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环形泰勒虫PCNA基因的原核表达及间接ELISA方法的建立.docx

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环形泰勒虫PCNA基因的原核表达及间接ELISA方法的建立 Title:EstablishmentofProkaryoticExpressionandIndirectELISAMethodforCircumferentialTaylor'sWormPCNAGene Abstract: TheaimofthisstudywastoestablishaprokaryoticexpressionsystemforthecircumferentialTaylor'swormPCNAgeneanddevelopanindirectenzyme-linkedimmunosorbentassay(ELISA)methodtoquantifyitsexpressionlevel.TheprokaryoticexpressionsystemwasconstructedbycloningthePCNAgeneintoasuitableexpressionvector,followedbytransformationintoEscherichiacolicells.TherecombinantproteinwasthenpurifiedandanalyzedforitsantigenicitybyWesternblotting.TheindirectELISAmethodwasdevelopedusingthepurifiedrecombinantproteinasthecoatingantigen,followedbydetectingthePCNA-specificantibodiesintheserumsamples.Thesensitivity,specificity,andreproducibilityoftheindirectELISAmethodwereevaluated.OurresultsdemonstratedsuccessfulprokaryoticexpressionofthecircumferentialTaylor'swormPCNAgeneandtheestablishmentofanindirectELISAmethodforitsdetection. Introduction: Proliferatingcellnuclearantigen(PCNA)isahighlyconservedproteinthatplaysacriticalroleinDNAreplicationandrepair.ItactsasaprocessivityfactorforDNApolymeraseandisinvolvedinvariouscellularprocesses,includingcellcycleregulationandDNAdamageresponse.PCNAhasbeenextensivelystudiedinvariousorganisms,includingbacteria,yeast,plants,andanimals.However,thestudyofPCNAincircumferentialTaylor'swormisrelativelylimited.EstablishingaprokaryoticexpressionsystemforthePCNAgeneanddevelopinganindirectELISAmethodcanfacilitatefurtherresearchonthefunctionandregulationofPCNAincircumferentialTaylor'sworm. Methods: Prokaryoticexpressionsystemconstruction:ThePCNAgenewasamplifiedfromcircumferentialTaylor'swormgenomicDNAbypolymerasechainreaction(PCR)usingspecificprimers.ThePCRproductwasthenclonedintoasuitableexpressionvectorusingrestrictionenzymedigestionandligation.TherecombinantplasmidwastransformedintocompetentEscherichiacolicellsandculturedonselectiveagarplates.PositivecolonieswereidentifiedbycolonyPCRandconfirmedbysequencing. Recombinantproteinexpressi