权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Identification of T cell epitopes of Trypanosoma cruzi antigen using HLA-B transgenic mice

使用 HLA-B 转基因小鼠鉴定克氏锥虫抗原的 T 细胞表位

基本信息

批准号：
06807025
负责人：
HIRAYAMA Kenji
金额：
$ 1.28万
依托单位：
Saitama Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06807025/
关键词：
Trypanosoma cruzi HLA Peptide vaccine Cysteine protease Cytotoxic T cell CD8 molecule Genetic variation Protective immunity 防御免疫トランスジェニックマウストリパノソーマ抗原ペプチド

项目摘要

Analysis of the protective immunity against infection with Trypanosoma cruzi (TC) in the experimental animalmodel using a HLA-B35 transgenic mouse (HLA-B35-TG).CD8 positive cytotoxic T cells are known to be major effector cells to kill the TC infected host cells. Those T cells recognize the invaders antigen with their own HLA-class lmolecules. Using chronically infected HLA-B35-TG,we tried to identify the T cell epitopes that HLA molecule presents to the CD8 cytotoxic T cells. Using the information of the binding motif of HLA-B35, we selected and synthesized 61 different peptides (8mer to 10 mer) from TC cysteine protease. After the binding assay, 5 peptides were chosen for in vitro stimulation experiments. Two months after the infection with TC,HLA-B35-TG mice were sacrificed to get the splenocytes. One million cells in 1 ml of culture medium were incubated with 10muM of each peptide and 30Units of IL-2. Ten dyas later, re-stimulation was done by adding irradiated peptide pulsed autologous splenocytes and IL-2. Totally 2wks. later, the viable cells (T cells) were examined for their cytotoxic activity against peptide pulsed target cells (HLA-B35 Transfectant) by Chromium 51 release assay. None of the peptides stimulated the cytotoxic T cells. though we repeated the experiments twice. Main reasons why there were no effective stimulation were considered as follows.(1) HLA-B35 might not be ideal for the analysis of immune response against the cysteine protease antigen due to row affintiy.(2) Human CD8 might have been essential to complete the HLA-peptide-T cell interaction.2. Search for the major T cell antigens recognized by human T cells from the patients in the endemic area. We have produced T cell clones reactive to epimastigote antigen. Using a T cell clone, 55KDa protein were purified whose molecular weight is almost the same as the cysteine protease. The amino acid sequencing are now on going study.

用HLA-B35转基因小鼠（HLA-B35- tg）建立克氏锥虫（TC）感染实验动物模型，分析其保护性免疫作用。已知CD8阳性细胞毒性T细胞是杀死TC感染宿主细胞的主要效应细胞。这些T细胞用它们自己的hla类分子识别入侵者抗原。利用慢性感染的HLA- b35 - tg，我们试图鉴定HLA分子呈现给CD8细胞毒性T细胞的T细胞表位。利用HLA-B35结合基序的信息，我们从TC半胱氨酸蛋白酶中选择并合成了61种不同的肽段（8 ~ 10 mer）。结合实验结束后，选择5个肽段进行体外刺激实验。感染TC 2个月后，处死HLA-B35-TG小鼠获得脾细胞。1 ml培养基中培养100万个细胞，每种肽10muM， IL-2 30Units。10天后，通过添加辐照肽脉冲的自体脾细胞和IL-2进行再刺激。完全两周之久。然后用51铬释放法检测活细胞（T细胞）对肽脉冲靶细胞（HLA-B35转染细胞）的细胞毒活性。这些肽都没有刺激细胞毒性T细胞。虽然我们重复了两次实验。没有有效的刺激措施的主要原因如下：(1) HLA-B35对半胱氨酸蛋白酶抗原具有较强的亲和性，可能不适合用于半胱氨酸蛋白酶抗原免疫应答分析。(2)人CD8可能是完成hla -肽- t细胞相互作用所必需的。寻找流行地区患者T细胞识别的主要T细胞抗原。我们已经生产出对表皮马鞭毛抗原有反应的T细胞克隆。利用T细胞克隆纯化了55KDa蛋白，其分子量与半胱氨酸蛋白酶基本相同。氨基酸测序正在进行中。