馬肝醇脫氫酶基因的克隆表達及活性研究.pdf

1、沈陽藥科大學碩士學位論文馬肝醇脫氫酶基因的克隆表達及活性研究姓名：韓云波申請學位級別：碩士專業(yè)：微生物與生化藥學指導教師：張怡軒陳代杰20090501沈陽藥科大學碩士學位論文AbstractAbstractInthisstudytwohorseliveralcoholdehydrogenaseisoenzymegenes(hladh—eandhladhs)wereamplifiedfromhorseliverwithRTPCRmetho

2、dAseriesofnewplasmidspLY105E／S，pLYl07E／S，pLYl08E／S，pLYl09E／S，pLYl10E／SandpLYl15E／Swereconstructedbyligatinghladh—e／sgenesintotheexpressionplasmidvectorpET30apET20b()，pkk223—3andpTrc99aAfterthesenewplasmidsweretransformed

3、toEcoliBL21(DE3)，therecombinantstrainswereincubatedinLBmediumat37℃，andtheninduced嘶nlIPTGatthefinalconcentrationoflmmolfor4hwhentheOD600reached0608Activitiesofbothrecombinanthorseliveralcoholdehydrogenaseisoenzymes(HLADHE

4、，HLADH—S)werestudiedInadditionthetwogeneswereclonedintoPichiapastorisexpressionvectorpPIC9，andinitialexpressionwascarriedoutafterintegratedintothegenomeofthePichiapastorisGSl15ThereWasaonebasedifferenceinamplifiedgeneshl

5、adh—eandhladh—Sfromthatreportedintheliterature，whereastheaminoacidsequencewereidenticalFurthermore，therecombinantexpressionplasmidspLYl05E／S，pLYl06E／S，pLYl07E／S，pLYl08E／S，pLYl09E／S，pLYl10E／SandpLYll5E／Swereconstructedand

6、inducedComparativelytherecombinantproteinHLADH—E／SexpressedinEcoliBL21(DE3)pLY109E／SwereofthebestWhenthestrainswereculturedinLBcultureunder30“CreachingaOD600valueatabout06，high—levelexpressionofHLADH—E／SWasavailablefollo

7、winglmmol／LIPTGinductionfor4hTheamountofHLADHE／Sanditsactiveproteinaccountfor20％ofallsolubleproteinsThesolublepartsofHLADHE／SwerepurifiedusingNickelcolumnTheoptimalreactiontemperature，optimalreactiontime，optimalreactions

8、ubstrateconcentrationandtheconcengationofcoenzymeweredeterminedVitalityofpurifiedenzymereached104103U／mgandtheseenzymesandtheirenzymaticprocessescouldbeappliedinindustrialproductionafterfurtherdevelopmentKeyword：horseliv