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Biological fluorescent probe for dynamically monitoring cholecystokinin and application thereof

A technology of cholecystokinin and fluorescent probe, applied in the field of biomedicine, can solve the problems of time-consuming, affecting the research and treatment process of brain diseases, low sensitivity, etc.

Pending Publication Date: 2021-08-10
黄燕婷
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the content of cholecystokinin in the brain is very small, and it is very easy to degrade, it is not easy to detect the content of cholecystokinin in the brain and the conditions for its release, which greatly affects the research and treatment of brain diseases.
[0004] Traditional cholecystokinin detection techniques, such as mouse cerebrospinal fluid puncture dialysis combined with liquid chromatography-mass spectrometry (LC-MS), are time-consuming, low-sensitivity, and low-success rate, and the concentration of cholecystokinin in the detection is maintained at the skin level. Molar level, much lower than its content in the brain (nanomolar) level
Moreover, most cholecystokinin was diluted by the artificial cerebrospinal fluid used in dialysis during the detection process, which could not reflect the change of cholecystokinin content in normal or diseased mice

Method used

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  • Biological fluorescent probe for dynamically monitoring cholecystokinin and application thereof
  • Biological fluorescent probe for dynamically monitoring cholecystokinin and application thereof
  • Biological fluorescent probe for dynamically monitoring cholecystokinin and application thereof

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preparation example Construction

[0021] The invention provides a bioluminescent probe for dynamically monitoring cholecystokinin, the fluorescent probe includes a recombinant adenovirus probe, and the preparation method of the recombinant adenovirus probe includes: adding pH-sensitive cholecystokinin protein molecules ( figure 2 ) is inserted into the specific site of the adeno-associated virus capable of recognizing specific Cre-protease.

[0022] The pH-sensitive cholecystokinin protein molecule of the present invention preferably utilizes figure 2 In the plasmid, Pish-Cck and pUV-15PhGFP are combined by restriction endonucleases BspQI and BstBI to form CCK-pHluorin. The combined CCK-pHluorin was inserted into the AAV9-EF1a-DIO element by enzyme digestion (restriction enzymes BmtI and EcoRI). The adeno-associated virus of the present invention also includes a CCK-pHluorin element, which can specifically detect the release rule of the CCK protein molecule at the neuron synapse according to the acid-base ...

Embodiment 1

[0036] 1. Entrust the virus manufacturing company under the Hong Kong University of Science and Technology to construct such figure 2 The indicated AAV9-EF1A-DIO-CCK-pHluorin, formed figure 1 The bioluminescent probes of the shown viral structures were dissolved in water to give 1×10 13 vg / ml CCK probe solution; take out 10 μL of CCK probe and put it in an ice box.

[0037] 2. Take out an equal amount of 10 μL optogenetic probe and put it in an ice box. The photosensitive probe used in this experiment is AAV9-Syn-ChrimsonR-tdt (1×10 12 vg / ml) probe was purchased from Addgene, USA. The excitation fluorescence of the light-sensing probe is 560nm, which is filtered by a 590nm band-pass filter to display red fluorescence.

[0038] 3. Mix the two probes 1:1 (v:v) and mix well to obtain a total of 20 μL of probe mixture.

[0039] 4. The object of this experiment is Grin1 at 6-8 weeks fl / fl The homozygous mice obtained by crossing with CCK-Cre, and the transgenic mice used wer...

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Abstract

The invention provides a biological fluorescent probe for dynamically monitoring cholecystokinin and application thereof, and relates to the technical field of biological medicines. The biological fluorescent probe (CCK probe) is provided with an element capable of specifically recognizing neurons carrying Cre-protease, so that the biological fluorescent probe can be expressed in the specific neurons; the CCK probe can explore the release rule of the neuronal synapse CCK neurotransmitter by comparing the change of the fluorescence intensity of the biological fluorescent probe. The CCK probe can mark the cholecystokinin in specific neurons and dynamically detect the release process of the cholecystokinin after neuronal synapse, can specifically recognize the cholecystokinin before and after the synapse, and is high in detection sensitivity, strong in anti-interference capability and low in trauma.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a bioluminescent probe for dynamically monitoring cholecystokinin and an application thereof. Background technique [0002] Cholecystokinin (CCK) is an essential protein hormone in the digestive system, which can stimulate the secretion of gastric acid in the stomach, bile in the liver, inhibit the absorption of sodium and water in the ileum, and stimulate the release of insulin and glucagon from the pancreas. Promotes digestion of fat and protein. At the same time, cholecystokinin is also an appetite suppressant, and the lack of cholecystokinin will cause overeating and lead to obesity. Recent studies have pointed out that cholecystokinin exists in the brain system at the same time, including the cerebral cortex, hippocampus, hypothalamus and amygdala, etc., but its central role is still unclear, and it is initially shown that cholecystokinin is closely related ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/16G01N21/64
CPCC12N7/00C07K14/595G01N21/6458G01N21/6428C12N2750/14121
Inventor 黄燕婷
Owner 黄燕婷
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