细胞表位的研究课件

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,第一部分,T,细胞表位的基本概念,1,第一部分T细胞表位的基本概念1,2,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,2抗原结合价 Antigenic valence,3,2,、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上不连续性排列，在空间上形成特定的构象。,32、抗原表位的类型顺序表位（sequential epit,T,细胞表位与,B,细胞表位,4,T细胞表位与B细胞表位4,5,3,、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。,53、共同抗原表位(common epitope)不同抗原间,细胞表位的研究课件,MHC class I,peptides of 8-10 amino acids,MHC class II,peptides of 13 amino acids,b2,-M,a,-chain,Peptide,a,-chain,b,-chain,Peptide,4.MHC,分子和抗原肽的相互作用,MHC class Ipeptides of 8-10 am,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,分子结合的氨基酸残基称为锚定残基。,抗原肽和MHC相互作用的分子基础,Y,I,MHC,分子,Y,I,MHC,分子,P,S,A,S,I,K,S,P,S,A,I,K,S,共用模体,YIMHC 分子YIMHC 分子PSASIKSPSAIKS共,MHC I,类分子胞外区的结构,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,抗原肽和HLA相互作用的分子基础MHC限制性;CD4与CD8,12,12,13,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,13T细胞表位的研究方法,14,T,细胞表位的研究意义,1.,检测,刺激细胞使用,Tetramer,技术,MHC,2.,疫苗,表位疫苗,3.,治疗,14T细胞表位的研究意义,15,第二部分,SARS-CoV S,蛋白,T,细胞表位的研究,15,16,16,1.SARS-CoV S DNA,疫苗,Zhi-yong Yang(NIH)Nature.2004,428(1),1.SARS-CoV S DNA 疫苗Zhi-yong Y,2.,免疫方法,8w female BALB/c,prime,at week 0,50ug DNA,boost twice,at week 3,6,2-3w,after prime,4-6w,after boost,1-2w after,boost,to observe the,effect of prime,immunization,to observe the,effect of boost,immunization,to observe the,effect of long term,immunization,:mouse sacrificed,50ug DNA,i.m,i.m,2.免疫方法8w female BALB/c prime50,3.,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，,2,10,6,/ml,3.单细胞悬液的准备 淋巴结脾肺剪碎（消化），研磨，200,4.SARS-CoV S,蛋白多肽,169,条,17-19aa,10aa,重叠,(NIH NIAID VRC),P1 S1-17,P2 S8-25,P3 S16-22,P168 S1200-1216,P169 S1207-1255,4.SARS-CoV S蛋白多肽169条,17-19aa,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 solid circles represent the results in the presence of peptides.,1.S抗原刺激以后IFN-（图A）和IL-2的产生（图,22,22,23,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.IFN-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 corner in each sample represent the percentage of positive cells.Representative results of three independent experiments were shown.,23Fig.2.Verification of pote,24,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.Peptides 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 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,.,24Fig.3.Identification of ne,25,Fig.4.Synergistic roles of peptide N50 and N60 in H-2,b,and H-2,d,restricted mice,BALB/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 performed to detect IFN-.“0”represents non-peptide contained negative control.Experiments were done in duplicate and representative results were shown.,25Fig.4.Synergistic roles of,26,26,27,Fig.6.N50 and N60 can elicit antigen specific immune responses in vivo,BALB/c mice were primed twice with the DNA vaccines.3 weeks later,mice were 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.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 representative resu