30708.新型dna堿基甲基化修復(fù)酶alkc的表達(dá)、純化、結(jié)晶與功能探討

1、華中農(nóng)業(yè)大學(xué)碩士學(xué)位論文新型DNA堿基甲基化修復(fù)酶AlkC的表達(dá)、純化、結(jié)晶與功能探討姓名：關(guān)文申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：農(nóng)業(yè)昆蟲與害蟲防治指導(dǎo)教師：張國安曹春陽20090601華中農(nóng)業(yè)大學(xué)2009屆碩士學(xué)位論文AbstractDNAbasedamageexistsinalmostalllivingspecies，bothprokaryotesandeukaryotesDNAglycosylasesrecognizespecificles

2、ionbasesinDNA，flippingitoutfiomDNAdoublehelix，hydrolyzingglycosylicbondbetweenthedamagedbaseanddeoxyriboseringFLually’DNAwillberepairedwithendonucleaseⅡI，DNApolymeraseandligaseDNAbasedamagealsowidelyexistsininsects。Ifitc

3、ouldnotberepairedorrepairedunsuccessfullyinsect’SsurvivalwillbethreatenedseriouslyItisadvantageoustodevelopnewpesticidesforinvestigatingthemechanismofbaseexcisionrepairHowevertheinitialmechanismforDNAglycosylasestotrigge

4、rraretargetlesionsintheseaofnomarlbasesisstillunknownAlkCWasdiscoveredasanovd3methyladenineDNAglycosylasefromBacilluscereusin2006，111cmechanismofitsspecificrecognizationhasnotbeenreportedTostudywhatdifferenceinspecificre

5、cogniztionmechanismbetweenAlkCandotherDNAmethylateddamageglycosylases，weinvestigatetheexpressionpurificationandfunctionofAlkC，usingmolecularbiologicaltechniquesandfluorecentassayTheXraychromatographyconditionforAlkCWasop

6、timizedalsointhisthesisThedetailedcontentWaslistedasfollows：1Theconstructionoftherecombinantplasmidsnerecombinantplasmidscontaining6xHistagsuchaspET28aAlkC，pSJ2AlkC，pSJ2AlkC27，pET28aAlkAandpET28aAAGwereconstructed2Theexp

7、ressionandpurificationofAlkC，AlkAandAAGnleexpressionconditionsofthetargetedproteinswereoptimized，whichshowedthetargetproteinscouldoverexpressinsupernantatlowtemperatureThroughaffinitychromatographyandgelfiltrationtheprot

8、einofAlkC，AlkAandAAGwereobtainedA墩C一27alsoexpressedinEeoliBL21(DE3)asinclusionbodies3FunctionalstudiesofAlkC111eenzymesactivityweredetectedatdifferentpHbyusingfluorencentassaywhichrevealedthattheactivitiesofAlkCandAlkAwe

9、realmostindipendentwi廿lpHThisindicatedthatAlkCmightspeciallyrecognizedamagedsite3methyladanineinDNAinasamewaytoAlkA4CrystallizatonscanningofAlkCWescannedalotofcrystallizationconditionsforAlkCTheresultsshowedfleeAlkCWasha