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Immunosuppressive polypeptide designed by acidic amino acid scanning method and application thereof

An amino acid, autoimmune technology, applied in the biological field

Active Publication Date: 2019-10-22
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is still challenging to screen and design high-efficiency and low-toxicity targeting potassium ion channel Kv1.3 polypeptide drugs

Method used

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  • Immunosuppressive polypeptide designed by acidic amino acid scanning method and application thereof
  • Immunosuppressive polypeptide designed by acidic amino acid scanning method and application thereof
  • Immunosuppressive polypeptide designed by acidic amino acid scanning method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Separation and purification of sequence recombinant polypeptides with different distribution of aspartic acid residues

[0060] The present invention adopts the genetic engineering technology (ZL200710053679.0; ZL201310208475.5; Structural Basis of a Potent Peptide Inhibitor Designed for Kv1.3Channel, a Therapeutic Target of AutoimmuneDisease. J Biol Chem,2008,283:19058-19065;Unusual binding mode of scorpiontoxin BmKTX onto potassium channels relies on its distribution of acidic residues.Biochem Biophys Res Commun,2014,447(1):70-76;Toxin design of acid residue-evolutioned fudition de novo peptide drugs forimmunotherapeutic target Kv1.3channel. Scientific Reports, 2015, 5:9881) to prepare a series of peptides, and separate recombinant peptides by reversed-phase high performance liquid chromatography. SEQ ID NO: 1 ~ 21 peptide separation chromatogram as shown in figure 1 shown.

Embodiment 2

[0061] Example 2: Identification of sequence recombinant polypeptides with different distribution of aspartic acid residues

[0062] Using mass spectrometry to perform mass spectrometry analysis on the polypeptide provided by the present invention with the amino acid sequence shown in SEQ ID NO: 1-21, the results are as follows figure 2 shown. After mass spectrometry analysis, the determined molecular weight of these peptides is consistent with the theoretical molecular weight deduced from the amino acid sequence.

Embodiment 3

[0063] Example 3: Analysis of the pharmacological activity of recombinant polypeptides with different distributions of aspartic acid residues on the potassium channel Kv1.3

[0064] HEK-293T cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Conditioned culture, the potassium channel Kv1.3 recombinant plasmid was used Sofast TM The transfection kit was used for transfection, and the transfected cells were selectively cultured on 0.8 mg / mL Geneticin medium. The pharmacological activity of the polypeptide having the amino acid sequence shown in SEQ ID NO: 1-21 is determined and analyzed by using the instrument for whole-cell patch clamp (EPC-10 dual-channel patch clamp amplifier HEKA, Elektronik, Lambrecht, Germany). analyze. The setting of experimental parameters, data acquisition and application of stimuli were all controlled by Pulse software (Elektronik, Lambrecht, Germany). The filter of the instrument is set to 10kHz (Bessel), and the ...

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Abstract

The invention discloses an immunosuppressive polypeptide designed by an acidic amino acid scanning method and application thereof, and relates to the technical field of biology. According to the immunosuppressive polypeptide designed by the acidic amino acid scanning method and the application thereof, a drug design technology of 'artificial control identification' of the mutual pattern of the polypeptide and potassium ion channel is applied, a molecular template BmKTX is dynamically adjusted through an aspartic acid scanning technology to identify a bonding interface of the potassium ion channel Kv1.3, the target potassium ion channel Kv1.3 can be diversely acted on, candidate immunosuppressive polypeptide drugs with highly similar sequences are designed, and amino acid sequences of the candidate immunosuppressive polypeptide drugs are shown in SEQ ID NO:1-21. The polypeptide performs well in various experimental models, the specific effect is that after the provided polypeptide is given for treatment, the symptoms of experimental autoimmune encephalomyelitis in rats are significantly improved; the degree of plantar swelling in the rats is significantly reduced compared with thatof a model negative control group. It is prompted that the immunosuppressive polypeptide has important development and application value in the treatment or prevention of related diseases of the potassium ion channel Kv1.3.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to molecular design, preparation and application of potassium ion channel Kv1.3 immunosuppressive polypeptide. Background technique [0002] The potassium ion channel Kv1.3 on the lymphocyte membrane has become a drug target for the treatment of autoimmune diseases. At present, scientific research institutions at home and abroad mainly use different methods such as drug screening, chemical modification, amino acid residue truncation, and peptide bonding interface regulation to develop immunosuppressive peptide drugs targeting potassium ion channel Kv1.3 (see application A review article on the development of potassium channel Kv1.3 peptide drugs jointly published by people and French researchers: Treating autoimmune disorders with venom-derived peptides.Expert Opin Biol Ther.2017,17(9):1065-1075). [0003] Based on the interaction between scorpion active polypeptides and potassium ion ...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12A61K38/17A61P37/06A61P37/02A61P9/00A61P17/00A61P29/00A61P9/10A61P19/02A61P3/10A61P1/16A61P25/02A61P17/06
CPCC07K14/43522A61P37/06A61P37/02A61P9/00A61P17/00A61P29/00A61P9/10A61P19/02A61P3/10A61P1/16A61P25/02A61P17/06A61K38/00
Inventor 吴英亮曹志贱李文鑫
Owner WUHAN UNIV
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