CYTOLYTIC T LYMPHOCYTE HELPER FACTOR

我们已经开发了一种体外细胞因子的测定方法， 原代小鼠细胞溶解性T淋巴细胞（CTL）的产生 对抗肿瘤细胞上的同种异体抗原 的因素之一 在该试验中的功能称为RDCHF。 我们最近用钠离子交换法将RDCHF纯化到一条带 十二烷基硫酸盐-聚丙烯酰胺凝胶电泳（SDS-PAGE）。 细胞因子可以从凝胶中洗脱，保留时间为20 min。 生物活性 从最初的10个初步序列中 RDCHF不同于所有测序的细胞因子。 我们还证明了至少存在一个RDCHF克隆 在cDNA文库中，我们已经从RDM 4细胞产生 RDCHF。 我们已经开始细分文库以分离cDNA 克隆 我们建议改进RDCHF纯化，以实现 获得纯材料的可重复技术。 我们将结合 放射性标记的RDCHF对来自已建立的细胞系或来自 荧光活化细胞分离的粗细胞制剂 分选仪（FACS）。 这样我们就能确定眼前的目标 因子的细胞。 我们还将结合放射性标记的RDCHF 与靶细胞上的受体共价结合，以研究 受体（S）。 我们还计划完成RDCHF cDNA克隆的分离。 我们将产生瞬时表达载体来表达 在COS细胞系中大量的细胞因子产生大量的 大量的蛋白质用于生物学研究。 我们将使用cDNA 克隆分离基因组克隆以研究基因的结构 为因子编码。 我们将进行杂交研究， 鉴定产生RDCHF的细胞，并研究 RDCHF基因。 我们还将尝试分离出一种 人类基因组中的基因。 一旦我们能够制造大量纯化的RDCHF， 试图制造这种细胞因子的单克隆抗体， 生物学研究和简化因子纯化。

We have developed an assay for cytokines needed for the in vitro generation of primary mouse cytolytic T lymphocytes (CTL) against alloantigens on tumor cells. One of the factors which functions in this assay is called RDCHF. We have recently purified RDCHF to a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cytokine could be eluted from the gel with retention of biological activity. From a preliminary sequence of the first 10 amino acids, RDCHF is different from all sequenced cytokines. We have also demonstrated that at least one RDCHF clone exists in a cDNA library we have made from RDM4 cells producing RDCHF. We have begun to subdivide the library to isolate cDNA clones. We propose to refine the RDCHF purification to achieve a reproducible technique for obtaining pure material. We will bind radiolabeled RDCHF to cells from established lines or from preparations of crude cells separated by fluorescent activated cell sorter (FACS). In this way, we will identify the immediate target cells of the factor. We will also bind radiolabeled RDCHF covalently to its receptor(s) on target cells to study the nature of the receptor(s). We also plan to complete the isolation of cDNA clones to RDCHF. We will generate transient expression vectors to express the cytokine in large amounts in the COS cell line to produce large amounts of protein for biological studies. We will use the cDNA clone to isolate genomic clones to study the structure of the gene coding for the factor. We will perform hybridization studies to identify the cells making RDCHF and to study the expression of the RDCHF gene. We will also attempt to isolate a homologous gene from the human genome. Once we can make large amounts of purified RDCHF, we will attempt to make monoclonal antibodies to this cytokine both for biological studies and for simplification for factor purification.