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1、本科生毕业设计相关内容 课题背景知识：CD8+T细胞免疫应答中起重要作用。CD8+CTL(CytotoxieTlymphoeyteepitopes)对靶细胞发挥 杀伤作用，必须能识别靶细胞表面与相应MHC-I类分子结合的特异性抗原表位，即CTL表位。课 题组已经根据抗原的一级结构，采用免疫信息学手段，对蛋白抗原的HLA-A * 2/ HLA-A3限制性 CTL表位进行了预测分析，初步筛选到表位。通过T2细胞结合力以及稳定性试验，对初步筛选的 表位进行验证，获取与HLA分子具有高结合力的表位，用于后续体外免疫活性检测。一、T2细胞结合力实验相关文献(一)理论知识1什么是T2细胞？ T2细胞是内源
2、性抗原提呈途径中必需的抗原多肽转运蛋白(TAP)缺陷的细 胞株1,为HLA-A2阳性的T, B淋巴细胞杂交瘤细胞，可用于研究抗原递呈过程和T细胞与MHC- 分子的相互识别2 。2为什么要做结合力试验？ MHC-I类分子与CTL表位的有效结合是特异性细胞免疫应答的一个重要环节。因为个体内仅存在少数几个型别的MHC-I类分子，由于每个MHC-I类分子可与一系 列同种类的抗原肽结合进而形成多种多样的M HC-肽复合物，这样就使机体免疫系统可以针对众多 抗原发生特异性的免疫应答。另外，在细胞外游离肽浓度近乎为零的环境中，MHC-1类分子还必须 使相应的抗原肽在细胞表面保留足够长的时间，实现特异性CD8
3、+T细胞对它的识别。有文献报道1 绝大多数CTL表位以高或中等亲和力与M HC-I类分子有效结合。3什么是T2细胞结合力实验？由于T2其自身不能提呈抗原肽到HLA-1类分子上，所以T2细胞 表面空载的HLA-A2分子表达极不稳定，表达后很快降解。但当有外源性抗原肽与之结合后，HLA-A2 的表达就被稳定。抗原肽与HLA-A2的结合力越强，则T2细胞表面HLA-A2分子的降解就越少，表 现为表达量越高。T2表面HLA-A2分子表达量的增加直观地反映了外来抗原肽与HLA-A2的结合力1 。4怎么设计候选表位肽T2细胞的结合力实验？ ( 1)阳性对照，文献中报道的大家公认的与T2细胞有强的结合力的多
4、肽；(2 )阴性对照，单纯的PBS ； ( 3 )背景对照，不用多肽冲击的T2细胞;(4 )肽与T2细胞的结合力的判定依据：A、浓度对比法来表示待测肽与HLA的相对亲和力(RA)。RA=诱导HLA表达20%的待测肽的浓度/诱导HLA表达20%的阳性肽的浓度,RA值越小，肽与HLA的亲和力越强3-4。B、间接免疫荧光法检测各肽与HLA-A2.1分子的结合情况。外源性多肽与到的MHC I类分子越多撮终以平均荧光强度为检测指标，以荧光系数(FI)作为衡量指标1-2, 5。 尽管不同的文献报道的FI的算法略有差别。(二)具体实验方法1 Measurement of peptide relative a
5、ffinity (RA) for HLAA*0201.(1) Briefly, T2 cells were incubated with various concentrations of peptides (0.1 -00M) for 16 hours and then stained with the mAb BB7.2 to quantify the surface expression of HLA-A*0201. For each peptide concentration, the HLA-A*0201-specific staining was calculated as the
6、 percentage of the staining obtained with 100 M of the reference peptide HIVpol589 (IVGAETFYV). The RA is determined as the ratio between the concentration of each peptide and the concentration of the reference peptide that induces 20% of HLA-A*0201 expression4 。(2) T2 cells (3 x105 cells/ml) were i
7、ncubated with various concentrations of peptidesranging from 100 to 0.1 M in serum-free RPMI 1640 medium supplemented with 100 ng/ml human 02m at 37C for 16 h. Cells were then washed twice and stained with the BB7.2 mAb, followed by FITC-conjugated goat anti-mouse Ig mAb to quantify the expression o
8、f HLA-A*0201. For each peptide concentration, the HLA-A*0201-specific staining was calculated as the percentage of the staining obtained with 100M of thereference peptide HIVpol589 (IVGAETFYV). The relative affinity (RA) is determined as: RA =concentration of each peptide that induces 20% of HLA-A*0
9、201-expression/concentration of the reference peptide that induces 20% of HLA-A*0201 expression. The definitive RA value for each peptide was determined from at least three independent experiments3 。2间接免疫荧光法检测各肽与HLA-A21分子的结合情况(1)首先将1x106/m的T2细胞用冰PBS、800r/min离心6 min，先涤3次后，再与10yg/ml的 候选肽37oC共孵育4h。孵育好的
10、细胞用冰PBS，同样离心条件洗涤3次，加入100川BB7.2杂交瘤 细胞培养上清液，水浴40min。PBS洗涤3次，加入100川1:100稀释的二抗FITC标记的羊抗鼠IgG 溶液，在冰上孵育40min，洗涤后于流式细胞仪检测平均荧光强度，波长488nm。阳性已知肽 MAGE-2112-120作为阳性对照，未加肽刺激的单纯T2细胞为背景对照。荧光系数(FI)=(样本平均 荧光强度-背景平均荧光强度)/背景平均荧光强度5 。(2) 首先将1x106 个T2细胞用冰PBS, 800r/min离心6 min,洗涤3次后，再与10 yg/ml待 测肽37oC共孵育4 h.孵育好的细胞用冰PBS,同样离
11、心条件洗涤3次，加入100 yL BB7.2杂交 瘤细胞培养上清液，冰浴40 min. PBS洗涤3次后，加入100 yL稀释度为1:100的FITC-羊抗鼠 IgG溶液，在冰上孵育40 min,洗涤后于流式细胞仪(FACS)检测平均荧光强度，波长488 nm.以单 纯的T2细胞加二抗为阴性对照,T2细胞加一二抗为背景对照。荧光系数(FI)=(样品平均荧光强 度-背景平均荧光强度)/( T2细胞荧光强度-背景荧光强度)x 100 2。(3) 首先收集T2细胞，用冰PBS洗三次后，调细胞浓度至1 x 106/ml,铺于96孔板中，10川/ 孔。再与50yM的候选多肽，2.5 pg/ml的(B2微
12、球蛋白于370C、5% CO2孵箱中共孵育18h。之后， 收集孵育好的细胞，用冰PBS洗三遍，加入2pl FITC标记的HLA-A2特异性的mAb BB7.2，放置4C 冰箱，孵育30mi n,之后PBS洗三遍，接着用流式细胞仪检测平均荧光强度。用已经证明的 HLA-A*0201限制性CD8+CTL表位肽SSp-1作为阳性对照，多肽O VA257-264作为阴性对照，未加肽 刺激的单纯T2细胞作为背景对照。结果判定:以荧光系数(FI)作为衡量指标。荧光系数(Fl)=(样本 平均荧光强度一背景平均荧光强度)/背景平均荧光强度。多肽的FI 1被认为是高亲和力的表位 1 。(4) T2 cells
13、were incubated for 3 h in humidified 5% CO2 chamber at 37C with each peptide. Anti-HLA-A2.1 monoclonal antibody, BB7.2, was added in saturating amount and incubated for 30 min at 4C. After two wash steps with cold PBS, fluorescein isothiocyanate (FITC)-labeled F(ab)2 fragments of goat anti-mouse IgG
14、were added and incubated for another 30 min at 4C. The cells were washed twice with cold PBS and fluorescence was measured on a FACScan flow cytometer The fluorescence index was calculated by the formula: FI=(mean fluorescence of sample-mean fluorescence of background)/(mean fluorescence of T2-mean
15、fluorescence of background)x1006 。(5 ) T2 cells were incubated with 50 pM of the synthesized peptides and 3 pg/ml of human 02-microglobulin in serum-free RPMI 1640 medium for 16 h at 37C/5% CO2. Expression of HLA-A*0201 on T2 cells was then determined with FACS Calibur Xow cytometer by staining with
16、 anti-HLA-A2 Ab derived from BB7.2 and FITC-labeled goat-antimouse IgG used as the second antibody. The data were analyzed using CellQuest software. The Xuorescence index (FI) was calculated as follows: FI = (mean FITC Xuorescence with the given peptide-mean FITC Xuorescence without peptide)/(mean FITC Xuorescence without peptide). Samples were measured in three replications.7(6 )我们实验室所做的T2细胞结合力试验:待测肽
