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1、MolecularImmunology44(2007)11151122CloningandcharacterizationofaTNF-likeproteinofPlecoglossusaltivelis(ayufish)MayaUenobea,b,ChieKohchia,c,NorikoYoshiokaa,AkihikoYuasad,HiroyukiInagawaa,c,e,KayokoMoriie,TakashiNishizawaa,YukinoriTakahashie,Gen-IchiroSomaa,c,∗aInstituteforHealtha
2、ndSciences,TokushimaBunriUniversity,Nishihama,Yamashiro-cho,Tokushima770-8514,JapanbSt.CatherineUniversity,660Houjyou,Matsuyama,Ehime799-2496,JapancInstituteforDrugDeliverySystems,TokyoUniversityofScience,2641Yamazaki,Noda,Chiba278-8510,JapandInstituteforFisheriesResearch,Tokus
3、himaAgriculture,ForestryandFisheriesTechnologySupportCenter,1-3Hiwasaura,Hiwasa-cho,Kaifu,Tokushima779-2304,JapaneNationalFisheriesUniversity,AppliedAquabiology,2-7-1Nagata-HonmachiShimonoseki,Yamaguchi759-6595,JapanReceived29April2006;receivedinrevisedform30June2006;accepted10
4、July2006Availableonline22August2006AbstractAyuTNFcDNAcontainsanopenreadingframeof708bpencoding235aminoacids.Polyadeniration(A)signalandeightAU-richsequenceswerepresentin858bp3UTR.SouthernblotanalysisindicatedthatayuTNFissingle-copygene.ThegenomicDNAsequenceofayuTNF,consistingo
5、ffourexonsandthreeintrons,wasshowntobeconservedwellthroughoutevolutionfromfishtomammals.TheaminoacidsequenceofayuTNFwasshowntohave3241%ofaminoacididentitytootherknownfishTNF,andabout30%ofaminoacididentitytomammalianTNFs.AphylogeneticanalysisbasedontheaminoacidsequenceofTNFindicat
6、edthatayuhasadistinctiveevolutionarypath.Also,tworesiduesofcysteineimportantfortheformationofthethree-dimensionalstructurewereconservedinayuTNF.Forthefunctionalanalysis,ayuTNFwasinsertedintoexpressionvectorpCold/TF,transferredintoChaperoneCompetentCellsBL21(pKJE7);thisproduceds
7、olublematureayurecombinantTNF.AyurecombinantTNFwasshowntoinducerespiratoryburstactivityfromayukidney.TheaboveresultsindicatethatayuTNFplaysanimportantroleinphylaxis,asitdoesinmammals.©2006ElsevierLtd.Allrightsreserved.Keywords:TNF;Innateimmunity;Phylaxis;Comparativeimmunology1.
8、Introductionpointedoutthatoveruseofantibioticsandchemi
