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植物表达的siRNA对乙型肝炎病毒表面抗原基因表达沉默的研究.docx

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植物表达的siRNA对乙型肝炎病毒表面抗原基因表达沉默的研究 Title:ResearchontheSilencingEffectofPlant-expressedsiRNAontheExpressionofHepatitisBVirusSurfaceAntigenGene Abstract: HepatitisBvirus(HBV)infectionremainsamajorglobalhealthchallenge,affectingmillionsofpeopleworldwide.ThedevelopmentofeffectivetherapeuticstrategiestocombatHBVisofgreatimportance.RNAinterference(RNAi)isapromisingtechnologythatcanspecificallytargetviralgenes,suppressingtheirexpressionandreplication.Inthisstudy,weinvestigatedthepotentialofplant-expressedsmallinterferingRNAs(siRNAs)tosilencetheexpressionoftheHBVsurfaceantigen(HBsAg)gene,aimingtodevelopanovelstrategyforHBVtherapy. Introduction: HBVisahighlyinfectiousvirusthatprimarilytargetstheliver,leadingtochronicliverinflammation,cirrhosis,andhepatocellularcarcinoma.TheHBsAggeneencodesviralsurfaceproteins,whicharecrucialforvirusinfectionandreplication.CurrentlyavailableantiviraltherapiesforHBVarelimitedandhavepotentialsideeffects.Therefore,alternativeapproachesareurgentlyneeded. siRNA-mediatedgenesilencinghasemergedasapowerfultoolfortargetedgeneregulation.PreviousstudieshavedemonstratedthesuccessfuluseofsiRNAsagainstvariousviralgenes,includingHBV.However,theefficientandsustainabledeliveryofsiRNAintohepatocytesremainsachallenge.Plant-basedexpressionsystemsofferapotentialsolutionastheyaresafe,cost-effective,andcanproducelargeamountsoftherapeuticproteins. Methods: 1.Constructionofplantexpressionvectors:HBsAg-specificsiRNAsweredesignedandclonedintoplantexpressionvectorsunderthecontrolofstrongpromoters. 2.Agrobacterium-mediatedtransformation:TobaccoorArabidopsisplantsweretransformedwiththeplantexpressionvectorsusingAgrobacterium-mediatedtechniques. 3.Confirmationoftransgenicplants:PCR,Southernblotting,andsequencingwereperformedtoconfirmthesuccessfulintegrationofthesiRNAconstructsintotheplantgenome. 4.DetectionofsiRNAexpression:RNAextractionandreversetranscriptionPCR(RT-PCR)wereconductedtodetecttheexpressionofsiRNAsintransgenicplants. 5.HBsAgexpressionanalysis:Westernblottingandim