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Novel antibacterial peptide and preparation method and application thereof

A technology for antimicrobial peptides and fungal infections, applied in antibacterial drugs, antifungal agents, medical preparations containing active ingredients, etc., can solve the problems of ParasinI without hemolyticity, low bactericidal activity, sequence modification and few functional applications

Active Publication Date: 2010-07-21
SHANGHAI HI TECH BIOENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in our experiments, we found that its bactericidal activity is low, and the natural sequence is difficult to apply clinically, but Parasin I is basically not hemolytic, and some of its related sequence modification and functional application development are still relatively small

Method used

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  • Novel antibacterial peptide and preparation method and application thereof
  • Novel antibacterial peptide and preparation method and application thereof
  • Novel antibacterial peptide and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 solid-phase chemical synthesis preparation and separation and purification of antimicrobial peptides

[0034] GKV-1 to GKV-12 were prepared according to the above sequence, while GramicidinS, parasinI and magaininII were prepared as controls.

[0035] Sequence of GramicidinS:

[0036] Val Orn Leu Phe Pro Val Orn Leu Phe Pro

[0037] Sequence of ParasinI

[0038] Lys Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr Arg Ser Ser

[0039] Sequence of magaininII:

[0040] Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe Val Gly Glu

[0041] Ile Met Asn Ser

[0042] In this example, solid-phase chemical synthesis was adopted, and the instrument used was the Pioneer polypeptide synthesizer produced by Applied Biosystems, Inc. of the United States. The synthesized peptide was sheared by high-concentration TFA, then purified by reverse column, and the purified peptide was identified by mass spectrometry. The specific test steps are as ...

Embodiment 2

[0052] Expression of embodiment 2 antimicrobial peptide GKV-4 gene in escherichia coli

[0053] Firstly, the GKV-4 gene encoding the antibacterial peptide was designed and synthesized, cloned into the pGEX-4T1 vector (purchased from Amersham Pharmcia Biotech), and then transformed into Escherichia coli JM109, induced by IPTG to express the GST-GKV-4 fusion protein, and then After cleavage by thrombin, the antimicrobial peptide GKV-4 is obtained.

[0054] ATP, IPTG, T 4 polynucleotide kinase, T 4 DNA ligase, Klenow enzyme, and restriction endonuclease are all products of BIOLAB unless otherwise specified, the rubber tapping recovery kit is a product of Shanghai Sangon Company, and oligonucleotides are synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Thrombin cleavage kit was purchased from SIGMA.

[0055] Molecular cloning operations such as DNA isolation, purification, PCR amplification, enzymatic hydrolysis, plasmid transformation of host bacteria...

Embodiment 3

[0062] Expression of embodiment 3 antimicrobial peptide GKV-4 gene in yeast cell

[0063] ATP, IPTG, T 4 polynucleotide kinase, T 4 DNA ligase, Klenow enzyme, and restriction endonuclease are all products of BIOLAB unless otherwise specified, the rubber tapping recovery kit is a product of Shanghai Sangon Company, and oligonucleotides are synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Thrombin cleavage kit was purchased from SIGMA.

[0064] The GKV-4 gene encoding the antibacterial peptide was designed and synthesized, which was digested with BamHI and ligated with the DNA sequence encoding GST, and then ligated into the plasmid pBluescriptSKII (purchased from Stratagene, USA) to transform Escherichia coli DH5α (purchased from Wuhan University strain Deposit Center), after the sequence of the extracted plasmid was confirmed to be correct by sequencing, the plasmid was digested with EcoRI and XhoI and connected to the pPIC9 vector (purchased from I...

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Abstract

The invention provides a novel group of antibacterial peptide, which has stronger bactericidal activity than the natural antibacterial peptide and good bactericidal effect on various pathogenic bacteria. The invention further discloses a preparation method of the group of antibacterial peptide, which can be synthesized in a solid phase chemical method, and also can be obtained by genetic engineering expression. The antibacterial peptide synthesized by the invention can be used for preparing medicaments for cuing diseases caused by gram-positive bacteria, gram-negative bacteria or fungal infection.

Description

[0001] This application is a divisional application of the original application with the application number 200410068195.X, the application date is November 16, 2004, and the invention name is a group of new antimicrobial peptides and their preparation methods and applications. technical field [0002] The present invention relates to a group of antibacterial peptides, its preparation method and its application in the preparation of medicines for treating bacterial, fungal and viral infections. Background technique [0003] Antimicrobial peptides are small molecular polypeptides with biological activity induced by organisms, generally composed of 20-60 amino acids, and the molecular weight is about 2000-7000D. With the rapid development of medical immunology and molecular biology, the study of antimicrobial peptides has increasingly become a hot topic in the field of biotechnology and biomedicine. So far, more than 200 kinds of antimicrobial peptides have been found in many ...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P31/04A61P31/10
Inventor 黄青山李国栋
Owner SHANGHAI HI TECH BIOENG
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