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Polypeptide for detecting activity of ATG4B (Autophagy-related protein 4B) in living cell and application thereof

A fusion peptide and reaction buffer technology, applied in the design of new small peptides, the application of cell autophagy level, and the synthesis field, can solve the problems of uncontrollable quality and complicated operation, and achieve strong objectivity, simple operation, and objective reflected effect

Active Publication Date: 2014-05-28
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of this method is that it can directly quantify the activity of ATG4B in cells, and it can be used for high-throughput detection; the disadvantage is that it is necessary to prepare a cell line stably expressing GFP-LC3-YFP recombinant protein, the quality cannot be controlled, and the operation is complicated

Method used

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  • Polypeptide for detecting activity of ATG4B (Autophagy-related protein 4B) in living cell and application thereof
  • Polypeptide for detecting activity of ATG4B (Autophagy-related protein 4B) in living cell and application thereof
  • Polypeptide for detecting activity of ATG4B (Autophagy-related protein 4B) in living cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1. Design of peptides

[0056] Based on the substrate sequence TFG of ATG4B, a membrane-penetrating peptide RKKRRQRRR and amino acid residue G are directly coupled at its N-terminus by a peptide bond to form a new type of polypeptide (fusion peptide) (synthesized by Shanghai Gil Biochemical Co., Ltd.) . The amino acid sequence of the polypeptide is RKKRRQRRRGTFG, and the molecular weight is 1701.02.

Embodiment 2

[0058] 2. Design and synthesis of peptide substrates

[0059] Label the N-terminal and C-terminal markers of the polypeptide to obtain a polypeptide substrate. The marker is a small molecular compound for detection, such as fluorescein or nitroaniline salt (pNA), and the marker in this embodiment uses fluorescein. The fluorescent colors of the N-terminal and C-terminal of the labeled polypeptide are different. The N-terminal fluorescence is used as a background reference signal, and the C-terminal fluorescence is used as a detection signal. It is also possible to label the C-terminus of the fusion peptide with fluorescein alone as the detection signal, and use the cell number or total protein amount as the background reference signal. The N-terminus of the polypeptide in this example is labeled with FITC green fluorescence, and the C-terminus is labeled with AMC blue fluorescence. Of course, the N-terminal and C-terminal of the polypeptide can also be labeled with other meth...

Embodiment 3

[0068] 3. Peptides can detect ATG4B activity in cell lysates

[0069] 1. Autophagy induction model

[0070] In order to detect whether the polypeptide of the present invention has the ability to detect ATG4B activity, a cell autophagy induction model was first constructed. Human cervical cancer cells Hela were treated with 1×10 5 Inoculated into 6-well cell culture plates at a ratio of 2 ml per well. After the overnight culture, the culture medium was removed, the control group was still cultured with normal medium, and the cells in the experimental group were washed with PBS and then replaced with HBSS (containing calcium, magnesium ions and 10mM Hepas buffer) medium for 2, 3, 4 hours respectively , as a model of cell starvation. After the induction, the cells in each group were collected, and after lysing, the changes of LC3 were determined by western blotting. The result is as Figure 6 As shown, with the increase of induction time, the expression of LC3-II protein inc...

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Abstract

The invention relates to a polypeptide for detecting the enzymatic activity of ATG4B (Autophagy-related protein 4B) in a living cell and an application thereof, in particular to the design and synthesis of a novel small peptide and the application of the small peptide in detecting the autophagy level of cells related to the activity of the ATG4B. The polypeptide disclosed by the invention is used for detecting the activity of the ATG4B in the living cell, and the detection has the advantages that the operation is simple and quick and the detection result is objective and accurate.

Description

technical field [0001] The present invention relates to the preparation and application of a novel polypeptide capable of specifically detecting ATG4B enzyme activity in living cells, in particular to the design and synthesis of a novel small peptide and the ability of the small peptide to detect the level of autophagy associated with ATG4B activity Applications. Background technique [0002] Autophagy-raleted protein 4B (Autophagy-raleted protein 4B, ATG4B) is a key enzyme in the process of cell autophagy, first produced by It was discovered and reported in 2003, and then Taichi et al. analyzed its structure, and gradually clarified its role and mechanism in the process of autophagy. [0003] 1. The basic composition and structure of ATG4B [0004] The full length of ATG4B consists of 394 amino acid residues, and its structure contains 8 α-helical structures and 13 β-sheet structures ( figure 1 ), consisting of two domains of flexibility and protease. Among them, D278,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12Q1/37
Inventor 连继勤何凤田倪振洪
Owner ARMY MEDICAL UNIV
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