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Application of znf383 protein in preparation of products inhibiting p53 protein activity

A product, an active technology, applied in the field of biomedicine

Active Publication Date: 2022-01-18
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The p53 gene is so far found to be the most relevant gene to human tumors. Mutations of the p53 gene have been found in more than 50% of human tumor tissues, and most of the tumors without p53 gene mutations have defects in the regulation mechanism of the p53 gene

Method used

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  • Application of znf383 protein in preparation of products inhibiting p53 protein activity
  • Application of znf383 protein in preparation of products inhibiting p53 protein activity
  • Application of znf383 protein in preparation of products inhibiting p53 protein activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Embodiment 1, the construction of plasmid and the preparation of oligomeric nucleic acid

[0097] 1. Construction of Myc-ZNF383 plasmid

[0098] The small fragment between the recognition sequences of the restriction endonucleases SalI and NotI of the pCMV-Myc plasmid was replaced with the DNA molecule shown in Sequence 2 in the sequence listing, and the resulting recombinant plasmid was the Myc-ZNF383 plasmid.

[0099] The Myc-ZNF383 plasmid expresses the ZNF383 protein shown in Sequence 1 in the Sequence Listing.

[0100] 2. Construction of Flag-p53 plasmid

[0101] The small fragment between the recognition sequences of the restriction endonucleases EcoRI and BamHI of the pCMV-Flag plasmid is replaced with the DNA molecule shown in sequence 4 in the sequence table, and the resulting recombinant plasmid is the Flag-p53 plasmid.

[0102] The Flag-p53 plasmid expresses the p53 protein shown in sequence 3 in the sequence listing.

[0103] 3. Preparation of oligonucleo...

Embodiment 2

[0110] Example 2, ZNF383 protein inhibits the activity of endogenous p53 protein in a dose-dependent manner

[0111] 1. Experiment 1

[0112] The experiment was repeated three times to take the average value, and the steps for each repetition were as follows:

[0113] 1. Cells (p53 + / + HCT116 cells or Hela cells) were seeded in 24-well plates containing 0.5 mL DMEM medium (8.0×10 per well 4 cells), and then placed at 37°C, 5% CO 2 Cultivate in an incubator, and when the fusion rate reaches 70-90%, they are randomly divided into four groups, and each group is set with three replicate wells, and the following treatments are carried out:

[0114] The first group: 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.4μg pCMV-Myc plasmid were added to each well for co-transfection;

[0115] The second group: add 0.1μg Myc-ZNF383 plasmid, 20ng pG13L plasmid, 0.2ng pRL-TK plasmid and 0.3μg pCMV-Myc plasmid to each well for co-transfection;

[0116] The third group: 0.2 μg Myc-ZNF383 p...

Embodiment 3

[0134] Example 3. ZNF383 protein depends on p53 protein to selectively down-regulate the expression of downstream apoptosis and immune-related target genes of p53 gene

[0135] 1. Experiment 1

[0136] The experiment was repeated three times to take the average value, and the steps for each repetition were as follows:

[0137] 1. Convert p53 + / + HCT116 cells were seeded in 6-well plates containing 2 mL of DMEM medium (2.0 × 10 per well 5cells), and then placed at 37°C, 5% CO 2 Culture in an incubator, and when the fusion rate reaches 70-90%, they are randomly divided into two groups, and each group is set with three replicate wells, and the following treatments are carried out:

[0138] Group 1: Add 2.0 μg pCMV-Myc plasmid to each well for co-transfection;

[0139] The second group: 2.0 μg Myc-ZNF383 plasmid was added to each well for co-transfection.

[0140] 2. 48 hours after step 1 was completed, the total RNA of each cell was extracted respectively, and then the first...

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Abstract

The invention discloses the application of ZNF383 protein in the preparation of products for inhibiting the activity of p53 protein. Experiments have shown that ZNF383 protein can inhibit the activity of p53 protein, down-regulate the expression of apoptosis-related target genes (such as puma gene or p53AIP1 gene), and down-regulate the expression of immune-related target genes (such as IRF5 gene, ISG15 gene or IFN-β gene) Quantity, reduce the expression level of puma protein, inhibit the signal pathway related to p53 protein and combine with p53 protein. Therefore, the ZNF383 protein has important application value in the preparation of products that inhibit the activity of p53 protein.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of ZNF383 protein in the preparation of products for inhibiting the activity of p53 protein. Background technique [0002] The KRAB zinc finger protein (KRAB-containing zinc finger protein, KZNF) family is the largest transcription factor / transcription regulator family in mammals, and its protein structure is mainly composed of the conserved KRAB (Krüppel -associated box) domain, the middle and the C-terminus contain multiple C2H2-type zinc finger structures that continuously recognize continuous DNA sequences. The KRAB domain recruits a variety of transcriptional repressors by binding to KAP-1 (KRAB-associated protein-1), forming a transcriptional repression complex, and the C-terminal zinc finger region binds to DNA and / or other transcription factors in the regulatory region of the target gene Finally, the transcriptional repression complex formed with KAP-1 is ancho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K48/00A61P35/00C07K14/47C12N15/12C12N15/113
CPCA61K38/1758A61K48/0058C12N15/113C07K14/4703C12N2310/14
Inventor 田春艳王建贺福初张秀园原艳芝
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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