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Application of ZNF383 protein in preparing product for inhibiting activity of p53 protein

A protein, product technology, applied in the field of biomedicine

Active Publication Date: 2017-05-10
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 preparing product for inhibiting activity of p53 protein
  • Application of ZNF383 protein in preparing product for inhibiting activity of p53 protein
  • Application of ZNF383 protein in preparing product for inhibiting activity of p53 protein

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 an application of ZNF383 protein in preparing a product for inhibiting the activity of p53 protein. An experiment proves that the ZNF383 protein can inhibit the activity of the p53 protein, can lower an expression quantity of apoptosis-associated target genes (such as puma genes or p53 AIPI genes), can lower the expression quantity of immune-associated target genes (such as IRF5 genes, ISG15 genes or IFN-beta genes), can lower an expression level of the puma protein, and can inhibit the binding of a p53 protein-associated signal pass and the p53 protein. Therefore, the ZNF383 protein has important application value in preparing the product for inhibiting the activity of the 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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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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