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Antibacterial polypeptide Pb2-1 or PCL-1 and preparation method and application thereof

A PCL-1, antibacterial polypeptide technology, used in antibacterial drugs, antifungal agents, chemical instruments and methods, etc., can solve problems such as poor stability and limitations, and achieve improved stability, reduced cost, and significant bactericidal effect. Effect

Active Publication Date: 2019-10-29
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The unique membrane destruction and bactericidal mechanism of cationic antimicrobial peptides is not easy to induce drug resistance of pathogens, which has great advantages over traditional antibiotics and is expected to become a new substitute for clinical treatment of infectious diseases; but its poor stability, especially Instability to trypsin limits its clinical application

Method used

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  • Antibacterial polypeptide Pb2-1 or PCL-1 and preparation method and application thereof
  • Antibacterial polypeptide Pb2-1 or PCL-1 and preparation method and application thereof
  • Antibacterial polypeptide Pb2-1 or PCL-1 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of antibacterial polypeptide Pb2-1, PCL-1:

[0045] (1) Using the polypeptide CATHPb2 as a template, intercept the region where its sequence is conserved and the α-helix is ​​highly concentrated, and intercept from the third position of the N-terminal to reduce the number of polypeptide amino acids from 29 to 13, and obtain the polypeptide Pb2- 1;

[0046] (2) An undecylcyclic peptide whose sequence is CWTKSIPPKPC, disulfide bonded into a ring, and has trypsin inhibitory activity is covalently linked to the N-terminal of Pb2-1 to obtain a polypeptide PCL- with an intramolecular disulfide bond 1.

[0047] (3) Polypeptides Pb2-1 and PCL-1 were synthesized by solid phase synthesis.

[0048] The amino acid sequence of the prepared Pb2-1 is: glycine-phenylalanine-arginine-lysine-phenylalanine-methionine-arginine-arginine-leucine-lysine - Lysine - Phenylalanine - Phenylalanine.

[0049] The amino acid sequence of the prepared PCL-1 is: cysteine-tryptophan-thre...

Embodiment 2

[0054] Determination of the purity (HPLC method) and mass spectrometry results of polypeptide Pb2-1 and PCL-1:

[0055] Example 1 Polypeptides Pb2-1 and PCL-1 were synthesized and purified to obtain finished products, which were identified by high performance liquid chromatography and mass spectrometry.

[0056] Liquid chromatography analysis conditions: C18 chromatographic column (4.6×250mm, 5 μm); mobile phase A is acetonitrile solution containing 0.1% trifluoroacetic acid, and mobile phase B is pure water containing 0.1% trifluoroacetic acid. The detection wavelength is 220nm; the flow rate is 1.0ml / min; the injection volume is 10μl, and gradient elution is performed. The elution condition is a linear gradient with increasing concentration: the initial concentration of the organic phase is 27%, which increases to 52% at 25 minutes, and the chromatographic column is washed with 100% organic phase from 25 minutes to 30 minutes.

[0057] attached by figure 1 , 3 It can be s...

Embodiment 3

[0060] Determination of antibacterial activity of polypeptide Pb2-1 and PCL-1 of the present invention in vitro

[0061] (1) Recovery and activation of strains

[0062] The experimental bacteria frozen at -20°C were transferred from the glycerol tube to the corresponding agar slant medium (bacteria were transferred to nutrient agar slant, fungi were transferred to Sabouraud dextrose agar slant). Bacteria were cultured at 37°C for 24 hours, fungi were cultured at 28°C for 48 hours, and placed in a refrigerator at 4°C for later use.

[0063] (2) Preparation of bacteria solution

[0064] Take a small amount of bacteria on the slant, transfer to 2ml of the corresponding liquid medium, place the bacteria at 37°C and the fungus at 28°C for 8 hours, and dilute to 10% with the corresponding liquid medium. 5 CFU / ml bacterial suspension, for later use.

[0065] (3) Drug preparation

[0066] Weigh the polypeptide Pb2-1 or PCL-1 prepared in Example 1 and dissolve them in normal saline...

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Abstract

The invention discloses an antibacterial polypeptide Pb2-1 or PCL-1 and preparation method and application thereof.The amino acid sequences of the antibacterial polypeptides Pb2-1 and PCL-1 are as shown in SEQ ID NO:1 and SEQ ID NO:2 respectively. According to the antibacterial polypeptide Pb2-1 or PCL-1 and the preparation method and application thereof, based on polypeptide CATHPb2 from Python-bivittatu, through structure-fuction relationship studies, polypeptide Pb2-1 withhigh alpha-helix degree is obtained, the N-end is in covalent linkage withasection of eleven cyclopeptidehaving trypsininhibitory activity to obtain thenovel structure polypeptidePCL-1 consisting 24 amino acids. The prepared polypeptide Pb2-1 and PCL-1, the alpha-helix degree and hydrophobicityofan efficacy moduleareincreased; also, the PCL-1 is connected withthe eleven cyclopeptide sequence so that while good in vitro antibacterial activity is realized, the stability to trypsin is also significantly improved, and the polypeptide Pb2-1 and PCL-1 can be applied to the treatment of bacterial and fungal infectious diseases.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drugs in biochemistry, and specifically relates to antibacterial polypeptide Pb2-1 or PCL-1 and its preparation method and application. Background technique [0002] Since the discovery of penicillin in 1928, people have gradually entered the era of controlling and treating bacterial infectious diseases. However, the abuse of antibiotics has accelerated the evolution of pathogenic bacteria, resulting in the emergence of a large number of drug-resistant bacteria and multi-drug resistant bacteria in recent years. Because the development of new antibiotics has not accelerated with the rapid increase of drug-resistant bacteria, and because antibiotics have low economic benefits and are difficult to apply for approval, many pharmaceutical companies have withdrawn from the research and development of antibiotics, resulting in a slowdown in the discovery of antibiotics year by year. The emergence of...

Claims

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Application Information

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IPC IPC(8): C07K14/46A61K38/17A61P31/04A61P31/10
CPCA61K38/00A61P31/04A61P31/10C07K14/46Y02A50/30
Inventor 周长林吴皓旻黄亚马菱蔓窦洁
Owner CHINA PHARM UNIV
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