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ANTI-PEPTIDE ANTIBODIES TO HUMAN T CELL RECEPTORS

人类 T 细胞受体的抗肽抗体

基本信息

批准号：
3133061
负责人：
ROBERT F SILICIANO
金额：
$ 14.37万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-04-01 至 1988-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3133061
关键词：
T lymphocyte antiantibody antireceptor antibody human subject immune complex immunogenetics immunoperoxidase major histocompatibility complex molecular cloning monoclonal antibody

项目摘要

Human T cell receptors have now been defined on inducer, suppressor and class I and class II specific cytotoxic T lymphocytes as 90KD T3-associated clonotypic molecules. These are comprised of one 49-51KD Alpha and 43KD Beta subunit which are disulfide linked. Peptide map analysis studies suggested that each subunit is comprised of constant and variable domains. Moreover, the precise molecular basis for this variability in the Beta subunit has been defined by DNA cloning studies, showing that similar to immunoglobulin, specific V, D, J and C segments fuse to form an active Ti Beta gene. In the thymus, such disulfide linked heterodimers are restricted in expression to a minor population of cells which are surface T3+. However, the precise molecular details regarding synthesis of Alpha and Beta subunits and the existence of cytoplasmic and surface forms has not been defined due to lack of appropriate reagents capable of reacting with the individual isolated subunits. To understand in greater detail the nature of human T cell receptor expression during intrathymic ontogeny, we will address three areas: 1) production and characterization of anti-peptide antisera to constant regions of the Ti Alpha and Beta subunit employing both heterologous immunization and monoclonal antibody techniques; 2) analysis of intrathymic ontogeny of Ti Alpha and Beta subunits with specific probes; and 3) utilization of anti-peptide antisera to further delineate the structure of the T3-Ti molecular complex. Firstly, peptides corresponding to regions of individual subunits likely to be immunogenic based on Kyte-Doolittle and Hopps and Wood predictions will be synthesized and utilized for production of heteroantisera and monoclonal antibodies. Reagents will then be screened against the immunizing peptide as well as denatured isolated subunits and subsequently assessed for reactivity with the native molecules on the surface of thymocytes and T lymphocytes. Secondly, once monoclonal and heteroantisera reagents are defined, the intrathymic expression of the subunits will be examined. Specifically, it will be determined whether surface and intracytoplasmic forms of the Ti Alpha and Beta subunits exist and where they appear in intrathymic T cell differentiation relative to one another and known T cell surface molecules including T3, T4, T8, T11 and T6. In addition, the localization of cells expressing one or both subunits will be defined. Finally, these anti-peptide reagents will be utilized to investigate some of the structural features of the T3-Ti molecular complex.

人类T细胞受体现已被确定为诱导剂、抑制剂和抑制剂。 I类和II类特异性细胞毒性T淋巴细胞作为90 KD T3相关克隆型分子这些由一个49- 51 KD的α和43 KD的二硫键连接的β亚基。肽图分析研究表明每个亚基由恒定和可变结构域组成。此外，β细胞中这种变异性的精确分子基础亚基已被定义的DNA克隆研究，显示类似的免疫球蛋白特异性V、D、J和C片段融合形成活性Ti 贝塔基因。在胸腺中，这种二硫键连接的异二聚体是仅限于表达于少数细胞群体， T3+。然而，关于阿尔法合成的精确分子细节和β亚基，细胞质和表面形式的存在，由于缺乏能够反应的适当试剂，与单个分离的亚基。为了更详细地了解我们研究了胸腺内个体发育过程中人类T细胞受体表达的本质，将涉及三个领域：1）生产和表征 Ti α和β亚单位恒定区的抗肽抗血清采用异源免疫和单克隆抗体技术; 2）Ti α和β的胸腺内个体发育分析亚基与特异性探针;和3）利用抗肽抗血清以进一步描绘T3-Ti分子复合物的结构。首先，对应于单个亚基的区域的肽可能根据Kyte-Doolittle和Hopps和Wood的预测，合成并用于生产异源抗血清和单克隆抗体抗体的然后针对免疫肽筛选试剂以及变性的分离亚基，随后评估与胸腺细胞和T细胞表面的天然分子的反应性淋巴细胞其次，一旦单克隆和异源抗血清试剂被定义，将检查亚基的胸腺内表达。具体地说，将确定表面和胞质内是否存在存在Ti α和β亚基的形式，它们出现在胸腺内T细胞相对于彼此和已知T细胞的分化表面分子包括T3、T4、T8、T11和T6。此外该将确定表达一种或两种亚基的细胞的定位。最后，这些抗肽试剂将被用于研究一些 T3-Ti分子复合物的结构特征。