一株PRRSV的分离及序列分析和XH株ORF5a的原核表达-豆柴文库

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一株PRRSV的分离及序列分析和XH株ORF5a的原核表达
Abstract:
Porcinereproductiveandrespiratorysyndromevirus(PRRSV)isasignificantpathogenofswine,resultingingreateconomiclossannually.Inthisstudy,astrainofPRRSVwasisolatedandanalyzed,andtheORF5ageneoftheXHstrainwasexpressedinprokaryotes.OurfindingsmayprovideusefulinformationforthepreventionandtreatmentofPRRSV.
Introduction:
PRRSVisclassifiedintotwogenotypes,EuropeanandNorthAmerican;eachgenotypeisfurtherclassifiedintodifferentsubtypeclusters.PRRSVcaninfectpigsofallages,resultinginsevererespiratoryandreproductivedisorders.TheORF5aproteinisavitalcomponentofPRRSV.Additionally,becauseofitsconservationamongstrainsanditsinvolvementinvaccineresistance,ORF5aisanattractivetargetforthedevelopmentofPRRSVvaccines.
MaterialsandMethods:
ThesampleofPRRSVwascollectedfromtissuesamplesofinfectedpigsandculturedintheMARC-145cellline.Afterinoculation,theinfectedcellswerecollectedandsubjectedtoreversetranscription-polymerasechainreaction(RT-PCR)analysistodetectthepresenceofPRRSV.ThevirusisolationwasconfirmedbyobservingcytopathiceffectsandviaRT-PCRamplification.ThePRRSVgenomewassequenced,andsequenceassemblyandanalyseswereperformedusingrelevantbioinformaticstools.
ForORF5aexpression,thegenewasamplifiedbyPCR,clonedintotheprokaryoticexpressionvectorpET28a(+),andtransformedintoE.coliBL21(DE3)cells.TherecombinantproteinwasinducedandpurifiedusingNi-NTAaffinitychromatography.Theexpressionoftheproteinwasconfirmedbywesternblotting.
Results:
AstrainofPRRSVwassuccessfullyisolatedfrominfectedtissuesandculturedintheMARC-145cellline.SequenceanalysisofthePRRSVgenomerevealedahighdegreeofsimilaritywithpreviouslyidentifiedPRRSVstrainsinNorthAmerica.TheORF5ageneoftheXHstrainwassuccessfullyamplifiedandclonedintothepET28a(+)vector.Therecombinantproteinwasexpressedandpurified,andtheexpressionwasconfirmedbywesternblotting.
Discussion:
OurstudysuccessfullyisolatedandsequencedastrainofPRRSV,providingvaluableinformationfortheunderstandingandcontrolofPRRSV.Additionally,thesuccessfulexpressi