EXPRESSION AND CHARACTERIZATION OF RECOMBINANT HUMAN INTERFERON ALPHA RECEPTOR

这项研究始于1991年6月，其长期目标是表达 并鉴定了最近分离到的基因编码的蛋白质 推测为I型干扰素受体。目前，人们对此知之甚少 关于受体蛋白的生化和功能性质。这个 从美国典型培养体系中获得了含有该基因的载体 收藏。全长插入，编码干扰素受体， 通过定点突变，被转化为编码 通过将一个氨基酸密码子改变为一个 在跨膜区之前停止密码子。这张插页是 亚克隆到名为PDR的真核表达载体(获得自 T.Kishimoto，日本大阪大学)，用于二氢叶酸的表达 还原酶阴性的中国仓鼠卵巢细胞(DG44，来源于L.Chasin， 哥伦比亚大学，纽约)。整个cdna已经被测序以证实 所做的改变。努力产生一种可溶性形式的受体 蛋白质的研究正在进行中。蛋白质产品将被提纯并用于 产生针对该蛋白质的多克隆和单克隆抗体。在……里面 此外，还将进行实验来评估这一能力 易于与多种类型的人干扰素-α结合的蛋白质 在我们的实验室可以买到。这些研究应该会导致加强 了解干扰素系统中的配体-受体相互作用。 尝试在细菌中表达的推论研究也在进行中 系统(主要在Nguyen博士的实验室中)，并制造抗体 这些产品。

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.