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

作　　者：高帆[1] 王一平[2] 方鑫[1] 毛群颖[1] 朱凤才[3] 杨静玉[4] 程刚[2] 鲁凤民[5] 梁争论[1] 李凤祥[1] 王军志[1] 

机构地区：[1]中国食品药品检定研究院,北京100050 [2]沈阳药科大学药学院,沈阳110016 [3]江苏省疾病预防控制中心,南京210009 [4]沈阳药科大学生命科学与生物制药学院,沈阳110016 [5]北京大学医学部感染病中心暨病原生物学系,北京100191

出　　处：《中国生物制品学杂志》2012年第1期1-5,共5页Chinese Journal of Biologicals

基　　金：国家科技支撑项目(2008BAI69B01)

摘　　要：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。Objective To screen the antigenic epitopes of inactivated enterovirus 71 （EV71 ） vaccine inducing cellular immunity in mice and investigate the mechanism of cellular immune response induced by EVT1 vaccine. Methods A total of 256 peptides were synthesized using the amino acid sequence of structural protein of EV71 BJ08 strain （genotype C4） as template, each con- sisting of 12 amino acid resdiues. The IFN＋ secretion level, expressed as the count of spot forming cells （SFCs）, from splenic mononu- clear cells（MNCs ） of BALB/c mice immunized with inactivated EV71 vaccine were detected by ELISPOT assay after stimulation with the screened peptides （80 p.g/ml）. The antigen-presenting pathway was investigated by removing CD4＋ or CD8~ T cells from MNCs and blocking MHC class Ⅱ or class I molecules. Results Three synthetic peptides screened from a total of 256 peptides stimulated the high secretion level of IFN＋ from splenic MNCs of mice. The mean SFC counts of IFN＋ secreted from splenic cells of mice stimulated with one dose of VP1-85, VP2-36 and VP3-71 were 22. 3, 12. 8 and 17.4, while those with two doses were 29. 3, 44. 1 and 28.5, respectively, which were significantly higher than those stimulated with HBsAg S^-39 peptide as control （2. 1 for one dose and 5. 2 for two doses）（P 〈 0. 01）. After removing the CD4＋ T cells from MNCs or blocking MHC class Ⅱ molecules, the mean SFC counts of IFN~ decreased significantly（P 〈 0. 05 ）. However, after removing CD8＋ T cells or blocking MHC class I molecules, the mean SFC counts showed no significant change （P 〉 0. 05）. Conclusion Three antigenic epitopes of EV71 inducing cellular immunity were successfully screened, all of which belonged to MHC class Ⅱ -restricted peptides, and were located in VPI, VP2 and VP3 domains respectively.

关 键 词：肠道病毒A型 人 疫苗 灭活 免疫 细胞 抗原表位 

分 类 号：R373.23[医药卫生—病原生物学] R392-33[医药卫生—基础医学]