sharp-2特异的rna干扰腺病毒载体构建

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1、浙江大学医学院硕士论文SileneingofSHARP一2geneusingadenoviralveetor一basedshortharPinRNAinterferingABSTRACTBaekground:RatetilianeerofsPlit一andhairy一relatedProtein一2(SHARP一2，alsoreferredtoastheStra13orDECI)15abasiehelix一100卜helixtranseriPtionalrePressorwhichPlaysimPortantrol

2、esinthetranseriPtionalregulationofcellularProliferation，differentiation，andoneogenesis.OurPreliminarystudieshasshownthatTGF一pcanPromoteSHARP一2exPression，andSHARP一2transeriPtionfaetoreouldPromotetheseeretionofIL一2.besidesinStra13knoekoutmieeINF一了andIL一2exPressi

3、onhasdeereased，thuswesPeeulateifSHARP一2genewillPlayanimPortantroleinorgantransPlantation.InordertoinvestigateSHARP一2genefuntion，wedrewassistancefromRNAinterferingtoindueeSHARP一2genesilencing.RNAinie成renee(RNAi)15inducedbydouble一StrandedRNA(dsRNA)andresultsinge

4、nesilencingthroughsequenee一speeifieeleavedbyDieerinio21一23bP51洲AduPlexeswhlchareseParatedbyamulti一ProteineomPlexcalled班SC，theantisensestrandwhiehremainsboundtotheRISC(RNA一indueedsileneingcomPlex)comPlexwilllocatehomologousmRNAsequeneeswithinthecytoPlasmandindu

5、eeeleavageofm洲A，therebyPreventingitstranslationintoProtein.ForRNAitobee士企etiveandelieitgenesileneingresPonse，thedoublestrandRNAmoleeulesmustbedeliveredintothetargetcell.UsingvectorstoexPresssherthairpinRNAs(shRNAs)thatareProeessedbyDieeriniosiRNAmoleeuleseanal

6、soindueetargetgenesilencing.Anadenoviralveetor一basedaPProaehhasnowbeendeveloPedforRNAi，whieheanachievelong一termsuPPressionofgeneexPressioninmanunalianeells.WehavewontheNSFCfund，andthisresearchasabasisfortheSHA即一2geneinorgantransPlantationresearehtoeonstnjctthe

7、SHA好一2gene一sPecifieRNAinierfereneeadenovirusvectorsystems·Objeetive:ToeonstrUetreeombinantadenovirus(Rad一hSHA即)exPressingshorthairpinRNA(SHA即一2一sllRNA).MethodS:浙江大学医学院硕士论文1.AcouPleofSHARP一2一ShRNAtemPlateDNAsequeneesweredesignedandsynthesized.TheannealedsllRNAt

8、emPlateswereinsertedintoPDC316一EGFP一U6ShuttlePlasmidtoconstrIJcttherecombinatplasmids(pDC316一SHARp一shRNA).2.ThePDC316一SHARP一shRNAShuttlePlasmidsandtheBackbonePlaZmidPBHGloxdelE13er