33 4 2017 6 CHINESEJOURNALOF VIROLOGY Vol.33 No.4 June 2017 M2e CRM197 1 1 2 1, 2 1,2 1,2 1,2 * 1,2, (1., 361102; 2., 361102) :, M2 (M2e),, (CRM197) CRM197,M2eCRM197(aa1~535) CRM197-N190 (aa1~190) CRM197-N389(aa1~389)C N M2eCRM197 ; (CRM197-N190)-M2eM2e-(CRM197-N190) M2e ; M2e CRM197,M2e CRM197N C ; GST,CRM197-N190 M2e, : ;M2e ;CRM197; ; :R373.1 :A :1000-8721(2017)04-0483-11 DOI:10.13242/j.cnki.bingduxuebao.003171,,WHO, WHO,,, 300 500,25 50, [1],, RNA [2] 3, (HA) (NA) (M), (NP), PB1 PB2 :2017-03-10; :2017-05-19 : ( :31670934), : : (1992-),,, Tel:0592-2880625,E-mail:liqiong0504@126. com * :(1976-) Tel: 0592-2880620,E-mail:shaowei@xmu.edu.cn 3 PA NS1 NS2,HA NA, 3 [3] -2(M2) M2e,,, 94%, [4] M2, 97 3, [5,6] M2,, ph [7] M2 O19 M2, M2
484 33 M2 (M2e)23, M2e CRM197 CRM197-N389 CRM197-N190 N C SDS-PAGE A,M2e, (AUC),, M2e-(CRM197-N190)(CRM197- [8],M2e,, N190)-M2e, M2e,, (HBc) M2e (O19 L18 S1 5D1) HSP70C (HSP70c),M2e CRM197, (OMPC) (KLH) CRM197-N389 CRM197-N190 N (tflic), M2e O19 [15] (CTA1-DD) Tol 5(TLR5), Balb/C I (STF2) GST-M2e, NSP4 [9], M2e CRM197,CRM197-N389,CRM197- AdC68Fiber N190, M2e,, H1N1 H5N1 H9N2 [10] (DT) β- 1 CRM197 M2e, β - CRM197 (GenBank: [11] (Tox) 197 AAV70486.1),CRM197-N389 (Cross-reactingmaterial197,CRM197) CRM197-N190,M2e DT,,, (GenBank:BAE47133.1) 535 58.4kD, A B A(aa1~190)N, ATCC(NO53281) C7( β 197) B 9 (aa201~384) DNA, β (aa385~535) [12] CRM197 CRM197-linker,N389-Linker N190-Linker 52 pto-t7 NdeI,, B BamHI pto-t7, pto-t7(crm197-lf) [13], pto-t7(n389-lf) pto-t7(n190-lf)bamhi CRM197 EcoRI M2e-FM2e-R, PHW2000(, [14] M2 ),M2e, M2e, pto-t7(crm197-m2e) pto-t7(n389-m2e)pto-t7(n190-m2e),,, M2e-LF,M2e-LR,CRM197-F, CRM197 (CRM197(aa1~535)) CRM197-R,N389-R N190-R, (CRM197-N389 (aa1 ~ 389) pto-t7(m2e-crm197) pto-t7 CRM197-N190(aa1~190) )M2e (M2e-N389)pTO-T7(M2e-N190) (GGGGSGGGGSGGGGS), Westernblot, CRM197-LF CRM197-LR N389-LR N190-LR, PCR 1
4 : M2e CRM197 485 1 PCR Table1 Theupperandlowerprimersusedtoamplifytheoverlappingfragmentforthefusionprotein PrimerName PrimerSequence(5 to3 ) CRM197-LF CRM197-LR N389-LR N190-LR M2e-F M2e-R M2e-LF M2e-LR CRM197-F CRM197-R N389-R N190-R CATATGGGCGCTGATGATGTTGTTGA GGATCCACCGCCACCGCTGCCACCGCCACCGCTGCCACCGCCACCGCTTTTGATTTCAAA GGATCCACCGCCACCGCTGCCACCGCCACCGCTGCCACCGCCACCAAATGGTTGCGTTTT GGATCCACCGCCACCGCTGCCACCGCCACCGCTGCCACCGCCACCACGATTTCCTGCACA GGATCCATGAGTCTTCTAACCGAGGT GAATTCTTAATCACTTGAACCGTTGCATC CATATGATGAGTCTTCTAACCGAGGT GGATCCACCGCCACCGCTGCCACCGCCACCGCTGCCACCGCCACCATCACTTGAACCGTT Notes:Theshadowfontdenotestherestrictionenzymecutingsite. IPTG 8h,8000g 5min,1 (50mmolTris ph 7.2,300 mmol/l NaCl), GGATCCGGCGCTGATGATGTTGTTGA GAATTCTTAGCTTTTGATTTCAAAAAATA GAATTCTTAAAATGGTTGCGTTTTATGCC GAATTCTTAACGATTTCCTGCACAGGCTT 2 CRM197M2e M2 N 713, ER2566 O19 (Invitrogen), LB M2, M2 L18 S1 LB,37 180rpm M2, OD 600nm 0.6, -β-d- L18 M2 (IPTG) 0.4 mmol/l,o19,l18s1,5d114c10 SonicsVCX750 2 12000rpm4 5min Table2 Themonoclonalantibodyusedinthisresearch Triton-100, 4 mol/l Antibody Epitope Source Reference, 14C10 CRM197 Ourlaboratory thisresearch 5D1 M2 Ourlaboratory thisresearch (PBS) 3 5 (mabs), 14C10 5D1 O19 L18 S1, 2 14C10( CRM197 ) 5D1( M2e ) 100μl, 20μl6 /, protein A (300 mmol/l Tris Cl, ph6.8,60%,1.2%,12%sds,/ PBS mabso19 L18S1 [15] Fu, O19 M2 CRM197 O19 M2(aa1~10) Merck & Co [15] L18 M2(aa1~10) Merck & Co [15] S1 M2(aa7~13) Merck & Co [15] 4 CRM197M2e 100mmol/Lβ- ), 10 min 12%SDS, L18 A/HK/ (NC ) 68, S1 5% 1 TN bufer (150 mmol/l M2 3 NaCl,10mmol/L Tris,pH8.0),NC, O19L18 1h; M2eCRM197 M2 N 10,S1 1h; TNT(50 mmol/l Tris,50 mmol/l
486 33 NaCl,0.05% Tween-20) 3, 10min; GST-M2e 6 ( IgG) 30 Balb/C 1ml min;tnt 3, 10min; 0.5μg 5μg, 5 (NBT)5--4--3- -, ; (BCIP) 0,2 4, 5 4 M2e BeckmanCoulter 8 XL-A, GraphPadPrism(GraphPad,SanDi- An-50Ti An-60Ti ego,ca) OD 280nm 130000rpm, sedfit c(s)c(m) 6 1 1 PBS 1μg/mL, 100μl 96 (,M2e CRM197 ),37 2h;,PBST(PBS+ CRM197-N389 CRM197-N190C N 0.05% Tween-20) 1; 180μl C N,37 2h;,3 4 (200ng/mL 2, ) M2e CRM197, 37 1h;PBST 5; 100μl GGGGSGGGGSGGGGS CRM197- (HRP) (1 M2e,(CRM197-N389)-M2e,(CRM197-N190)- 5000),37 30min;PBST 5; M2e,M2e-CRM197,M2e-(CRM197-N389),M2e- 100μlHRP ( ),37 (CRM197-N190) 15min; 50μl2 mol/l ;, OD 450nm 7 PBS GST-M2e96 ELISA, CRM197, (Linker,L), 80%, 3 CRM197/N389/N190-M2e Table3 FusionproteinCRM197/N389/N190-M2e(schematic) Fusionprotein CRM197(535aa) Linker(15aa) M2 (97aa) CRM197-M2e 1~535 GGGGSGGGGSGGGGS 1~23 (CRM197-N389)-M2e 1~389 GGGGSGGGGSGGGGS 1~23 (CRM197-N190)-M2e 1~190 GGGGSGGGGSGGGGS 1~23 4 M2e-CRM197/N389/N190 Table4 Fusionprotein M2e-CRM197/N389/N190 (schematic) Fusionprotein M2 (97aa) Linker(15aa) CRM197(535aa) M2e-CRM197 1~23 GGGGSGGGGSGGGGS 1~535 M2e-(CRM197-N389) 1~23 GGGGSGGGGSGGGGS 1~389 M2e-(CRM197-N190) 1~23 GGGGSGGGGSGGGGS 1~190 SDS-PAGE Westernblot,1,M2e CRM197 CRM197-N389 CRM197- N190, 62kD 45kD 25kD
4 : M2e CRM197 487 : (GST-M2e) (A) (B), SDS-PAGE Westernblot ; 5D1 14C10 M2e CRM197 ; M Marker; H 10min; N Notes:Thefusionproteinsweresubjectedtonon-reducingSDS-PAGE(figureA)andreducingSDS-PAGE(figureB).GST-M2eserved asacontrolprotein.monoclonalantibody(mab)5d1againstm2eandmab14c10againstcrm197wereusedtodetectthefusionproteins. Thelanesmarked M wereloadedwithamolecularweight-calibrationproteinmarker.thelanesmarked H indicatethatsampleswere heatedinboiledwaterfor10min.thelanesmarked N denotethatthesampleswerenotheated. 1 SDS-PAGEWesternblot Figure1SDS-PAGEandwesternblotingoffusionproteins
488 33 5D1( M2e ) 14C10( 2 CRM197 ), CRM197 M2e (2),,M2e CRM197 N, β- M2e CRM197C,,, CRM197-M2e(CRM197-N389)-M2e,(CRM197-N190)-M2e : ;A C,BN Notes:Maingraphsshowthedistributionofthesedimentationcoeficientoffusionproteins.Insetgraphsindicatethecorresponding molecular-weightprofiles.graftingofthem2epeptideontothec-terminusofcrm197isdisplayedinfigureaandontothen-terminusis displayedinfigureb. 2 Figure2 Sedimentationcoeficientandmolecularweightinsolutionsoffusionproteinsmeasuredby sedimentation-velocityexperimentsusinganalyticalultracentrifugation, 3, CRM197-M2e (CRM197-N389)-M2e M2e-CRM197 M2e- (CRM197-N389) CRM197-M2e (CRM197-N389)-M2e (CRM197-, N190)-M2e O19,, C L18 (CRM197-N190)-M2e M2e-(CRM197-, S1 5D1 14C10 AUC N190),, C, 3, O19, M2e M2e N ELISA 3, C N M2e-
4 : M2e CRM197 489 : O19 L18 S1 5D114C10 ELISA ; M2e :O19 L18 S1 5D1, CRM197 14C10; OD450nm A C,BN Notes:ThereactivityprofileoffusionproteinswithdiferentmAbsagainstM2epeptideandCRM197,respectively.mAb5D1,S1, CRM197 M2e-(CRM197-N389) M2e-(CRM197-, N O19 N190)5 C L18,O19,14C10,and1E6wereusedinELISAs.mAbO19,L18,S1and5D1againstM2e,and14C10againstCRM197.Thehorizontal axisdenotestheconcentrationofthecoatingprotein.theperpendicularaxisdenotestheod450.graftingofthem2epeptideontothec-ter- minusofcrm197isdisplayedinfigureaandontothen-terminusinfigureb. 3 M2e CRM197 ELISA Figure3 ReactivityofthefusionproteinsagainstM2e-specificmAbsandCRM197-specificmAbsbyELISA,M2e CRM197N M2e
490 33 CRM197, 0.5μg M2e M2e, 5μg, N L18O19 C 4 1~2, Balb/C M2e (4) 1 5μg0.5μg M2e- M2e (CRM197-N190) M2e, 56CRM197 CRM197-N190 M2e CRM197 CRM197-N389 5μg, N,CRM197-N190 M2e,10 4 ~ 10 5, C 1~2 M2e, : GST-M2e,5μg0.5μg Balb/C ; GST-M2e96 ; (), Notes:PurifiedfusionproteinswereformulatedwithaluminumadjuvantandtheninoculatedtoBalb/C miceattwodoses(5μgand0.5 μg).gst-m2eservedasthecontrolprotein.theantibodyeliciationwasdeterminedbygst-m2e-coatedelisa.thehorizontalaxisde- notesthetimingofimmunization.theperpendicularaxisdenotesthem2e-specificantibodytiter. 4 Figure4 Reactiontitersofantiserumraisedbyfusionproteins
4 : M2e CRM197 491,M2e [20] Neirynck M2e-HBc,M2eVLP,CRM197, M2e [21] M2e, M2e, M2e,CRM197 M2e (HA2, NP M1), [16] [17] [22] M2e, [18] DynaVax8M2e,CRM197 CpG,VaxInnate4M2e 1924,Landsteiner,,, M2eCRM197 CRM197-N389 CRM197-N190N C,, M2e 2Cys,CRM197 4 CRM197 Cys,CRM197-N389 2 Cys,CRM197- CRM197, N190 1 Cys Westernblot, D, M2e 20 60, (TIV) 65,, M2eCRM197 20 90,C 2009,, O19,N O19 Fu, O19 [19] M2e,M2e M2, N,O19, M2eM2 M2e CRM197 N, M2e N 2~9,M2eCRM197N 99% M2e M2e, M2 10 5 GST-M2e 2,M2e, CRM197 M2e,,,M2e,,, M2e, :, [1]GrohskopfL A,Sokolow LZ,BroderK R,OlsenSJ, M2e KarronR A,JerniganDB,BreseeJS.Preventionand Filete M2econtrolofseasonalinfluenzawithvaccinesrecommenda-, M2e tionsoftheadvisorycommiteeonimmunizationprac- GCN4(Generalcontrolnondepressible4) tices -UnitedStates,2016-17influenzaseason[J].Mm-
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4 : M2e CRM197 493 AntigenicityandImmunogenicityofInfluenzaM2eProteinFusedwith IntramolecularAdjuvantCRM197 LIQiong 1,WANG Kaihang 1,ZHOU Lizhi 2,CHEN Tingting 1,ZHANG Xiao 2, CHEN Yixin 1,2,GU Ying 1,2,XIA Ningshao 1,2,LIShaowei 1,2* (1.NationalInstituteof Diagnosticsand Vaccine DevelopmentinInfectious Diseases, Schoolof LifeSciences,Xiamen University,Xiamen361102,China;2.State Key Laboratoryof Molecular Vaccinology and Molecular Diagnostics,Schoolof Public Health,Xiamen University,Xiamen361102,China) [KH-*2D] Abstract:Newstrategiesareneededurgentlytodevelopbroad-spectrumvaccinesagainsttheinfluenzavirus andtoreducetheriskofpandemics.theconserved M2eproteinisapotentialcandidatefordevelopmentof auniversalvaccineagainstinfluenzaviruses.inthis work,m2e wasfusedtothe N-orC-terminusof CRM197(aa1~535),CRM197-N389(aa1~389)andCRM197-N190(aa1~190)bygeneengineering.Data fromsds-pageandwesternblotingshowedthatthesefusionproteinsformed multimers,andthatthe monomeranddimerbandswerethemostprominent.moreover,aucdatademonstratedthatthetetram- ersofsomefusionproteinswerepresentinsolutionsthatmightreflectthenativeconformationofthem2e protein.thefusionproteinof M2efusedtotheN-terminusofCRM197,CRM197-N389,andCRM197- N190showedexcelentreactivitytothecharacterizedmonoclonalantibodiesO19andL18ascomparedwith M2efusedtotheC-terminusofCRM197,CRM197-N389,andCRM197-N190.Furthermore,theimmuno- genicityofthesefusionproteinswasevaluatedin mice.resultsdemonstratedthattheimmunogenicityof M2ewasenhancedsignificantlywhenbeingfusedtoCRM197-N190thantoGST.Thesefindingsprovide somecluesforthedevelopmentofa M2e-based,broad-spectruminfluenzavaccine. Keywords:Influenzavirus;M2eprotein;CRM197;Antigenicity;Immunogenicity Funding:Thepresent work wassupportedby agrantfrom NSFC (31670934). *Corresponding author:li Shaowei,E-mail:shaowei@xmu.edu.cn