NCI CLINICAL INVESTIGATOR AWARD

绵羊红细胞受体(又名p50、t11或CD2)是一种T 淋巴细胞特异性细胞表面糖蛋白介导 抗原、凝集素和辅助细胞非依赖性T淋巴细胞 激活。抗CD2抗体已经被证明是 以IL-2依赖的方式诱导或阻断T细胞激活。 赞助商实验室的目标是克隆CD2，以便 研究这一重要的结构与功能的关系 分子。为此，我们分离了5个重组的cdna。 来自lambda GT-11文库的插入片段，(A)范围从1000- 1500bp，(B)经Southern分析为交叉反应，(C)编码 与多价抗CD2抗血清反应的蛋白质决定簇 和(D)杂交到仅在 CD2阳性的T细胞，CD2阴性的B或T细胞。这 该奖项将用于验证我们的cdna分离株的身份。 检测COS-7成纤维细胞中CD2表位的研究 用pcEXV-3-CD2构建体腺病毒载体。此向量包含 SV40早期启动子和增强子序列。我们比计划 CD2阳性与阴性白血病淋巴组织共转染的研究 PSV-2-neo和pcEXV-3-CD2构建的细胞系。我们的目标 是(A)稳定表达细胞的转染体的分离 表面CD2，(B)完整CD2的流式细胞仪分析显示 转基因细胞中的表位和(C)转染体的检测 CD2依赖的抗CD2抗体、凝集素和 有丝分裂原。如果我们成功地分离出转基因细胞 功能CD2，我们提出了未来的实验以(A)映射 功能重要的CD2表位，如T112和T113，(B) 通过选择性突变确定涉及哪些结构 在CD2介导的信号转导中，(C)如果是特异性的 跨膜突变可以改变CD2的功能 对于p185(neu蛋白)和(D)转染者是否有IL-2 依赖CD2介导的反应性。

The sheep erythrocyte receptor (aka p50, T11 or CD2) is a T lymphocyte specific cell surface glycoprotein which mediates antigen, lectin and accessory cell independent T lymphocyte activation. Anti-CD2 antibodies have been shown to either induce, or block T cell activation in an IL-2 dependent manner. The goal of the sponsors laboratory is to clone CD2 in order to study the structure-function relationship of this important molecule. To that end, we have isolated 5 recombinant cDNA inserts from a lambda GT-11 library which (a) range from 1000- 1500 bp, (b) are cross-reactive by Southern analysis, (c) code for protein determinants reactive with a polyvalent anti-CD2 antisera and (d) hybridize to 1.7 and 1.3 kb RNA transcripts found only in CD2 positive T cells but not CD2 negative B or T cells. This award will be applied to verify the identity of our cDNA isolates as CD2 by detecting CD2 epitopes in COS-7 fibroblasts transfected with pcEXV-3-CD2 constructs. This vector contains the SV40 early promotor and enhancer sequences. We than plan on cotransfecting CD2 positive and negative leukemic lymphoid cell lines with pSV-2-neo and pcEXV-3-CD2 constructs. Our aims are (a) the isolation of stable transfectants which express cell surface CD2, (b) demonstration by FACS analysis of intact CD2 epitopes in transfected cells and (c) assay of transfectants for CD2 dependent responsiveness to anti-CD2 antibodies, lectins and mitogens. If we are successful in isolating transfectants with functional CD2, we propose future experiments to (a) map functionally important CD2 epitopes such as T112 and T113, (b) determine by selective mutagenesis which structures are involved in CD2 mediated signal transduction, (c) if specific transmembrane mutations can alter the function of CD2 as seen for p185 (neu protein) and (d) whether transfectants exhibit IL-2 dependent CD2 mediated responsiveness.