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Title
Expression of P30, The Major Surface Antigen of
Toxoplasma gondii in Baculovirus-insect System
and The Evaluation of Immune Response Induced by
P30
Author(s)
Chen, Xiao-Guang, Ma, Xin, Wu, Kun, Peng, HongJuan, Shen, Shu-Man, Liu, Guo-Zhang, Chan, SiuMing
Citation
The Journal of Protozoology Research, 8(1): 1927
Issue Date
URL
Rights
1998-01
http://ir.obihiro.ac.jp/dspace/handle/10322/275
The Research Center for Protozoan Molecular
Immunology
帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
J．Pro10∠UOl．REゝ．、8、1927（1998）
C叩．γrJgん将J997，r力e伽∫ビ〟rぐ力CビJZrPりわrJ’r（フJ〝ヱ（沼〃〟oJβぐ〟／〟r仙川〟〃〝J〃g．V
ExpressioれOfP30′TheMajorSurfa⊂eAn也gemof払叫抽捌相計川繭
inBaculoviruSrinsectSystemandTheEvaluation of
ImmlmeResponseInduced吋P30
XIAO−CUANG CHENl′XTN MAl，KUN WUl′HONC−JUAN PENGl，
SHU−MAN SHENl，GUO−ZHANG LIUIAND SIU−MINC CHAN2
ーD叩〟抽〃川上呼P〟r打SノーけJogy′T／−ビFfr5‖レ仙hryん王ビ血〝JU∫血ビr5巾′C一冊JgZ柚1510575′CJ‖〃〟′
ご伽p〟r仙川f呼ZoロメogyノTノー＝仙加血相叶肋−g長0′′g′伽ゆ血椚鋸南′〃珊gK刑g
Recei、−edlヰM且〉’1998ノA（、C叩l亡d】6June199さ‡
Keい、・nrds＝恥xnp】asma糾川dii，m勾nrsuぬαan【i旦en，bacu】0Virus．exp■℃SSions〉StCm，immun亡▲
ABSTRACT
The m叫OrSurface antigen（P30）ofthe Toxqplasmagondliwasexpressed
by aninsect cellculture systerninfected with recombinant baculoviruS・About
750ygofpurifiedP30（95％purity）wasobtainedfromacultureoflO8insect
Sf21cells．The reconlbinant P30was used toimmunize mice toinduceimmune
response・MicelnJeCtedwiththerecombinantproteinproduced specinchumoral
andceIlularimmuneresponses・ImmuJlizationwithP30alsoprolongedtheperiod
OfsurvivalofmiceinfectedbyToxqpLasmaparasites．
INTRODUCT10N
TbxqpLasmagondiiisanintracellularprotozoanparasite・1tiswidelyknown
tobeinfectiveinhuman，agrlCulturalanddomesticanimals．Itwasestimatedthat
as many as one third of the world population show serological evidence of
infectjon（Hughes1985）．BecauseofthepossibleLonBincubationperiodinsidethe
host，the symptom fortoxoplasmosisis usual］y suppressedand undeteeted・1tis
OneOfthemostcoTTlmOnOPpOrtunisticinfectiousdiseasesfoumdinpatients who
areimmunosuppressed．About 6，10％ of the ATDS patient developed
toxoplasmosisL Ofallthe encephalitis occurredin HIV posltive patients，about
50％is due to theinfection causedby T．gondii．1n chronicinfection，infected
patien［sshowssymptomsofmalaise，fever，SOrethroatandheadache・1npregnant
WOmen andanimalsinfectedwithT．gondiE，it maycause stillbirthand abortion
J夕
EXPRESStON OF P301N BACULOVIRUS SYSTEM
（Qian1993）・Thesurvivinginfantmaysufferfromhydrocephalus，intracerebral
Calcincations，COnVulsions，blindnessandmentalretardation．Becauseofthehigh
infectionrateandtheserioushealththreatcausedbytheTbxqpklSma，thereisan
urgeneyfor theproduction ofadiagnostickitandan effective vaccineforthe
preventionandcurlngOfthedisease．rIbproduceaneffective subunitvaccinefor
theTbxqplasma，SeVeralsurfaceantlgenSOfthe T．gondiihave beenjdentified．
One ofthe surface antlgen，P30，has beenisolated from the tachyzoite and
identinedtohavepotentialuseassubunitvaccine（Bur】etal・198B）・Thisprotein
ishighlyantlgenicandalsopresentinmoststrainsofTbxtplasmaLItcanstimulate
bothce11ularandhumoralimmune response（Partanenet al．1984；Khan etal．
1988），inducingtheproductionofIgG，IgM，IgAandIFN−Y．Monoclonalantibody
（mAb）against P30hasalsobeenreportedto beprotective．Wehave previousIy
expressed P30inbacterialexpression system（Chen etal．1994），POOl−reaCtivity
With anti−P30mAb for this recombinant protein was obtained・Therefore，We
attemptedtoexpresstheproteinin aninsectbacllloviruSeXpreSSion system．The
foJlowingresearch reportsthecloningoftheToxoplasma（ZSIstrain）P30gene
intoabacu10Virusexpressionsyste汀LTheabi）ityofthepurinedrecombinantP30
toinduceimmuneresponsewasfurthertestedbyimmunizlngthemicewiththe
immunostimu］a11ngCOmplexcontainlngtherecombinantprotein．
MATERIALS AND METHODS
P山∫川′d〃JI（JJ叫Jt・J（，rんI／∫けd血
Plasmid containing the P30gene（pBV220−P30）was constructed from a
pBV220plasmidvector（Chenetal．1994）．Thehostfor thep］asmidisaXLrl
bL11ebacterialcell（Strategene，USA）．
r力ぐ〝高、（山川れJん血
BaculoviruS eXpreSSion system was from a BaculoGold Transfection kit
（Pharmingen，USA）．T4 DNAligase was from GIBCO−BRL（USA）and
restrictionenzymeswerefrom Sigma（USA）．0thercommonbi∝hemicalswere
analytica）gradesproducedinChina．
C‘｝那Jr以仁血乃q′血けα〃垂rvgcわrco搾Jβ血7〝gPjOge乃ピ
TheP30genewasrecoveredfromthepBV220−P30withEcoRIandBamHl
digestion andinsertedintoabaculoviruS VeCtOrPAcHCしApreviously cutwith
EcoRlandBglII．Afterconfirmationofinsertion，theDNAconstruetwasused
forthefollowingtransfection．TheP30containedtransfervector（pAcHCL−A−30）
was co−tranSfectedwithbaculovirus DNA toinsectce11．Recombinantbaculovirus
WaSPurified by plaque assayasdescribedin the manuaJofthe transfectionkit
gβ
EXPRESS10■N OF P3OIN BACULOVIRUS SYSTEM
（Pharmingen，USA）．PCRtestwasrurtherusedtoconfirm recombination（Chen
etal．1996）．
P〟r折c（ブJわ〝q／Pj（げmm∼〃∫gCJceJJ
lnsect Sf21cells wereinfected with recombinant pAcHCL−A−3O and
Cu］turedforthreedaysa1270c・Thece11swerecollectedaftercentrifLLgationand
SuSpendedinalysisbuffer・Afterincubationinicefor45min，（hesupernatantwas
passed through a column containlng Ni−NTA agarose・The fusion protejn
COmPOSedofpoly−HistagandP30waspurifiedaccordingtotheinstntctionofthe
kit（Pharmingen，USA）・Theproteinwasthenconcentratedinaspinspeedvacand
portionofwhichwasusedinaSDS−PAGEanalysis，AmAbDE53（providedfrom
Prof・Boothroyd，Stanford University，USA）was usedin the Westem blot
analysis．
J川一り川‥ハ∫・・心・＝石高＝・いJ‥㌧朝J・り川JJ一、い
）mmunostimulating Complex（ISCOM）wasprepared basedon Lovgren’s
method（Lovgren etal・1987）．Briefly，Cholesterol（250mg／ml），Phosphatidyl
（250mg／mL），QuilA（0．I％）and recombinant P30were mixedin PBS and
Centrifugedin alO−50％sucrose gradient at200，000g for18hrs・Thelayer
containing the lSCOM was collected and the concentration of protein was
determined・ExperimentalmicewereimmunizedwithO・1FLg，l・0〟gandlOJLgOf
P30inJSCOMbysubepidermalirtIeCtion・ThecontrolmicewerelqeCtedwith
ISCOMcontainingonlyQuilA・Fora】1groups，aboosteriqectionofthe same
VOlumeoflSCOMcontainlngdifferentamountofP30orcontroIwasperformed
arler（iweeks．
／〃り小り∫‥・・、／叫J、・・・十牒−・、
One week after the boosterlIIJeCtion，blood samples from experiment
mice were taken for measurement of the specific antibodies agalrlSt P30b
ELISA．
CJ王〟JJビ〃ggJ／モ斥cJわ〃
Tachyzoites of the Tbxqplasma virulent s（rain ZSIwerelrUeCted
intraperitonealyataconcentrationof104intoexperimentalmice・Thesurvival
rateofthemicewasrecorded．
∫ん〟緑ど（・止肌叫血
ALlOn−paramelric U−teStWaSuSedtoanalyzethe significantdifferencesin
experimental and control groups.
EXPRESSION OF P301N BACULOVIRUS SYSTEM
RESULTS
Figurelshows the construCtion of the pAcHCL−A−P30・Digestion of
plasmidwithbothEcoRIandBamHIhaveconfirmedthatthecorrectsizeofthe
insert・After co−tranSftction and plaque assay puri茄cation，three different
recombinantviruS WaS Selected．These clones were also shown to contain P30
insertsasdetectedbyPCR．
lEc。Rほ如＝■
P
紛＼T一
FigurelConstructionoftherecombinanttransfervectorpAcHCL−ArP30
Figure2showsthat P30gene expressedin Sf21celleffectively・After
furtherpurificationofP30withaNi−NTAcolumn，P300fover95％purltyWaS
obtained・Thepuritycanbeincreaseto98％bypassingthepur浦edP30through
thecolumnagain・WealsooptlmizedtheproductionofP30usingdifferentMOI
（3−10）toinfectinsectce11．Itwasfbund thataMOlof5producedthehighest
expressionofP30inlOBinsectcells・Over750JLgOfhighlypurifiedP30was
obtained．
M 1 2 3 4 M 粥亜
Figure2 SDS−PAGEana】ysis
on theモXpreSSion products of
円Oin Sf2t cell．bnes l：
NomalSf21ce】l，2：S佗Icell
飽8
185
14．了
∴−
infected by wiJd type ViruS
AcNPV，3：Sf2lcelli血tedby
recombinarltVirusAcNPV−P30，
4：Pu正爪ed recombinant P30，
M：Standard mo［ecular weight
marker．
EXPRESSION OF P3O［N BACULOVIRUS SYSTEM
The same samples were suttIeCted to Western blot analysIS With a mAb
DE52．TheresultsindicatedthattheantibodyrecognizedtheP30proteininboth
thesamplesfromthetransfectedinsectcellsandthepurifiedrecombinantfusion
P30（Fig．3）．
Figure 3 Westem blot analysis
On the expression products of
P30．bnesl：NomalSr2t
CeIl、2：Sf2JceLlinfected by
wildtypeVirusAcNPV，3＝Sf21
ce］LirIfected by recombirlant
virus AcNPV−P30．4：Purified
recombinant P30，M：Standard
mo］ecularweightm且rker・
lmmuneresponsesinexperimentalmicewereexaminedLAntibodytitersfor
P300fmicefromgroupl，2，and3wererangedfroml＝6・400tol：25，600・
The control group showed negative reaction to the antigen. The titers in
experimentalgroupsshoweddose−dependentresponse（Thblel）■Thelymphocyte
stimulationtestshowedthattheSIoftheexperimentalgroupsweresignincantly
higherthanthatofthecontrolgroup（P＜O・01）・Therealsoappearedadose−effect
responseindifferentexperimentalgroups（Fig・4）・
Table］Antlbodyresponseinexperjmentalmice・
Antibodytiter＊
Jrnmu11izeddose
0．り唱
6，400，6，400，6，400，12，800，6，400
1．0／Jg
12，800，12，800，6，400，25，600，25，600
仙0′一g
25，600，】2，SOO，25，600，25，600，25，600
−，−，−，died，一
lO．叫gquilÅ
3，200
Positivecontrol＊＊
Negativecontrol＊＊＊
＊Highestdilutionwithabsorbanceshigherthanthoseofnegativecontro】s・
＊＊Pooledser且fromchrorlicallyinfectedmice・
＊＊＊Pooledserafrom†】Oma】mice．
∴7
EXPRESSlON OF P30 IN BACULOVIRUS SYSTEM
4
Figure4Resultofinvitro
3
2
崇芯Uγ uヱ首tコ∈−︺S
Jymphocytestimula（iontest．
Cotumnsl：10．0ノ瑠OfQuilA
group，2：0・l〝gOfrecombi−
nantP30group，3：1．OメイgOf
recombinantP30gro11p，4：
10．0鵬O一丁ecombiれanlP30
grOuP■
一l
1
2
4
3
Groups
Fjgur巳5Resultofch山k喝e
exPerlm蝕t・l＝10．0〝gOf
QuilAgroup，2：0．1鵬Of
recombi−n且血P30group，3：
Ⅰ・OJ‘gOfre拍mbin油tP30
group，4：10・D〃gOf
recombinantP30group・
1011121ユ14151617181920ヱ1苫22ユ242ち2（】
DAYS APTERINFECTION
TheZSlisavirulentTbxpplα珊aStrain．1nfectionoflO6taehyzoitesvia
abdominali叫eCtionisleぬal，thぢinfectedmice wil】diewithinthree daysafter
lnjeetion．UsinglO4tachyzoitestocha）1engemice，themortalityusuallyoccurs祉
day14qAsshowninFig・5，WhenP30−immunizedmicewerechallengedwi血IO4
tachyzoites，aVerageSllrVivaltimeforthesemicewasextended．Fore克ample，the
micelqeCtedwithlQpgofP30，theaveragesurvivaltimewasproJongedto20．4
days，Moreover，thedifferenceinsurvivaltimebetweentheP30immu11izedmice
andthecoTltrOImiceishighIysign摘cant（p＜0．01）．AJthoughthesurvivaJtimeof
P30iruected mice were extended afterthey were challenged by7b叩払sma
tachyzoites，themicedidnotdevelopfullimmunerespひnSetOtOXOplasmosisas
P30immuniヱation could not completely protect the mice＆ga）nSt Tb岬ぬsmm
2〃
EXPRESS［ON OF P30JN B＾CULOVLRUS SYSTEM
infection．
DISCUSSION
ThemaJOrSurfaceantigen（P30）oftheTbxtplasmahasbeenprovedtohave
goodantlgenicltyandhavepotentialusea5SubunitvaccineagalnSttOXOPlasmosis・
Most previotlS reSearCh usedimmunoafnnity approach topurify P30from the
protozoadirectly．ThequalitywasnotuniformandquantltyObtainedwaslimited・
The recombinant DNA approach to produceJarge amount of P30can provide
sufficient material for the diagnosis and production of subunit vaccine for
7brqplasma．Although we have previous produced bacterialexpressed
recombinantP30，thepurifiedP30hadpoorreactivity・Thiscouldbeduetothe
lackofsufficientproteinprocesslngmeChanismsuchasposトtransla†ionprocesslng
inthebacterialexpressionsystem．Our studyrepresentsthe firstreportonthe
expression ofaTbx（pJasma surfaceantlgenin abacu］oviruS eXpreSSion system．
OurstudyshowsthatP30canbeexpressedeffectivelylninsectce11．Thepurified
P30（95％）can also be recognized by mAb against P30・This baculoviruS
expressedP30ishigh）yantLgenic．Thisapproachcanprovide sufficientma（erial
for the production of a vaccine and a diagnostic kit・The recombinant P30
producedinthisstudyisafusionproteinrTheN−terminalofthefusionproteinis
asmallpeptideconsistLngOf6histidineresidues．ThispeptidecaJlberemovedby
enzymaticcleavage．BeeauseitwouldnotaffecttheantigenicltyOftheP30，ithas
notbeenremovedinthisstudy・
Previous studies showthat there were contrastlng reSultswhen P30was
used toimTnunize mice．These resultsmight attribute to the toxiclty Of the
Freund’sadjuvant．OurstudiesusingLiposome andQuilAasadjuvantshowed
improved result to boostimmunity of experimental animals（Bulow and
Boothroyd1991；Mowatetal，1991），ISCOMisarecentIyrecognizedadjuvant・
Somereportsdescribed thatIACOMcouldenhancetheimmunLty Ofmicewhen
injectedwith P30（Lunden et al．1993；Lunden］995），In our study，We also
Showed that P30inISCOM a］so stimu］ated theimmune response ofmice to
7bxLPla．mainfection・lnthecha］1engestudy，althoughsurvivaJofthe7bxoplasma
infected mice was proIonged，COmPlete mortality eventua11y occurred．This
SuggeS［sthattheiruectionofP30cannotcompletelyprotectthemieefromalethal
Thrqplasmainfection．ZSIstrainisconsideredasoneofthemostviru1entstrains
Ofallthe Throp／asmaparasites．DirectinfectionbyperitoneallnJeCtionishighly
StreSS札11toexperimentalanimals・SinceonlyonedoseoftachyzoiteswaslqeCted，
further studyisneededtooptlmizethedoseandscheduleofimmunization・The
natura）infectiverouteofTbxopLasmaismostlyviamouthbythecystoroocyst・
Therefore，thebestcha11engewaylSOralinfectionwithcystoroocyst．0wingto
gぎ
EXPRESS】ON OF P301N BACULOVIRUS SYSTEM
thedifflcultiesirLCO11ectlngandcountlngOfcystoroocyst，thepresentstudyuses
tachzoitestogetthepreliminarydataofcha］lengeexperiment．
ACXNOWLEDGEMENTS
WethankDr．）．C．Boothroydto supplythemonoclonalantibodyagalnSt
P30（DE52）．This study was supported by the National NaturalScience
Foundation of China（No．39400115）and the NaturalScience Foundation of
Gua一喝Dong，China（No．940292）．
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