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Ligand oligopeptide specific combining human epidermal growth factor receptor EGFR

An epidermal growth factor and receptor technology, applied in the fields of molecular biology and medicine, can solve the problem of low affinity

Inactive Publication Date: 2005-10-05
REGENEX PHARMA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, although a known GE7 ligand oligopeptide has a certain affinity with EGFR, iodine binding experiments show that the peptide has a low affinity with EGFR receptors

Method used

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  • Ligand oligopeptide specific combining human epidermal growth factor receptor EGFR
  • Ligand oligopeptide specific combining human epidermal growth factor receptor EGFR
  • Ligand oligopeptide specific combining human epidermal growth factor receptor EGFR

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0072] Preparation method of targeted non-viral vector

[0073] Usually, the above-mentioned (a), (b), (c) components or their complexes are mixed together to obtain the targeting non-viral vector of the present invention. Wherein, the mixing ratio of (a), (b) and (c) components is usually 0.5-1:1-2:0-1, preferably 0.8-1:1-1.5:0.5-1.

[0074] In addition, when administered in the form of (a)-(b) complex and (b)-(c) complex, the mixing ratio of (a)-(b) complex: (b)-(c) complex Usually 0.8-1.2:0.8-1.2, preferably 0.9-1.1:0.9:1.1.

[0075] exogenous DNA

[0076] The exogenous DNA that can be used in the present invention is not particularly limited, and can be various therapeutic or preventive DNAs, such as target genes, antisense oncogenes, anticancer genes, suicide genes, apoptosis genes, cytokine genes, or A combination thereof, or a eukaryotic expression vector DNA containing the above genes. Proto-oncogene antisense sequences include proto-oncogene (ras H 、ras K 、ras ...

Embodiment 1

[0094] Embodiment 1 chemically synthesized polypeptide and its mass spectrometry analysis

[0095] In this example, GE9, GE10 and GE11 were prepared by artificial synthesis.

[0096] Taking the polypeptide GE9 [SEQ ID NO: 1] as an example, its synthesis was carried out on a polypeptide synthesizer (Applied biosystems Inc.) by a solid-phase synthesis method.

[0097] 1). Polypeptide solid-phase synthesis

[0098] Select resin: Fmoc-Lys (Boc) Wang resin, dosage: 0.46mmol / g

[0099] a. Deprotection group (Fmoc): remove twice (5' and 15') with 20% piperidine in DMF; the resin is washed 3 times with DMF, 3 times with MeOH, and 3 times with DCM.

[0100] b. Fmoc-amino acid connection

[0101] Fmoc-AA / Bop / HoBt / DIEA=1:1:1:2 (corresponding to 5 times excess moles of resin)

[0102] After condensation for 1 hour, the resin was washed 3 times with DMF, 3 times with MeOH, and 3 times with DCM.

[0103] c. Kaiser test

[0104] If the resin is blue, reconnect until it is colorless. R...

Embodiment 2

[0115] Example 2 GE9 and SMMC7721 Cell Surface EGFR Receptor Specific Binding Test

[0116] Commonly used SMMC7721 cells were seeded in 96-well plates, 5×10 3 Cells / well, cultivate overnight until the cells adhere to the wall, wash 3 times with PBST (PBS+0.1% Tween20), add pre-cooled binding buffer (PBS+20mM HEPES+2.5mg / ml BSA, pH7.4) to the competition group 20 μl of diluted GE9 or EGF (containing 20 pmol of GE9 or EGF) in each well, and 20 μl of binding buffer in the non-competition group, and three parallel wells in each group; at the same time, iodine-labeled GE9, diluted to 1 ml with binding buffer, and adding 20 μl in each well ( contains 125 IGE9 0.02 pmol). Bind for 3 hours at 4°C. Wash with PBST for 3 times, add 100 μl of 0.25% trypsin, digest for 10 minutes, transfer to a measurement tube, wash with 100 μl of PBST, add the washing solution to the corresponding measurement tube, and count gamma.

[0117] See the experimental results figure 2 . It indicated that...

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Abstract

The present invention provides a gene transfer system mediated by epidermal growth factor receptor (EGFR). It comprises (a) ligand oligopeptide specifically combined in EGFR, (b) polycation polypeptide or protein and optional (c) endocytic corpuscular release oligopeptide and (d) exogenous DNA. The described gene transfer system can effectively make the exogenous gene be target-introduced into the tumor cell expressing EGFR in vivo and in video so as to inhibit the growth of tumor.

Description

technical field [0001] The present invention relates to the fields of molecular biology and medicine. Specifically, the present invention relates to a class of ligand oligopeptides that specifically bind to human epidermal growth factor receptor EGFR and the DNA sequence encoding the polypeptides. The present invention also relates to the use of the DNA and the ligand oligopeptide. Background technique [0002] The mechanism of tumor occurrence and development is mainly due to the activation of oncogenes or the inactivation of tumor suppressor genes. EGFR is a widely expressed proto-oncogene, and its changes (deletion, high expression, etc.) play an important role in the occurrence and development of tumors. [0003] EGFR is a single transmembrane receptor, consisting of 1186 amino acid residues, with a molecular weight of 170kD, and has an extracellular region, a transmembrane region and an intracellular region. The extracellular region is the region where the receptor b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C07K5/00C12N15/09C12N15/12
Inventor 顾健人李宗海徐宇虹吴向华
Owner REGENEX PHARMA LTD
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