INTRACELLULAR MODULATION OF SPECIFIC INTRACELLULAR PROTEINS

我们想要在细胞内调节表达的蛋白质 翻译级。我们的方法是提供一种替代方案 反义技术。为了完成这项任务，我们有分子上的重新- 改造了丙种球蛋白重链基因的DR1或DR3区域 通过随机化过程获得片段以产生重组蛋白 约10(12)个不同免疫球蛋白片段的文库 不同的抗原识别部位。可以对图书馆进行筛选以找到 通过融合蛋白质来识别给定蛋白质的个体克隆 与M13p8重组。我们已经证明，我们可以分离出一种新的 在大型随机文库中发现的工程化重组蛋白 与SV40大T抗原特异性紧密结合。在这 在特殊情况下，大T抗原被选为靶标是因为 大T抗原是一种转录因子，已被发现 在间皮肿瘤细胞系中选择性地连续转化。 Hc基因的前导序列被移除，并被一个 核仁或核仁靶向序列以促进Re-Re的运动 工程化的HC连接到T抗原发挥作用的细胞核。在……里面 此外，癌细胞内的癌基因或转录因子是 潜在的感兴趣目标。该方法将允许一种新的方法 寻找与修复或抵抗有关的未知蛋白质 电离辐射。这种方法学为基因治疗提供了一条新的途径。 用于治疗癌症或病毒感染的细胞。该项目需要 免疫球蛋白基因结构、基因和蛋白的知识和应用 融合表达系统，定点突变，聚合酶链 反应、核苷酸测序、质粒和载体构建、蛋白质 筛选和其他分子生物学技术。

We want to regulate expressed proteins within cells at the post- translational level. Our approach is to provide an alternative to antisense technology. To accomplish the task we have molecularly re- engineered the DR1 or DR3 region of gamma globulin heavy chain (HC) gene fragment by randomization processes to produce a recombinatorial protein library of approximately 10(12) different immunoglobulin fragments having different antigen recognition sites. The library can be screened to find an individual clone that recognizes a given protein by fusing the recombinant to the M13p8. We have shown that we can isolate a newly engineered recombinant protein found within the large random library that binds specifically and tightly to SV40 large T-antigen. In this particular case the large T-antigen was chosen as a target because the large T-antigen is a transcriptional factor that has been found to transform selectively and continually in a mesothelial tumor cell line. The leader sequence of the HC gene was removed and replaced by either a nuclear or nucleolar targeting sequence to promote movement of the re- engineered HC to the nucleus where the T-antigen is functioning. In addition, oncogenes or transcriptional factors within cancer cells are potential targets of interest. The method will allow for a new approach to find yet unknown proteins that contribute to repair of or resistance to ionizing radiation. This methodology provides a new path to gene therapy for treatment of cancer or virus infected cells. The project requires knowledge and use of immunoglobulin gene structure, gene and protein fusion expression systems, site directed mutagenesis, polymerase chain reaction, nucleotide sequencing, plasmid and vector construction, protein screening, and other molecular biologic techniques.