鳞翅目昆虫细胞系的DNA指纹图谱法鉴定及对SfaMNPV敏感性的测定

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1、华中师范大学博士学位论文鳞翅目昆虫细胞系的DNA指纹图谱法鉴定及对SfaMNPV敏感性的测定姓名：沙丁申请学位级别：博士专业：动物学指导教师：彭建新20090501AbstractCompletecharacterizationofcelllinesisessentialforthecellbanksandfortheregulatoryrequirementsinbiopharmaceuticalproduction．Characterizationofacelllineprovidesinfor

2、mationregardingthelineageorspeciationofthecellline，geneticstabilityofcells，andcross—contaminationofcelllines．Celllinecharacterizationproceduresclassicallyincludeanalysisofdistinguishingmarkersofthecells．Insectcelllinesarebeingwidelyusedinresearchandind

3、ustryforcultivationofinsectvirusesandproductionofrecombinantproteins．Comparedtothemammaliancelllines，theextentofcross—contaminationandmislabelingisreportedhigherincasesofinsectcelllines．TheoverallgoalofthisworkWastheidentificationof17lepidopteraninsect

4、celllinesbyDNAAmplificationfingerprintingandtestsnsceptibilityofinsectcelllinestoinfectionbysyngraphafatcifeILlmultiplenuclearpolyhedrosisviruses(SfaMNPV)．DNAamplificationfmgerprinting(DAF)、析tllarbitrarilyselectedprimersWasusedtoobtainDNAfingerprintpro

5、filestodistinguishamong17lepidopteraninsectcelllines．Thefmgerprintingpatternisastablecharacteristicofthecelllinebecausehighandlowpassagesgeneratedthesameprofile．TheDNAfromeachcelllinewasamplified，andPCRproductswereanalyzedbyagarosegelelectrophoresis．Al

6、lcelllinescouldbedistinguishedfromeachotherwithfollowingexception：BCIRL·-HA·-AMlestablishedfromHeliothisarmigeraproducedthesameprofileasLaphygmaexigua(Le-H-HNU7)．Thiscaseofidenticalpatternsisbelievedtobeduetocrosscontaminationofcultures．Ofthetotal17cel

7、llines，sixcelllineshasbeentestedforsusceptibilitytoSyngraphafatciferamultiplenucleocapsidnucleopolyhedrovirus(SfaMNPV)infectionbyuseofatypicalendpointassayprocedure．CelllinesfromTrichoplusianiBTI—TN5B1-4(TnH5一S)，Spodopterafrugiperda(sf9BCIRLclone)，Spod

8、opteralitura(SL-ZSU一1)，PlutellaxylostellaBCIRL—PX2一HNU3，LymantriadisparIPLB-Ld652，andHelicoverpazea(BCIRL—rlZ-AM1)in96-welltissuecultureplateswereinfected、析mdilutionsofvirussuspensions(SfaMNPV)．EachcelVviruscombinationWasincubatedattemp