IMMUNOGLOBULIN-RELATED T-LYMPHOCYTE SURFACE RECEPTORS

这项研究的目的是获得关于 确定的抗原特异性表面组分的结构性质 小鼠和灵长类T细胞系。总体目标是获得详细的 有关生物化学和分子生物学特性的信息 与血清学相关的T细胞表面成分 免疫球蛋白-VH区域标志物(VT)，这将允许直接 与其他可能的T细胞受体，如表面复合体的比较 受主要组织相容性复合体的限制。具体目标是： (1)对VT相关的T细胞分子进行结构表征。这个 这里的主要方法将是从大量同质、 在体外培养的T细胞系(大于50克)中制备 分子的免疫亲和层析。特定条件 将使用多肽键的裂解，并分离片段 采用高效液相色谱分离。(2)与VT相关的限制 T细胞系70-n2表达和合成的决定簇是遗传的 作为人类群体中的常染色体显性基因。我们将确定 特定于该标记的基因的遗传连锁。(三)为确保 获得完整的序列信息后，我们将使用 我们定义的品系中携带VT的T细胞受体的问题 分子生物学技术。我们将使用我们的特定抗血清来 分离含新生多肽链的完整多聚核糖体 细胞株，分离mRNA并产生cDNA，然后克隆和 对该基因进行测序。另一种战略将是建设 利用总mRNA和表达载体pUC8构建的cDNA文库 允许合成抗原性产品。我们将对文库进行筛选 靶向T细胞表面的镍翻译DNA探针 感受器。然后用我们的抗血清对阳性克隆进行筛选 确定我们产生的VT相关产品的抗血清是否 最近与可能的T细胞受体基因的产物发生反应 已排序。(CS)

This research is directed towards obtaining detailed information on the structural properties of the antigen-specific surface component of defined murine and primate T-cell lines. The overall goal is to obtain detailed information regarding the biochemical and molecular biological properties of T-cell surface components which are serologically related to immunoglobulin-VH region markers (VT), which will allow direct comparison with other possible T-cell receptors such as surface complexes restricted by the major histocompatibility complex. The specific aims are: (1) to characterize the VT-related T-cell molecules structurally. The major approach here will be to begin with large quantities of homogenous, in vitro-grown T-cell lines (greater than 50 grams) and to prepare the molecules by immune affinity chromatography. Conditions for specific cleavage of peptide bonds will be used and the fragments will be separated by high performance liquid chromatography. (2) The restricted VT-related determinant expressed and synthesized by the T-cell line 70-N2 is inherited as an autosomal dominant within human populations. We will determine the genetic linkage of the gene specifying this marker. (3) In order to ensure that complete sequence information is obtained, we will approach the problem of the VT-bearing t-cell receptor of our defined lines using the techniques of molecular biology. We will use our specific antisera to isolate intact polyribosomes bearing nascent polypeptide chains from our cell lines, isolate mRNA and produce cDNA followed by cloning and sequencing of the cDNA. An alternative strategy will be the construction of a cDNA library using total mRNA and the expression vector pUC8 which allows synthesis of antigenic products. We will screen the cDNA library with nicktranslated DNA probes directed against putative T-cell surface receptors. The positive clones will then be screened with our antisera to determine whether the antisera we have generated to VT-related products react with the products of the putative T-cell receptor genes recently sequenced. (CS)