CYTOLYTIC T LYMPHOCYTE HELPER FACTOR

我们开发了一种检测体外所需细胞因子的方法

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.