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Mutant based on key protein ATG7 of autophagy and application thereof

A key protein and mutant technology, applied in the field of biotechnology and anti-aging treatment, to achieve the effects of controllable purity, improved cell proliferation status, and simple preparation method

Active Publication Date: 2021-02-23
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current approaches to slowing aging are mostly based on molecules that affect metabolic pathways rather than functional proteins that play active regulatory roles

Method used

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  • Mutant based on key protein ATG7 of autophagy and application thereof
  • Mutant based on key protein ATG7 of autophagy and application thereof
  • Mutant based on key protein ATG7 of autophagy and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 is based on the preparation of a mutant of the key autophagy protein ATG7.

[0024] A mutant based on the key autophagy protein ATG7, the amino acid sequence of which is SEQ ID No1, wherein the 411st and 413th lysines are mutated to glutamine, and the 513th, 514th and 516th amino acids are mutated to arginine.

[0025] The preparation method of the mutant based on the key autophagy protein ATG7 is as follows: using a eukaryotic expression system, overexpressing the plasmid of the mutant ATG7 with a flag tag in HEK293T cells, collecting the cells after 48 hours of expression, extracting the protein, Add flag-beads to the protein extract, mix overnight at 4°C, collect flag-beads at 4°C, use flag-peptide to compete for the elution of the ATG7 protein mutant, and the eluent is the purified ATG7 protein mutant.

Embodiment 2

[0026] Example 2 is based on the pharmacological study of mutants of the key autophagy protein ATG7.

[0027] 1. Establishment of senescent cell model and experimental grouping.

[0028] Firstly, primary mouse embryonic fibroblasts (MEFs) were cultured. Take 13.5-day-old mouse embryos, separate the trunk into 1×PBS; discard the PBS, add 0.125% trypsin, and digest in a 37°C incubator for 15-20min. Planting medium (DMEM+15% serum) was used to stop digestion, centrifuged at room temperature, and the precipitate was resuspended and planted in a 6cm2 cell culture dish. After genotype identification, ATG7- / - MEFs were used to infect lentivirus negative control and flag-ATG7 mutants, respectively.

[0029] 2. Detection of cell nucleus morphology.

[0030] MEF cells stably overexpressing the negative control and flag-ATG7 mutants were subcultured in vitro, and poly-lysine was coated with cell slides in a 12-well plate at room temperature for 2 hours. The first-generation MEFs pass...

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Abstract

The invention belongs to the field of biotechnology and anti-aging treatment, relates to a mutant based on a key protein ATG7 of autophagy and an application thereof in anti-aging treatment, and particularly relates to simulation of a protein modification state through mutation at amino acid sites and an application of the mutant in anti-aging treatment. The mutant based on the key protein ATG7 ofautophagy has an amino acid sequence as shown in SEQ ID No 1, in which the lysines at positions 411 and 413 are mutated into glutamines, and amino acids at positions 513, 514 and 516 are mutated intoarginines. The mutant based on the the key protein ATG7 of autophagy is applied to preparation of drugs for targeting senescence delaying or preventing and treating senescence related diseases. Experiments prove that the mutant based on the the key protein ATG7 of autophagycan play a pharmacological role in delaying senescence by improving the functions of cell nuclei, improving the proliferationstate of cells and improving the metabolic state of cells.

Description

technical field [0001] The invention belongs to the field of biotechnology and anti-aging treatment, and relates to a mutant based on the key autophagy protein ATG7 and its application in anti-aging treatment, in particular to simulating protein modification state through amino acid site mutation and its role in anti-aging application in therapy. Background technique [0002] Aging, as an inevitable natural life process, is a ubiquitous phenomenon in nature and is accompanied by the gradual decline of tissue and organ functions. With the process of human aging, the risk of senile diseases such as cardiovascular disease, neurodegenerative disease, cancer, diabetes, etc. also increases. Therefore, delaying aging is of great significance for the prevention and treatment of aging-related diseases. With the continuous exploration of aging mechanisms, aging is closely related to genetic and epigenetic changes. Therefore, obtaining suitable protein modification targets has becom...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/17A61P39/06
CPCA61K38/00A61P39/06C07K14/47
Inventor 王卓曹流郭文东宋晓宇周婷婷
Owner 中国医科大学
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