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Preparation method and application method for indicator for monitoring activity of protease in real time

A technology of proteolytic enzymes and real-time monitoring, applied in biochemical equipment and methods, chemical instruments and methods, microbial measurement/inspection, etc., can solve the problems of not providing an experimental tool

Inactive Publication Date: 2012-07-11
李兵辉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, biochemistry does not provide an experimental tool to quantitatively and accurately monitor the intracellular or extracellular physiological and pathological changes at the molecular level in experiments, so as to determine the dynamic behavior of living systems

Method used

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  • Preparation method and application method for indicator for monitoring activity of protease in real time
  • Preparation method and application method for indicator for monitoring activity of protease in real time
  • Preparation method and application method for indicator for monitoring activity of protease in real time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Taking the key enzyme caspase-3 in apoptosis as an example to illustrate the monitoring of this proteolytic enzyme, Caspase-3 plays an important role in apoptosis, and can be monitored conveniently and in real time by the method we describe The specific operation of Caspase-3 activity in cells is as follows (the fluorescent protein used in this example is Venus):

[0037] (1) Construct plasmid:

[0038] (a) The free N-terminal and C-terminal of the fluorescent protein are shortened by molecular biology techniques to ensure a shorter sequence for the function of the fluorescent protein;

[0039] (b) Cut it between 154 and 155 of the fluorescent protein to form two parts, N-terminal and C-terminal;

[0040] (c) Connect the original and shortened N-terminal and C-terminal, and insert the recognition site DEVD-G (Asp, Glu, Val, Asp, Gly) of caspase-3 at the same time, which is PAI;

[0041] (d) Link the cDNA expressing the protein (PAI) to a sui...

Embodiment 2

[0052] Example 2: Purify PAI-C3 containing Caspase-3 hydrolysis site (DEVDG, amino acid sequence) as the substrate of Caspase-3, mix Caspase-3 sample and PAI-3 substrate in the reaction system, and detect the reaction system The fluorescence intensity in the sample can be used to know the activity of Caspase-3 in the sample, and the results are shown in Table 2.

[0053] Table 2: In vitro detection of Caspase-3 activity using PAI-C3 as substrate

[0054]

[0055] PAI-C3 was transformed with Venus fluorescent protein (a yellow fluorescent protein) as a template; the reaction system was 2 ml of phosphate buffer (pH7.5); in the calculation of fluorescence intensity, all background fluorescence was deducted, and the unit was an arbitrary value; the result It shows that the fluorescence intensity of PAI-C3 is directly proportional to the amount of Caspase, indicating that the activity of Caspase-3 can be detected very accurately with PAI-C3 as the substrate.

Embodiment 3

[0056] Example 3: Insert HIV-1 Protease hydrolase recognition sequence Ser-Gln-Asn-Tyr-Pro-Val-Val into PAI (using Venus as a template), express and purify the protein PAI-HP in a bacterial system; PAI -HP was used as the substrate of HIV-1 Protease to detect its activity; the results are shown in Table 3.

[0057] Table 3: Monitoring the activity of HIV-1 Protease with PAI-HP as substrate

[0058]

[0059]The fluorescence intensity of the substrate PAI-HP is directly proportional to the amount of HIV-1 Protease in the reaction system, showing that the fluorescence intensity of PAI-HP can truly reflect the activity of HIV-1 Protease; ) plays an important role when invading the host, and inhibiting the activity of HIV-1 Protease can prevent the reproduction of HIV virus. Therefore, inhibitors of HIV-1 Protease can be used as important drugs for the treatment of AIDS; using PAI-HP as a substrate can facilitate A high-throughput screening platform for HIV-1 protease inhibitor...

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Abstract

The invention discloses a preparation method and an application method for an indicator for monitoring activity of protease in real time. The activity of the protease is monitored by reforming a fluorescent protein structure, inserting the reformed fluorescent protein structure into an acting site of the protease and observing existence and strength change of fluorescence of the fluorescent protein. The preparation method comprises the following steps of: performing circular displacement on the fluorescent protein, connecting ends N and C to change the spatial structure of the fluorescent protein by using the fluorescent protein, and generating new ends N and C, wherein the fluorescent protein subjected to the circular displacement is the protease activity indicator (PAI), the PAI is changed to consist of two peptide fragments of the ends N and C of the fluorescent protein under the action of the corresponding protease, and the two peptide fragments generate fluorescence by fluorescence complementation. The change of the protease in the physiological and pathological processes is monitored in vivo in real time by using the indicator as a tool, the activity of the protease in a sample is monitored in vitro, and the method is sensitive, intuitive, convenient and time-saving.

Description

technical field [0001] The invention relates to a preparation method and an application method of a monitoring indicator, in particular to a preparation method and an application method of an indicator for real-time monitoring of proteolytic enzyme activity. Background technique [0002] With the continuous advancement of science and technology, biochemistry has gradually become the main means to explore the basic principles of anabolism and catabolism in living cells, enabling people to understand the functions of various enzymes and reveal the structure of proteins from the atomic level through crystallography. Lay the foundation for research and effective use. However, biochemistry does not provide an experimental tool to quantitatively and accurately monitor the intracellular or extracellular physiological and pathological changes at the molecular level in experiments, so as to determine the dynamic behavior of living systems. To gain these insights, new tools, both exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12Q1/37G01N21/64
Inventor 李兵辉张矫
Owner 李兵辉
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