两种虎纹捕鸟蛛昆虫毒素的分离纯化及生物学活性鉴定

ISSN100727626CN1123870PQChineseJournalofBiochemistryandMolecularBiology2003819(4):488492,3(,410081)2,22.239811024171112.,2,6.22,;,2,2;2,2I.2,2,,Q5,Q965.9PurificationandCharacterizationofHuwentoxin2andHuwentoxin2,TwoNovelInsecticidalNeurotoxinsfromtheChineseBirdSpiderSelenocosmiahuwenaDAIJie,LIANGSong2ping3(CollegeofLifeSciences,HunanNormalUniversity,Changsha410081,China)AbstractHuwentoxin2andHuwentoxin2wereisolatedfromthecrudevenomofthespiderSelenocosmiahuwenabythecombinationofionexchangeandreverse2phaseHPLC.Themassesofthetwotoxinsare3981102and4171112byMALDTI2TOFMS.Thesequencesofthetwotoxinsareverysimilarexceptthatsixresiduesaredifferent.Thebiologicalfunctionsofthetwotoxinsaresimilartoo.Theyparalyzelocust,causedeathinmousecentralnervoussystem(CNS)inhighdosage,butinlowdosagehuwentoxin2cancauseconvulsion2reaction,whilehuwentoxin2cannot.Bothofthemcanblockneuromusculartransmissioninanisolatedmousephrenicnervediaphragmpreparation,andalsoreactcooperativelytotheactivityofhuwentoxin2I.Keywordshuwentoxin2,huwentoxin2,aminoacidsequence,biologicalfunction:2002209211,:2002210224(No.39370152)3:Tel:[phone];Fax:[phone]E2mail:[e-mail],,19769,Received:September11,2002;Accepted:October24,2002SupportedbyNationalNaturalScienceFoundationofChina(No.39370152)3CorrespondingauthorTel:86273128872556;Fax:[phone]E2mail:[e-mail](Selenocosmiahuwena),[1].,,61.HWTX233N2;HWTX237;HWTX233;HWTX235;HWTX2V;HWTX240;SHL2,D2[26].©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[79].,HWTX2[10],(inhibitorcysteineknotmotif)[1113].300,10kD[14].,.2(huwentoxin2,HWTX2)2(huwentoxin2,HWTX2),.111111111[1].,.,..11112(PITC)N2(TFA)AppliedBiosystems.(ACN)..11211211HWTX2HWTX2HWTX2HWTX2.(Waters,20mm100mm)Waters650EAdvancedProteinPurificationSystem,150mg.50mlWaters20102HPLC(YWGC18,10mm250mm)..11212[15]MALDI2TOFMS(BrukerPROFLEXTM),;510Hz;1(ISP1)20100kV;2(ISP1)18185kV.:1l9lCCA(2cyano242hydrox2ycinnamicacid)011%TFA250%,1l,,.11213HWTX2HWTX2NEdmanPE2ABI2491Gas2liquidPhaseSequencer.PTH2HPLC,HWTX2HWTX2.11214112141120l(1822g),48h.20l.1121412,4520l,,48h..1121413(intracerebroventricular,i.c.v)[16].1822g5mm5mm,12mm10l,,48h.21(SHL2),.1121414[17].:1822g,,,223(Tyrodessolution),95%O25%CO2,3ml,3032.012Hz2ms,012Hz012ms.Rm6240B().30min.2211HWTX2HWTX2Fig.1A,HWTX2,,,,HWTX2HWTX2,.(3)2HPLC(Fig.1B),23174minHWTX2,20184min(a)21166min(b)HWTX2HWTX2.HWTX2HWTX2MALTI2TOFMS,HWTX2,3981102(Fig11C)4171112(Fig11D).9844:©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(A)IonexchangeHPLCofthevenomofSelenocosmiahuwena.Column,(CM,Waters,20mm100mm);BufferA,011molPLNaH2PO4;BufferB,011molPLNa2HPO4;BufferC,1molPLNaCl;BufferD,H2O.Gradient:040%CP0250minand40%80%P50260min;A,2%;B,18%;Flowrate:6mlPmin;280nm.(B)RP2HPLCofHWTX2,HWTX2.Column(YWG,C18,10mm250mm);BufferA,011%TFAinH2O;BufferB,011%TFAinacetonitrile.Gradient:5%45%Bin50min;Flowrate,2mlPmin,280nm.(C)MALDI2TOFmassspectraofHWTX2.(D)MALDI2TOFmassspectraofHWTX2212HWTX2Edman35(Table1),6,3986183,6,63.HWTX2Edman36(Table1),6,4177103,63.,HWTX2HNTX2()ESTX[18]venomprotein1(Mexicanredkneetarantula)[19].HWTX2HWTX26,HWTX2HWTX2HNTX27,HWTX2NHWTX21,36(Table1).HWTX2HWTX2HWTX2(Table1),123225426,HWTX2.HWTX2HWTX2,HWTX21415ES,HWTX2DKP,HWX2LOOP,P2.HWTX218P,HWTX2K,HWTX2HWTX2.Table1AlignmentoftheHuwentoxin2withthesequenceofotherspidertoxinsToxinAminoacidsequenceHomologytoHWTX2(%)HWTX22FECSISCEIEKKGES2CKPKKCKGGWKCKFNMCVKV3HWTX22FECSFSCEIEKEGDKPCKKKKCKGGWKCKFNMCVKV8611HWTX2LFECSFSCEIEKEGDKPCKKKKCKGGWKCKFNMCVKV8318HNTX2LFECSVSCEIEKEGNKDCKKKKCKGGWKCKFNMCVKV8318ESTXIFECVFSCDIEKEGKPCKPKGEKKCTGGWKCKIKLCLKI6010Venomprotein1IFECVFSCDIEKEGKPCKPKGEKKCTGGWKCKIKLCLKI601021321311(200g20g)HWTX2HWTX2,.21312(60gPg)HWTX2(n=6)HWTX2(n=6).,5min,,15min,,2d.2.213131822g50g09419©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(n=4)25gHWTX2(n=4)50gHWTX2(n=4)25gHWTX2(n=4)50gSHL2.SHL2,50gHWTX231h,,.25gHWTX248h.HWTX22,.50gHWTX210min,,.,10s.20min4,.25gHWTX220min,,,,20s.3h,.HWTX2HWTX22(CNS).HWTX2,HWTX2,2.HWTX2HNTX2,.4(Table1),HWTX212141518.12KHWTX2E,.21314HWTX2HWTX2Fig12.15molPL21HWTX2HWTX2,21120min(n=3,Fig.2A),HWTX2HWTX2,HWTX23217214min(€xs,n=6,Fig12B),HWTX24810218min(n=6,Fig.2C).HWTX2HWTX2[5].HWTX2HWTX2HWTX2,,216molPLHWTX2113molPLHWTX2113molPLHWTX2113molPLHWTX2Fig.2EffectofHWTX2andHWTX2onneuromusculartransmissioninisolatedmousephrenicnervediaphragmpreparationTwitchresponseofthediaphragmtoelectricalstimulationinthepresenceof(A)15molPLSHL2;(B)15molPLHWTX2;(C)15molPLHWTX2Fig.3CooperationofHWTX2orHWTX2withHWTX2onneuromusculartransmissioninisolatedmousephrenicnervediaphragmpreparationTwitchresponseofthediaphragmtoelectricalstimulationinthepresenceof(A)216molPLHWTX2;(B)113molPLHWTX2and113molPLHWTX2;(C)113molPLHWTX2and113molPLHWTX2113molPLHWTX2,3(Fig13).2510216min(n=6,Fig13A)1810215min(n=6,Fig13B)2010213min(n=6,Fig13C).HWTX2HWTX2HWTX2,2HWTX2.HWTX2HWTX2,(CNS)2.