CYTOLYTIC T LYMPHOCYTE HELPER FACTOR

溶细胞T淋巴细胞辅助因子

• 批准号: 3172812
• 负责人: DAVID P FAN
• 金额: $ 18.06万
• 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 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3172812
• 关键词: T lymphocyte; complementary DNA; cytokine; genetic manipulation; high performance liquid chromatography; immunochemistry; interferons; interleukin 2; leukocyte activation /transformation; major histocompatibility complex; monoclonal antibody; tissue /cell culture; tumor antigens

Recently we have developed an assay for cytokines needed for the in vitro generation of primary murine cytolytic T lymphocytes (CTL) against alloantigens on tumor cells. The factors which function in the assay are called CHF for CTL helper factor. This assay can detect previously uncharacterized cytokines including a factor of 30,000 molecular weight (RDCHF) made by the cell line RDM4. We have been able to concentrate this cytokine by adsorption to Sepralyte and to obtain a good degree of purification using reverse phase HPLC. We will continue to purify this molecule to a single major protein species for use in biological experiments and for obtaining an N terminal amino acid sequence. We will confirm that the protein we begin to sequence is actually RDCHF by cloning the molecule using recombinant DNA techniques. We have already accomplished the key step in this procedure, the expression of the molecule in Xenopus laeis oocytes. Thus we are able to assay the mRNA specific for the cytokine. Our next steps are: (1) Construction of cDNA libraries. (2) Identification of RDCHF cDNA. (3) Analysis of the nucleotide sequence of RDCHF cDNA. (4) Expression of recombinant RDCHF cDNA in mammalian cells. Once we have obtained reasonably homogeneous RDCHF, we will test it for its biochemical properties. We will also test its biological properties including the binding of radiolabeled RDCHF to different cells involved in CTL generation, the addition of RDCHF to spleen cells depleted of certain cell types, and the function of RDCHF in other help deficient systems for the generation of CTL in vitro. We have found novel molecules with CHF activity from not only RDM4 but also other cell lines such as EL4 and ASFTL-1. We propose to repeat the studies described above with these cytokines.

最近，我们已经开发了一种体外细胞因子所需的测定法， 产生原代鼠细胞溶解性T淋巴细胞（CTL）， 肿瘤细胞上的同种异体抗原。 在测定中起作用的因素是 称为CHF的CTL辅助因子。 该检测方法可以检测先前 未表征的细胞因子，包括分子量为30，000的因子 （RDCHF）由细胞系RDM4产生。 我们已经能够通过吸附到Sepralyte上来浓缩这种细胞因子 并使用反相HPLC获得良好的纯化度。 我们 将继续将这种分子纯化成单一的主要蛋白质种类， 在生物学实验中的用途以及用于获得N末端氨基酸的用途 顺序 我们将确认我们开始测序的蛋白质实际上是RDCHF， 使用重组DNA技术克隆所述分子。 我们已经 完成了这一过程中的关键步骤，表达了分子 非洲爪蟾卵母细胞中。 因此，我们能够检测特异于 细胞因子 我们下一步的工作是：（1）cDNA文库的构建。 (2)RDCHF cDNA的鉴定。 (3)核苷酸序列分析 的RDCHF cDNA。 (4)重组RDCHF cDNA在哺乳动物细胞中的表达。 一旦我们获得了合理均匀的RDCHF，我们将测试它的 生化特性 我们还将测试它的生物特性 包括放射性标记的RDCHF与不同细胞的结合， CTL生成，将RDCHF添加到某些CTL缺失的脾细胞中， 细胞类型，以及RDCHF在其他帮助缺乏系统中的功能， 体外CTL的产生。 我们发现了具有CHF活性的新型分子，不仅来自RDM 4， 其他细胞系如EL4和ASFTL-1。 我们建议重复研究 与这些细胞因子一起使用。