冠状病毒Coronavirusl论文-2017 [Methods in Molecular Biology] Reverse Genetics of RNA Viruses Volume 1602 __ Efficient Reverse Genetic Systems for.pdf

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1、Chapter5EfficientReverseGeneticSystemsforRapidGeneticManipulationofEmergentandPreemergentInfectiousCoronavirusesAdamS.Cockrell,AnneBeall,BoydYount,andRalphBaricAbstractEmergentandpreemergentcoronaviruses(CoVs)poseaglobalthreatthatrequiresimmediateintervention.Rapidinterventionnecessitatestheca

2、pacitytogenerate,grow,andgeneticallymanipulateinfectiousCoVsinordertorapidlyevaluatepathogenicmechanisms,hostandtissuepermissibility,andcandidateantiviraltherapeuticefficacy.CoVsencodethelargestviralRNAgenomesatabout28–32,000nucleotidesinlength,andtherebycomplicateefficientengineeringofthegeno

3、me.Deconstructingthegenomeintomanageablefragmentsaffordstheplasticitynecessarytorapidlyintroducetargetedgeneticchangesinparallelandassortmutatedfragmentswhilemaximizinggenomestabilityovertime.Inthisprotocolwedescribeawell-developedreversegeneticplatformstrategyforCoVsthatiscomprisedofpartition

4、ingtheviralgenomeinto5–7independentDNAfragments(dependingontheCoVgenome),eachsubclonedintoaplasmidforincreasedstabilityandeaseofgeneticmanipulationandamplification.CoronavirusgenomesareconvenientlypartitionedbyintroducingtypeIISorIIGrestrictionenzymerecognitionsitesthatconferdirectionalcloning

5、.Sinceeachrestrictionsiteleavesauniqueoverhangbetweenadjoiningfragments,reconstructionofthefull-lengthgenomecanbeachievedthroughastandardDNAligationcomprisedofequalmolarratiosofeachfragment.Usingthismethod,recombinantCoVscanberapidlygeneratedandusedtoinvestigatehostrange,genefunction,pathogene

6、sis,andcandidatetherapeuticsforemergingandpreemergentCoVsbothinvitroandinvivo.KeywordsCoronavirus(CoV),Reversegenetics,Severeacuterespiratorysyndromecoronavirus(SARS-CoV),MiddleEastrespiratorysyndromecoronavirus(MERS-CoV),Emerging,Preemergent,Batcoronavirus,Porcineepidemicdiarrheavirus(PEDV)1I

7、ntroductionHumancoronaviruses(HCoVs)werefirstidentifiedinthe1960s(HCoV-229EandHCoV-OC43)andwereprimarilyassociatedwithmildupperrespiratorytractinfectionswiththepotentialtoprogresstoasevererespiratorydiseaseinyoungchildren,theelderly,and