INTRACELLULAR MODULATION OF SPECIFIC INTRACELLULAR PROTEINS

我们希望在细胞内调节表达的蛋白质， 翻译水平。 我们的方法是提供一种替代方案， 反义技术 为了完成这个任务我们从分子上重新- 工程化γ球蛋白重链（HC）基因的DR 1或DR 3区 通过随机化过程片段化以产生重组蛋白质 约10（12）种不同免疫球蛋白片段的文库， 不同的抗原识别位点。 图书馆可以通过筛选来找到 一种单独的克隆，通过融合 重组到M13 p8。 我们已经证明，我们可以分离出一个新的 在大型随机文库中发现的工程重组蛋白， 与SV 40大T抗原特异性紧密结合。 在这 在特定情况下，选择大T抗原作为靶标，因为 大T抗原是一种转录因子，已发现其 在间皮瘤肿瘤细胞系中选择性和持续转化。 HC基因的前导序列被移除并被替换为 核或核仁靶向序列，以促进re-RNA的运动。 将HC工程化到T抗原发挥作用的细胞核中。 在 此外，癌细胞内的癌基因或转录因子， 潜在的目标。 该方法将允许一种新的方法 去寻找未知的蛋白质， 电离辐射 该方法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、基因和蛋白质知识和应用 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.