characterization

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1、Molecularandfunctionalcharacterizationofanovelstefinanalogueinlargeyellowcroaker(Pseudosciaenacrocea)XinhuaChen*E-mail:Chenxinh@tom.com,JingqunAo,ShuyingLi(KeyLaboratoryofMarineBiogeneticResources,ThirdInstituteofOceanography,SOA,Xiamen,P.R.China)Abstract：Inthispaper,wereportthemol

2、ecularcloningofanovelstefinanaloguefromthespleenoflargeyellowcroakerPseudosciaenacrocea(Lycstefin).Theopenreadingframe(ORF)of297nucleotides(nt)ofLycstefinencodesaproteinof99aminoacids(aa)withaputativemolecularweightof11kDa,inwhichnosignalpeptideandpotentialN-glycoslationsitearepred

3、icted.ThededucedLycstefinpossessesthestructuralfeaturesofthemammalianstefins,includingtwoconservedmotifsknowntointeractwiththeactivesitesoffamilyC1cysteinepeptidases:oneglycineintheN-terminalregion(G6)andGln-Xaa-Val-Xaa-Glymotif(Q48LVAG52).Itshares32-47.5%aasequenceidentitytotheseq

4、uencesfoundinmammalsandotherfishspeciesandisrichincysteineresidues(sevencysteines).GenomicanalysisrevealedthatLyccysgene,757ntlong,consistedofthreeexonsandtwointrons.TheLycstefingenewasconstitutivelyexpressedinvarioustissuesexaminedalthoughatdifferentlevels.Uponstimulationwithpoly(

5、I:C)orinactivatedtrivalentbacterialvaccine,Lycstefintranscriptwassignificantlyup-regulatedinspleenandheadkidneywhiledown-regulatedinblood.Immuno-electronmicroscopyshowedthatLycstefinwasmainlylocalizedinthecytoplasmofspleencellsoflargeyellowcroaker,andalsointhenucleus.RecombinantLyc

6、stefinproteinfusedwithglutathioneS-transferase(rLycstefin)wasshowntohavestronginhibitoryactivityagainstpapainwithaKiof1.3´10-13M.TheinvivoexperimentsrevealedthatLycstefincouldnotmodulatetheexpressionlevelsoflargeyellowcroakertumornecrosisfactor-α2(TNF-α2)andinterleukin-10inspleenan

7、dheadkidney.Toourknowledge,thisisthefirstreportonthemolecularandfunctionalidentificationofastefinanalogueinbonyfish.Keywords:largeyellowcroakerPseudosciaenacrocea;stefin;Subcellularlocalization;protease-inhibitoryactivity;expressionmodulation177斜带石斑鱼免疫球蛋白D和T重链基因的克隆与表达分析徐镇聂品（中国科学院水生

8、生物研究所，武汉430072）石斑鱼（Epineph