凡納濱對蝦組織蛋白酶C基因的克隆、表達(dá)與定量分析.pdf

1、廣東海洋大學(xué)碩士學(xué)位論文凡納濱對蝦組織蛋白酶C基因的克隆、表達(dá)與定量分析姓名：林詩天申請學(xué)位級別：碩士專業(yè)：水產(chǎn)養(yǎng)殖指導(dǎo)教師：吳灶和簡紀(jì)常20090501MolecularcloningandexpressionofcathepsinCin■Lttopenaeusvanna，咒PZAbstractCathepsinC(dipeptidyl—peptidaseI，DPPI)isawidelyexpressedinalmostalllife

2、formsItisalysosomalcysteineproteinasebelongingtothepapain刪amily、析tllthecapacityofremovingdipeptidessoqu訓(xùn)allyfromtheaminoterminusofpeptideandproteinsubstratesThisellzymehasbeenpostulatedtoinvolvincellgrowthandtheactiVatio

3、nofseveralgranuleassociatedse血eproteases，suchas鱸咖珊鶴AandB，cathspsinG，neutrophilelastaseandchymasesSomehumandiseasesarecausedbythemutationandinactivationofcathepsinCBeacauseofthespecialityofcathepsinC，furtherstIldi圓oninter

4、actionbetwealcathspsinCandimmunesystemofshrimpwouldgreatlyimprovetheshrimp’Sabni哆ofdefendingpathogenandsupporttheoreticalprooftoshrimp’SimrrlunomechanismThecDNAencod啦cathepsincfromthehaemocytesofLitopenaeusvannamei(desig

5、natedLVcathepsinC)WasclonedbyrapidamplicationofcDNAends(PACE)techniquesThefulllengthofLVcathepsinccDNAWas2026bp，andallopenreadingflame(OItF)of1356bpencodingapolypepetideof451aminoacidsresidues麗matheoreticalmolecularweigh

6、tof5076kDaandanestimatedisoelectricp0缸o(hù)f601Theid∞fityofLVcathcpsincsharedllighsimilarity誦t11pena饑珞mondonandMarsupenaeusjaponicasbyBLASTanalysisAimtosetupahighlevelprokaryoticexpressionofcathepsinCofLitopenaeusvannameiinE

7、coliBL21(DE3)，thecathepsinCgoneofLitopenaeusvannameiWasclonedintoprokaryoticexpressionvectorpET32TherecombinationvectorpET32CatC，wastransformedintoEcoliBL21(DE3)andinduced5htoexpressby口TG(1mmol／L)TheproteinproductWasiden

8、tifiedbySDSPAGETheresultshowedthatwe期烈x宅edininducingthecathepsinCproteinFluorescentquantitativerealtimePCRWasusedtoexaminetheexpressionofLVcathepsincgeneinhaemocytesofLitopenaeusvannameiafterthechallengewithbacteriaVibri

9、oalginolyticusThereWasacleartimedspcndentexpressionpatternofLVcathepsincafterbacterialchallenge，andthemRNAexpressionreachedamaximumlevelat4hpostchallenge，andthenreturnedtocontrollevelafter8hTheup—regulatedmRNAexpressiono