T细胞表位的研究医学知识讲解培训课件

1、T细胞表位的研究医学知识讲解T细胞表位的研究医学知识讲解第一部分T细胞表位的基本概念2T细胞表位的研究医学知识讲解第一部分T细胞表位的基本概念2T细胞表位的研究医学知识讲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T细胞表位的研究医学知识讲解3抗原结合价 Antigenic valence 42、抗原表位的类型顺序表位（sequential epitope)/

2、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。T细胞表位的研究医学知识讲解42、抗原表位的类型顺序表位（sequential epitT细胞表位与B细胞表位5T细胞表位的研究医学知识讲解T细胞表位与B细胞表位5T细胞表位的研究医学知识讲解63、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。T细胞表位的研究医学知识讲解63、共同抗原表位(comm

3、on epitope)不同抗原间7T细胞表位的研究医学知识讲解7T细胞表位的研究医学知识讲解MHC class Ipeptides of 8-10 amino acidsMHC class IIpeptides of 13 amino acidsb2-Ma-chainPeptidea-chainb-chainPeptide4.MHC分子和抗原肽的相互作用8T细胞表位的研究医学知识讲解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

4、的氨基酸残基称为锚定残基。 9T细胞表位的研究医学知识讲解 抗原肽和MHC相互作用的分子基础 9T细胞表位的研YIMHC 分子YIMHC 分子PSASIKSPSAIKS共用模体10T细胞表位的研究医学知识讲解YIMHC 分子YIMHC 分子PSASIKSPSAIKS共MHC I 类分子胞外区的结构11T细胞表位的研究医学知识讲解MHC I 类分子胞外区的结构11T细胞表位的研究医学知识抗原肽和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12T细胞表位的研究医学知识讲解抗原肽和HLA相互作用的分子基础MHC限制性;CD4与CD813T细胞表位的研究医学知识讲

5、解13T细胞表位的研究医学知识讲解14T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T细胞表位的研究医学知识讲解14T细胞表位的研究方法T细胞表位的研究医学知识讲解15T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T细胞表位的研究医学知识讲解15T细胞表位的研究意义T细胞表位的研究医学知识讲解16第二部分SARS-CoV S蛋白T细胞表位的研究T细胞表位的研究医学知识讲解16T细胞表位

6、的研究医学知识讲解17T细胞表位的研究医学知识讲解17T细胞表位的研究医学知识讲解1. SARS-CoV S DNA 疫苗Zhi-yong Yang (NIH) Nature. 2004, 428 (1 )18T细胞表位的研究医学知识讲解1. SARS-CoV S DNA 疫苗Zhi-yong Y2.免疫方法8w female BALB/c primeat week 050ug DNAboost twiceat week 3, 62-3wafter prime4-6w after boost1-2w afterboostto observe the effect of prime immuni

7、zationto observe the effect of boost immunizationto observe the effect of long term immunization:mouse sacrificed50ug DNAi.mi.m19T细胞表位的研究医学知识讲解2.免疫方法8w female BALB/c prime503. 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 20T细胞表位的研究医学知识讲解3. 单细胞悬液的准备 淋巴结脾肺剪碎（消化），研磨，2004. SARS-CoV S蛋白多肽169条, 17-19aa

8、, 10aa重叠 (NIH NIAID VRC)P1 S1-17P2 S8-25P3 S16-22P168 S1200-1216P169 S1207-125521T细胞表位的研究医学知识讲解4. SARS-CoV S蛋白多肽169条, 17-19aa1. 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

9、.22T细胞表位的研究医学知识讲解1. S抗原刺激以后IFN- （图A）和IL-2的产生（图23T细胞表位的研究医学知识讲解23T细胞表位的研究医学知识讲解24Fig. 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 immunize

10、d 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 st

11、imulated control. Numbers at the corner in each sample represent the percentage of positive cells. Representative results of three independent experiments were shown. T细胞表位的研究医学知识讲解24Fig. 2. Verification of pote25Fig. 3. Identification of new synthetic 10aa CD4 and CD8 epitopes The overlapped amino

12、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.

13、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

14、 negative control.T细胞表位的研究医学知识讲解25Fig. 3. Identification of ne26Fig. 4. Synergistic roles of peptide N50 and N60 in H-2b and H-2d restricted miceBALB/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 adminis

15、trated 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 show

16、n.T细胞表位的研究医学知识讲解26Fig. 4. Synergistic roles of27T细胞表位的研究医学知识讲解27T细胞表位的研究医学知识讲解28Fig. 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 divided into four groups (n=4), injected by peptides (N50 and N60, 50

17、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

18、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 results were shown.T细胞表位的研究医学知识讲解28

19、Fig. 6. N50 and N60 can elic29T细胞表位的研究医学知识讲解29T细胞表位的研究医学知识讲解30T细胞表位的研究医学知识讲解30T细胞表位的研究医学知识讲解第三部分SARS-CoV S蛋白CTL表位残基的初步研究31T细胞表位的研究医学知识讲解31T细胞表位的研究医学知识讲解32T细胞表位的研究医学知识讲解32T细胞表位的研究医学知识讲解锚定位残基33T细胞表位的研究医学知识讲解锚定位残基33T细胞表位的研究医学知识讲解34T细胞表位的研究医学知识讲解34T细胞表位的研究医学知识讲解35T细胞表位的研究医学知识讲解35T细胞表位的研究医学知识讲解T细胞表位的研究医学知识讲解培训课件37T细胞表位的研究医学知识讲解37T细胞表位的研究医学知识讲解38T细胞表位的研究医学知识讲解38T细胞表位的研究医学知识讲解39T细胞表位的研究医学知识讲解39T细胞表位的研究医学知识讲解40T细胞表位的研究医学知识讲解40T细胞表位