SOLUBLE T CELL RECEPTOR EXPRESSIONS IN ESCHERICHIA COLI

大肠杆菌中可溶性 T 细胞受体表达

• 批准号: 3455896
• 负责人: ELIZABETH SALLY WARD
• 金额: $ 10.29万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455896
• 关键词: Escherichia coli; T cell receptor; X ray crystallography; chimeric proteins; complementary DNA; crosslink; gene expression; genetic manipulation; histocompatibility antigens; hybridomas; immunologic assay /test; interleukin 2; laboratory mouse; molecular cloning; monoclonal antibody; oligonucleotides; passive immunization; plasmids; polymerase chain reaction; protein structure function; receptor binding; receptor expression; site directed mutagenesis; western blottings

Detailed knowledge at the molecular level concerning the interaction of T cell receptors (TCRs) with cognate peptide-MHC complexes would facilitate the development of immunotherapy for the treatment of, inter alia, autoimmune disease and cancer. The initial aim of this study is to express TCRs in soluble form as either single Valpha, Vbeta domains, Fv-like Valpha-Vbeta heterodimers, Fab-like ValphaCalpha:Vbeta-C beta heterodimers or as Fab-like Valpha-CH1:Vbeta-Ckappa immunoglobulin-chimeras, using Escherichia coli as a host. Using murine experimental allergic encephalomyelitis (EAE) as a model system, TCR genes will be isolated from a well characterized encephalitogenic T call hybridoma (1 934.4), which is associated with EAE induction in the H-2u mouse. This system has been chosen as the TCRs, restricting MHC molecules and peptide antigen have been defined, and murine disease models are available for the testing of the efficacy of immunotherapeutic reagents. The recombinant TCRs will be expressed and secreted into the E. coli culture supernatant, and the functional activity of the recombinant TCR fragments will be analyzed using direct and competition binding assays. The latter will be designed to assess the ability of the TCR fragments to interfere with the interaction of the 1934.4 hybridoma with cognate peptide-MHC. This E. coli expression system could allow the production of functional Fv-like or Fab-like TCRs in sufficient yields to allow crystallographic analyses to be carried out. In addition, site-directed mutagenesis experiments will be focused towards the identification of the TCR residues which are functionally important in binding peptide-MHC. This, together with the high resolution structural work, will be followed by random mutagenesis of regions of the TCR involved in binding, with the aim for generating TCRs of higher affinity. Such TCRs could be of considerable utility in the blocking of pathogenic TCRs in EAE, and will be used in passive immunization to assess their efficacy in both preventing and reversing this disease in the H-2u mouse. Thus, the ultimate aim of this project is to use data from the proposed structure-function studies of recombinant, soluble TCRs to develop immunotherapeutic strategies, using murine EAE as the model. These strategies could in the long term be applicable to the therapy of autoimmune disease in humans.

在分子水平上关于T的相互作用的详细知识 细胞受体(TCR)与同源肽-MHC复合体将促进 免疫疗法的发展，除其他外，用于治疗 自身免疫性疾病和癌症。这项研究的最初目的是 以可溶性形式将TCR表达为单个Valpha，Vβ结构域， 类Fv的Valpha-Vβ杂二聚体，类Fab的ValphaCalpha：Vβ-Cβ 异二聚体或类Fab的Valpha-CH1：Vbeta-Ckappa 免疫球蛋白-嵌合体，以大肠杆菌为宿主。用小鼠 实验性变态反应性脑脊髓炎模型系统 基因将从一种具有良好特征的脑源性T呼叫中分离出来 杂交瘤(1 934.4)，与H-2U中EAE的诱导相关 老鼠。该系统已被选为TCR，限制MHC 分子和多肽抗原已经被定义，小鼠疾病 已有模型可用于测试免疫治疗的疗效 试剂。重组的TCRs将被表达并分泌到 大肠埃希氏菌培养上清，并测定其功能活性 重组TCR片段将使用直接和竞争分析 有约束力的分析。后者将被设计用于评估 干扰1934.4杂交瘤细胞相互作用的TcR片段 与同源多肽-MHC。 这种大肠杆菌表达系统可以生产出 功能类似Fv或Fab的TCR具有足够的产量，以允许 进行结晶学分析。此外，站点定向 诱变实验将集中于鉴定 TCR残基在结合肽-MHC中具有重要的功能。 这将与高分辨率的结构工作一起进行。 通过随机突变参与结合的TCR区域，与 目的是产生更高亲和力的TCR。这样的TCR可能是 在阻断EAE中的致病TCRs方面有相当大的效用，并将 用于被动免疫，以评估其在两种疾病中的效果 在H-2U小鼠中预防和逆转这种疾病。 因此，该项目的最终目标是使用来自 重组可溶性TCRs的结构与功能研究 以小鼠EAE为模型，发展免疫治疗策略。 从长远来看，这些策略可能适用于 人类中的自身免疫性疾病。