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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斜帶石斑魚免疫球蛋白D和T重鏈基因的克隆與表達分析徐鎮(zhèn)聶品（中國科學院水生
8、生物研究所，武漢430072）石斑魚（Epineph
