舞茸中一种新型抗HSV-1蛋白的分离鉴别及其功能确定

声明：本文摘自AntiviralResearch75(2007)250-257，中文名《抗病毒研究》，是一本关于病毒性疾病预防和治疗研究的杂志，由国际抗病毒研究协会（ISAR）官方出版，主要收录与人类及动物病毒感染控制相关的研究报告，综述以及评论等。为方便广大读者阅读，我们对论文摘要进行了中文翻译，译文仅供参考，如有不足之处，敬请指正。-----本文摘自舞茸D-fraction官方网站【参考译文】Isolation,identificationandfunctionofanovelanti-HSV-1proteinfromGrifolafrondosa舞茸中一种新型抗HSV-1蛋白的分离、鉴别及其功能确定摘要用40%硫酸铵沉淀法和DEAE纤维素离子交换色谱法从舞茸子实体中提取一种新型的抗病毒蛋白，并命名为GFAHP。这种蛋白质能在体外抑制1型单纯疱疹病毒（HSV-1）的复制，其50%抑制浓度（IC50）为4.1μg/ml，治疗指数29.3。在小鼠模型中，更高浓度的GFAHP（125和500μg/ml）也能显著减轻由HSV-1诱导的睑缘炎、新血管形成以及基质角膜炎。小鼠眼角膜外用GFAHP能显著减少病毒的产量（实验组为3.4log10PFU，对照组为4.19log10PFU）。我们证实，GFAHP在阻止HSV-1穿透进入肾细胞的同时能直接使HSV-1失活。凝胶电泳显示GFAHP的分子量为29.5kDa。用胰蛋白酶水解GFAHP后，从MALDI-TOF-MS测得的肽质量指纹谱（PMF）和电喷雾质谱法分析得知GFAHP的N端序列是一个含11个氨基酸的多肽，即NH2-REQDNAPCGLN-COOH,这与任何一种已知的氨基酸序列不匹配，这说明GFAHP很有可能是一种新型的抗病毒蛋白。据我们所知，这是关于舞茸的抗HSV蛋白的首个报道。关键词：舞茸；蛋白质；抗病毒活性；鉴定；HSV-1论文原文AntiviralResearch75(2007)250–257Isolation,identificationandfunctionofanovelanti-HSV-1proteinfromGrifolafrondosaChang-QingGu,Jun-WenLi∗,FuhuanChao,MinJin,Xin-WeiWang,Zhi-QiangShenInstituteofEnvironmentandHealth,1DaliRoad,Tianjin300050,PRChinaReceived15November2006;accepted22March2007AbstractAnovelantiviralproteinwaspurifiedfromanextractofGrifolafrondosafruitingbodiesusingaprocedurethatincluded40%ammoniumsulfateprecipitationandDEAE-celluloseionexchangechromatography,anddesignatedGFAHP.Thisproteininhibitedherpessimplexvirustype1(HSV-1)replicationinvitrowithanIC50valueof4.1μg/mlandatherapeuticindex29.3.HigherconcentrationsofGFAHP(125and500μg/ml)alsosignificantlyreducedtheseverityofHSV-1inducedblepharitis,neovascularization,andstromalkeratitisinamurinemodel.TopicaladministrationofGFAHPtothemousecornearesultedinasignificantdecreaseinvirusproduction(meanvirusyields:3.4log10PFUinthetreatedgroupand4.19log10PFUinthecontrolgroup).WeprovedthatGFAHPdirectlyinactivatesHSV-1whilesimultaneouslyinhibitingHSV-1penetrationintoVerocells.GelelectrophoresisshowedthatGFAHPhadamolecularweightof29.5kDa.GFAHPwastrypticdigestedandanalyzedfromthePMFofmatrixassisteddesorptionionization-timeofflightmassspectrometry(MALDI-TOF-MS)andnanoelectrosprayionizationtandemmassspectrometry.TheN-terminalsequenceofGFAHPconsistedofan11aminoacidpeptide,NH2-REQDNAPCGLN-COOHthatdidnotmatchanyknownaminoacidsequences,indicatingthatGFAHPislikelytobeanovelantivirusprotein.Toourknowledge,thisisthefirstreportthatcharacterizesananti-HSVproteinfromG.frondosa.©2007ElsevierB.V.Allrightsreserved.Keywords:Grifolafrondosa;Protein;Antiviralactivity;Identification;HSV-11.IntroductionFungiareanimportantsourceofanti-microbialandimmuno-suppressivecompounds.Manyhavealsobeentestedforantibacterialactivityandsomemushroomswererecentlyshowntopossessantiviralactivity.Thebasidiomycete,Fomesfomentarius,forexample,hasantiviralactivityagainsttobaccomosaicvirus(Aokietal.,1993)andanextractoftheedibleJapaneseLentinusedodeswasfoundtoinhibitreplicationofherpessimplexvirus(HSV),westernequineencephalitisvirus,poliovirus,measlesvirus,andmumpsvirus(Sorimachietal.,1990;Sarkaretal.,1993).Thesterolsfromtheediblemushroom,Hypsizigusmarmoreus,haveapotentinhibitoryeffectonEpstein-Barrvirusactivationinducedbythetumorpromoter12-O-tetradecanoylphorbol-13-acetate(TPA)inRajicells(Akihisaetal.,2005)andganodermadiolandapplanoxidicacidGfromG.pfeifferipossessantiviralactivityagainstinfluenzavirustypeA(IVA)(Mothanaetal.,2003).InvitroantiviralactivityagainstIVAwasalsoshownformycelialextractsofKuehneromycesmutabilis(Menteletal.,1994).Screeningothermushroomspeciesmayhelptoidentifyadditionalantiviralfactors.Theediblemushroom,G.frondosa,isabasidiomycetefungusbelongingtothePolyporaceaefamily.Fruitbodiesandliquid-culturedmyceliumfromthismushroomarereportedtocontainusefulantitumorpolysaccharides(Borchersetal.,1999;Adachietal.,1994),andshowsomepromiseasimmunomodulatoryagents.Theymayalsobebeneficialforthetreatmentofhyperlipidemia,hypertension,andhepatitis(Mayell,2001).ItisreportedthatD-Fraction,apolysaccharideextractedfromthemaitakemushroom,G.frondosa,activatesmacrophages,dendriticcells,andTcells,inhibitstumorcellgrowth(Kodamaetal.,2002,2005;Inoueetal.,2002),enhancesthecytotoxicityofNKcellsbyinducingtheproductionofIL-12(Kodamaetal.,2002),andimprovesthesymptomsandsecondarydiseasescausedbyHIV(Nanbaetal.,2000).WeshowedthattheD-FractionalsohastheabilitytoinhibitHBVinvitro(Guetal.,2006).Ingeneral,itisthepolysaccharidesfrommushrooms,likeG.frondosa,thatcontainantiviralactivity(CollinsandNg,1997;Leeetal.,2003,2004;Shietal.,2006;Hsiehetal.,2006;Nieetal.,2006;Cuietal.,2007).Inthisstudy,wedescribeanovelproteinfromG.frondosa,namedGFAHPwhichhasantiviralactivity.Toourknowledge,thisisthefirstisolationandcharacterizationofaproteinfromthismushroomspeciesthathasantiviralfunction.2.Materialsandmethods2.1.VirusandcellcultureHightitervirusstocksweregrowninVerocellsasdescribedpreviously(Grauetal.,1989).Dr.WangattheNationalInstituteforViralDisease,ChineseCenterforDiseaseControlandPreventionprovidedtheHSV-1strain,KOS,andtheVerocells.VerocellswereculturedinDulbecco’sModifiedEaglesMedium(DMEM)with8%fetalbovineserum(FBS)at37℃with5%CO2.Virusstockswerepre