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Active peptide capable of being specifically combined with TfR1 and application of active peptide

An active peptide, specific technology, applied in the field of active peptides, can solve problems such as limitations

Inactive Publication Date: 2015-05-27
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The transferrin receptors of the blood-brain barrier are thought to be almost completely saturated with transferrin, which limits the use of transferrin as a drug brain-targeting carrier

Method used

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  • Active peptide capable of being specifically combined with TfR1 and application of active peptide
  • Active peptide capable of being specifically combined with TfR1 and application of active peptide
  • Active peptide capable of being specifically combined with TfR1 and application of active peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Through the method of phage display, the transferrin receptor-specific binding peptide CPU-312-01 was obtained:

[0033](1) The nucleotide sequence of the transferrin receptor-specific binding peptide CPU-312-01 was obtained by screening the phage random peptide library:

[0034] A. Biopanning of phage random 7-peptide library: Dilute the transferrin receptor to 100 μg / ml with coating solution (0.1 M NaHCO3, pH 8.6), add 150ul to one well of the microplate In the middle, rotate repeatedly until the surface is completely wet, keep wet, 4 ℃ overnight. Pour off the coating solution, clap the plate to remove the residual solution, fill up with blocking solution (0.1 M NaHCO3, pH 8.6, 5 mg / ml BSA), and react 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 well coated with transferrin receptor,...

Embodiment 2

[0043] Example 2 Fluorescent microplate reader detects the uptake experiment of cells coupled with FITC-binding peptide and FITC:

[0044] 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 10% calf serum, pipet gently with a pipette to form a single cell suspension; add 8×10 4 cells in a 24-well plate, and add 500 μl RPMI-1640 medium containing 10% serum, at 37 °C, containing 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% C0 2 Cultured in a constant temperature incubator; at the same time, FITC and LO2 cells (normal liver cells) were used as negative controls, and th...

Embodiment 4

[0045] Example 4 Fluorescent microscope observation of phagocytosis of cells coupled to FITC-binding peptides and FITC:

[0046] 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 10% calf serum, pipette gently to make a single cell suspension, add 8×10 4 cells in a 24-well plate, and add 500 μl RPMI-1640 medium containing 10% serum, at 37 °C, containing 5 % CO 2 Cultivate in a constant temperature incubator for 24 hours to the state of monolayer cells, add 500 μl of RPMI-1640 medium containing 10% calf serum, 50um coupled FITC-binding peptide to each well, and place in 3 duplicate wells at 37°C, 5% C0 2 Cultured in a constant temperature incubator; at the same time, FITC and LO2 cells (normal liver cells) were used as negative controls. After 4 hours, the medium was aspirated, washed twice with PBS,...

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Abstract

The invention discloses an active peptide capable of being specifically combined with TfR1. The active peptide has an amino acid sequence as shown in SEQ ID NO:1. The in-vitro experiment shows that the active peptide can be combined with cells capable of high-expressing transferrin receptors, and coupled fluorescein isothiocyanate (FITC) can be transferred into cells specifically. Therefore, the combined peptide disclosed by the invention can be applied to a medicine targeted carrier which takes the transferrin receptors as target points, and has significant meanings for development of tumor target medicines, target treatment on brain-derived diseases, prediction on the transferrin receptors as targets, and tumor happening and development diagnosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to an active peptide capable of specifically binding to TfR1 and its application. Background technique [0002] Transferrin (also known as transferrin, TRF, siderophilin) ​​is the main iron-containing protein in plasma, responsible for carrying iron absorbed by the digestive tract and released by the degradation of red blood cells. TRF-Fe 3+ The complex form enters the bone marrow for the production of mature red blood cells. Transferrin receptor (TfR) is an important protein molecule that mediates iron metabolism in the body, and plays a key role in iron transport, transformation and utilization. In recent years, with further research on transferrin receptors, it has been found that, in addition to the function of transporting iron, transferrin and its receptors are also related to cell growth and proliferation and the metabolism of tumor cells. Two different types of trans...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11A61K47/42A61P35/00A61P25/00G01N33/68G01N33/574
Inventor 纪雪梅甘牡丹顾梦月邵明盛力
Owner CHINA PHARM UNIV
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