A group of new antibiotic peptides, preparation method thereof and use thereof

A technology for antimicrobial peptides and fungal infections, which can be applied in the direction of antibacterial drugs, antifungal agents, and medical preparations containing active ingredients. It can solve the problems of sequence modification and functional applications, ParasinI has no hemolytic property, and low bactericidal activity.

Active Publication Date: 2012-03-28
SHANGHAI HI TECH BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A group of new antibiotic peptides, preparation method thereof and use thereof
  • A group of new antibiotic peptides, preparation method thereof and use thereof
  • A group of new antibiotic peptides, preparation method thereof and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 solid-phase chemical synthesis preparation and separation and purification of antimicrobial peptides

[0032] GK V-1 to GK V-12 were prepared according to the above sequence, while GramicidinS, parasinI and magaininII were prepared as controls.

[0033] Sequence of GramicidinS:

[0034] Val Orn Leu Phe Pro Val Orn Leu Phe Pro

[0035] Sequence of ParasinI

[0036] Lys Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr Arg Ser Ser

[0037] Sequence of magaininlI:

[0038] Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe Val Gly Glu

[0039] Ile Met Asn Ser

[0040] 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 a...

Embodiment 2

[0050] Expression of embodiment 2 antimicrobial peptide GK V-4 gene in escherichia coli

[0051] First, design and synthesize the GKV-4 gene encoding the antibacterial peptide, clone it into the pGEX-4T1 vector (purchased from Amersham Pharmcia Biotech), then transform Escherichia coli JM109, induce the expression of GST-GKV-4 fusion protein by IPTG, and then After cleavage by thrombin, the antimicrobial peptide GK V-4 is obtained.

[0052] 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.

[0053] Molecular cloning operations such as DNA isolation, purification, PCR amplification, enzymatic hydrolysis, plasmid transformation of host bacteria, fragment rec...

Embodiment 3

[0060] Expression of embodiment 3 antimicrobial peptide GK V-4 gene in yeast cell

[0061] 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.

[0062] The GK V-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 Bacillus Species Collection Center), the extracted plasmid was sequenced to prove that the sequence was correct, and the plasmid was digested with EcoRI and XhoI and connected to the pPIC9 vector (purchase...

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Abstract

The present invention provides a group of new antibacterial peptides which has stronger bactericidal activity than that of natural antibacterial peptides and has good killing effect on various pathogenic bacteria. The present invention also discloses a method for preparing the antibacterial polypeptides which can be synthesized by a solid phase chemical method or obtained through expression by a genetic engineering method. The synthesized antibacterial peptides of the present invention can be used for preparing medicines for treating diseases caused by the infection of gram-positive bacteria,gram-negative bacteria or fungi.

Description

[0001] This application is a divisional application with the application number 200910223410.1, the application date being November 16, 2004, and the title of the invention 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 animals (especial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K38/10A61P31/04A61P31/10
Inventor 黄青山李国栋
Owner SHANGHAI HI TECH BIOENG
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