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Method for screening carbohydrate chip of carbohydrate binding protein competitive inhibitor

A competitive inhibitor and protein-binding technology, applied in the field of biomedicine, can solve the problems of undeveloped sugar chips, and achieve the effects of high feasibility, small protein dosage, and simple and rapid operation.

Pending Publication Date: 2022-06-07
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Whether Glycoarrays are effective enough to assess this binding competitiveness has not yet been explored

Method used

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  • Method for screening carbohydrate chip of carbohydrate binding protein competitive inhibitor
  • Method for screening carbohydrate chip of carbohydrate binding protein competitive inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Comparison of the competitiveness of novel coronavirus S protein and antithrombin AT-binding heparin

[0017] 1. Pretreatment of heparin chip:

[0018] The chip immobilized with heparin (the immobilized amount of heparin is 0.5 ng, the diameter of the spot is about 150-200 μm) is put into ultrapure water, soaked for about 30 seconds, and then taken out. After centrifugation and draining, the chips were carefully mounted on the chip holder. The chip holder will separate the sugar microarrays on the chip into independent square well spaces with a volume of about 150μL.

[0019] 2. Close:

[0020] Add 100 μL of TBST (Tris-HCl-Tween) buffer (150 mM NaCl, 20 mM Tris-HCl) containing 1% BSA (bovine serum albumin) by mass volume concentration (g / ml) to the square well space of each chip holder , pH=7.4, 0.05% Tween-20), blocked at room temperature for 1 h. After the BSA solution was aspirated, 100 μL of TBST was added for washing three times in total.

[0021] 3. Use TBST ...

Embodiment 2

[0026] Competitive comparison of heparin cofactor II and novel coronavirus S protein binding heparin

[0027] 1. Pretreatment of heparin chip:

[0028] The chip immobilized with heparin (the immobilized amount of heparin is 0.5 ng, the diameter of the spot is about 150-200 μm) is put into ultrapure water, soaked for about 30 seconds, and then taken out. After centrifugation and draining, the chips were carefully mounted on the chip holder. The chip holder will separate the sugar microarrays on the chip into independent square well spaces with a volume of about 150μL.

[0029] 2. Close:

[0030] Add 100 μL of TBST (Tris-HCl-Tween) buffer (150 mM NaCl, 20 mM Tris-HCl) containing 1% BSA (bovine serum albumin) by mass volume concentration (g / ml) to the square well space of each chip holder , pH=7.4, 0.05% Tween-20), blocked at room temperature for 1 h. After the BSA solution was aspirated, 100 μL of TBST was added for washing three times in total.

[0031]3. Using TBST buffer...

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Abstract

The invention relates to a carbohydrate chip method for screening a carbohydrate binding protein competitive inhibitor. On the basis that a carbohydrate chip technology is used for screening protein receptors, the inhibitor is added to cause the change of the fluorescence intensity of the known binding protein, so that the screening of the inhibitor of the known protein and the comparison of the competitiveness of the inhibitor are realized. According to the invention, the original screening function of the sugar chip technology is broken through, and the application of the sugar chip technology is expanded. The method can be used for carrying out primary affinity comparison on different sugar binding proteins and screening inhibitors with relatively strong competitiveness, and is simple, convenient and rapid to operate, small in protein dosage and high in feasibility. In addition, the method can also be applied to other biological chips, such as protein chips and gene chips.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a sugar chip method for screening sugar binding protein inhibitors. It is used for the screening of carbohydrate-binding protein inhibitors, the comparison of binding sugar competitiveness and the preliminary judgment of binding sugar sites. [0002] technical background [0003] Biochip is a biomolecular lattice formed by immobilizing biomolecules, such as oligonucleotides, DNA, RNA, proteins, polypeptides, polysaccharides, oligosaccharides, etc., on a substrate according to the principle of specific interaction between biomolecules. It is then hybridized with the substance to be tested (either fluorescently labeled itself or specifically bound to a fluorescently labeled antibody), and the number of sample molecules and the information of binding ligands can be obtained by detecting the hybridization signal intensity of each probe molecule. Among them, sugar chip technology ha...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/68G01N21/64
CPCG01N33/56983G01N33/68G01N21/6428
Inventor 梁鑫淼郑义闫竞宇李佳齐郭志谋
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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