STRUCTURAL - BIOLOGICAL CHARACTERIZATION OF RECOMBINANT HUMAN IFN ALPHA RECEPTOR

重组人干扰素α受体的结构-生物学特征

• 批准号: 3770350
• 负责人: N Y NGUYEN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3770350
• 关键词: biological signal transduction; glutathione transferase; immune response genes; immunological substance; interferon alpha; molecular cloning; monoclonal antibody; neoplastic growth; protein purification; protein structure; receptor binding; receptor expression; recombinant DNA; virus replication

Studies were conducted to express and purify the extracellular domain and subdomains of human IFN alpha 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 in receptor-ligand binding assays. Soluble Form of GST-Receptor: Induction of protein expression with 0.1 mM IPTG at 30 degrees C, sequential disruption with french press in Tris pH 9.0 buffer containing DTT, EDTA, PMSF and CHAPS (or Triton) released HuIFN- R soluble form from inclusion bodies. Expression and Isolation of HuIFN-R Subdomains: Three subdomains (residues 1-103, 93-260 and 224-401) were expressed in PRSET vector and served to identify several hybridoma colonies which reacted with specific regions found in each fragment. Cloning and monoclonal antibody production from those colonies are near completion. Antibody Production: Polyclonal antibodies against GST-receptor produced 25 - 30% inhibition of both IFN alpha binding to receptor and IFN biological activity. Testing of inhibition of IFN biological properties by monoclonal antibodies is in progress. 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.

进行研究以表达和纯化胞外结构域， 人IFN α受体（HuIFN-R）的亚结构域并产生抗体 用于研究信号转导机制。 HuIFN-R的表达和纯化：编码细胞外因子的基因 HuIFN-R结构域与谷胱甘肽-S-转移酶融合表达 （GST）。 GST-受体是从包涵体中分离的， （可溶形式）和存在（不溶性形式）8 M尿素。 整体复苏 约为1毫克/升细胞培养物。 不溶性和可溶性 抑制5 - 10 U的抗病毒和抗增殖活性 IFN α的受体-配体的Kd值约为10 - 9， 结合测定。 GST-受体的可溶性形式：用0.1 mM IPTG在30 ° C下，在Tris pH中用法式压榨机连续破坏 9.0含有DTT、EDTA、PMSF和CHAPS（或Triton）的缓冲液释放HuIFN-γ 从包涵体中可溶出来。 HuIFN-R亚结构域的表达和分离：三个亚结构域（残基 1 - 103、93 - 260和224 - 401）在PRSET载体中表达，并用于 鉴定了几个与特定区域反应的杂交瘤菌落 在每一个碎片中。 克隆和单克隆抗体生产 这些殖民地已经接近完成。 抗体产生：产生抗GST-受体的多克隆抗体25 - IFN α与受体结合和IFN生物学抑制均为30% 活动单克隆抗体对干扰素生物学特性的抑制试验 抗体正在进行中。 这些调查的结果将大大丰富我们的 了解干扰素和其他细胞因子 调节免疫反应并控制病毒和肿瘤生长， 他们的受体。