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家蚕中β-半乳糖苷酶基因的克隆表达.docx

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家蚕中β-半乳糖苷酶基因的克隆表达 Abstract β-Galactosidaseisanessentialenzymethatbreaksdownlactose,asugarfoundinmilk,intoglucoseandgalactose.Inthispaper,weclonedandexpressedtheβ-galactosidasegene(LacZ)fromthedomesticsilkworm(Bombyxmori).TheB.moriLacZgenewasamplifiedbyPCR,clonedintoapET-28a(+)expressionvector,andthentransformedintoE.coliBL21(DE3)cells.TherecombinantproteinwaspurifiedandcharacterizedusingSDS-PAGEandwesternblotting.Thepurifiedproteinexhibitedenzymaticactivitytowardslactose,confirmingthesuccessfulexpressionandpurificationoftheB.moriLacZgene.ThisworkprovidesafoundationforfurtherstudyofthepropertiesandregulationofthisenzymeinB.mori. Introduction Asamemberoftheglycosidehydrolasefamily2(GH2),β-galactosidase(EC3.2.1.23)isanessentialenzymethatcatalyzesthehydrolysisoflactoseintoglucoseandgalactose(1,2).Thisenzymeiswidespreadinnatureandplaysacriticalroleinthedigestionanddairyindustry.β-galactosidaseisalsoimportantinthestudyofcarbohydratemetabolismandgenetics,suchaslactasepersistenceandgalactosemia. Thedomesticsilkworm,Bombyxmori,isaneconomicallyimportantinsectinChina,withalonghistoryofbeingusedforsilkproduction.B.morialsoservesasamodelorganismforfunctionalgenomicsandmolecularbreeding.Theβ-galactosidasegeneinB.morihasnotbeenextensivelystudiedyet,althoughitisexpectedtoplayacrucialroleinlactosemetabolism.Inthisstudy,weclonedandexpressedtheB.moriβ-galactosidasegenetofacilitatefurtherresearchonthisenzymeinthesilkworm. Materialsandmethods Materials Silkwormlarvaewereobtainedfromthelaboratoryandrearedonanartificialdiet.PCRreagentsandthepET-28a(+)expressionvector(Novagen)werepurchasedfromTakaraBio(Shiga,Japan),andE.coliBL21(DE3)cellswerepurchasedfromTiangenBiotech(Beijing,China).Allotherreagentswereanalyticalgradeandpurchasedfromcommercialsources. PCRamplificationandgenecloning TotalRNAwasextractedfromthesilkwormmidgutusingTRIzolreagent(Invitrogen)accordingtothemanufacturer'sinstructions.ThequantityandpurityoftheRNAweredeterminedusingaNanoDropspectrophotometer(ThermoFisherScientific,W