtainedthatreactedexclusively
with Glehedaand accordingly wassuitablefor
immunodetectionstudies.
1 Materialsandmethods
1.1 Preparationofantigens
TheproteinGlehedawasisolatedasdescribedby
Wangetal[1].Robiniapseudoacaciaagglutinin(RPA)
waspurifiedfromthebarkoftheR.pseudoacaciatree
asdescribedpreviously[5].
1.2 Immunizationoftherabbitsandpreparation
ofantiserum
AntibodiesagainstGlehedawereraisedinamale
NewZealandwhiterabbit.Therabbitwasinjected
subcutaneouslywith1mghighlypurifiedGleheda
dissolvedin0.2mol/LNaClandemulsifiedin1mlof
Freund!scompleteadjuvanttoenhancetheresponseto
theimmunogen.Fourboosterinjectionswith1mg
purifiedproteinin0.2mol/LNaClweregivenwith
10-dayintervalstoobtainaprolongedpersistenceof
theimmunogenintissuesandacontinuousstimulation
oftheimmunesystem.Tendaysafterthefinal
injection,50mlbloodwascolectedfrom anear
marginalveinandkeptovernightatroomtemperature
toalowclotingoftheblood.Thecrudeantiserum
wascolectedbycentrifugation(3000gfor5min)and
theglobulinfractionisolatedbythreeroundsof
selectiveprecipitationwithammoniumsulfate(40%
saturation).Afterthefinalprecipitation,theproteins
weredissolvedin25ml0.2mol/LNaCl.Atthisstage,
thespecificityoftheantibodieswascheckedby
Westernblotanalysisofacrudeextractfrom
G.hederacealeaves.
1.3 ImmobilizationoflectinsonSepharose4B
PurifiedGlehedaandRPA werecoupledto
Sepharose4B usingthedivinylsulfonemethod.
Sepharose4B waspouredinaBüchnerfunnel,
washedwith20volumesofdistiledwaterand
equilibratedwitha0.5mol/LNa2CO3(pH11).50ml
ofsetledgelwassuspendedin50mlofthesame
carbonatebuferand supplemented with 5ml
divinylsulfone(Aldrich,Bornem,Belgium)toactivate
thevinylgroupofSepharose4B.Afterincubationfor
3hatroomtemperaturewithoccasionalstiring,the
suspensionwastransferedtoaBüchnerfunneland
washedwith250mlcarbonatebufer(pH 11)
folowedby1Ldistiledwatertoremoveunreacted
divinylsulfone.10mlaliquotsofthe activated
Sepharose4Bweresubsequentlysuspendedin20ml
ofa0.5mol/LsolutionofNa2CO3/NaHCO3(pH10)
containing50mgoftheproteintoimmobilize.The
mixtureswereincubatedforapproximately15hat
37℃ undercontinuousagitationtoalowtheproteins
toreactwiththeactivatedSepharose.Aftercompletion
ofthecoupling,thesuspensionwastransferedinto
singlefrited25mlcolumns(InternationalSorbent
TechnologyLtd.(IST),MidGlamorgan,UK)and
alowedtosetlebydrainingthecouplingmedium
undergravity.Analiquotoftheflowthroughwas
colectedanditsA280wasmeasuredtocheckthe
couplingeficiency.Undertheconditionsdescribed
here,>98%oftheproteinswereimmobilizedtothe
Sepharose4Bmatrix.Aftersetling,theSepharose4B
gelwaswashedwith50mlNa2CO3/NaHCO3(pH10)
buferandrinsedwith50mlH2O.Toblockthe
remainingfreeactivatedvinylgroups,thematrixwas
equilibratedwitha0.2mol/LsolutionofTris-HCl
(pH8.7),keptinthisbuferforatleast6handwashed
with50ml0.2mol/LNaClcontaining0.01%Na-azide
asanantimicrobialagent. Columnswereused
immediatelyorstoredat2℃untiluse.
1.4 Afinitychromatographyofantiserum on
immobilizedGleheda
AfinitychromatographyonimmobilizedGleheda
wasdoneusingasinglefrited25mlcolumnfiled
with10mlofGleheda-Sepharose4B.Thecolumnwas
mountedonanISTVacMasterSampleProcessing
Station.Theglobulinfractionpreparedfromthecrude
antiserumwasloadedonthecolumnataflowrateof
approximately0.5ml/min.Afterloading,thecolumn
waswashedwith0.2mol/LNaCluntiltheA280fel
below0.01.Boundantibodiesweredesorbedwitha
1114· ·
王卫芳等：抗欧亚活血丹凝集素特异性多克隆抗体的制备2006;33(11)
solutionof5mmol/Lunbufered1,3-propanediamine
(pH11).Topreventinactivationoftheantibodies,
1mlaliquotsoftheeluatewerecolectedin0.2ml
0.4mol/Lphosphatebufer(pH 7.4) containing
1mol/L NaCl.The presence of anti-Gleheda
antibodies in the fractions was checked by
immunodifusionagainstpurifiedGleheda.Fractions
containingtheantibodieswerepooledandthepH
adjustedto7.5with0.1mol/LH3PO4.Thespecificity
oftheantibodieswascheckedbyWesternblotanalysis
ofacrudeextractfromG.hederacealeaves.
1.5 Afinitychromatographyofantiserum on
immobilizedRPA
The antibody fraction isolated by afinity
chromatographyonimmobilizedGlehedawasbrought
toafinalconcentrationof0.2mol/LNaClandloaded
onacolumnofRPA-Sepharose4B(usingthesame
typeofcolumnandequipmentasdescribedabove)ata
flowrateof0.5ml/min.Afterloading,thecolumn
waswashedwith0.2mol/LNaCluntiltheA280fel
below0.01,andelutedasdescribdedin2.4.Theflow
throughandeluatewascolectedinfractionsof1ml.
Thepresenceofanti-Glehedaantibodiesin the
fractionswascheckedbyimmunodifusionagainst
purifiedGleheda. Fractionscontainingantibodies
againstGlehedawerepooledanddialyzedat4℃
against20mmol/LTris-HCl(pH8.7)for24h(with3
changesofbufer).Thespecificityoftheantibodies
wascheckedbyWesternblotanalysisofacrude
extractfromG.hederacealeaves.
1.6 Exchangechromatographyofantiserum on
QFastFlow
Theantiserum fractionobtainedbysuccessive
afinitychromatographyonimmobilizedGlehedaand
RPAwasappliedonacolumn(1.6cm×10cm,
approximately20mlbedvolume)ofQFastFlow
(Amersham Biosciences, Uppsala, Sweden)
equilibratedwith20mmol/LTris-HCl(pH8.7).After
loadingthecolumnwaswashedwith50mlofthe
samebuferandtheIgGfractionelutedwith0.1mol/L
NaClin20mmol/LTris-HCl(pH8.7).Fractions
(1ml)werecolectedandtheA280determined.The
presenceofanti-Glehedaantibodieswascheckedina
doubleimmunodifusionassay.Fractionscontaining
theantibodieswerepooledandspecificityofthe
antibodiescheckedbyWesternblotanalysisofacrude
extractfromG.hederacealeaves.Thisfinalantibody
preparationwasdividedinsmalportionsandstoredat
-20℃untiluse.
1.7 Doubleimmunodifusionassay
Doubleimmunodifusionassaysweredonein
smalpetridishes(5cmindiameter)filedwith8ml
PBScontaining1%agarose,4%(w/v)polyethylene
glycol(Mr6000)and0.1mol/Lgalactose(toprevent
aspecificbindingofthelectin).Sixwels(4mmin
diameter)suroundingonecentralwelweremadeinto
thesolidifiedgelandfiledwith10μlsamplesof
proteinsorantibodies.Plateswereincubatedat37℃
for15h.Theformationofprecipitinlineswaschecked
visualy.Alternatively,theplateswerewashedwith
0.2mol/LNaClfor24htoelutetheunprecipitated
proteins,fixedinamixtureofwater/methanol/acetic
acid(5∶5∶1,v∶v∶v)andtheremainingprecipitin
linesstainedwith0.05% CoomassieBriliantblue
R250inthesamemixture.
1.8 Westernblotanalysis
Crudeextractswerepreparedbyhomogenizing
G.hederacealeavesin4volumes(w/v)of20mmol/L
unbufered1,3-diaminopropane(pH11)pergoffresh
weight. Homogenateswerecentrifuged(10min,
12000g)andthesupernatantsusedimmediatelyor
frozenat-20℃ untiluse. Extractsandpurified
proteinswerereducedwith2-mercaptoethanoland
separatedbySDS-PAGEin15% (w/v)acrylamide
gels[6].Afterelectrophoresis,gelswereelectrobloted
onanImmobilonPmembrane(Milipore,Bedford
MA,USA)usingasemidryblotingsystemfrom
BioRAD (Hercules, CA). Tocheckthebloting
eficiency,thegelswerefixedandtheremaining
proteinsstainedwithCoomassiebriliantblue.Priorto
theimmunodetectionthefreebindingsitesonthe
membranewereblockedwith5%(w/v)bovineserum
albumininTSB(10mmol/LTris,150mmol/LNaCl,
0.1% TritonX-100, pH 7.6) for1hatroom
temperature.Then,themembranewaswashedwith
TSBfor5minandconsecutivelytreatedwithprimary
antibody(overnightincubationatroomtemperature),
goat-anti-rabbitantibody (1hincubationatroom
temperature)andperoxidase-anti-peroxidase-complex
(1hincubationatroom temperature).Aftereach
treatmentthemembranewaswashed3timeswithTSB
for5min. Justbeforetheimmunodetectionthe
membranewaswashedfor5minwith0.1mol/L
Tris-HCl(pH7.6).Theperoxidasereactionwascaried
outinafreshsolutionof0.1mol/LTris-HCl(pH7.6)
containing0.7mmol/L3,3!-diaminobenzidinetetra-
hydrochlorideand0.01%(v/v)H2O2.Thereactionwas
stoppedbywashingthemembraneindistiledwater.
1115· ·
生物化学与生物物理进展 Prog.Biochem.Biophys. 2006;33(11)
Fig.1 Doubleimmunodifusionassaysofdiferentantibodypreparations
Afterformationofprecipitinlines,solubleproteinswereelutedbyextensivewashingofthegelswith0.2mol/L
NaCl.Afterwardsthegelwasstainedwith0.05%CoomassieBriliantblueR250.(a)Determinationofthetiterofthe
crudeantiserum.10μlofa1g/LsolutionofGlehedawasappliedinthecentralwel.Peripheralwelswerefiled
with10μlcrudeantiserum(wel1)and2-,4-,8-,16-and32-folddilutedantiserum(wels2,3,4,5and6,
respectively).(b)Reactionofthecrudeantiserum(centralwel)withpureGleheda(welL),crudeextractsfroma
lectin-positive(wel+)andalectin-negative(wel-)plant.(c)Analysisofthereactivityofdiferentantibody
fractionsobtainedinthediferentstepsofthepurificationprocedure.Glehedawasappliedinthecentralwel.The
flow-throughandboundfractionsfromtheafinitychromatographyonimmobilizedGlehedawereloadedinthe
wels1and2,respectively.Flow-throughandboundfractionsfromtheafinitychromatographyonimmobilized
RPAwereappliedintowels3and4,respectively.Wels5and6wereloadedwiththefractionselutedwith
0.1mol/LNaCland0.5mol/LNaCl,respectively,fromtheQFastFlowcolumn.
1
2
34
5
6
(a) (b) (c)
1 2
3
45
6
-
-
+
+
L
L
Fig.2 Analysisofthespecificityofdiferentpreparationsofanti-GlehedaantibodiesbyWestern
blotanalysis
Lanes1,2and3wereloadedwithpureGleheda(0.1μg),and10μlcrudeextractfromleavesofalectin-positiveanda
lectin-negativeplant,respectively.Alsampleswerereducedwith2%ofβ-mercaptoethanol.Antibodyfractionswere
usedinadilutionof1∶500.Thefolowingantibodyfractionswereused:(a)Crudeantiserum.(b)Fractionboundon
Gleheda-Sepharose4B.(c)Flow-throughoftheafinitychromatographyonRPA-Sepharose4B.(d)Fractionelutedwith
0.1mol/LNaClfromtheQFastFlowcolumn.(e)Fractionelutedwith0.5mol/LNaClfromtheQFastFlowcolumn.
Numbersontherightsideofpicture(e)showthesizeofproteinmarker.
(a) (b) (c) (d) (e)
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
2 Resultsanddiscussion
Onthedayofthefourthinjectionoftherabbit
withGleheda,afewdropsofbloodwerecolectedand
thepresenceofanti-Glehedaantibodiescheckedbya
double immunodifusion assay against purified
Gleheda.Sincethetiteroftheantiserum already
exceeded1∶8(Figure1a),therabbitwasbled10days
afterthefourthinjectionandthecrudeantiserum
prepared.
A preliminary double immunodifusion test
demonstratedthattheglobulinfractionobtainedby
ammonium sulfate precipitation yielded clear
precipitinlineswithpureGlehedaandacrudeextract
fromaplantwithahighleveloflectinexpressionbut
was unreactive towards an extract from a
lectin-negativeplant(Figure1b),indicatingthatthe
antiserum containsantibodiesthatwerespecificaly
directedagainstnativeGleheda.Totestthepossible
cross-reactivitywithotherunrelatedproteins, a
Westernblotcontainingthepurifiedantigen(Gleheda)
andcrudeleafextractsfromalectin-positiveanda
lectin-negativeplantwasdevelopedwiththesame
globulinfraction.AsshowninFigure2a,theantiserum
reactednotonlywiththeGlehedapolypeptidesbut
alsowithseveralotherproteins.Accordingtothe
60
28/26
ku
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王卫芳等：抗欧亚活血丹凝集素特异性多克隆抗体的制备2006;33(11)
paternobtainedwiththelectin-negativeextract,the
strongestnonspecificreactionoccurswithaproteinof
approximately60kuoftheextract(Figure2a).
Therefore,amethodhadtobedevelopedtoisolatethe
monospecificantibodiesfrom thecrudeglobulin
fraction.
Inthefirstatempttoincreasethespecificity,the
antibodiesthatreactedwithGlehedawereselectively
isolated from the globulin fraction by afinity
chromatography on immobilized Gleheda. This
approach yielded a relatively large quantity of
anti-Glehedaantibodies(Figure3a),whichreacted
verywelwithGlehedainadoubleimmunodifusion
assay(Figure1c, wel2). Howeverwhenthis
afinity-purifiedfractionwasusedinaWesternblot
analysis,itstilexhibitedanextensivecross-reactivity
withmanyunrelatedpolypeptidesotherthanthelectin
subunits(Figure2b).SimilartotheWesternblot
paternobtainedwiththecrudeantiserum(Figure2a),
theWesternblotpaternwiththeafinity-purified
antibody fraction (Figure 2b) clearly showed
cross-reactivity especialy with apolypeptideof
approximately60kuincrudeextractfromleavesofa
lectin-positiveandalectin-negativeplant.Itappears,
therefore,thatafinitychromatographyonimmobilized
Glehedayieldsafractionthatishighlyrichin
anti-Glehedabutalsoinantibodiesthatcross-react
with unrelated proteins and especialy with a
polypeptideofapproximately60ku.
Themostplausibleexplanationforthestrong
cross-reactivity isthe presence ofa subsetof
antibodiesthatisnotdirectedagainstthepolypeptide
chainsbutagainsttheN-glycanstructuresofthe
G.hederacealectin.Itiswel-known,indeed,that
serologicalcross-reactivitybetweenunrelatedplant
proteinsoftenreliesonthepresenceofstructuraly
identicalN-glycans. Manyplantproteinscontain
N-glycanswithaxyloseresidueβ-1,2linkedtothe
coremannoseresidueandafucoseresidueα-1,3
linkedtotheasparagine-linkedGlcNAc-residue.These
structuresarecommoninplantN-glycansbutdonot
occurinN-glycansfrommammalianorigin,andhence
areverypotentantigens[7,8].Asdescribedinthepaper
ofWangetalin2003[1],the26and28kusubunitsof
GlehedacontainoneandtwoN-glycans,respectively.
ThoughthestructureoftheseN-glycansonGlehedais
notknown,onecanreasonablyexpectontheanalogy
oftheN-glycanspresentonthesubunitsoftherelated
lectinsfrom theLamiaceaespeciesClerodendron
trichotomum[9] andSalviasclarea[10] thatGleheda
containsβ-1,2linkedxylose,andα-1,3linkedfucose
residues.Accordingly,thereisareasonablechance
thatthestrongcross-reactivityoftheafinity-purified
anti-Glehedaantibodyfractionisduetothepresence
ofantibodiesthatarespecificalydirectedagainstthe
N-glycans.Ifso,thereisapossibilitytoremovethe
cross-reactingantibodiesbyafinitychromatography
onanimmobilizedplantglycoproteinashasbeen
demonstratedforthepreparationofmonospecific
antibodiesagainstaglycosylatedtype2ribosome-
inactivatingproteinfromelderbery(Sambucusnigra)
bark[11].Inthisparticularcase,theantiserum was
depletedfromN-glycanbindingantibodiesbyspecific
retention on acolumn ofimmobilized Robinia
pseudoacacia agglutinin (RPA), which is a
glycosylatedlectinbelongingtothefamilyoflegume
lectins[6].
SinceRPAdoesnotbindrabbitimmunoglobulins
through protein-carbohydrate-interactions, the
technique developed forthe elderbery type 2
ribosome-inactivatingproteincouldinprinciplealso
be applied forthe purification ofanti-Gleheda
antibodiesforremovalofthoseantibodiesthat
cross-reactwithN-glycans. Inaddition, afinity
chromatographyonimmobilizedRPA couldalso
increasethespecificityoftheantiserumbyselective
removaloftheantibodieswiththehighestafinityfor
thepolypeptidechainofthedistantlyrelatedlegume
lectin (thuseliminating antibodiesreacting with
epitopescommontolegumelectins).Therefore,the
antibodyfractionisolatedbyafinitychromatography
onimmobilizedGlehedawasloadedonacolumnof
immobilizedRPA.AsshowninFigure3b,mostofthe
proteinswerenotretainedindicatingthattheydidnot
reactwiththeN-glycansoftheimmobilizedRPA.
Onlyasmalfractionboundtothecolumnandcould
beelutedbyincreasingthepH.
Doubleimmunodifusiontestsrevealedthatonly
theantibodiespresentintheflowthroughreactedwith
purifiedGleheda(Figure1c,wel3).However,a
subsequentspecificityassaybyWesternblotanalysis
demonstratedthatdespiteanobviousincreasein
specificity(ascomparedtothatoftheafinity-purified
antibodyfractionobtainedafterGleheda-Sepharose
4B)theflowthroughfractionstilexhibitedastrong
cross-reactivityespecialywitha60kupolypeptidein
thecrudeextractsofleaves(Figure2c).Obviously,the
depletion of cross-reacting glycan-recognizing
1117· ·
生物化学与生物物理进展 Prog.Biochem.Biophys. 2006;33(11)
Fig.3 Overviewofthechromatographystepsincludedinthepurification
protocolofmonospecificanti-Glehedaantibodies
(a)AfinitychromatographyonGleheda-Sepharose4B.(b)Afinitychromatographyon
RPA-Sepharose4B.(c)Anion-exchangechromatographyonQFastFlow.
antibodiesonanRPA-afinitycolumnincreasesthe
specificityoftheantibodypreparationbutdoesnot
yieldmonospecificanti-Glehedaantibodies.
In a finalatemptto obtain monospecific
antibodies,thepreparationobtainedbysuccessive
afinitychromatographyonimmobilizedGlehedaand
immobilized RPA was further purified by
ion-exchangechromatographyonacolumnofQFast
Flow.AfterbindingoftheantibodiesinTrisbuferthe
IgGfractionwaselutedwith0.1mol/LNaClandthe
remainingproteinswith0.5mol/LNaCl.Boththe
largeIgGfractioninthe1stpeakandthesmalfraction
inthe2ndpeak(Figure3c)gaveapositivereaction
upondoubleimmunodifusionagainstGleheda(Figure
1c,wel5and6).However,Westernblotanalysis
revealedthattheIgGpresentinthefirstpeakreacted
exclusivelywiththeGlehedapolypeptides(Figure2d)
whereastheantibodiespresentinthesecondpeak
exhibitedastrongcross-reactivityespecialywiththe
60kuproteininthecrudeextractofleaves(Figure2e).
3 Conclusion
Ourobjectivewastodevelopahighlyspecific
polyclonalantibodyagainstGlehedaforapplicationin
immunologicalstudies. Usingacombinationof
ammonium sulfateprecipitation, doubleafinity
chromatographyandaion-exchangechromatography,
apreparationofmonospecificpolyclonalantibodies
thatreacted exclusivelywith Glehedacould be
preparedfrom acrudeantiserum.Sincethefinal
preparationexhibitednocross-reactivitytowardsany
unrelatedproteinsinacrudeextractitisalsosuitable
forimmunodetectionandimmunolocalizationstudies.
Thepresentantibodypurificationprotocolisnotonly
simple,butisalsospecificandcostefective,doesnot
require expensive facilities. Hence itmay be
applicabletothepreparationmonospecificpolyclonal
antibodiesagainstotherglycosylatedplantproteins.
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1118· ·
王卫芳等：抗欧亚活血丹凝集素特异性多克隆抗体的制备2006;33(11)
*比利时Flanders科学研究资助项目(G.0113.01),广东省自然科学基资助项目(04010899),教育部留学归国人员科学基金资助项目(2004-6),韶
关市科技计划资助项目(2004-02),中山大学生物防治国家重点实验室资助项目(2004-2006)和中国博士后科学基金资助项目(2005-2006).
**通讯联系人.Tel:0755-26036381,Fax:0755-26036740,E-mail:wfwang1@yahoo.com.cn
收稿日期：2006-06-17，接受日期：2006-07-28
抗欧亚活血丹凝集素特异性多克隆抗体的制备 *
王卫芳 1,3)**VANDAMMEEls2)何朝族 1)
(1)清华大学深圳研究生院生命学部，深圳 518055;
2)DepartmentofMolecularBiotechnology,FacultyofBioscienceEngineering,GhentUniversity,Gent9000,Belgium;
3)韶关学院英东生物工程学院生物工程系，韶关512005)
摘要 欧亚活血丹外源凝集素(Gleheda)是分离自欧亚活血丹(Glechomahederacea)叶片中的一种糖基化植物新蛋白.如同其他
糖基化蛋白，通过免疫学方法探测Gleheda的过程中通常受到一些不相干糖蛋白的妨碍，为此制定了抗Gleheda特异性多克
隆抗体的纯化方案.免疫血清蛋白经硫酸铵选择性沉淀后，分别以Gleheda和刺槐外源凝集蛋白(RPA)结合在Sepharose4B
作为亲和配体，采用亲和层析法连续纯化2次，然后进一步采用离子交换层析QFastFlow提纯.经每一步骤提纯得到的抗体
组分对Gleheda的特异性，均同时采用双向免疫扩散检验和Westernblot分析.结果表明，以Gleheda为配体，亲和纯化制备
得到的抗体组分对叶片粗提物中的许多植物(糖)蛋白仍然表现交叉反应.为除去由植物糖蛋白中的聚糖所引起这些非特异性
交叉反应抗体，接着以RPA为配体再次进行亲和纯化，Westernblot分析显示，抗体的特异性得到提高但并非除去了所有非
特异性交叉反应的抗体.最后进一步采用离子交换层析制备得到仅抗Gleheda蛋白的特异性抗体组分，此抗体组分适用于免
疫探测研究.该抗体纯化制备程序简易而高效，而且不需要昂贵的设备.
关键词 特异性多克隆抗体，抗体制备，欧亚活血丹外源凝集素(Gleheda)
学科分类号 Q5-3
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