BIOLOGY OF THE CD2/E-RECEPTOR COMPLEX

CD2/E-受体复合物的生物学

• 批准号: 3130640
• 负责人: Malek Kamoun
• 金额: $ 18.32万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3130640
• 关键词: T lymphocyte; chemical binding; chemical structure function; electroporation; erythrocytes; gel electrophoresis; gene deletion mutation; gene expression; genetic mapping; genetic markers; human subject; immune adherence reaction; immunochemistry; interleukin 2; leukocyte activation /transformation; membrane activity; membrane proteins; mitogens; molecular cloning; monoclonal antibody; phosphorylation; protein engineering; protein sequence; receptor; tissue /cell culture; transfection; transposon /insertion element

This proposal is a continuation of our previous studies aimed at analyzing the properties and the function of the CD2/E-receptor pathway of human T cell activation. Our interest is to study the structure-function relationship of CD2 and to define and better understand the molecular and physiological events associated with the triggering of CD2 receptors by mitogens and monoclonal antibodies. We will examine 1) the phosphorylation of CD2 molecules on human T cells in the presence of regimens known to affect cell activation, including the triggering by mitogenic combinations of anti-CD2 monoclonal antibodies. We will analyze the phosphoamino acid composition of 32P CD2. 2) Furthermore, we will pursue the analysis and functional testing of CD2 cDNA clones using DNA transfection techniques. We will attempt to reconstitute an active CD2 receptor by DNA transfer in CD2-negative lymphoid T cell lines. We already have demonstrated that a full-length CD2 cDNA clone can be transiently expressed in Cos-7 cells, using the pcEXV-CD2 vector containing the SV40 promoter. Surface CD2 molecules of transfectant cells expressed the adhesion sites as well as activation sites defined by monoclonal antibodies. We will attempt to isolate stable lymphoid cell line transfectants expressing high levels of cell surface CD2 using the expression vector pBC12/CMV/CD2 containing the human cytomegalovirus immediate-early promoter as well as other expression vectors. Positive selection for stable transfectant will be achieved by co- transfectant plasmids (pcEXV-Neo) containing the mammalian selectable marker neo. The generation of deletion mutants of CD2 cDNA using Exonuclease III digestion or site specific mutagenesis will allow us to examine specific amino acid sequences necessary for the proper functioning of CD2, in particular, the ligand binding site (T111), the activation site (T112/T113), and the role of particular amino acids in the cytoplasmic region, including those modified by phosphorylation, as well as the unusual clusters of basic residues and prolines. Many described roles of CD2 in T cell activation and differentiation make the study of CD2 structure-function extremely important for understanding and potentially manipulating the immune response.

这一建议是我们以前研究的延续， 分析CD 2/E受体的性质和功能 人T细胞活化的途径。 我们的兴趣是研究 CD 2结构-功能关系，并定义和更好地 了解与之相关的分子和生理事件 有丝分裂原和单克隆抗体对CD 2受体的触发 抗体的 我们将研究1）CD 2的磷酸化 在已知的方案存在下， 影响细胞活化，包括促有丝分裂的触发 抗CD 2单克隆抗体的组合。 我们将分析 32 P CD 2的磷酸氨基酸组成。 2)而且我们 将继续进行CD 2 cDNA克隆的分析和功能测试 使用DNA转染技术。 我们将尝试重建 通过DNA转移在CD 2阴性淋巴T细胞中激活CD 2受体 细胞系 我们已经证明了全长CD 2 cDNA克隆可以在Cos-7细胞中瞬时表达，使用 含有SV 40启动子的pcEXV-CD 2载体。 表面CD 2 转染细胞的分子表达粘附位点， 以及由单克隆抗体限定的活化位点。 我们将 尝试分离稳定的淋巴样细胞系转染子 使用所述表达来表达高水平的细胞表面CD 2， 人巨细胞病毒载体pBC 12/CMV/CD 2 立即早期启动子以及其它表达载体。 稳定转染子的阳性选择将通过共转染来实现。 转染质粒（pcEXV-Neo）含有哺乳动物 选择标记neo。 CD 2缺失突变体的产生 使用核酸外切酶III消化或位点特异性诱变的cDNA 将使我们能够检查特定的氨基酸序列， 对于CD 2的正常功能，特别是配体， 结合位点（T111），激活位点（T112/T113），和作用 在细胞质区域的特定氨基酸，包括 那些被磷酸化修饰的，以及不寻常的簇， 碱性残基和脯氨酸。 许多人描述了CD 2在T细胞活化中的作用， 分化使CD 2结构-功能的研究变得极其重要 对于理解和潜在地操纵免疫系统 反应