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: 3455897
• 负责人: ELIZABETH SALLY WARD
• 金额: $ 9.71万
• 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 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455897
• 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.

在分子水平上对T 细胞受体（TCR）与同源肽-MHC复合物将促进 用于治疗，阿利亚是， 自身免疫性疾病和癌症。 这项研究的最初目的是 将TCR以可溶形式表达为单个Valpha、Vbeta结构域， Fv样Valpha-Vbeta异二聚体，Fab样ValphaCalpha：Vbeta-C beta 异二聚体或Fab样Valpha-CH 1：Vbeta-C κ 免疫球蛋白嵌合体，使用大肠杆菌作为宿主。 使用鼠 实验性变态反应性脑脊髓炎（EAE）作为模型系统，TCR 基因将从充分表征的致脑炎性T细胞中分离， 杂交瘤（1934.4），其与H-2u中的EAE诱导相关 老鼠. 该系统已被选为TCR，限制MHC 分子和肽抗原已经被定义， 模型可用于测试免疫系统的功效 试剂 重组TCR将被表达并分泌到细胞中。 E.大肠杆菌培养物上清液，和功能活性的 重组TCR片段将使用直接和竞争分析法进行分析。 结合测定。 后者的目的是评估 干扰1934.4杂交瘤相互作用的TCR片段 同源肽-MHC。 这个E。大肠杆菌表达系统可以生产 - 以足够的产量产生功能性Fv样或Fab样TCR， 进行晶体学分析。 此外，现场指导 诱变实验将集中于鉴定 在结合肽-MHC中具有重要功能的TCR残基。 这一点，连同高分辨率的结构工作，将遵循 通过随机诱变参与结合的TCR区域， 目的是产生更高亲和力的TCR。 这样的TCR可以是 在阻断EAE中的致病性TCR方面具有相当大的效用，并且将 用于被动免疫，以评估其在两种情况下的效力 预防和逆转H-2u小鼠的这种疾病。 因此，本项目的最终目的是使用来自 提出的重组可溶性TCR的结构-功能研究， 以小鼠EAE为模型，开发免疫策略。 从长远来看，这些策略可能适用于治疗 自身免疫性疾病