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An anti-endotoxin coupled biomolecule and its preparation and application

A biomolecular and anti-endotoxin technology, applied in the field of biomedicine, can solve the problems of large absolute dosage and high production cost, and achieve the effect of improving affinity, good neutralization of LPS characteristics, and excellent direct antibacterial activity.

Inactive Publication Date: 2019-06-21
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its biological half-life is less than 1 hour, and there are problems such as high production cost and large absolute dosage.

Method used

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  • An anti-endotoxin coupled biomolecule and its preparation and application
  • An anti-endotoxin coupled biomolecule and its preparation and application
  • An anti-endotoxin coupled biomolecule and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This example relates to the solid-phase synthesis of Fmoc-CLP-19-CTC to illustrate the fragment synthesis method of CLP-19-amPEG of the present invention. The following operations are only for illustration, but not limitation of the present invention.

[0046] 1.1 Synthesis of Fmoc-CLP-19-CTC peptide resin

[0047] Weigh 6.8g (2mmol, Sub 0.314mmol / g) Fmoc-Lys(Boc)-CTC Resin and pour it into the synthesis column, weigh 55mL DCM-DMF to swell. After 30 minutes, the solvent was extracted, and the Fmoc protecting group of the polypeptide synthetic amino acid resin was removed with 35 mL of 30% hexahydropyridine in DMF. After 15 minutes, it was washed with DCM, MeOH, DMF, DCM, and DMF in sequence to remove all the hexahydropyridine. Wash with DCM and DMF in sequence to remove piperidine. Weigh 5g of Fmoc-Lys(Boc)-OH in a conical flask, add DMF to dissolve, add 19mL of DIC, 1.183g of HOBt and 55mL of 25% diisopropylethylamine in DMF. Ninhydrin detection reaction process. A...

Embodiment 2

[0059] This example relates to the inhibition test of CLP-19-amPEG and Ac-CLP-19-amPEG prepared in Example 1 against some human pathogens.

[0060] 1. Experimental strains

[0061] The strains used in the experiment were standard bacteria Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213 and Pseudomonas aeruginosa ATCC27853.

[0062] 2. Media and Reagents

[0063] Mueller-Hinton (MH) culture medium (liquid, solid), China Institute for the Control of Pharmaceutical and Biological Products products (lot number 010925); Phosphate buffer saline ((PBS): dipotassium hydrogen phosphate 8.17g, potassium dihydrogen phosphate 0.87g, add Distilled water 1000mL, adjust pH to 6.0 with 0.1N NaOH.

[0064] 3. Instrument

[0065] Multi-point inoculation instrument (manufactured by Kurahiko Co., Ltd., Japan)

[0066] Electronic balance (Sartorius, Germany)

[0067] pH meter (Beckman 21, USA)

[0068] Adjustable pipette (eppendorf, USA)

[0069] 4. Experimental method

[0070]...

Embodiment 3

[0080] This example relates to the effect of amPEG-modified allosteric peptides CLP-19-amPEG and Ac-CLP-19-amPEG of the present invention on LPS-induced inflammation of RAW264.7 cells. When the cells are attacked by the endotoxin LPS, they will actively stress and produce an inflammatory response, producing a series of inflammatory factors such as tumor necrosis factor TNF-a, interleukin IL-6, IL-8 and so on. To clarify the effects of CLP-19 and amPEG-modified allosteric peptides on the TNF-a level of LPS-induced inflammation in RAW264.7 cells, this experiment was carried out.

[0081] 1.1 Materials and main instruments

[0082] 1.1.1 Synthetic peptides: CLP-19-amPEG, Ac-CLP-19-amPEG, CLP-19, amPEG (molecular weight 500)

[0083] 1.1.2 Other reagents

[0084] Cell line: RAW264.7 cell line was provided by the Department of Immunology, Third Military Medical University;

[0085] LPS 7261: product of Sigma; MDEM medium: product of Invitrogen;

[0086] Quantitative PCR kit: pr...

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Abstract

The invention relates to an antiendotoxin coupling biological molecule. The coupling biological molecule is a compound shown in the formula Y-CLP-19-amPEG, wherein CLP-19 is a horseshoe crab antiendotoxin factor simulation peptide molecule, Y is selected from one of hydrogen, acyl group, an alkyl group of C1-C18, an aryl group, a benzyl group and an allyl group, and the molecular weight of amPEG is 500, 1000, 2000, 4000, 6000, 12000 or 20000. The direct antibacterial activity of CLP-19 obtained through the molecule and modified by PEG is better than or equivalent to that of unmodified CLP-19; the affinity of the modified CLP-19 and LPS is remarkably improved, a better LPS neutralizing characteristic is achieved, a remarkable progress is made compared with the CLP-19, and a novel sepsis treating candidate medicine is expected to be developed.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-endotoxin coupled biomolecule and its preparation and application Background technique [0002] Endotoxin, also known as lipopolysaccharide (LPS), is one of the main components of the outer membrane of Gram-negative bacteria and is released into the extracellular environment when cells proliferate or die. Free LPS can be recognized by LPS binding protein (LPS binding protein, LBP) and form a complex with the pattern recognition receptor (PRR) on the host cell membrane - Toll-like receptor 4 (TLR4), MD-2 and CD4 complexes Binding leads to activation of MyD88-dependent and MyD88-independent signaling pathways. In macrophages, this pathway can promote the release of various inflammatory factors, such as: TNF-α[10-12], IL-1β[13-16] and IL-6, etc. Under normal circumstances, the immune response triggered by LPS recognition by host cells can be precisely regulated, including cytoki...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08A61K38/10A61K47/60A61P31/04C07K1/06C07K1/04
CPCA61K38/10C07K7/08
Inventor 刘耀夏培元吕军孙凤军李頔杨雅
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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