INTRACELLULAR MODULATION OF SPECIFIC INTRACELLULAR PROTEINS

特定细胞内蛋白质的细胞内调节

• 批准号: 3752415
• 负责人: A RUSSO
• 金额: --
• 依托单位: DIVISION OF CANCER TREATMENT
• 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/3752415
• 关键词: chemical binding; chimeric proteins; fusion gene; gamma globulin; gene expression; genetic library; genetic regulation; immunoglobulin genes; nucleic acid sequence; plasmids; polymerase chain reaction; posttranslational modifications; recombinant proteins; site directed mutagenesis

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.

我们想要调控细胞内表达的蛋白