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Polypeptide HIP-15 capable of antagonizing hnRNPK protein RNA binding activity and application thereof

A technology that combines activity and protein, applied to polypeptides, hybrid peptides, peptide/protein components containing positioning/targeting motifs, etc., to achieve the effects of inhibiting cell proliferation, inhibiting leukemia, and improving efficiency

Pending Publication Date: 2022-06-21
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, hnRNPK-specific inhibitors have not been reported

Method used

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  • Polypeptide HIP-15 capable of antagonizing hnRNPK protein RNA binding activity and application thereof
  • Polypeptide HIP-15 capable of antagonizing hnRNPK protein RNA binding activity and application thereof
  • Polypeptide HIP-15 capable of antagonizing hnRNPK protein RNA binding activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Synthesis of embodiment 1 antitumor polypeptide

[0033] Synthesize an anti-tumor polypeptide by solid-phase synthesis, which includes a tumor cell killing domain and a membrane-penetrating domain, wherein the tumor cell killing domain sequence is TIKLFQECCPHSTDR (SEQ ID No.1), and the membrane-penetrating domain sequence is YGRKKRRQRRR (SEQ ID No.2), the transmembrane domain is connected to the N-terminal of the tumor cell killing domain, the obtained sequence is: the amino acid sequence is YGRKKRRQRRR-TIKLFQECCPHSTDR (SEQ ID No.3), named HIP-15. For the convenience of research, we linked FITC labeled with fluorescein isothiocyanate to the C-terminus of the anti-tumor polypeptide, and linked biotin-labeled Biotin to the N-terminus.

[0034] The tumor suppressor polypeptide HIP-15 synthesized by the biological company has been tested by High Performance Liquid Chromatography (HPLC), and the purity reaches 97.3%. The polypeptide was dissolved in sterile PBS buffer (Sigm...

Embodiment 2

[0037] Example 2 Detection of cellular localization of HIP-15

[0038]Human leukemia cell line HL-60 (China Center for Type Culture Collection) was used for detection. Suspension cells grown in logarithmic phase were added to 24-well plates at approximately 15,000 cells per well. Cultured in 10% fetal bovine serum (HyClone Company), RPMI1640 medium (Gibco Company), and 30 μM polypeptide HIP-15 was added, and maintained for 48 hours. A staggered peptide with the same amino acid composition as the polypeptide was randomly rearranged as a control peptide. Aspirate the cell suspension in each well, adjust the speed of the centrifuge (Fresco21, ThermoFisher Company) to centrifuge at 1000 rpm for 5 minutes, add 500 μl of 4% paraformaldehyde (Sigma-Aidrich Company) after washing with 1x PBS, and fix the cells at room temperature for 20 minutes , discard the paraformaldehyde, wash twice with 1x PBS, 5 min each. Add 300 μl of 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aidrich Compan...

Embodiment 3

[0040] Example 3 MTT colorimetric analysis to detect the inhibitory effect of HIP-15 on the growth of leukemia cells

[0041] The effect of the polypeptide on the proliferation activity of different tumor cells was detected by MTT method. The human leukemia cell line HL-60 was used for detection. A staggered peptide with the same amino acid composition as the polypeptide was randomly rearranged as a control peptide. HL-60 cells in the logarithmic growth phase were taken, and 5000 cells were planted in each well of a 96-well cell culture plate, and 10 replicate wells were set for each sample. After 24 hours, the HIP-15 polypeptide was added. The concentrations of the polypeptide and its control polypeptide were 10 μM, 15 μM, 20 μM, and 30 μM, respectively; the action time was 24 hours, 48 ​​hours, and 72 hours, respectively. After reaching the calibration time, centrifuge in a 96-well plate centrifuge (Eppendorf), discard the culture supernatant, and wash the sample wells wi...

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Abstract

The invention discloses a polypeptide HIP-15 capable of antagonizing hnRNPK protein RNA binding activity and application of the polypeptide HIP-15, and belongs to the technical field of molecular biology.The polypeptide HIP-15 comprises a leukemia tumor cell killing structural domain and a transmembrane structural domain, the amino acid sequence of the leukemia tumor cell killing structural domain is shown as SEQ ID No.1, and the amino acid sequence of the transmembrane structural domain is shown as SEQ ID No.2. The transmembrane structural domain of the anti-leukemia tumor polypeptide has no cytotoxicity, but has obvious effects of inhibiting leukemia cell proliferation and clone formation after being connected with the leukemia cell killing structural domain. The anti-leukemia tumor polypeptide disclosed by the invention not only can be independently used as an anti-leukemia tumor biological treatment medicine, but also is expected to be combined with other treatment modes to inhibit leukemia.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a polypeptide HIP-15 capable of antagonizing the RNA binding activity of hnRNPK protein and its application. Background technique [0002] Leukemia is a cancer of the bone marrow that causes an abnormally high number of white blood cells to be produced. It is a malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, differentiation disorder, and apoptosis inhibition, and infiltrate other non-hematopoietic tissues and organs. Hematopoietic function. Leukemia seriously affects human health. Although the existing anti-leukemia methods have certain curative effects, they still have problems such as low curative effect, poor selectivity, large toxic and side effects, and easy drug resistance. Therefore, searching for new anti-l...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K19/00A61K38/10A61P35/02
CPCC07K7/08A61P35/02C07K2319/10A61K38/00Y02A50/30
Inventor 周浩陈莉娟刘伟朱建华陈雪星李紫平
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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