28040.d1蛋白酶的克隆表達、純化、生物活性測定及抗體的制備

1、華中師范大學碩士學位論文D1蛋白酶的克隆表達、純化、生物活性測定及抗體的制備姓名：李慧申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：祁超李偉國20100501⑨碩士學位論文MASTER’STHESISAbstractmcarboxyterminalprocessingproteaseofDIprotein(CtpA)，whichisencodedbytheCtpAgeneinchloroplasts，isresponsiblef

2、orprocessingofprecursorD1protein(pD1)、ⅣimashortC—terminalextensionCtpAclipsthepDlpeptideextensiontoformthematureDIprotein(mD1)，whichisusedintheassemblyofphotosystemIICOreSotheCtpAplaysanimportantroleinphotodamage—repairc

3、ycleinphotosystemII，andispredictedtobeanexcellenttargetforthediscoveryofageneralbroad—spectrumherbicideTheresearchaimofthisworkiStoconstructtheprokaryoticexpressionvectorofspinachCtpAandexpresstherecombinantproteinbygene

4、engineeringmethodWeamplifiedtheCtpAgenewithstandardPCRmethodfromspinacheDNAandconstructeditintopET23aandpET28avectorstogivetwecombinantexpressionplasmidsRecombinantpET23aCtpAandpET28aCtpAfusionproteinswithHistagwereexpre

5、ssedassolubleproteininEscherichiacoliBL21(DE3)afterinduction、耐tIlO2mmol／Lor01mmol／LIPTGat8“t2for7211Wepurifiedtherecombinantproteins、析tIltheNiNTAa伍nitychromatographyandverifiedtheproteinbySDSPAGEandWesternblottingwitllan

6、tihisantibodyHydrolysisactivityofpET28aCtpAWasassayedbyHPLCmethod、骯thasynthetic24一metoligopeptidecorrespondingtocarboxylterminalofprecursorD1protein，andgaveatotalactivityof110nmol／(nagmin)WeusedthepurifiedCtpAproteinasan

7、tigentoimmunerabbitandBalb／cmicefortheproductionofpolyelonalantibodyandmonoclonalantibodywiththetiterof1：100000byELISArespectivelyandgainedfourhybridomacellsTheresultsobtainedinthispaperprovidedthefeasibilityofhighthroug