Substrate, method and kit capable of detecting enzymatic activity of metalloproteinase ADAMTS-13 by utilizing MALDI-TOF-MS

A technology of enzyme activity and kit, which is applied in the field of medical molecular biology, can solve the problems of complex testing process, time-consuming, large molecular weight of recombinant protein, etc., to improve survival rate and quality of life, reduce treatment cost, and detect accurately Effect

Pending Publication Date: 2020-11-24
刘学博 +8
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Problems solved by technology

This recombinant protein has a large molecular weight, and the testing process is complex and time-consuming. It is difficult to meet the needs of large-scale TTP screening, and it is not suitable for the clinical needs of emergency diagnosis and treatment of patients in emergency rooms.

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  • Substrate, method and kit capable of detecting enzymatic activity of metalloproteinase ADAMTS-13 by utilizing MALDI-TOF-MS

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Embodiment Construction

[0046] In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0047] The invention discloses a method for detecting ADAMTS-13 enzyme activity in serum by using MALDI-TOF-MS, comprising the following steps:

[0048] Include the following steps:

[0049] (1) Expression and purification of ADAMTS-13 digestion substrate GST-vWF82-6XHis recombinant protein:

[0050] 1.1. The 82 amino acid fragments D1587-R1668 of vWF and 6 histidines (6XHis) at the C-terminus were inserted into the bacterial expression vector pGEX6p-1 with GST protein at the N-terminus;

[0051] 1.2. Select positive clones and transfect BL21-expressing bacteria;

[0052] The BL21 strain can eff...

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Abstract

The invention belongs to the technical field of medical molecular biology and protein polypeptide detection, and particularly relates to a substrate, a method and a kit capable of detecting the enzymatic activity of metalloproteinase ADAMTS-13 by utilizing MALDI-TOF-MS. The method comprises the following steps: (1) performing expression and purification of an ADAMTS-13 enzyme digestion substrate GST-vWF82-6XHis recombinant protein; (2) performing enzyme digestion reaction; and (3) enriching the ADAMTS-13 enzyme digestion product by a metal chelating chip, identifying and detecting the ADAMTS-13 enzyme digestion product by MALDI-TOF-MS, and obtaining the enzyme digestion activity of ADAMTS-13 in the serum sample by analysis software. The substrate, the method and the kit for in-vitro quantitative determination of ADAMTS-13 enzyme activity are good in sensitivity and accuracy, simple and convenient to operate, low in price and suitable for wide patient screening.

Description

technical field [0001] The invention belongs to the technical field of medical molecular biology and protein polypeptide detection, and in particular relates to a substrate, method and kit capable of detecting the enzymatic activity of metalloprotease ADAMTS-13 by using MALDI-TOF-MS. Background technique [0002] MALDI-TOF-MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) mainly consists of three parts: the first part is a metal slide, which can be directional selected as a metal chelate chip, and the second part is a time-of-flight mass spectrometer, which can also be called The mass spectrum reader, the third part is the system-specific analysis software. [0003] The detection method adopts a metal chelating chip loading chip, and the surface of the metal chelating chip can chelate positive divalent metal ions Ni, and can specifically capture protein polypeptides with histidine groups. [0004] The mass spectrometer is a matrix-assisted la...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/62G01N33/573
CPCG01N27/628G01N33/573G01N2800/222G01N2800/226G01N2800/34
Inventor 刘学博马龙华谭爱国王文静张佳华周游江游于海波李宁
Owner 刘学博
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