CYTOLYTIC T LYMPHOCYTE HELPER FACTOR

溶细胞T淋巴细胞辅助因子

• 批准号: 3172817
• 负责人: DAVID P FAN
• 金额: $ 19.45万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3172817
• 关键词: Anura; T lymphocyte; antibody dependent killer cell; complementary DNA; cytokine; flow cytometry; genetic manipulation; high performance liquid chromatography; histocompatibility antigens; laboratory mouse; laboratory rat; leukocyte activation /transformation; molecular cloning; monoclonal antibody; nucleic acid probes; nucleic acid sequence; protein sequence; radiotracer; receptor; tissue /cell culture; transposon /insertion element; tumor antigens

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.

我们已经开发了一种体外培养所需的细胞因子的分析方法。 原代小鼠细胞溶解T淋巴细胞(CTL)的产生 对抗肿瘤细胞上的同种异体抗原。其中一个因素是 此检测中的功能称为RDCHF。 我们最近用钠将RDCHF提纯为单条带 十二烷基硫酸酯-聚丙烯酰胺凝胶电泳法(SDS-PAGE)。 细胞因子可以从凝胶中洗脱出来，保留 生物活性。从前10个人的初步序列 氨基酸，RDCHF与所有已测序的细胞因子不同。 我们还演示了至少存在一个RDCHF克隆 在我们用RDM4细胞制作的cDNA文库中， RDCHF。我们已经开始对文库进行细分，以分离出cdna 克隆人。 我们建议提纯RDCHF以实现 获得纯净材料的可重复性技术。我们将捆绑 放射性标记的RDCHF到已建立的品系或来自 荧光激活细胞分离粗细胞的制备 分类器(FACS)。通过这种方式，我们将确定直接的目标 该因子的细胞。我们还将绑定放射性标记的RDCHF 共价结合其受体(S)对靶细胞性质的研究 受体(S)。 我们还计划完成RDCHF的cDNA克隆的分离。 我们将生成瞬时表达载体来表达 COS细胞系中大量的细胞因子产生大量的 用于生物学研究的蛋白质数量。我们将使用cDNAs 克隆：分离基因组克隆以研究基因的结构 对因子进行编码。我们将进行杂交研究，以 RDCHF形成细胞的鉴定及其表达的研究 RDCHF基因。我们还将尝试分离出一种同源的 人类基因组中的基因。 一旦我们能够生产大量纯化的RDCHF，我们将 尝试制作这种细胞因子的单抗，以用于 生物学研究和简化因子纯化。