MOLECULAR IMMUNOLOGY

分子免疫学

• 批准号: 3853561
• 负责人: T S PAPAS
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 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/3853561
• 关键词: bacteriophage lambda; genetic library; genetic techniques; immunoglobulin genes; immunoglobulins; molecular cloning; monoclonal antibody; oncoproteins; recombinant DNA

Recombinant DNA technology has greatly increased our ability to study gene structure and function. This technology can now be applied to monoclonal antibody (Mab) production. Monoclonal antibodies facilitate biochemical analysis of proteins by their highly-specific recognition of a single epitope. Conventional methods for generating Mabs are not capable of efficiently surveying the induced antibody response to a given antigen. For example, an individual animal has 5-10,000 different B-cell clones, each capable of producing unique antibodies to an antigen. However, with the current cell-fusion technique, only a few hundred different antibodies can be produced. Recombinant DNA technology allows the immunoglobin variable region genes to be amplified, which provides for the generation of a large CDNA library using the bacteriophage lambda-immuno-zap expression vectors. This library is much easier to access than hybridomas produced from cell fusion. Screening is also greatly enhanced, considering immunoglobin gene products of at least 50,000 clones or 1-10,000,000 antibodies can be readily examined in one day, compared to screening hybridomas, which is labor-intensive, time-consuming and expensive. Once produced, these expression vectors can be transfected into mammalian cells or used for making transgenic mice. Currently, we have cloned the heavy and light chain genes of the pan-ets (T-7) antibody directed against the human ETS2 oncoprotein. The light chain has been success-fully inserted into the lambda-Lcl expression vector and a library has been constructed. Work continues with progress to construct the heavy chain library.

重组DNA技术大大提高了我们的研究能力 基因结构与功能 这项技术现在可以应用于 单克隆抗体（Mab）生产。 单克隆抗体促进 通过对蛋白质的高度特异性识别来进行蛋白质的生化分析。 单个表位。 用于产生Mab的常规方法不是 能够有效地测量对给定的抗体的诱导的抗体应答， 抗原的 例如，单个动物具有5- 10，000种不同的B细胞， 克隆，每个克隆都能够产生针对抗原的独特抗体。 然而，利用目前的细胞融合技术， 可以产生不同的抗体。 重组DNA技术允许 待扩增的免疫球蛋白可变区基因， 为了使用噬菌体产生大的cDNA文库， 免疫抑制表达载体。 这个图书馆更容易 比从细胞融合产生的杂交瘤更容易获得。 筛选也是 大大增强，考虑到免疫球蛋白基因产物至少 50，000个克隆或1- 10，000，000个抗体可以很容易地在一个 与筛选杂交瘤相比，这是劳动密集型的， 既耗时又昂贵。 一旦产生，这些表达载体 可以转染到哺乳动物细胞中或用于制备转基因 小鼠 目前，我们已经克隆了pan-ets的重链和轻链基因 (T-7)针对人ETS 2癌蛋白的抗体。 光 链已成功插入到Lcl表达式中 载体和文库的构建。 工作继续取得进展 以构建重链文库。