Probe and method for detecting membrane of glucose transporter 4

A technology of glucose transport and probe, applied in biochemical equipment and methods, measurement devices, biological tests, etc., can solve the problem of difficult to achieve high-throughput screening of drugs, difficult to quantify the amount on the membrane, difficult to obtain cell membrane components, etc. question

Inactive Publication Date: 2012-07-04
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 components; these will affect the results of the assay, and it is difficult to achieve high-throughput screening of drugs
The method of using GFP to mark GLUT4 to detect GLUT4 also has some shortcomings. GLUT4 protein is a multi-transmembrane protein, and both the amino and carboxy ends are in the cytoplasm. Located on the cytoplasmic side of GLUT4 vesicles, it will emit light under 488nm wavelength laser excitation, so it is difficult to quantify the amount of GLUT4 on the membrane after insulin stimulation

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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

[0055] Hereinafter, the present invention will be described in detail with reference to Examples, which are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The scope of the invention is specifically defined by the appended claims.

[0056] 1. Plasmid construction

[0057]Firstly, total RNA was extracted from 3T3-L1 cells (ATCC CL-173, USA) with RNA small extraction kit RNeasy Mini Kit (Qiagen, GmbH, Germany), and GLUT4 was expanded by RT-PCR kit (Qiagen, GmbH, Germany). The cDNA encoding gene (GenBankAccession Number: NM_009204),

[0058] GLUT4 upstream primer sequence:

[0059] 5'-TACCGCTCGAGACAAGATGCCGTCGGGTTTTCCAGCAGATCG-3';

[0060] GLUT4 downstream primer sequence:

[0061] 5'-GCTAGGATCCCCGTCATTCTCATCTGGCCCTAAGTATTCAAG-3'.

[0062] PCR conditions used: 1. 50°C for 30 minutes; 2. 95°C for 3 minutes; 3. 95°C for 30 seconds; 4. 55°C for 30 seconds; 5. 72°C for 1 minute and 30 seconds; The amplified P...

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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 kind of fusion protein probe, more specifically relate to the probe that is used to detect glucose transporter 4 (Glucose Transporter4, GLUT4) upper membrane (that is, to the translocation of cell membrane), and a kind of detection glucose transporter 4 ( GLUT4) high-throughput screening method on the membrane. 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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