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Polypeptide for restraining EGF induced tumor cell proliferation through targeted EGFR

A tumor cell proliferation and tumor cell technology, applied in the field of peptides targeting EGFR to inhibit EGF to promote tumor cell proliferation, can solve the problems of high production cost of monoclonal antibodies, strong toxic and side effects of small molecule inhibitors, and many adverse reactions of patients, etc. Achieve the effect of easy to achieve large-scale production, short peptide sequence, and low production cost

Active Publication Date: 2019-01-11
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, tumor molecular targeting drugs targeting EGFR are mainly divided into two categories according to their properties: one is monoclonal antibodies, and currently on the market in my country are cetuximab, panitumumab, and nimotuzumab anti-antibody, etc., but monoclonal antibodies have the problem of high production costs; the other is small molecule inhibitors, such as gefitinib, erlotinib and lapafenib, which have been on the market, but the toxicity of small molecule inhibitors Strong side effects, patients with more adverse reactions after medication

Method used

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  • Polypeptide for restraining EGF induced tumor cell proliferation through targeted EGFR
  • Polypeptide for restraining EGF induced tumor cell proliferation through targeted EGFR
  • Polypeptide for restraining EGF induced tumor cell proliferation through targeted EGFR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Construction of pET-30a / EGFR-ECD expression vector

[0024] The DNA sequence encoding the 25th to 645th amino acids (aa25-645) of the EGFR extracellular domain (EGFR-ECD, EGFR extracellular domain) fragment was extracted from Genebank (https: / / www.ncbi.nlm.nih.gov, login No.: CCDS5514.1), as shown in SEQ.ID.NO.2, and design PCR primers based on it:

[0025] Forward primer (SEQ.ID.NO.3): 5'-GCTGATATC GGATCC ATGCTGGAGGAAAAGAAAGT-3'

[0026] and reverse primer (SEQ.ID.NO.4): 5'-ACGGAGCTC GAATTC TCAGGACGGGATCTTAGG-3', the underlined parts are the restriction sites BamHI and EcoRI at both ends respectively. The PCR product of the target gene EGFR-ECD and the vector pET-30a (Novagen, #69909-3) were digested with BamHI and EcoRI at 37°C for 1 h, then ligated with T4 DNA ligase at 16°C for 12 h. Transform the ligation product into DH5α competent cells (full gold, CD201), wait for a single colony to grow on the kanamycin (50 µg / mL) resistant LB plate, pick a ...

Embodiment 2

[0028] Example 2: Induced expression and refolding purification of EGFR-ECD

[0029] Induced expression of EGFR-ECD: Transform the successfully constructed recombinant plasmid pET-30a / EGFR-ECD into the host strain BL21(DE3) (full gold, CD601), use the kanamycin resistance plate to screen recombinants, and pick single The colonies were cultured in LB liquid medium containing kanamycin for 10 h. The culture was inoculated in LB liquid medium at a volume ratio of 1:100, and cultured to OD at 37°C with vigorous shaking 600 =0.5-0.6, add 0.1 M IPTG to make the final concentration 0.5 mM, and induce at 25°C for 4 h.

[0030] Verification of the expression of the target protein: Centrifuge the bacterial solution obtained from the above induced expression at 5000 rpm for 10 min, remove the supernatant, and resuspend the bacteria in the lysate (50 mM Tris -HCl pH7.5, 500 mM NaCl, 10% glycerol, 1% TritonX-100, 1 mM protease inhibitor PMSF, 1 mg / mL lysozyme), placed on ice and sonicate...

Embodiment 3

[0034] Example 3: Phage display panning for biologically active peptides specifically binding to EGFR

[0035] Immobilization of target molecules: Add 100 µL of target molecule EGFR-ECD solution with a concentration of 100 µg / mL (the base solution is 0.1 MpH7.4 TBS) into a 96-well plate, place on a shaker and vibrate slightly, and place in a humidified container Incubate overnight at 4°C. The target molecule solution was removed and washed 6 times with TBS-T (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% [v / v] Tween-20). Finally, block with blocking solution (0.1 M NaHCO3 pH8.6, 5 mg / mL BSA, 0.02% NaN3) for 1 h.

[0036] Binding of phage random peptide library to target molecules: remove the blocking solution and wash 10 times with TBS-T (containing 0.1% [v / v] Tween-20). Phage library or amplified phage (purchased from NEB (Beijing) Co., Ltd., E8111, Ph.D.-12 Phage Display Peptide Library) diluted with TBS-T (containing 0.1% [v / v] Tween-20) , the titer of phage was 10 9 -10 11 ...

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Abstract

The invention relates to a polypeptide for restraining EGF induced tumor cell proliferation through targeted EGFR and belongs to the technical field of biological medicines. According to the invention, a biological active polypeptide TUZG20 is acquired by taking EGF (Epidermal Growth Factor) as a target molecule, elutriating by adopting a phage display technology, taking EGF as an active ingredient, specifically eluting and combining with the phage of the target molecule. The invention proves that the biological active polypeptide TUZG20 is capable of obviously restraining the gastric carcinoma cell proliferation process caused by EGF. The polypeptide provided by the invention has a short sequence, is easy for compounding and large-scale production, is capable of restraining the tumor-promoting action of EGF, has a potential of subsequently developing into anti-cancer drugs and has an important application value at the aspect of research and development of anti-cancer drugs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a polypeptide targeting EGFR and inhibiting EGF to promote tumor cell proliferation. Background technique [0002] EGFR (Epidermal Growth Factor Receptor) is a member of the epidermal growth factor receptor family, widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes and other cells. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation. Epidermal Growth Factor (EGF, Epidermal Growth Factor) is an active cytokine in the human body, which stimulates the growth, proliferation and differentiation of cells by binding to its receptor EGFR. EGF is a protein with a molecular weight of 6 kD. It has 53 amino acid residues and three intramolecular disulfide bonds. It has an isoelectric point of 4.6 and is stable to heat. It is one of the most stable proteins...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K38/10A61P35/00
CPCA61K38/00A61P35/00C07K7/08
Inventor 屠志刚李濡妍刘晗青卢子文徐涵任德万
Owner JIANGSU UNIV
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