PHOTOPROTEINS AS LABELS IN BIOLUMINESCENCE ASSAYS

光蛋白作为生物发光测定中的标记

• 批准号: 6636061
• 负责人: Sylvia Daunert
• 金额: $ 19.53万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636061
• 关键词: bioluminescence; calcium indicator; chimeric proteins; fusion gene; immunologic assay /test; ligands; lysine; method development; molecular cloning; nucleic acid sequence; plasmids; polymerase chain reaction; protein sequence; reagent /indicator; site directed mutagenesis

The objective of this proposal is to demonstrate that naturally occurring photoproteins and their mutants can be used in the design and development of highly sensitive luminescent assays capable of small volume analysis. In particular, we propose to prepare and characterize new genetically engineered variants of aequorin, obelin, and other photoproteins, so that they are capable of emission of light at different wavelengths than those of the corresponding wild-type photoproteins. These photoproteins will be then employed as reagents in the development of assays for physiologically important molecules and for the high-throughput screening of biopharmaceuticals. By using the different variants of the photoproteins, array detection of biomolecules will be investigated. Research performed by our group during the current budget period demonstrated the usefulness of employing recombinant conjugates in the highly sensitive detection of biomolecules. We now propose to design and develop new methods based on the bioluminescence of photoproteins that are suitable for the microanalysis of important biomolecules, such as vitamins, drugs, peptides, etc., in nL-sized samples.

该提案的目的是证明天然存在的发光蛋白及其突变体可用于设计和开发能够进行小体积分析的高灵敏度发光测定法。 特别是，我们建议制备和表征水母发光蛋白、奥贝林和其他发光蛋白的新基因工程变体，以便它们能够发射与相应野生型发光蛋白不同波长的光。 然后，这些发光蛋白将用作试剂，用于开发生理学重要分子的检测方法和生物药物的高通量筛选。 通过使用发光蛋白的不同变体，将研究生物分子的阵列检测。 我们小组在当前预算期间进行的研究证明了采用重组缀合物在生物分子的高灵敏度检测中的有用性。 我们现在建议设计和开发基于光蛋白生物发光的新方法，适用于 nL 大小样品中重要生物分子（如维生素、药物、肽等）的微量分析。