Novel cell-penetrating peptide and uses thereof

A kind of penetrating peptide, a new type of technology, applied in the field of new penetrating peptides, can solve the problems of complex structure of penetrating peptides, difficult synthesis, purification, mass production, etc.

Inactive Publication Date: 2009-04-22
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In comparison, artificially synthesized amphiphilic membrane-penetrating peptides such as MPG and PEP-1 can carry proteins through the cell membrane through non-covalent bonds, but due to the complex structure of these membrane-penetrating peptides, it is very difficult to synthesize, purify, and mass-produce

Method used

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  • Novel cell-penetrating peptide and uses thereof
  • Novel cell-penetrating peptide and uses thereof
  • Novel cell-penetrating peptide and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Taking RRRRRREW (R at the C-terminus) as an example to illustrate the method of synthesizing peptides by solid-phase method

[0016] Add 1g of Rinkamide-MBHA resin (0.74mmolN / g) into a 50mL round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 20mL DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then add 20mL of 20% piperidine / DMF solution, stir for 30 minutes, and filter with suction. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 1.44g (2.22mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.46g ( 2.22mmol) dicyclohexylcarbodiimide and 0.30g (2.22mmol)}HOBt (1-hydroxybenzotriazole) and 20mL DMF, stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color test, and the result showed that the condensation reaction was complete. Suctio...

Embodiment 2

[0020] Take RRRRRREW (R at C-terminal), RRRRRRREW (R at N-terminal), RRRRREWW (R at C-terminal), RRRRREWW (R at N-terminal), RRRRRRW (R at C-terminal), RRRRRRW (R at N-terminal) as examples Compared with the classic membrane-penetrating peptide RRRRRRRRR, the membrane-penetrating activity of the synthetic polypeptide of the present invention is illustrated.

[0021] RRRRRREW, RRRRRREWW, RRRRRRW were synthesized by solid-phase method in our laboratory, and RRRRRRRR was purchased from AnaSpec Company; the N-terminals were all labeled with FITC fluorescein.

[0022] ECV-304 was cultured in RPMI 1640 medium containing 10% calf serum, 100mg / L each of ampicillin and streptomycin. At 37°C, 5% CO 2 , cultured normally under fully saturated humidity, and subcultured once every 3 days.

[0023] Take a six-well plate, add a cover glass, and inoculate ECV-304 cells at a density of 2×10 5 Each well was cultured overnight. After the cells adhered to the wall, replace with fresh culture m...

Embodiment 3

[0026] Enteric-coated insulin delivery system delivered by RRRRRREWW (R at C-terminal)

[0027] 10mg of insulin (potency 29.3IU / mg, the same below) was dissolved in a small amount of dilute hydrochloric acid, added to 2mL of PBS buffer, freeze-dried, added an appropriate amount of diluent, and filled into enteric-coated capsules for rats.

[0028] 100mg RRRRRREWW was added to 2mL PBS buffer, freeze-dried, and filled into enteric-coated capsules for rats.

[0029] 10 mg of insulin was dissolved with a small amount of dilute hydrochloric acid, 2 mL of PBS buffer and 100 mg of RRRRRREWW were added, freeze-dried, and divided into enteric-coated capsules for rats.

[0030] The SD diabetic rats were randomly divided into 3 groups, 6 in each group, and fasted for 12 hours before administration. The first group was the insulin control group, which was orally administered insulin enteric-coated capsules (dose 20IU / Kg); the second group was the RRRRREW control group. Group RRRRREWW ent...

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Abstract

The invention relates to a novel cell-penetrating peptide. The cell-penetrating peptide is characterized in that the cell-penetrating peptide has the capacity for penetrating cell membranes; each peptide consists of three parts which can be shown as X-Y-Z, wherein X represents an alkali amino acid cluster consisting of 6 to 9 continuous arginine residual radials; Y represents o to 1 glutamic acid residual radials; Z represents 1 to 2 tryptophan residual radials; and the alkali amino acid cluster can be positioned in C end or N end. The application of the cell-penetrating peptide is to participate in carrying a physiological active substance or a detection reagent to penetrate the cell membranes.

Description

Technical field: [0001] The invention belongs to the field of biomedicine, specifically, it is a novel membrane-penetrating peptide and its application. Background technique: [0002] In recent years, some polypeptides with cell membrane penetration ability, such as TAT, MPG, PEP-1, penetratin, oligoarginine, oligolysine, etc., have gradually become the focus of attention. They can pass through the cell membrane without the cell membrane being damaged. . These natural or synthetic peptides are water-soluble, low-lying, and enter various cell membranes through non-phagocytosis. Transmembrane transport, it is also called protein transduction domain (protein transduction domain, PTD). The membrane-penetrating peptides have very high translocation efficiency for biomacromolecules and do not consume energy. The membrane-penetrating mechanism of membrane-penetrating peptides is not yet fully understood, but these membrane-penetrating peptides are very similar in membrane-penetra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K1/00A61K9/20A61K9/48A61K9/14A61K9/08A61K9/52A61K38/28A61K47/42A61P3/10
Inventor 周建平张振海吕慧侠张磊
Owner CHINA PHARM UNIV
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