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Method for separating antibiotic peptide and separated antibiotic peptide

A technology of antimicrobial peptides and leading peptides, applied in chemical instruments and methods, peptides, hybrid peptides, etc., can solve the problems of reduced antibiotic efficacy, increased bacterial resistance, and ineffectiveness, achieving efficient separation and good bioaccessibility , strong antibacterial activity

Inactive Publication Date: 2004-12-29
刘秋云 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the widespread use of antibiotics, two problems have arisen clinically: one is that bacterial drug resistance has increased year by year, resulting in reduced efficacy or even ineffectiveness of some antibiotics, and multi-drug resistance continues to worsen
Antimicrobial peptides are generally 10 to 40 amino acids in length and have strong immunogenicity. They are effective for the first use of patients, and the effect will gradually decrease after use, so the dosage must be continuously increased.

Method used

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  • Method for separating antibiotic peptide and separated antibiotic peptide
  • Method for separating antibiotic peptide and separated antibiotic peptide
  • Method for separating antibiotic peptide and separated antibiotic peptide

Examples

Experimental program
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Effect test

Embodiment 1

[0028] (1) Construct the fusion DNA fragment of yeast α mating factor leader peptide and random peptide for secretory expression of random peptide. The two primers are GCGAATTCAAAAATGAGATTTCCTTCAA. CGTCTAGATCA(N) 9 TCTTTTCTCGAGAGAT. Amplified with PfuDNA polymerase with high-fidelity activity. The amount of amplified template used was 50ng of plasmid pPICZαA (product of Invitrogen, containing yeast α mating factor leader peptide DNA sequence) per 1ml of PCR reaction solution. The amplification conditions are: pre-denaturation at 94°C for 5 minutes; 15 cycles of 30 seconds at 94°C, 30 seconds at 50°C, and 20 seconds at 72°C; fill-in at 72°C for 5 minutes. The amplified product was precipitated with 1 / 10 2.5M NaAc, pH 5.2 and 2.5 times absolute ethanol at 13000 rpm for 7 minutes. Wash with 70% ethanol and precipitate at 13000rpm for 3 minutes. Hair dryer to dry. Suspended in sterile distilled water. Digested overnight with EcoR I and Xba I, the total volume was 30 μl. He...

Embodiment 2

[0038] (1) Construct the fusion DNA fragment of yeast α mating factor leader peptide and random peptide for secretory expression of random peptide. The two primers are GCGAATTCAAAAATGAGATTTCCTTCAA. CGTCTAGATCA(N) 9 TCTTTTCTCGAGAGAT. Amplified with Pyrobest DNA polymerase with high-fidelity activity. The amount of amplification template used was 100 ng of plasmid pPICZαA (product of Invitrogen, containing the DNA sequence of the leader peptide of yeast α mating factor) per 1 ml of PCR reaction solution. The amplification conditions are: pre-denaturation at 94°C for 5 minutes; 18 cycles at 94°C for 30 seconds, 30 seconds at 49°C, and 30 seconds at 72°C; fill-in at 72°C for 5 minutes. The amplified product was precipitated with 1 / 10 2.5M NaAc, pH 5.2 and 2.5 times absolute ethanol at 13000 rpm for 7 minutes. Wash with 70% ethanol and precipitate at 13000rpm for 3 minutes. Hair dryer to dry. Suspended in sterile distilled water. Digested overnight with EcoR I and Xba I, the...

Embodiment 3

[0046] (1) Construct the fusion DNA fragment of yeast α mating factor leader peptide and random peptide for secretory expression of random peptide. The two primers are GCGAATTCAAAAATGAGATTTCCTTCAA. CGTCTAGATCA(N) 9 TCTTTTCTCGAGAGAT. Amplified with PfuDNA polymerase with high-fidelity activity. The amount of amplified template used was 50ng of plasmid pPICZαA (product of Invitrogen, containing yeast α mating factor leader peptide DNA sequence) per 1ml of PCR reaction solution. The amplification conditions are: pre-denaturation at 94°C for 5 minutes; 20 cycles of 94°C for 30 seconds, 51°C for 30 seconds, and 72°C for 20 seconds; 72°C for 5 minutes to fill in. The amplified product was precipitated with 1 / 10 2.5M NaAc, pH 5.2 and 2.5 times absolute ethanol at 13000 rpm for 7 minutes. Wash with 70% ethanol and precipitate at 13000rpm for 3 minutes. Hair dryer to dry. Suspended in sterile distilled water. Digested overnight with EcoR I and Xba I, the total volume was 30 μl. ...

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Abstract

The invention relates to a method of isolating antibacterial peptides and the isolated peptides thereof. The method comprises: constructing a fusion DNA sequence of yeast alpha mating factor leader sequence and a random peptide, inserting the sequence into a saccharomyces cerevisiae vector which contains an inducible promotor, digesting the unlinked linear DNA with exonuclease III and mungbean sprout nuclease, electrotransforming the yeast vector after purification, culturing the transformant 24h and covering the transformant with the upper medium which contains E. coli K12D31, and detecting the bacterial inhibition ring and the bacterial inhibition rate after inducing the expression by liquid medium, selecting the positive clones and amplifying by PCR, and determining the sequence. The method of the present invention can be used easily and effectively to isolate antibacterial oligopeptides and polypeptides and can be used as an alternative way to find antibiotics.

Description

technical field [0001] The invention relates to a method for separating antibacterial peptides and the separated antibacterial peptides. Background technique [0002] Antibiotics are microbial secondary metabolites that can selectively inhibit or kill other microorganisms or tumor cells at low concentrations, as well as similar compounds and structural modifications prepared by chemical or biological methods. The antibacterial effects of various antibiotics are mostly antibacterial, and a few have bactericidal or lytic effects. The antibacterial effect of antibiotics mainly acts on the physiological aspects of fungi, inhibiting certain links or certain enzyme systems of microbial cell metabolism. The mechanism of action of antibiotics can be summarized into the following types: (1) inhibition of nucleic acid synthesis. (2) Inhibit protein synthesis. (3) Change the permeability of the cell membrane. (4) Interfering with the formation of cell walls. (5) Act on the energy ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81C12P21/02
Inventor 刘秋云赫然
Owner 刘秋云
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