敲除meq的鸡马立克氏病毒强毒株对超强毒的免疫保护作用

ResearchPaperActaMicrobiologicaSinica50(3):380-386;4March2010ISSN0001-6209;CN11-1995/Q:(30671571)3Tel:[phone];Fax:[phone];E2mail:[e-mail]:(1985-),,,,E2mail:sushuaisdau@163com:2009210213;:2009211224meq1,2,1,1,3,2,13(1,271018)(2,100081)(3,100081):meq(MDV)1SPF1505,30,SPF1,152000PFU/GX0101meq,22000PFU/CVI988/Rispens,345d,123500PFU/MDVvvrMd590d,,,,90dGX0101meq5,GX0101meqvvrMd51,CVI988/RispensvvrMd529/42,vvrMd5387%,25%,GX0101meqCVI988/Rispens12vvrMd5100%89%MDVmeq2GX0101meq,SPF,CVI988/RispensMDV:;meq;;;:R392:A:000126209(2010)0320380207(Mareksdisease,MD),(Mareksdiseasevirus,MDV),,,,MDV2,3,,T,MDV3,,MDVCVI988/RispensSB1(herpesvirusofturkey,HVT)FC126[1-2]MDV,()[3-4]MD,,:meq./(2010)50(3),MDV,;,MD,,MDV[5],(Cosmid)[6-9](Bacterialartificialchromosome,BAC)[10-11]MDVvvrMd5,BACmeq2rMd5meqmeqMDV[12],,SPF,rMd5meqMDV[13],BAC,BACMDV[14-16],2001MDV,GX0101[17],GA()Md5()[18],BACMDVGX0101bac2GX0101[19]CVI988/Rispens,,Md5meq,meq2rMd5meq,,,MDVmeq,MDVMDVGX0101BAC,GX0101meq,111111111SPFMDV:9-10SPF,MDVCVI988/RispensrMd5[6](vv)MDV11112:Biosharp,,IgGSigma,Invitrogen,OXOIDTpMD182TTaKaRa,OMEGAQiagenPCRAppliedBiosystemsPCREppendorfBio2Rad112GX0101meqBAC,MDVGX0101BAC,bac2GX0101DNA,,Jarosinski[20],(kanamycinresistancegene,kanr)bac2GX0101meq:,Meq2Kana2F:52AGAAACATGGGGCATAGACGATGTGCTGCTGAGAGTCACAATGCGGATCAGTGTAGGCTGGAGCTGCTTC23,Meq2Kana2R:52CTTGCAGGTGTATACCAGGGAGAAGGCGGGCACGGTACAGGTGTAAAGAGCATTCCGGGGATCCGTCGAC23,5MDVmeq50bp(),320(),,kanr34bpFlp(flprecognitiontarget,FRT)PKD13,PCRMDVmeq50bpmeq:Meq2F:52CATAGACGATGTGCTGCTG23;Meq2R:52GTGCTGGAATGTTAAGAATAAA23,MDVmeq,1020bpmeq1160bp,200bpDpn1hPCR,2000V/100/183ShuaiSuetal./ActaMicrobiologicaSinica(2010)50(3)25FGX01012BACEL250[19],LB,3216h,PCRkanrmeq[10],011%Flp,FLP34bpFRT,,FRT[20]PCRkanrmeq.meqBACDNAPCRCEF[21],meqGX0101meq()113CVI988/RispensMDVCVI988/Rispens(CEF),MD,96,MD,:(3:2)8min,,PBS1,10,96,375%CO245min,PBS3,,FITCIgG(Sigma,PBS)50L,3745min,,PBS3,,50%,MDV114meqGX0101SPFGX0101meq,1SPF1505,30,5SPF112000PFU/GX0101meq,22000PFU/CVI988/Rispens,3,4,52000PFU/GX0101meq5,123500PFU/MDVvvrMd5,,,HE90,,,,5,10%,4,HE,GX0101meqvvrMd5CVI988/RispensvvrMd5,MDMD100(PI)[13],PIMDMDMD100,,2211GX0101meqmeqmeqPCR,meqGX01011160bp,meqGX0101meq200bp,PCR(1),GX0101meqmeq1GX0101GX0101meqmeqPCRFig.1PCRidentificationfornormalanddeletionthemeqoncogeneofGX010111:GX0101;2:GX0101meq;3:DL2000marker4:Negativecontrol.212GX0101meqvvrMd5,vvrMd5,,,,(2)GX0101meqCVI988/RispensvvrMd5,90,GX0101meq,CVI988/Rispens6GX0101meq283:meq./(2010)50(3),,5290,,,CVI988/RispensMDV(),,GX0101meqvvrMd5vvrMd52GX0101meqCVI988/RispensvvrMd5Fig.2MortalitycurvesaftervvMDV2challengeinchickensvaccinatedwithGX0101meqorCVI988/Rispensandthecontrolgroup.A:Themortalitycurveofthefirstexperiment;B:Themortalitycurveoftherepeatexperiment.Eachchickenwasinoculatedwith2000PFUoftheindicatedvirusesat1dayofageandmaintainedinisolationfor13weeks.Non2immunizationgroupservedasthenegativecontrol.Thethreeexperimentalgroupswerechallengedwith500PFUofvvrMd5strainonday5afterimmunization.Themortalitiesofdifferentgroupswererecordedweekly.DeadchickensduringtheexperimentwereevaluatedforMDV2specificgrosslesions.213GX0101meqvvrMd5,,,GX0101meq,GX0101meqvvrMd5(1)GX0101meq,5,,0/30,GX0101meqvvrMd50/30,GX0101meqvvrMd50/30,CVI988/Rispens9/42,,100/1083,,GX0101meq,CVI988/Rispens,,,,,1GX0101meqMDVTable1PreventiveeffectofGX0101meqvaccinationontumorogenesisinducedbyvvMDV.VirusDied/no.testedTumors/no.testedExperiment1rMd525/308/30GX0101meq+rMd50/300/30GX0101meq0/300/30Experiment2rMd527/307/30GX0101meq+rMd50/300/30GX0101meq0/300/30Eachchickenwasinoculatedwith2000PFUoftheindicatedvirusbysubcutaneousrouteandchallengedwith500PFUofvvrMd55dayslater.3vvrMd590(H.E,200)Fig.3Comparisonsofspleenhisto2sectionsfromchickenssacrificed90daysafterchallengewithvvrMd5indifferentgroups(H.E,200).A:VaccinatedwithCVI988/RispensandthenchallengedwithvvrMd5ingroup2;B:VaccinatedwithGX0101meqandthenchallengedwithvvrMd5ingroup1;C.OnlychallengedwithvvrMd5ingroup3;D.Normalcontrolingroup41383ShuaiSuetal./ActaMicrobiologicaSinica(2010)50(3)214GX0101GX0101meq5,8GX0101meqMDV100%,41580114(Log10),6GX010141250129(Log10),,GX0101meq,GX0101215meqGX0101SPFSPFvvrMd5,GX0101meqCVI988/RispensSPF97%(2),GX0101meq,vvrMd5,,,,,,GX0101meqCVI988/RispensvvrMd5SPF11%MD,MD,GX0101meqCVI988/RispensrMd5100%89%2GX0101meqSPFTable2PathogenicityandimmunoprotectiveefficacyofGX0101meqinSPFchickensVaccinesChallengedwithExperiment1Experiment2SummaryMDmortality/%MDlesions/%PIMDmortality/%MDlesions/%PIMDmortality/%MDlesions/%PIaGX0101meqrMd50/30(0)0/30(0)1000/30(0)0/30(0)1000/60(0)0/60(0)100aCVI988/RispensrMd54/30(13)4/30(13)872/30(617)3/30(10)896/60(10)7/60(11)89b-rMd525/30(83)30/30(100)-27/30(90)28/30(93)-52/60(87)58/60(97)---0/30(0)0/30(0)-0/30(0)0/30(0)-0/60(0)0/60(0)-GX0101meq-0/30(0)0/30(0)-0/30(0)0/30(0)-0/60(0)0/60(0)-Note:One2day2oldSPFchickenswerevaccinatedwiththeGX0101meqorCVI988/Rispensvaccineandchallenged5dayslaterwithvvrMd5strain.Mortalitywereobservedfor13weeksafterchickenschallengedwithvvrMd5strain.,andbothd