一个低成本,灵敏的记者系统,使用膜连接的胡卜过氧化酶进行高效的基因表达分析
Mu-Shen Chang1, Chia-Yi Lee2, En-Shuo Liu3
1PhD Program in Life Science, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.
Analytical chemistry
|September 16, 2023
概括
一种新的膜绑定卜过氧化酶 (mHRP) 记者系统为高吞吐量分析提供了昂贵的光酶基质的经济有效的替代方案. 该系统提供了可比的灵敏度和更好的稳定性,减少了测试时间和成本.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 测试开发 测试开发
背景情况:
- 记者基因测试对于高通量查和信号通路分析至关重要.
- 对于常见的火虫光酶 (FLuc) 记者系统的基板的高成本限制了它们的广泛应用.
- 需要有负担得起和高效的记者基因测试替代品.
研究的目的:
- 开发和评估一种新的,具有成本效益的记者基因系统,使用膜连的胡卜过氧化酶 (mHRP).
- 在灵敏度,稳定性和成本方面,将mHRP系统的性能与传统的FLuc测试进行比较.
主要方法:
- 工程细胞在它们的表面表达与膜结合的胡卜过氧化酶 (mHRP).
- 使用像ABTS和TMB这样的基质评估mHRP酶活性.
- 将mHRP与FLuc报告系统的信号灵敏度和稳定性进行比较.
- 评估了测试持续时间,检测极限和成本效益.
主要成果:
- mHRP表现出与FLuc测定相比较的信号灵敏度.
- mHRP系统的基质半衰期比d-Luciferin长五倍以上.
- 测试时间缩短,检测极限延长了mHRP系统.
- 与FLuc测试相比,每次测试的成本显著降低 (三分之一至二十八分之一).
结论:
- 该mHRP报告员系统提供了一个稳定的,方便的,和显著更实惠的替代FLuc测试.
- 这种系统消除了对细胞溶解和昂贵基质的需求.
- mHRP是高通量分析,药物查和信号研究的一个有前途的工具.
相关概念视频
02:11Reporter Genes
11.5K
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
11.5K
03:31Labeling DNA Probes
8.2K
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
8.2K