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Application of MYDGF protein in preparation of telomerase expression and cell senescence regulating agent

A technology for cell aging and expression regulation, which is applied in the field of telomerase to achieve the effect of delaying cell aging, delaying the decline of expression, and improving activity

Active Publication Date: 2020-08-18
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 telomerase expression and cell senescence regulating agent
  • Application of MYDGF protein in preparation of telomerase expression and cell senescence regulating agent
  • Application of MYDGF protein in preparation of telomerase expression and cell senescence regulating agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Influence 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. Anneal and connect the PX330 vector, transform and pick bacteria for sequencing, select the correct clone, amplify, culture and extract the plasmid, and transiently transfer 293T cells for T7E1 enzyme digestion to identify gRNA that has positive cleavage. The candidate gRNA complementary DNA was connected to different resistance lentiviral packaging vector pLenti-gRNA-puro, and 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, added 1 μg / mL doxycycline doxycycline to induce culture for 3-6 days, observed cell growth and detected the target protein by Western blotting to verify the knockout effect. Three MYDGF knockout mutan...

Embodiment 2

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

[0061] 1. MYDGF eukaryotic protein purification

[0062]Transient MYDGF-FLAG plasmids were cultured in 293T cells in a 15cm plate. Filter the culture medium with a 0.22um filter head and separate the supernatant. The obtained cells were isolated and purified using Anti-FLAG M2 affinity gel (company: Sigma). Take 200ul gel, take 1mL 4 ℃ pre-cooled PBS solution to wash the column. Load the sample, flow naturally until all the supernatant passes through the column, and collect the flow-through for detection. Add 1mL PBS solution to wash away unbound protein. Add 300 ul of PBS solution containing 3×Flag (100 ug / mL) to elute the target protein, wash 4 times, and collect the washing solution for protein detection. Add 600ul 0.1M Glycine-HCl (pH3.5) for washing, and collect the filtrate for detection. Add 1mL PBS solution to wash away Glycine-HCl, and collect the filtrate for...

Embodiment 3

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

[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, and PET21b-MYDGF-C -His is Amp resistance. Pick a single clone into the corresponding 8mL resistant LB medium, culture and activate at 37°C, 120rpm for 12h. Take 6 mL of activated bacterial liquid, add it to 300 mL of LB medium with corresponding resistance, and incubate at 37°C and 120 rpm for 4-6 hours. During this period, the bacterial liquid is taken for OD600 absorbance value detection. When OD600 reaches 0.4-0.6, the strain enters logarithmic growth At this time, IPTG was added to a final concentration of 0.1 mM for induction. Afterwards, the bacterial solution was transferred to a shaker at 16°C at 120rpm and cultivated overnight (more than...

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Abstract

The invention discloses an application of MYDGF protein in preparation of a telomerase expression and cell senescence regulating agent. Research finds that the MYDGF protein acts on a human telomerasecatalytic subunit TERT promoter to activate transcriptional expression of TERT and improve the activity of telomerase. The MYDGF purified protein is added into the human mesenchymal stem cells cultured in vitro, so that telomerase can be activated, reduction of telomerase TERT expression in the replicative aging process is delayed, and cell aging is delayed. Therefore, the protein MYDGF has an important regulation and control function in the telomerase activation and aging process, an effective cell telomerase activation and anti-aging regulation and control agent can be developed for the MYDGF, the cell aging process is delayed, and the protein MYDGF has an important application prospect.

Description

technical field [0001] The present invention relates to the technical field of telomerase, more specifically, relates to the application of MYDGF protein in the preparation of telomerase expression and cell senescence regulator. Background technique [0002] my country has entered an aging society, and the degree of aging continues to intensify. 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 solving the problem of population aging. The basis of individual aging is cellular senescence. Cellular senescence refers to the irreversible growth arrest of originally proliferating cells. Normal cells cultured in vitro undergo a limited number of divisions, gradually shorten telomeres, accumulate DNA damage, and change cell morphology and physiological metabolic activities, showing slowed cell growth, increased β-galactosidase activity, and increased secretion of inf...

Claims

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Application Information

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Patent Type & Authority Applications(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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