Anti-inflammatory peptide with membrane penetration effect

A short peptide, membrane-penetrating technology, applied in the field of medicine, can solve problems such as physiological dysfunction, and achieve the effect of short peptide sequence and easy synthesis

Inactive Publication Date: 2010-06-16
THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as we all know, the application of NF-κB antagonists must face the following unavoidable problems: ①Safety: Since NF-κB i

Method used

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  • Anti-inflammatory peptide with membrane penetration effect
  • Anti-inflammatory peptide with membrane penetration effect
  • Anti-inflammatory peptide with membrane penetration effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The operating steps of embodiment 1, Lys (D-Pro) ValCit preparation method

[0024]The main operation steps of solid-phase peptide synthesis are as follows: 1. Resin swelling: Put Wang-Resin resin into a reaction tube, add DMF (15ml / g) for 30min. 2. Deprotection: remove DMF, add 20% piperidine DMF solution (15ml / g) for 5min, remove and add 20% piperidine DMF solution (15ml / g) for 15min. 3. Detection: Take out the piperidine solution, take more than a dozen resins, wash them three times with ethanol, add ninhydrin, KCN, and phenol solution one drop each, heat at 105°C-110°C for 5 minutes, and turn dark blue to indicate a positive reaction. 4. Washing: DMF (10ml / g) twice, methanol (10ml / g) twice, DMF (10ml / g) twice. 5. Condensation: Add a three-fold excess of protected amino acids and a three-fold excess of activator HBTU, dissolve them with as little DMF as possible, add to a reaction tube, immediately add a ten-fold excess of NMM, and react for 30 minutes. 6. Washin...

Embodiment 2

[0025] Example 2, ELISA method to detect polypeptide binding to nuclear factor-κB p65 subunit and nuclear factor-κB cis-acting element Competitive Inhibition Experiment

[0026] The synthesis of the peptide sequence was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., with a purity of >95%. On a 96-well enzyme-linked plate coated with nuclear factor-κB cis-acting elements (purchased from Mercury TM Add 150 μl / well of transcription factor blocking solution to Transfactor p65kits), incubate at room temperature for 15 minutes; discard the transcription factor blocking solution, add 50 μl / well of detection samples diluted with transcription factor blocking solution, and the samples contain 10 ng / μl of p65 standard protein as positive Control, p65 standard protein containing 10ng / μl was added with serial concentrations of peptides at the same time, p65 standard protein containing 10ng / μl was added with the same series of negative peptides at the same time, incubated a...

Embodiment 3

[0028] Example 3. Inhibition experiment of polypeptide on expression of nuclear factor-κB responsive reporter gene.

[0029] RAW 264.7 cells were cultured in 1640 medium, and the cell density was adjusted to 0.5-2.5×10 the day before transfection 5 Cells / mL, overnight culture; take 1mL of overnight culture cells or 0.5×10 5 The amount of cells per well was added to a 24-well culture plate; 3 μL GeneJuice transfection reagent was added to 100 μL serum-free 1640 medium, vortexed to mix, and left at room temperature for 5 min; 1 μg p4kB-Luc plasmid was added to the GeneJuice / 1640 medium mixture, gently Mix by blowing and blowing, and place at room temperature for 5-15 minutes; add the above mixture dropwise to the complete medium, shake gently to mix the mixture and cells thoroughly; culture in a 5% CO2 incubator at 37°C; after 5 hours of transfection, use complete The culture medium was exchanged for 1 h, and then the cells were washed 3 times with PBS, and finally the cells ...

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Abstract

The invention relates to an anti-inflammatory peptide with membrane penetration effect, in particular to a mutant peptide which is used for treatment and takes a nuclear factor-kappa B (NF-kappa B) rho65 subunit antagonistic peptide as a matrix. The mutant peptide has membrane penetration effect. More particularly, the invention relates to a small molecular peptide Lys(D-Pro)ValCit which not only can antagonize the activity of a nuclear factor-kappa B rho65 subunit, but also can act on other unknown targets. The invention also relates to the preparation and application of the anti-inflammatory peptide.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a remodeled peptide for treatment, which uses nuclear factor-κB (NF-κB) p65 subunit antagonist peptide as a parent, and the restructured peptide has membrane-penetrating effect. More specifically, it relates to a small molecular peptide Lys(D-Pro)ValCit that can not only antagonize the activity of nuclear factor-κB p65 subunit, but also act on other unknown targets. The present invention also relates to this anti-inflammatory short peptide preparation and application. Background technique [0002] Since nuclear factor-κB (nuclear factor-κB, NF-κB) was accidentally discovered in 1986, its importance has been increasingly concerned and has become one of the most active research fields. Typical NF-κB is a heterodimer formed by two subunits of polypeptide chain p50 and p65. When cells are in a resting state, NF-κB mainly combines with κB inhibitory proteins (IκBs, of which IκBα and IκBβ pla...

Claims

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Application Information

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IPC IPC(8): C07K5/11A61K38/07A61P29/00A61P11/06A61P13/12A61P19/02A61P19/04A61P31/00A61P37/02
Inventor 范霞梁华平魏强王希杨雪
Owner THE THIRD AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIV OF PLA
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