STRUCTURAL AND BIOLOGICAL CHARACTERIZATION OF A RHIFN ALPHA RECEPTOR

进行了表达和纯化胞外结构域和 人干扰素-α受体亚区(Hu干扰素-R)及其抗体的制备 用于研究信号转导机制。表达式和 编码人干扰素-R胞外区基因的纯化 人干扰素受体与谷胱甘肽S转移酶融合表达。 在缺乏GST受体的情况下，从包涵体中分离GST受体 (可溶形式)和存在(不可溶形式)的8M尿素。总括 细胞培养回收率约为1毫克/升。两者都是不可溶的 而可溶性形式抑制了抗病毒和抗增殖 5-10U的干扰素活性，其Kd值约为 受体-配基结合实验：10-9M。芘标记的结合作用 用荧光法测定干扰素-AB对纯化受体蛋白的作用 极化。结果表明，荧光的饱和增强。 随着受体蛋白浓度的增加，各向异性。AS 预期，通过预先孵育标记的干扰素- AB的未标记干扰素超量10倍。GST受体的可溶性形式： 在30oC下用0.1 mM IPTG诱导蛋白质表达，序贯 在含有DTT、EDTA的Tris pH 9.0缓冲液中用法国印刷机中断， PMSF和CHAPS(或Triton)以可溶性形式从 包涵体。大约20%的HuIFNR是在 细胞裂解物的上清液。80%的HUIFNR表达 仍然留在包涵体中，只在 存在8M尿素。人干扰素受体亚区的表达和分离 三个亚域，命名为M1、M2和M3(残基1-103、93-260和224- 401)在PRSET载体中表达，用于鉴定单抗 与每个片段中发现的特定表位发生反应的抗体。 在三个子域中，只有两个，M2和M3似乎表现出 抗病毒和抗繁殖活性。此外，只有M3展出 与芘标记的干扰素-AB的饱和结合，其Kd值与 对纯化的受体融合蛋白进行了Kd测定。 抗体产生：产生抗GST受体的多克隆抗体 干扰素与受体和干扰素结合的抑制率为25%~30% 生物活性。几种单抗免疫沉淀 天然的细胞受体，并特异性识别细胞 蛋白质印迹图上的受体。所有的项目都已经完成。 这些调查的结果将大大丰富我们的 对干扰素和其他细胞因子作用机制的理解 通过调节免疫反应和控制病毒和肿瘤的生长 它们的受体。

Studies were conducted to express and purify the extracellular domain and subdomains of human IFN-a receptor (HuIFN-R) and to generate antibodies for the investigation of signal transduction mechanisms. Expression and Purification of HuIFN-R: The gene encoding the extracellular domain of HuIFN-R was expressed as fusion with glutathione-S-transferase (GST). The GST-Receptor was isolated from inclusion bodies in the absence (soluble form) and presence (insoluble form) of 8 M urea. Overall recovery was approximately 1 mg/liter of cell culture. Both insoluble and soluble forms inhibited the antiviral and antiproliferative activities of 5 -10 U of IFN alpha and showed a Kd value of approximately 10-9 M in receptor-ligand binding assays. The binding of pyrene-labeled IFN-aB to the purified receptor proteins was measured using fluorescence polarization. Results indicated a saturable increase in the fluorescence anisotropy with increasing concentrations of receptor proteins. As expected, reduced binding was obtained by preincubating the labeled IFN- aB with a 10-fold excess of unlabeled IFN. Soluble Form of GST-Receptor: Induction of protein expression with 0.1 mM IPTG at 30oC, sequential disruption with french press in Tris pH 9.0 buffer containing DTT, EDTA, PMSF and CHAPS (or Triton) released HuIFN-R in soluble form from inclusion bodies. Approximately 20% of HuIFNR was obtained in the supernatant of cell lysate. Eighty per cent of the expressed HUIFNR still remained in the inclusion bodies and were solubilized only in the presence of 8M urea. Expression and Isolation of HuIFN-R Subdomains: Three subdomains, named M1, M2 and M3 (residues 1-103, 93-260 and 224- 401) were expressed in PRSET vector and served to identify monoclonal antibodies which reacted with specific epitopes found in each fragment. Of the three subdomains, only two, M2 and M3 appeared to exhibit antiviral and antiproloferative activities. Further, only M3 exhibited saturable binding to pyrene-labeled IFN-aB with a Kd comparable to the Kd which was determined for the purified receptor fusion-proteins. Antibody Production: Polyclonal antibodies against GST-receptor produced 25 - 30% inhiition of both IFN alpha binding to receptor and IFN biological activity. Several monoclonal antibodies immunoprecipitated the natural cellular receptor and specifically recognized the cellular receptor on western blots. All the projects have been completed. Findings from these investigations will significantly enrich our understanding of the mechanisms by which interferons and other cytokines modulate immune responses and control viral and neoplastic growth through their receptors.