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Application of BRSK2 in producing antidiabetic medicine

An anti-diabetic and drug technology, applied in the field of biomedicine, can solve problems such as the effect of BRSK2 that has not been reported yet

Inactive Publication Date: 2008-02-27
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, so far, no one has reported the effects of BRSK2 on organs or tissues other than the brain and nerves

Method used

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  • Application of BRSK2 in producing antidiabetic medicine
  • Application of BRSK2 in producing antidiabetic medicine
  • Application of BRSK2 in producing antidiabetic medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 The specific steps of Northern hybrid expression of BRSK2 in pancreas are:

[0047] 1.1 cDNA probe preparation and purification

[0048] (1) Using specific primers of BRSK2 gene to amplify the PCR product from the constructed and sequenced plasmid. After the PCR product was purified with the QIAquick PCR purification kit, the concentration of the purified DNA was measured with a GeneQuant II UV spectrophotometer.

[0049] (2) After the DNA template was heat-denatured at 100°C for 2 minutes, the random primer labeling kit was used to incorporate [α- 32 P] dCTP. After the labeling reaction, EDTA was added to a final concentration of 20mmol / L to stop the reaction, heat denatured at 100°C for 5min, and placed on ice.

[0050] (3) Purify the probe with MicroSpin G-25 purification column. Before and after purification, 1 μl of the sample was taken and diluted 100 times, and 3 μl of the diluted solution was spotted on Whatman DE81 filter paper, and the radiation ...

Embodiment 2

[0057] Embodiment 2 prepares antibody

[0058] BRSK2 protein is used to immunize animals to produce antibodies, the specific method is as follows. The recombinant molecules are separated by chromatography for further use. It can also be separated by SDS-PAGE gel electrophoresis, and the electrophoresis bands are excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100 [mu]g / 0.2 ml emulsified protein. Fourteen days later, mice were boosted by intraperitoneal injection of the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 µg / 0.2 ml. Give booster immunizations at least three times every 14 days. The specific reactivity of the obtained antiserum was assessed by its ability to precipitate the human BRSK2 gene translation product in vitro. As a result, it was found that the antibody can specifically precipitate the protein of the present invention.

Embodiment 3

[0059] Example 3 BRSK2 Western hybridization experiment in pancreas

[0060] 3.1 Extraction of mouse tissue proteins

[0061] (1) All excised tissues were quickly frozen into liquid nitrogen tanks and stored at -80°C.

[0062] (2) Take 50-100 mg of tissue samples and add 400 μl of cell lysate (containing protease inhibitors).

[0063] (3) Grind the above-mentioned tissue sample with a grinder until it becomes a suspension.

[0064] (4) Add 100 μl of 5X protein loading buffer to 400 μl of the suspension, and boil at 100° C. for 10 minutes.

[0065] (5) Centrifuge at 12,000 rpm for 5 minutes at 4°C, and the supernatant can be used for sample loading.

[0066] 3.2 Western detection of endogenous expression of BRSK2 in various tissues of mice

[0067] (1) Prepare SDS-PAGE separating gel and stacking gel.

[0068] (2) Perfusion of the gel (the process is as follows)

[0069] Perfuse separation gel-→add absolute ethanol to remove air bubbles-→stand at room temperature and wait...

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Abstract

The invention discloses an application of BRSK2 to prepare antidiabetic drug in the biological medical domain, wherein the diabetes mellitus is a group of clinical syndrome interacted by genetic and environmental factor, which bases on hyperglycemia as main mark to lead the harm of multiple systems due to lingering illness; the BRSK2 can inhibit the function of insulin secretory as new antidiabetic drug and target, which provides a new approach and way to treat and test diabetes.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a new application of a gene related to neuron differentiation, namely BRSK2. Background technique [0002] BRSK2 (Brain Selective Kinase 2) protein kinase was classified into the AMPK (AMP-activated Protein Kinase) kinase subfamily by Manning et al. in 2002 according to the conservation of amino acids in the kinase domain. There are 14 members in this subfamily, including: AMPKα1, AMPKα2, BRSK1 / SAD-A, BRSK2 / SAD-B, MARK1, MARK2, MARK3 / Par-1A / C-TAK1, MARK4, NUAK1 / ARK5, NUAK2 / SNARK, QIK, QSK, SIK and MELK. [0003] Among these homologous kinases, AMPK is considered a probe that monitors cellular energy status. Depletion of ATP and an increase in AMP concentration, or more precisely, an increase in the AMP:ATP ratio, leads to the activation of AMPK. After activation, AMPK will close the anabolic pathway that consumes ATP and activate the catabolic pathway that synthesizes ATP, thereby regu...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/17A61K39/395A61P3/10
Inventor 余龙汤文文郭泽坤袁健唐丽莎
Owner FUDAN UNIV
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