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A kind of antibacterial peptide and its preparation method and application

An antibacterial peptide, peptide amino acid technology, applied in antibacterial drugs, peptides, pharmaceutical formulations and other directions, can solve the problems of high cost, low yield, limitations, etc. Effect

Active Publication Date: 2021-05-14
JINLING INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using yeast and Escherichia coli as genetic engineering bacteria to express, the existing technology has problems such as low yield and high cost
Therefore, its practical application is greatly limited

Method used

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  • A kind of antibacterial peptide and its preparation method and application
  • A kind of antibacterial peptide and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Fermentation obtains the thalline containing expression recombinant protein

[0048] Construction of recombinant genetic engineering bacteria BL pET30a ABP-1 of antibacterial peptide ABP-1:

[0049] The amino acid sequence of the antimicrobial peptide ABP-1 is:

[0050] HHHHHHSSSSGSSSS(DDDDKYYEERRWQWRGSGRWQWRRFFFKKRSSGSS) 10 Among them, HHHHHH is the nickel chelation column purification label; SSSSGSSSS and SSGSS are transition peptides, and DDDDK is the cleavage site of enterokinase.

[0051] According to the codon preference of Escherichia coli, the nucleotide sequence encoding the above antibacterial peptide ABP-1 is artificially synthesized as shown in SEQID NO.2. Among them, catatg and ggtacc are restriction endonucleases NdeI and KpnI enzyme cutting sites.

[0052] After the nucleotide sequence was digested with restriction endonucleases NdeI and KpnI, it was inserted into the NdeI and KpnI of the multiple cloning site of the Escherichia coli expr...

Embodiment 2

[0070] Embodiment 2: Purification to target protein

[0071] After the IPTG-induced expression of the bacterial cells obtained in step 4 of Example 1, the bacterial cells collected by centrifugation were ultrasonically lysed, and the inclusion body precipitates were collected by centrifugation. After the inclusion bodies were washed with washing solution (50mmol / L phosphate buffer, 1% Triton 100, 2mol / L urea, pH7.8), centrifuge at 10000r / min for 20min to collect the inclusion body precipitates, and then use the extraction solution (50mmol / L phosphate buffer, 8mol / L urea and 2mmol / L DTT, pH7.8) after fully dissolved, pass through an ultrafiltration membrane with a molecular weight of 50,000 to collect the filtrate for later use.

[0072] Nickel Sepharose FF or Nickel NTA Sepharose FF prepacked column, equilibrate to baseline with equilibration buffer, and equilibrate to baseline with 8M urea-2mmol / L DTT-equilibrium buffer pH7.8.

[0073] Take the above ultrafiltrate and load t...

Embodiment 3

[0077] The antimicrobial peptide ABP-1 sample prepared in Example 2 was inserted into LB medium for filtration and sterilization to prepare a 2000 μg / mL antimicrobial peptide mother solution. Then it was diluted to obtain 1000 μg / mL and 200 μg / mL antimicrobial peptide solutions respectively.

[0078] The antimicrobial peptide solution prepared above was transferred to cultured Escherichia coli ATCC 25922, Bacillus subtilis ATCC9372, Salmonella ATCC14028, Pseudomonas aeruginosa ATCC27853, and Staphylococcus aureus ATCC25923.

[0079] Specifically, PCR tubes were taken and grouped according to different concentrations of antimicrobial peptide (ABP-1), target bacteria inhibited, sodium diacetate control and kana control, see Table 1 for details. Add 100 μL prepared different concentrations of antibacterial peptide ABP-1 and 100 μL prepared Escherichia coli ATCC 25922, Bacillus subtilis ATCC9372, Salmonella ATCC14028, Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus ATCC259...

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Abstract

The invention discloses an antibacterial peptide and its preparation method and application. The antibacterial peptide includes an amino acid sequence: RRWQWRGSGRWQWRR. It solves the following problems: (1) most of the existing antibiotics produce drug resistance and cannot effectively kill pathogenic bacteria; (2) the natural resources of antimicrobial peptides are limited and the antibacterial spectrum is not wide, the output is limited, and the preparation cost is high; ( 3) The antibacterial activity of the protein sample was improved. (4) When used in combination with other antibacterial preparations, its antibacterial activity can be significantly improved to solve the problem of high cost or environmental pollution in the existing polypeptide preparation technology. The invention includes the amino acid sequence of the antibacterial peptide and its application. The product prepared by the invention can be used to prepare products against Gram-negative and positive bacteria, and to produce veterinary medicine, feed additives, and preservatives.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antibacterial peptide and its preparation method and application. Background technique [0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are a class of natural or synthetic peptides with a small molecular weight and certain antibacterial and immune effects. Its special amino acid composition makes it suitable for hydrophobicity and chargeability, so it can combine with negatively charged substances on the surface of pathogenic microorganisms to destroy the cell membrane structure, or enter the cell interior and combine with nucleic acid, protein and other macromolecular substances, and finally Disrupt normal physiological functions of cells and lead to their death. The mechanism of action of antimicrobial peptides can be divided into two categories: membrane damage and intracellular damage. Among them, amino acid sequence, lipid membrane, p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/70A61K38/10A61P31/04
CPCA61K38/00A61P31/04C07K7/08C12N15/70
Inventor 戴鼎震陈俊红陈涛蒋加进方光远陆雨楠沙奕羽黄翔宇
Owner JINLING INST OF TECH
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