冠状病毒Coronavirusl论文-2001 [Advances in Experimental Medicine and Biology] The Nidoviruses Volume 494 __ Cloning Of A Transmissible Gastroente.pdf

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1、CloningOfATransmissibleGastroenteritisCoronavirusFull-LengtheDNAJOSEM.GONZALEZ,FERNANDOALMAZAN,ZOLTANPENZES,ENRIQUECALYO,ANDLUISENJUANESDepartmentofMolecularandCellBiology.CentroNacionaldeBiotecnologia.CS/C.CampusUniversidadAut6noma.Cantoblanco.28049Madrid.Spain1.INTRODUCTIONTounders

2、tandgenefunctionandexpressionincoronavirusesitwouldbeofinteresttoobtainacDNAencodingafull-lengthinfectiousRNA.Thishasnotyetbeenpossibleduetothelargesizeofthecoronavirusgenomeandtheinstabilityofplasmidscarryingcoronavirusreplicasesequences.Inthisreportwedescribetheconstructionofatrans

3、missiblegastroenteritisvirus(TGEY)full-lengthcDNA.Forthispurpose,westartedfromacDNAencodingthedefectiveRNADI-C,thatwasstablyandefficientlyreplicatedbythehelpervirus(IzetaetaI.,1999).DuringthecompletionofthecDNAanORFlafragmentthatwastoxictothebacteriawasidentified.Advantageofthisfindi

4、ngwastakenbyreintroducingthetoxicfragmentintotheviralcDNAinthelastcloningstep.Toenhancethestabilityoftheviralsequence,thecDNAwasclonedasabacterialartificialchromosome(BAC),asystemthathasbeenusefultostablyclonelargeDNAfromavarietyofcomplexgenomicsourcesintobacteria.Thecytomegalovirus(

5、CMV)immediate-earlypromoterwasplacedupstreamofthecDNAtomakeuseofatwo-stepamplificationsystemthatcouplesRNApolII-driventranscriptioninthenucleuswiththeamplificationbytheviralreplicaseinthecytoplasm.TheNidoviruses(CoronavirusesandArteriviruses).EditedbyEhudLavietal.,KluwerAcademic/Plen

6、umPublishers,200I.533534Cloninf?ofaTGEVfull-lenf?thcDNA2.MATERIALSANDMETHODS2.1Cells,Viruses,PlasmidsandBacteriaStrainsTheTGEVstrainsPUR46-MADandPUR46-CIIweregrownandtiteredasdescribed(Sanchezetal.,1999).PlasmidpACNR1180waskindlyprovidedbyJ.D.Tratschin(InstituteofVirologyandImmunopro

7、phylaxis,Mittelhaiisern,Switzerland).PlasmidpBeloBAC11waskindlyprovidedbyH.ShizuyaandM.Simon(CaliforniaInstituteofTechnology,Pasadena,CA).PlasmidpRL-CMVwasobtainedfromPromega.E.coliDHIOBstrainwasobtainedfromGIBCO/BRL.2.2PlasmidDNAPreparation,SequenceAnalysisandPlasmidStabilityTheprep

8、arationofpla