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A fluorescent polypeptide substrate for detecting human gelatinase mmp-2 and its application

A MMP-2, fluorescent peptide technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, microbial determination/inspection, etc., to achieve the effects of long storage time, good stability, clear specificity and pertinence

Active Publication Date: 2022-02-18
FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, substrate similarity exists among MMPs families

Method used

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  • A fluorescent polypeptide substrate for detecting human gelatinase mmp-2 and its application
  • A fluorescent polypeptide substrate for detecting human gelatinase mmp-2 and its application
  • A fluorescent polypeptide substrate for detecting human gelatinase mmp-2 and its application

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Embodiment

[0049] Embodiment: The fluorescent polypeptide substrate of the present invention is chemically synthesized by Beijing Saibaisheng Bioengineering Co., Ltd., recombinant human MMP-2 and 9 are purchased from American R&D Systems Company, APMA and MMP-2, MMP-9 activity titration Fluorescent substrate XIV was purchased from Anaspec Company of the United States, and GM6001 (broad-spectrum MMPs inhibitor) was purchased from MCE Company of the United States.

[0050] 1. Synthesis and storage of fluorescent peptides

[0051] Chemically synthesize the fluorescent peptide substrate, the sequence is shown as Peptide II, the synthetic fluorescent peptide substrate is purified by high pressure liquid chromatography, and the molecular weight of the fluorescent peptide substrate is detected by mass spectrometry, as shown in figure 1 , figure 2 shown. Fluorescent peptide substrates are stored in a freeze-dried powder form at -20°C. DMSO dissolves the lyophilized powder of the fluorescent ...

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PUM

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Abstract

The invention discloses a fluorescent polypeptide substrate for detecting human gelatinase MMP‑2. The fluorescent polypeptide substrate includes an amino acid sequence as shown in Peptide II, and the first valine in the amino acid sequence is bound to the fluorescent group 5-carboxyfluorescein, and the 11th lysine is bound to the fluorescent quenching group 5 ‑Carboxytetramethylrhodamine. The fluorescent polypeptide substrate of the present invention reacts with human MMP-2, and its enzymatic reaction kinetic constant Km is 315 μ M, Kcat / Km: 2565M ‑1 ·S ‑1 ; Human MMP-9 with an enzyme activity concentration of less than 6 μM has almost no reaction with the fluorescent polypeptide substrate of the present invention. The fluorescent polypeptide substrate for detecting human MMP-2 activity of the present invention does not react with the same family of human gelatinase MMP-9, and has certain specificity. The method for detecting human MMP-2 and screening human MMP-2 inhibitors of the present invention is simple and quick to operate and has good application prospects.

Description

technical field [0001] The invention relates to a fluorescent polypeptide substrate capable of detecting human gelatinase-2 (MMP-2) containing a specific sequence or motif and an activity detection method thereof, belonging to the field of biotechnology. [0002] technical background [0003] Matrix metalloproteinases (Matrix Metalloproteinases, MMPs) can almost degrade various protein components of the extracellular matrix, and are a class of Zn-dependent 2+ Endopeptidase family, mainly derived from various tissues and cells such as brain tissue, endothelial cells, fibroblasts and smooth muscle cells [1] . According to substrate specificity, sequence similarity and domain composition, MMPs can be divided into gelatinases (MMP-2, MMP-9), collagenases (MMP-1, MMP-8, MMP-13, MMP-18), Stromal lytic enzymes (MMP-3, MMP-10, MMP-11), matrix lytic factors (MMP-7, MMP-26), membrane-type matrix metalloproteinases (MT-MMPs; (MMP-14, MMP-15, etc. ) and other secreted MMPs (MMP-19, MM...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/37G01N21/64
Inventor 孟照辉万雯叶雨佳
Owner FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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