Probe and method for detecting membrane of glucose transporter 4

A technology for glucose transport and probe, applied in biochemical equipment and methods, measurement devices, biological tests, etc., can solve the problems of difficult to achieve high-throughput screening of drugs, lack of high-throughput detection methods, and troublesome operation.

Inactive Publication Date: 2008-09-17
北京普赛资产管理有限责任公司
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Problems solved by technology

[0010] (2) The use of drug screening methods to screen out new drugs with anti-insulin resistance from existing chemical drug libraries, plants, and traditional Chinese medicine libraries has good development prospects, but there is a lack of convenient and reliable high-throughput detection methods
The method of using biochemical antibody detection has many disadvantages, such as cumbersome operation; the required sample volume is large, and it is difficult to obtain relatively pure cell membrane compo

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  • Probe and method for detecting membrane of glucose transporter 4
  • Probe and method for detecting membrane of glucose transporter 4
  • Probe and method for detecting membrane of glucose transporter 4

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Embodiment Construction

[0036] We used two fluorescent proteins, pHluorin and TDimer2, to label amino acids 1–131 of the insulin-responsive aminopeptidase (IRAP) protein that fully co-localized with GLUT4 (exactly similar experimental results were also obtained using full-length IRAP, data not shown). We fused the pH-sensitive fluorescent protein pHluorin in front of IRAP131 (1-131 amino acids of the IRAP protein), and fused the red fluorescent protein TDimer2 insensitive to pH behind it to obtain the fusion protein TDimer2-IRAP-pHluorin. pHluorin does not emit light in an acidic environment, but emits green fluorescence when excited by 488nm light in a neutral or near-neutral environment. The constructed fluorescent protein is introduced into fat cells (lines) or muscle cells (lines) for expression through liposome or electroporation, and a stable cell line is established. Before stimulation by drugs such as insulin, pHluorin is in the acidic GLUT4 vesicles, so the cells only observe the red fluores...

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Abstract

The invention relates to a probe and method for detecting the glucose transporter protein (GLUT4) membrane, to realize high pass screening. The red fluorescence protein TDimer2 for probe and pH-sensitive fluorescence protein pHluorin mark the insulin-sensitive IRAP protein (TDimer2-IRAP-pHluorin) to establish fat cell system and skeletal muscle cell system for stably expressing the TDimer2-IRAP-pHluorin protein and the effect of the insulin on the target cell can be adjudged by simply measuring the fluorescence ratio. Thus, the high pass screening of the medicine for activating the GLUT4 membrane can be performed by detecting the ratio variance of the pHluorin and TDimer2 fluorescence intensity after medicine activating. The method is simple and easy for high pass screening and industrialization with high sensitivity.

Description

technical field [0001] The present invention relates to a fusion protein probe, more specifically to a probe for detecting glucose transporter 4 (Glucose Transporter 4, GLUT4) on the membrane (that is, to the transport of the cell membrane), and a method for detecting glucose transporter 4 (GLUT4) on-membrane high-throughput screening method. The probe and method can be used to quickly and efficiently screen out the GLUT4 membrane-promoting drugs that can regulate the blood sugar concentration in the body and treat diabetes from the existing chemical drug library, traditional Chinese medicine library and other resource libraries. Background technique [0002] Diabetes and GLUT4 [0003] As we all know, glucose is the basic energy source of animals, and its metabolism, synthesis and storage are all subject to strict and complex regulation. In humans, the blood sugar concentration is strictly controlled between 4-7mM. Deviation from this interval will lead to diseases, for...

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Application Information

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IPC IPC(8): G01N33/52G01N33/53C12Q1/37G01N21/00
Inventor 徐涛徐平勇范俊梅姜丽
Owner 北京普赛资产管理有限责任公司
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