28513.恥垢分枝桿菌ercc3基因相互作用蛋白的串聯(lián)親和純化

1、福建農(nóng)林大學(xué)碩士學(xué)位論文恥垢分枝桿菌ercc3基因相互作用蛋白的串聯(lián)親和純化姓名：鄭嘉熙申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：汪世華20100401福建農(nóng)林大學(xué)碩士學(xué)位論文TandemAffinityPurificationofProteinInteractedwithMycobacteriumsmegmatisercc3ABSTRACT：EukaryoticXPB／Ercc3isasubunitofproteincom

2、plexTFIIHwhichparticipateintranscriptioninitiate，promotorclearanceandnucleotideexcisionrepair(NER)ofDNAdamageXPBusesitsATP—dependenthelicaseactivityandssDNAdependentATPaseactivitytoplayaroleintheopeningofDNAdoublestrandi

3、ntranscriptionandremoveofdamagecontainedDNAfragmentinNERXPBalsoparticipatecellcycleregulationandotherregulationpathwayasamolecularsensorandresponsortoalotofcellpathwaysInprokaryoticlifeMycobacteriumwefoundthehomologueofe

4、ukaryoticercc3，butnoothertranscriptionandNERrelativegenewhichinteractandCOworkwitheukaryoticercc3MycobacteriumhasitsprokaryotetranscriptionandNERsystemofitselfSothefunctionofercc3geneinMycobactefiumisstilltoaquestionalis

5、sueByaMycobacterimrecombineersystembaseonphagegene，weinsertedTAPtag(proteinATEVproteasesitecalmodulinbindingprotein)intoCteminalofercc3geneinMsmegmatisgenomeAsaresulttherecombinantMsmegmatisexpressercc3tapgeneasthewildty

6、peercc3gene，sincetheyalebothsimplecopyandtranscriptedbythesamepromotorUsedthemethodofTAP(randomaffinitypurification)，wepurifiedtheErcc3TAPanditsinteractionproteinfromcellextractionAfterSDS—PAGEandsilverstain，thepurificat

7、ionofinteractionproteinwasidentifiedbyLCMSTheLCMSresultrevealedalistofpossibleinteractionproteinwithErcc3，includeDNAdirectedRNApolymerasep’andpsubunit(pDCandrpoB)，andrecombinantproteinrecAByinvitrotestsuchasfarwesternand

8、pulldown，wefoundErcc3interactiondirectlywithRPOCinvitroAndotherexperiencefoundErce3alsointeractwithhypotheticalproteinATPaseandMSMEG_5705Butwedidn’tfinddistinctphenotypeonercc3delectedstrainFromthedataabove，wecandeduceth

9、atercc3(withitsinteractionpartner)mayparticipateintranscriptioninitiation，particularlytranscriptioninsomespecialenvironmentKeywords：MycobacteriumsmegmatisXPB／erce3tandemaffinitypurifictiontranscriptioninitiationproteinin