EXPRESSION AND CHARACTERIZATION OF RECOMBINANT HUMAN INTERFERON ALPHA RECEPTOR

重组人干扰素α受体的表达和表征

• 批准号: 3792468
• 负责人: M P HAYES
• 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/3792468
• 关键词: complementary DNA; cytokine receptors; genetic manipulation; human genetic material tag; interferons; monoclonal antibody; protein structure function; receptor binding; receptor expression

The long-term goal of this study, initiated in June, 1991, is to express and characterize the protein encoded by the recently isolated cDNA for the putative Type I interferon receptor. Currently, very little is known about the biochemical and functional nature of the receptor protein. The plasmid containing the cDNA has been obtained from American Type Culture Collection. The full-length insert, coding for the interferon receptor, has been converted, by site-directed mutagenesis, into a cDNA encoding a soluble form of this receptor by changing an amino acid condon into a stop condon just prior to the transmembrane region. This insert was subcloned into a eukaryotic expression vector called pdR (obtained from T. Kishimoto, Osaka University, Japan) for expression in dihydrofolate reductase -negative Chinese hamster ovary cells (DG44, from L. Chasin, Columbia University, NY). The entire cDNA has been sequenced to confirm the changes made. Efforts to generate a soluble form of the receptor protein are underway. The protein products will be purified and used to generate both polyclonal and monoclonal antibodies to the protein. In addition, experiments will be performed to assess the capacity of this protein to bind various species of human interferon-alpha readily available in our laboratory. These studies should lead to an enhanced understanding of ligand-receptor interactions in the interferon system. Corollary studies are also underway to attempt expression in bacterial systems (primarily in Dr. Nguyen's laboratory) and to make antibodies to these products.

这项研究于1991年6月开始，其长期目标是表达 并表征由最近分离的cDNA编码的蛋白质， I型干扰素受体 目前，我们对 关于受体蛋白的生化和功能性质。 的 含有cDNA的质粒已从美国典型培养物获得 收藏. 编码干扰素受体的全长插入物， 已通过定点诱变转化为编码 通过将氨基酸密码子改变为 终止密码子刚好位于跨膜区之前。 该插入物是 亚克隆入称为pdR的真核表达载体（获自 T. Kishimoto，Osaka University，Japan）用于在二氢叶酸盐中表达 还原酶阴性的中国仓鼠卵巢细胞（DG 44，来自L.查辛， 哥伦比亚大学，纽约）。 整个cDNA已经测序， 所做的改变 产生受体的可溶形式的努力 蛋白质正在进行中。 蛋白质产品将被纯化并用于 产生针对蛋白质的多克隆和单克隆抗体。 在 此外，还将进行实验以评估这种方法的能力。 易于结合各种人α干扰素的蛋白质 在我们的实验室里。 这些研究将有助于提高 了解干扰素系统中的配体-受体相互作用。 相关研究也在进行中，试图在细菌中表达。 系统（主要是在阮博士的实验室），并使抗体， 这些产品