猪Hepcidin基因在毕赤酵母中的表达及活性研究.docx

猪Hepcidin基因在毕赤酵母中的表达及活性研究
猪Hepcidin基因在毕赤酵母中的表达及活性研究
摘要：
猪Hepcidin基因是与铁代谢有关的重要基因，其表达不足或异常会影响身体的铁代谢平衡。本研究利用毕赤酵母作为表达载体，成功构建了pYES2.1/Hepcidin重组质粒，并将其转化到毕赤酵母中进行表达。结果表明，在诱导条件下，Hepcidin蛋白在毕赤酵母中表达并可成功纯化。进一步的实验表明，Hepcidin可以有效地抑制酵母中的铁吸收，说明其具有活性。本研究为深入探究Hepcidin在铁代谢中的作用机制提供了基础。
关键词：猪Hepcidin基因、毕赤酵母、表达、活性
Introduction
Hepcidinisasmallpeptidehormonethatregulatesironmetabolisminthebody.Itismainlyproducedintheliverandplaysanimportantroleinmaintainingironhomeostasisbycontrollingtheabsorption,recycling,andstorageofiron.Hepcidindeficiencyordysfunctioncanleadtovariousiron-relateddisorders,suchashemochromatosis,anemia,andinflammation.Therefore,thestudyofHepcidinhasimportantimplicationsforthepreventionandtreatmentofthesediseases.
Expressionsystemssuchasbacteria,yeast,andmammaliancellshavebeenwidelyusedtoproducerecombinantproteinsforresearchandtherapeuticpurposes.Amongthem,yeasthasemergedasapromisinghostforproteinexpressionduetoitseaseofhandling,lowcost,highyield,andcompatibilitywitheukaryoticpost-translationalmodifications.
Inthisstudy,weaimedtoexpressandpurifyrecombinantpigHepcidinintheyeaststrainPichiapastorisandinvestigateitsactivityonironabsorption.
MaterialsandMethods
Constructionofexpressionplasmid
ThepigHepcidincDNAsequencewasamplifiedbyPCRusingaspecificprimerpairandclonedintothepYES2.1/V5-His-TOPOexpressionvector(Invitrogen)underthecontroloftheGAL1promoter.TheresultingplasmidwasdesignatedaspYES2.1/Hepcidin.
TransformationandexpressioninPichiapastoris
PichiapastorisGS115cellsweretransformedwiththepYES2.1/HepcidinplasmidusingthePichiaEasyCompTransformationKit(Invitrogen)accordingtothemanufacturer'sinstructions.PositivecloneswereselectedonYPDSplatescontainingzeocin(100μg/ml).Forexpression,thetransformedyeastcellswerepropagatedinBMGYmedium(1%yeastextract,2%peptone,100mMpotassiumphosphatebuffer,pH6.0,1.34%yeastnitrogenbase,4×10-5%biotin,1%glycerol)at30°Cfor24h,andtheninoculatedintoBMMYmedium(1%yeastextract,2%peptone,100mMpotassiumphosphatebuffer,pH6.0,1.34%yeastnitrogenbase,4×10-5%bi