肠道病毒71型灭活疫苗诱导小鼠细胞免疫抗原表位的筛选Screening of antigenic epitopes of inactivated enterovirus 71 vaccine inducing cellular immunity in mice
高帆;王一平;方鑫;毛群颖;朱凤才;杨静玉;程刚;鲁凤民;梁争论;李凤祥;王军志;
摘要(Abstract)：
目的筛选肠道病毒71型(Enterovirus 71,EV71)灭活疫苗免疫小鼠后诱导的细胞免疫抗原表位,探讨EV71疫苗诱导的细胞免疫应答机制。方法以EV71 BJ08株(C4基因型)病毒结构蛋白氨基酸序列为模板,合成覆盖EV71 VP1～VP3的256条合成肽(每条肽12个氨基酸),分别以其为刺激物(80μg/ml),采用ELISPOT法测定EV71灭活疫苗免疫小鼠脾单个核细胞(Mononuclear cell,MNC)诱导IFNγ分泌的斑点形成细胞数(Spot-forming cell,SFC),筛选细胞免疫抗原表位。从脾MNC中去除CD4+或CD8+T细胞及封闭MHCⅡ类分子或MHCⅠ类分子,分析抗原提呈途径。结果通过对256条合成肽的筛选,获得3条可刺激小鼠脾MNC高水平分泌IFNγ的合成肽。以3条合成肽作为刺激物时,小鼠脾MNC分泌IFNγSFC均值(单针免疫组:22.3、12.8、17.4;两针免疫组:29.3、44.1、28.5)较乙肝表面抗原对照肽S28-3(9单针:2.1;两针:5.2)显著升高(P<0.01)。当MNC中去除CD4+T细胞或封闭MHCⅡ类分子后,IFNγSFC均值显著下降(P<0.05);而去除CD8+T细胞或封闭MHCⅠ类分子时,IFNγSFC均值未见显著性差异(P>0.05)。结论已成功筛选出3个EV71细胞免疫抗原表位,分别位于VP1、VP2和VP3区域,3条合成肽均属MHCⅡ类限制性多肽。
关键词(KeyWords)： 肠道病毒A型,人;疫苗,灭活;免疫,细胞;抗原表位
基金项目(Foundation): 国家科技支撑项目(2008BAI69B01)
作者(Author): 高帆;王一平;方鑫;毛群颖;朱凤才;杨静玉;程刚;鲁凤民;梁争论;李凤祥;王军志;
Email:
DOI: 10.13200/j.cjb.2012.01.6.gaof.013
参考文献(References)：
- [1]Lin TY,Chang LY,Hsia SH,et al.The 1998 enterovirus 71 out-break in Taiwan:pathogenesis and management[J].Clin InfectDis,2002,34(Suppl2):S52-S57.
- [2]Yang F,Ren L,Xiong Z,et al.Enterovirus 71 outbreak in thePeople’s Republic of China in 2008[J].J Clin Microbiol,2009,47(7):2351-2352.
- [3]Mao LX,Wu B,Bao WX,et al.Epidemiology of hand,foot,andmouth disease and genotype characterization of enterovirus 71 inJiangsu,China[J].J Clin Virol,2010,49(2):100-104.
- [4]Palacios G,Oberste MS.Enteroviruses as agents of emerging in-fectious diseases[J].J Neurovirol,2005,11(5):424-433.
- [5]金奇.医学分子病毒学[M].北京:科学出版社,2001:606-613.
- [6]Foo DG,Alonso S,Phoon MC,et al.Identification of neutralizinglinear epitopes from the VP1 capsid protein of enterovirus 71 usingsynthetic peptides[J].Virus Res,2007,125(1):61-68.
- [7]Foo DG,Alonso S,Chow VT,et al.Passive protection againstlethal enterovirus 71 infection in newborn mice by neutralizing an-tibodies elicited by a synthetic peptide[J].Microbes Infect,2007,9(11):1299-1306.
- [8]Li X,Mao C,Ma S,et al.Generation of neutralizing monoclonalantibodies againse enterovirus 71 using synthetic peptides[J].Biochem Biophys Res Commun,2009,390(4):1126-1128.
- [9]毛群颖,何鹏,于祥,等.人肠道病毒71型中和抗体检测方法的实验室评价[J].中国生物制品学杂志,2010,23(8):885-888.
- [10]姚昕,毛群颖,黄维金,等.肠道病毒71型北京分离株全基因组序列分析[J].中华流行病学杂志,2009,30(7):729-732.
- [11]Foo DG,Macary PA,Alonso S,et al.Identification of humanCD4+T-cell epitopes on the VP1 capsid protein of enterovirus 71[J].Viral Immunol,2008,21(2):215-224.
- [12]Cello J,Strannegdrd O,Svennerholm B.A study of the cellularimmune response to enteroviruses in humans:identification ofcross-reactive T cell epitopes on the structural proteins of en-teroviruses[J].J Gen Virol,1996,77(Pt9):2097-2108.