Application of mydgf protein in preparation of regulators for telomerase expression and cell senescence

A technology of cell aging and protein expression, applied in the field of telomerase, to achieve important application prospects, delay the decline of expression, and improve the effect of activity

Active Publication Date: 2022-06-07
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the role of MYDGF protein in the regulation of telomerase activity and cell senescence.

Method used

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  • Application of mydgf protein in preparation of regulators for telomerase expression and cell senescence
  • Application of mydgf protein in preparation of regulators for telomerase expression and cell senescence
  • Application of mydgf protein in preparation of regulators for telomerase expression and cell senescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Effect of MYDGF expression on telomerase activity in cells

[0042] 1. Method

[0043] 1. Knockout of MYDGF in wild-type Hela cells

[0044] Design and order gRNA template single-stranded complementary DNA. The PX330 vector was annealed and ligated, the strains were transformed and sequenced, the correct clone was selected, amplified and cultured to extract the plasmid, and the plasmid was transiently transferred to 293T cells for T7E1 digestion to identify the positive cut gRNA. The candidate gRNA complementary DNA was ligated to the lentiviral packaging vector pLenti-gRNA-puro with different resistances, and the 293T packaging lentivirus was used to infect HeLa cells stably expressing pLenti-inducible Cas9-neo. Stable cell lines were obtained by screening with 1 μg / mL puromycin puromycin, and 1 μg / mL doxycycline was added to induce and culture for 3-6 days. The cell growth was observed and the target protein was detected by western blotting to verify the k...

Embodiment 2

[0060] Example 2 MYDGF eukaryotic protein purification and its telomerase activity on HeLa and MSC

[0061] 1. MYDGF eukaryotic protein purification

[0062]The MYDGF-FLAG plasmid was transiently transfected into 293T cells and cultured in 15cm plates. Filter the medium with a 0.22um filter and separate the supernatant. The obtained cells were separated and purified using Anti-FLAG M2 affinity gel (company: Sigma). Take 200ul gel and take 1mL of 4 ℃ pre-cooled PBS solution to wash the column. Samples were loaded, and all supernatants passed through the cartridge at a natural flow rate, and the flow-through was collected for detection. Unbound protein was washed away by adding 1 mL of PBS solution. Add 300ul PBS solution containing 3×Flag (100ug / mL) to elute the target protein, wash 4 times, and collect the washing solution for protein detection. Add 600ul of 0.1M Glycine-HCl (pH3.5) to wash, and collect the filtrate for detection. Add 1 mL of PBS solution to wash off Gly...

Embodiment 3

[0067] Example 3 Purification of MYDGF prokaryotic protein and its telomerase activity on MSCs

[0068] 1. MYDGF prokaryotic protein purification

[0069] The obtained prokaryotic expression vectors (PET28-N-His-MYDGF, PET21b-MYDGF-C-His) were transformed into BL21 (DE3) competent cells, wherein PET28-MYDGF-N-His was Kana resistant, PET21b-MYDGF-C -His is Amp resistance. Pick a single clone into the corresponding 8 mL of resistant LB medium, and cultivate and activate it for 12 h at 37 °C and 120 rpm. Take 6 mL of activated bacterial liquid, add it to 300 mL of LB medium with corresponding resistance, and cultivate at 37 °C and 120 rpm for 4-6 h. During this period, take the bacterial liquid for OD600 absorbance detection. When the OD600 reaches 0.4-0.6, the strain enters logarithmic growth. period, at which point IPTG was added to a final concentration of 0.1 mM for induction. Afterwards, the bacterial liquid was transferred to a 16°C shaker at 120 rpm and cultured overnig...

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Abstract

The invention discloses the application of MYDGF protein in the preparation of telomerase expression and cell senescence regulator. The research of the present invention finds that the MYDGF protein acts on the human telomerase catalytic subunit TERT promoter, activates the transcription and expression of TERT, and improves the activity of telomerase. In human mesenchymal stem cells cultured in vitro, adding MYDGF purified protein can activate telomerase, delay the decline of telomerase TERT expression during replicative aging, and delay cell senescence. Therefore, the protein MYDGF has an important regulatory function in the process of telomerase activation and aging, and effective regulatory agents for cell telomerase activation and anti-aging can be developed for MYDGF to delay the process of cell aging, which has important application prospects.

Description

technical field [0001] The present invention relates to the technical field of telomerase, more particularly, to the application of MYDGF protein in the preparation of telomerase expression and cell senescence regulators. Background technique [0002] my country has entered an aging society, and the degree of aging continues to increase. Therefore, the development of anti-aging or anti-aging drugs is not only of great help to the treatment of aging-related diseases, but also of great significance to solve the problem of population aging. The basis of individual aging is cellular aging. Cellular senescence refers to the irreversible growth arrest of cells that were originally proliferating. Normal cells cultured in vitro undergo a limited number of divisions, gradually shorten telomeres, accumulate DNA damage, change cell morphology and physiological metabolic activities, and show slowed cell growth, increased β-galactosidase activity, and increased secretion of inflammator...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/18A61K9/19A61P35/00A61P39/06C07K14/475
CPCA61K38/18A61K9/19A61K9/0019A61P39/06A61P35/00C07K14/475
Inventor 黄燕松阳洲李婷婷李楚君陈柯辛尹丽
Owner SUN YAT SEN UNIV
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