CYTOLYTIC T LYMPHOCYTE HELPER FACTOR

最近，我们开发了一种体外培养所需的细胞因子的分析方法。 抗小鼠原代细胞溶解T淋巴细胞(CTL)的制备 肿瘤细胞上的同种异体抗原。在检测中起作用的因素有 CTL辅助因子称为CHF。这个测试可以检测到之前 未知的细胞因子，包括一个分子量为30,000的因子 (RDCHF)由细胞系RDM4制成。 我们已经能够通过吸附到Sepralyte来浓缩这种细胞因子 并用反相高效液相色谱法获得了较好的纯度。我们 将继续将这种分子提纯成单一的主要蛋白质物种 用于生物实验和获得N端氨基酸 序列。 我们将通过以下方式确认我们开始测序的蛋白质实际上是RDCHF 使用重组DNA技术克隆分子。我们已经这么做了 完成了这个过程中的关键步骤，分子的表达 在非洲爪蛙卵母细胞中。因此，我们能够分析特异性的mRNA。 细胞因子。我们下一步的工作是：(1)构建cDNA文库。 (2)RDCHF基因的鉴定。(3)核苷酸序列分析 RDCHF基因的表达。(4)重组RDCHF基因在哺乳动物细胞中的表达。 一旦我们获得了相当均匀的RDCHF，我们将对其进行测试 生化特性。我们还将测试它的生物学特性 包括放射性标记的RDCHF与参与的不同细胞的结合 CTL的产生，RDCHF的加入使脾细胞耗尽某些 细胞类型，以及RDCHF在其他帮助不足的系统中的功能 CTL的体外生成。 我们不仅从RDM4中发现了具有CHF活性的新分子，而且还发现了 其他细胞系，如EL4和ASFTL-1。我们建议重复这些研究 上面描述了这些细胞因子。

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.