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Dimer polypeptide with antibacterial and immunomodulating dual functions and application thereof

An immunomodulatory and dimer technology, applied in the field of biomedicine, can solve problems such as low hemolytic activity, and achieve the effects of high safety, low production cost and convenient artificial synthesis

Inactive Publication Date: 2019-07-30
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Most of the current AMPs are natural sequences, and there are relatively few AMPs based on artificial design and preparation, and there are very few AMPs with excellent pharmacological activity and low hemolytic activity. peptides, so that it can be used as a candidate biopharmaceutical is of great significance

Method used

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  • Dimer polypeptide with antibacterial and immunomodulating dual functions and application thereof
  • Dimer polypeptide with antibacterial and immunomodulating dual functions and application thereof
  • Dimer polypeptide with antibacterial and immunomodulating dual functions and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] [Example 1] Preparation of intermediate KATIFGLAAWALCLA with antibacterial and immunomodulatory bifunctional polypeptide

[0044] In the synthesis of the intermediate polypeptide sequence, 9-fluorenylmethoxycarbonyl (FMOC) was used to protect the amino terminal group, and 4-methyl-benzhydrylamine resin HCl, MBHA resin was selected. It is a solid phase carrier, and HOBt / DCC is a condensation agent, which extends the peptide chain from the carboxyl terminal to the amino terminal. Cleavage it from MBHA resin with a mixture of 96% trifluoroacetic acid, 2% water and 2% triisopropylsilane (TIA) (both in mass percentage). After repeated precipitation with diethyl ether, it was purified by preparative RP-HPLC. Use a C18 reverse-phase preparative column (20mm×250mm, 5 μm); mobile phase: 1‰ trifluoroacetic acid, 0%-60% (volume percent) acetonitrile as the mobile phase, and gradient elution at a flow rate of 1.0 mL / min. Detected by RP-HPLC, the retention time (retention time, RT...

Embodiment 2

[0045] [Example 2] Preparation of intermediate RKCKEKIGKEFKR with antibacterial and immunomodulatory bifunctional polypeptide

[0046] In the synthesis of the intermediate polypeptide sequence, 9-fluorenylmethoxycarbonyl (FMOC) was used to protect the amino terminal group, and 4-methyl-benzhydrylamine resin HCl, MBHA resin was selected. It is a solid phase carrier, and HOBt / DCC is a condensation agent, which extends the peptide chain from the carboxyl terminal to the amino terminal. Cleavage it from MBHA resin with a mixture of 96% trifluoroacetic acid, 2% water and 2% triisopropylsilane (TIA) (both in mass percentage). After repeated precipitation with diethyl ether, it was purified by preparative RP-HPLC. Use a C18 reverse-phase preparative column (20mm×250mm, 5 μm); mobile phase: 1‰ trifluoroacetic acid, 0%-60% (volume percent) acetonitrile as mobile phase, gradient elution at a flow rate of 1.0mL / min. Detected by RP-HPLC, RT is 7.94min, and its purity>95% ( image 3 ) a...

Embodiment 3

[0047] [Example 3] Preparation of Dimeric Polypeptides with Antibacterial and Immunomodulatory Functions

[0048] The intermediate polypeptide KATIFGLAAWALCLA prepared in Example 1 and the intermediate polypeptide RKCKEKIGKEFKR prepared in Example 2 were used as raw materials. Take 1mg and 1.5mL EP tubes respectively, dissolve them with 1 mL Tris-HCl buffer solution with a concentration of 0.2M (pH value 9.0), and gently blow the solution with a pipette gun to accelerate the dissolution of the polypeptide. Put the EP tube containing the peptide solution into a constant temperature shaker at 26°C-28°C, 80 rpm and incubate for about 48h. Centrifuge at room temperature at 12 000 rpm for 3 min at high speed, and take the supernatant. Purified by preparative RP-HPLC. Use C18 reverse-phase preparative column (20mm×250mm, 5μm); mobile phase: 1‰ trifluoroacetic acid, 0%-70% (volume percent) acetonitrile as mobile phase, gradient elution at a flow rate of 1.0mL / min. Detected by RP-H...

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Abstract

The invention discloses a dimer polypeptide with antibacterial and immunomodulating dual functions and application thereof, and belongs to the field of biomedicine. The polypeptide is obtained by thesteps that two stage are divided for preparation by means of a conventional polypeptide solid-phase synthesis technique, then a disulfide bond is formed by thiol pairing of cysteine side chains, and finally semi-preparative reverse high performance liquid chromatography is adopted for purification. Pharmacological experimental results show that the polypeptide has a good inhibition effect on Gram-positive bacteria; the expression of proinflammatory cytokines TNF-alpha, IL-1beta, IL-6 and IL-17 in murine macrophages RAW264.7 induced by lipopolysaccharide can also be inhibited, and the expression of an anti-inflammatory cytokine IL-10 is promoted. Meanwhile, the bifunctional polypeptide shows low hemolytic activity, the artificial preparation is convenient, the pharmacological activity is definite, and the polypeptide has further development value and industrialization potential.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a dimer polypeptide with dual pharmacological functions of antibacterial and immune regulation, a preparation method and application thereof. Background technique [0002] Since Alexander Fleming (Alexander Fleming) discovered penicillin from Penicillium in 1928, the widespread use of natural and synthetic antibacterial agents has made bacterial infectious diseases well treated on the one hand, but on the other hand On the one hand, it brings huge selection pressure (the ability to resist antibiotics) to the strain. [0003] On August 5, 2016, China released the "National Action Plan to Contain Bacterial Antimicrobial Resistance (2016-2020)" (Guo Wei Yi Fa [2016] No. 43), becoming one of the few comprehensive plans to combat antibiotics and drug resistance one of the countries. The China Antimicrobial Surveillance Network (CHINET), led by the Institute of Antibiotics of Hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/04C07K1/06C07K1/20C07K1/14A61K38/10A61P31/04A61P37/02
CPCA61K38/00A61P31/04A61P37/02C07K7/08
Inventor 沈秉正曹志贱喻研宋金春涂毅冯莉芳祝成亮高翔黄亚冰高越
Owner WUHAN UNIV
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