CLONING AND EXPRESSION OF HUMAN INTERLEUKIN-3

该项目的目标是开发一种商业上可行的 用于产生白细胞介素-3（IL-3）的人等效物的系统 作为治疗剂用于治疗 化疗或放疗导致造血功能受到抑制 治疗或正在从骨髓移植中恢复的患者。 同相 I，将cDNA克隆到GEM-1体外转录载体中。 一 在E.杆菌 复制平板接种后 质粒DNA将从文库的子集制备并线性化 限制性内切酶。 RNA将从cDNA转录， 每个池使用SP 6或T7 RNA聚合酶。 RNA池将被翻译为 在青蛙卵母细胞中。 将通过组织培养评估IL-3的表达 生物测定。 产生IL-3活性的那些文库亚群将 进一步细分直至分离出IL-3的纯克隆。 表达 生物活性IL-3在第一阶段将提供信息， 确定获得生产水平表达式的可行性， 第二阶段。 第三阶段将涉及扩大生产和纯化 IL-3的商业水平和试验的性能，以确定安全性， 药物的剂量和用途。

The goal of the project is the development of a commercially feasible system for the production of the human equivalent of interleukin-3 (IL-3) for use as a therapeutic agent in the treatment of patients whose hematopoiesis has been suppressed as a result of chemo- or radiation therapy or who are recovering from bone marrow transplantation. In Phase I, cDNA will be cloned into a GEM-1 in vitro transcription vector. A plasmid library will be established in E. coli. After replica plating plasmid DNA will be prepared from subsets of the library and linearized with restriction endonucleases. RNA will be transcribed from the cDNA in each pool using SP6 or T7 RNA polymerase. The RNA pools will be translated in frog oocytes. Expression of IL-3 will be assessed by tissue culture biological assays. Those library subsets which produce IL-3 activity will be further subdivided until pure clones of IL-3 are isolated. Expression of biologically active IL-3 in Phase I will provide information for determining the feasibility of obtaining production level expression in Phase II. Phase III will involve scale-up of production and purification of IL-3 to commercial levels and performance of trials to determine safety, dosage and usefulness of the drug.