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1、第三章 T细胞表位Antigen 广州医学院免疫教研室 黄 俊,1,第一部分T细胞表位的基本概念,2,3,抗原结合价 Antigenic valence 能与抗体分子结合的抗原表位的总数。,半抗原 单价抗原完全抗原 多价抗原,表位 Epitope 抗原决定基 Antigenic Determinant 决定抗原特异性的特殊化学基团，是与抗体或抗原受体结合的基本结构单位。,1、抗原表位的概念,4,2、抗原表位的类型,顺序表位（sequential epitope)/线性表位 由连续性线性排列的短肽构成。构象表位(conformationtial epitope)/非线性表位 短肽或多糖残基在序列
2、上不连续性排列，在空间上形成特定的构象。,T细胞表位与B细胞表位,5,6,3、共同抗原表位(common epitope),不同抗原间含有的相同或相似的表位。,交叉反应(cross-reaction)抗体或致敏淋巴细胞对具有相同或相似表位的不同抗原的反应。,MHC class I,peptides of 8-10 amino acids,MHC class II,peptides of 13 amino acids,4.MHC分子和抗原肽的相互作用,抗原肽和MHC相互作用的分子基础*锚定残基（anchor residue）在抗原肽-MHC分子复合物中，抗原肽的两个或两个以上专司和MHC分子结合
3、的氨基酸残基称为锚定残基。,Y,I,MHC 分子,共用模体,MHC I 类分子胞外区的结构,抗原肽和HLA相互作用的分子基础,MHC限制性;CD4与CD8表位的差别；人和其他动物的差别,13,14,T细胞表位的研究方法1.多肽片段的来源降解；表达；人工合成2.MHC分子的结合直接结合；预测3.抗原特异性T细胞的来源感染或者免疫动物或病人筛选方法细胞系的建立4.检测指标活化；增殖；细胞因子；杀伤作用,15,T细胞表位的研究意义1.检测刺激细胞使用Tetramer技术 MHC2.疫苗表位疫苗3.治疗,16,第二部分SARS-CoV S蛋白T细胞表位的研究,17,1.SARS-CoV S DNA 疫
4、苗,Zhi-yong Yang(NIH)Nature.2004,428(1),2.免疫方法,8w female BALB/c,primeat week 0,50ug DNA,boost twiceat week 3,6,2-3wafter prime,4-6w after boost,1-2w afterboost,to observe the effect of prime immunization,to observe the effect of boost immunization,to observe the effect of long term immunization,:mous
5、e sacrificed,50ug DNA,i.m,i.m,3.单细胞悬液的准备,淋巴结,脾,肺,剪碎（消化），研磨，200目筛网过滤，裂解红细胞，2106/ml,4.SARS-CoV S蛋白多肽,169条,17-19aa,10aa重叠(NIH NIAID VRC),P1 S1-17P2 S8-25P3 S16-22P168 S1200-1216P169 S1207-1255,1.S抗原刺激以后IFN-（图A）和IL-2的产生（图B）,结 果 与 讨 论,The empty circles represent the results in the absence of peptides and
6、 solid circles represent the results in the presence of peptides.,23,24,Fig.2.Verification of potential SARS CoV S epitopes Potential SARS CoV S epitopes P50,P51,P59 and P60 in pool 3,and P141,P144,P151 and P152 in pool 8 were used to stimulate splenocytes from SARS CoV S DNA immunized BALB/c mice.I
7、FN-production was detected by ELISA(A)and ELISPOT(B),respectively.Each symbol represents the results of an individual experiment(n=37).In addition,intracellular cytokine staining(C)was performed to determine CD4+or CD8+T cell population.“0”represents the non-peptide stimulated control.Numbers at the
8、 corner in each sample represent the percentage of positive cells.Representative results of three independent experiments were shown.,25,Fig.3.Identification of new synthetic 10aa CD4 and CD8 epitopes The overlapped amino acids between P50 and P51(N50),and between P59 and P60(N60)were synthesized.Pe
9、ptides N50 and N60 were used to stimulate splenocytes from SARS CoV S DNA vaccine-immunized mice.P50 and P60 were used as positive controls,respectively.ELISA(A)and ELISPOT(B)were performed as described above.Furthermore,N50 and 60 were serial diluted to stimulate splenocytes from the DNA immunized
10、mice,ELISPOT(C)was performed to detect numbers of antigen specific IFN-producing cells.Experiments were carried out in duplicate and representative results were shown.“0”represents non-peptide-cultured negative control.,26,Fig.4.Synergistic roles of peptide N50 and N60 in H-2b and H-2d restricted mi
11、ceBALB/c and C57BL/6 mice were immunized by SARS CoV S DNA vaccine as described previously.1-2 weeks after final boost vaccination,N50 and N60 were administrated alone or combined to stimulate splenocytes from both kinds of heterogeneous mice at the same times.ELISA(A),ELISPOT(B)and FACS(C)were perf
12、ormed to detect IFN-.“0”represents non-peptide contained negative control.Experiments were done in duplicate and representative results were shown.,27,28,Fig.6.N50 and N60 can elicit antigen specific immune responses in vivoBALB/c mice were primed twice with the DNA vaccines.3 weeks later,mice were
13、divided into four groups(n=4),injected by peptides(N50 and N60,50 g of each)plus CpG ODN(25 g),peptide,CpG ODN and PBS,respectively.1-2 weeks after boost vaccination,single cell suspensions were prepared from lymph node(LN),spleen and lung.Cells were stimulated by peptide N50 and N60 plus anti-CD28.
14、ELISA(A)and ELISPOT(B)were performed to detect the antigen specific IFN-levels and cells in each group.FACS was performed to detect the frequencies of antigen specific IFN-and/or IL-2 producing cells in CD4+(C)and CD8+(D)T cell populations,respectively.Experiments were done in duplicate and represen
15、tative results were shown.,29,30,第三部分SARS-CoV S蛋白CTL表位残基的初步研究,32,锚定位残基,34,35,36,37,38,39,40,41,42,第四部分SARS-CoV S蛋白B细胞表位的研究,43,44,45,图2 含B细胞表位的多肽组的筛选Fig.2 Scanning peptide pool which contained potential S protein B cell epitope,46,图3 5号多肽组中B细胞表位的筛选Fig.3 Scanning B cell epitope in pool 5,47,图4 使用Bcipep数据库预测B细胞表位Fig.4 predict SARS-CoV S protein B cell epitope by Bcipep data,48,49,50,THANK YOU,
