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Polypeptide with specificity inhibiting HB-EGF promoting tumor cell migration and infiltration

A polypeptide sequence, ovarian cancer cell technology, applied in the field of active polypeptide invention, can solve problems such as heavy workload and low screening efficiency, and achieve the effect of short polypeptide sequence, broad market and prospects, and high clinical value

Active Publication Date: 2016-08-10
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current screening methods for peptide drugs mainly include biochemical separation of natural products, structural modification on existing natural peptides, etc., often with a large workload and low screening efficiency

Method used

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  • Polypeptide with specificity inhibiting HB-EGF promoting tumor cell migration and infiltration
  • Polypeptide with specificity inhibiting HB-EGF promoting tumor cell migration and infiltration
  • Polypeptide with specificity inhibiting HB-EGF promoting tumor cell migration and infiltration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Phage display panning for polypeptides that specifically bind to HB-EGF

[0017] Target molecule immobilization: 600 μl of target molecule HB-EGF (Pepro Tech Biological Company, USA) solution (dissolved in 0.1 M NaHCO 3 pH 8.6) was added to a six-well plate (NEST Sciences), placed on a shaker with gentle shaking, and incubated overnight at 4°C. The target molecule solution was removed and washed 6 times with TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% [v / v] Tween-20). Finally, block solution (0.1 M NaHCO 3 pH 8.6, 5 mg / ml BSA, 0.02% NaN 3 ) closed for 1 h.

[0018] Binding of phage random peptide library to target molecules: Remove the blocking solution and wash 10 times with TBST (0.1% [v / v] Tween-20). After the phage library or the amplified phage was diluted with TBST (0.1% [v / v] Tween-20), the titer of the phage was 10 9 ~10 11 In between, the diluted phage was added to the six-well plate to allow it to bind to the target molecule, and incubate...

Embodiment 2

[0026] Example 2. Cell culture and passage and cell scratch experiment

[0027] SKOV3 cells (American Type Culture Collection, USA) were cultured in RMPI-1640 (Life Technologies, USA) medium containing 10% fetal bovine serum and penicillin-streptomycin, and the growth status of the cells should be observed regularly every day and passaged in time.

[0028] Day 1: Seed boards.

[0029] from CO 2The subcultured SKOV3 cells were taken out of the incubator (Thermo Fisher Co., Ltd.), the original medium was discarded, and 2ml of PBS (Sinopharm Chemical Reagent Co., Ltd.) was added to wash, and the supernatant was sucked off with a Pasteur tube. Add 0.5 ml trypsin (Sigma-Aldrich (Shanghai) Trading Co., Ltd.) for digestion, transfer to a microcentrifuge tube (Haimen Leimin Experimental Equipment Business Department), centrifuge at 200 g for 5 min, and suck off the supernatant in the Pasteur tube . Pipette and mix 1 ml of RMPI-1640 medium containing 10% fetal bovine serum and penic...

Embodiment 3

[0032] Example 3. Cell Invasion Experiment

[0033] Day 1: ECM Matrigel (Sigma, USA) in a -20°C refrigerator was moved to a 4°C freezer and thawed overnight.

[0034] The next day: Pre-cool the ready-to-use 24-well plate, 10 μl pipette tip and Transwell chamber (Corning Life Sciences, USA), use pre-cooled fetal bovine serum-free medium and penicillin-streptomycin-containing medium and ECM matrigel 1 : 8 dilutions (in a pre-cooled microcentrifuge tube), after dilution, add 40 μl of ECM diluent to the Transwell chamber, and then put in 5% CO 2 , 37 ℃ cell culture incubator overnight.

[0035] Day 3: Transwell experiment.

[0036] from CO 2 Take out the subcultured SKOV3 cells from the incubator, add 1 ml of PBS, and suck off the supernatant with a Pasteur tube. Add 0.5 ml of trypsin for digestion, transfer to a microcentrifuge tube, centrifuge at 200 g for 5 min, aspirate the supernatant in the Pasteur tube, and add serum-free medium to resuspend the cells. Count with a hem...

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Abstract

The invention discloses a polypeptide with specificity inhibiting HB-EGF promoting tumor cell migration and infiltration and belongs to the technical field of biological medicine. According to the polypeptide, heparin-binding epidermal growth factors (HB-EGF) serve as target molecules, by means of three rounds of screening of the phage display technology, the bioactive polypeptide TUZG7 is obtained through specificity elution with HB-EGF as the effective component and phages of the target molecules. The bioactive polypeptide TUZG7 remarkably inhibits migration, filtration and other processes promoted by ovarian cancer cells of HB-EGF. The polypeptide is short in sequence, is easy to synthesize, can achieve large-scale production, can inhibit the cancer promoting function of HB-EGF, has the potential in being used for preparing anti-cancer medicine, and has important application value in the aspect of anti-cancer medicine research and development.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to the invention of an active polypeptide screened by phage display technology that can specifically inhibit HB-EGF to promote tumor cell migration and infiltration. Background technique [0002] Heparin-binding epidermal growth factor (HB-EGF), a member of the epidermal growth factor (EGF) superfamily, is a type I transmembrane protein that can be produced by macrophages, smooth muscle cells, and endothelial cells. The gene encoding HB-EGF is located on human chromosome 5. The gene is 14 kb in length and contains 6 exons and 5 introns. [0003] HB-EGF plays an important role by combining with the epidermal growth factor receptor (EGFR) on the cell surface, and participates in pathophysiological processes such as embryo implantation, wound healing, smooth muscle hyperplasia, atherosclerosis and tumorigenesis. As a ligand of EGFR, HB-EGF can promote the proliferation of various ...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P35/00A61P35/04
CPCA61K38/00C07K7/08
Inventor 刘晗青张雅菲阮玲玲屠志刚赵志聪尚东胜吴燕芳沈艳婷廉彩霞卢子文鲁永金张佩珊梁智全
Owner JIANGSU UNIV
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