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TfR (transferrin receptor) specific binding peptide and application thereof

A transferrin-binding peptide technology, applied in the direction of peptides, drug combinations, non-active ingredients of polymer compounds, etc., can solve problems such as limitations

Inactive Publication Date: 2014-07-02
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The transferrin receptor TfR of the blood-brain barrier is thought to be almost completely saturated with transferrin, which is limited as a drug brain-targeting carrier

Method used

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  • TfR (transferrin receptor) specific binding peptide and application thereof
  • TfR (transferrin receptor) specific binding peptide and application thereof
  • TfR (transferrin receptor) specific binding peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Through the method of phage display, the transferrin receptor TfR specific binding peptide CPU-312-03 was obtained:

[0029] (1) The nucleotide sequence of the transferrin receptor TfR-specific binding peptide CPU-312-03 was obtained by screening the phage random peptide library:

[0030]A. Biopanning of phage random 7-peptide library: Dilute transferrin receptor TfR to 100 μg / ml with coating solution (0.1M NaHCO3, pH8.6), add 150ul to a well of the microplate, repeat Spin until surface is completely wet, leave wet overnight at 4°C. Pour off the coating solution, clap the plate to remove the residual solution, fill up with blocking solution (0.1M NaHCO3, pH8.6, 5mg / ml BSA), and act at 4°C for at least one hour. Pour out the blocking solution, wash 6 times with 0.1% TBST [TBS+0.1% (v / v) Tween-20], and tap the plate each time. Dilute 10 μl of the original phage peptide library with 100 μl TBST, add it to the wells coated with the transferrin receptor TfR, incubate at ro...

Embodiment 2

[0038] In silico simulation of transferrin receptor TfR binding to binding peptide:

[0039] The model of transferrin receptor TfR and binding peptide was established by zDock docking software, and then the binding situation of transferrin receptor TfR and binding peptide was simulated by discovery studio2.5 (DS2.5) software, transferrin receptor TfR The complex model formed with the binding peptide as figure 1 , where the bars represent the transferrin receptor TfR and the spheres represent the binding peptide. figure 2 is the amino acid residue of the interaction between the transferrin receptor TfR and the binding peptide simulated by software. The results showed that the binding peptide was wrapped in the binding region binding to the transferrin receptor TfR, wherein the 533,559 glutamic acid (GLU533, GLU559), 538,560,562 aspartic acid ( ASP538, ASP560, ASP562), tightly interact with arginine (ARG4) and histidine (HIS6) of the binding peptide.

Embodiment 3

[0041] Fluorescent microplate reader to detect the uptake of coupled FITC-binding peptides and FITC by cells:

[0042] Take HepG2 cells (liver cancer cells) in the logarithmic growth phase, digest with an appropriate amount of 0.25% trypsin solution, and shake gently to make the digestive juice evenly act on the cells. RPMI-1640 medium with calf serum, blow gently with a pipette to form a single cell suspension; add 8×10 4 Put the cells in a 24-well plate, add 500 μl RPMI-1640 medium containing 10% serum, and incubate at 37°C with 5% CO 2 Cultivate in a constant temperature incubator for 24 hours to the state of monolayer cells; 1h, 2h, 3h, 4h before the test, add 500μl of RPMI-1640 medium containing 10% calf serum, 50um coupled with FITC-binding peptide, each time Spot 3 replicate wells and place at 37°C, 5% CO 2 Cultured in a constant temperature incubator; at the same time, FITC and LO2 cells (normal liver cells) were used as negative controls, and the fluorescence was de...

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Abstract

The invention discloses a TfR (transferrin receptor) specific binding peptide CPU-312-03 and an application thereof. The binding peptide sequences and nucleotide sequences of CPU-312-03 are respectively GLSRAHQ and GGGCTTTCTCGGGCTCATCAG. Vitro experiments shows that the binding peptide can be bound with cells capable of highly expressing the transferrin receptor TfR, and the coupled fluorescein isothiocyanate (FITC) can be inoculated into cells in a target mode. The binding peptide disclosed by the invention can be used in the drug targeting vector with the transferrin receptor TfR as a target site and is of great significance for the development of tumor targeting medicines, the targeted therapy of brain-derived diseases and predicting and diagnosing of the occurrence and development of tumors with transferrin receptor TfR as a target.

Description

technical field [0001] The invention belongs to a transferrin receptor TfR antibody, in particular to a transferrin receptor TfR specific binding peptide and application thereof. Background technique [0002] Two different types of transferrin receptors are known, namely transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2). [0003] TfR1 is expressed in many cells, such as red blood cells, hepatocytes, monocytes and the blood-brain barrier. TfR1 is composed of homodimers cross-linked by two disulfide bonds. Each monomer contains 760 amino acids, with a molecular weight of 90-95 kDa, including a large extracellular C-terminal region (671 amino acids), a single transmembrane region (28 amino acids) and a short N-terminal region (61 amino acids). The C-terminal region is an ectodomain that contains the binding site for transferrin (Tf), three N-linked glycosylation sites and one O-linked glycosylation site. The glycosylation of these sites is Required for TfR1 fu...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11A61K47/42A61P35/00G01N33/68
Inventor 刘煜甘牡丹顾梦月邵明叶靖宇
Owner CHINA PHARM UNIV
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