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Natural antimicrobial peptides and application thereof

An antibacterial peptide, a natural technology, applied in the field of biomedicine, can solve the problem of high production cost of antibacterial peptides, and achieve the effect of significant bacteriostatic activity, broad application value and market prospect, and good bactericidal effect.

Inactive Publication Date: 2018-03-30
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fungal defensins are easy to produce in large quantities through gene expression, which solves the problem of high production cost of antimicrobial peptides
However, only 0.46% of the extant antimicrobial peptides come from fungi

Method used

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  • Natural antimicrobial peptides and application thereof
  • Natural antimicrobial peptides and application thereof
  • Natural antimicrobial peptides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Discovery of natural antimicrobial peptides and Plectasin similar to fungal defensins

[0038] Through searching, two polypeptide sequences derived from Scedosperium apiospermum and Emmonsia parva UAMH 139 were found from the fungal proteome data of the UniProt database (http: / / www.uniprot.org / ) (A0A084GET2, A0A0H1BJF3). The group of polypeptide sequences and their functional regions are respectively shown in SEQ ID NO: 1-2 and SEQ ID NO: 3-4.

[0039]The group of polypeptide full-length gene sequences was compared with the blastX software (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) of the NCBI website, and the results showed that the The encoded product of the gene may be the precursor of antimicrobial peptide of fungal defensin family. The polypeptide precursor sequences encoded by this group of genes were further compared with the protein blast software (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRA...

Embodiment 2

[0040] Embodiment 2 antimicrobial peptide P5, P6 gene synthesis

[0041] Antimicrobial peptide P5-P6 gene sequences (SEQ ID NO: 5-6) were designed according to the codon preference of Pichia pastoris. In order to ensure the integrity of the sequence during expression, an XhoI restriction site and a Kex2 restriction site were added to the 5' end of the mutant gene sequence, and a TAA, TAG terminator sequence and XbaI restriction site were added to the 3' end. The above sequence was completed by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 3

[0042] Example 3 Construction of Pichia pastoris inducible expression vector pPICP5-pPICP6

[0043] The synthetic gene fragment and vector pPICZαA were double-digested with restriction endonucleases XhoI and XbaI respectively, the vector fragment of pPICZαA and the antimicrobial peptide gene fragment were recovered, and ligated to obtain vectors pPICP5-pPICP6;

[0044] The detailed construction process of the vector pPICP5~pPICP6: use the restriction endonuclease XhoI and XbaI to carry out double enzyme digestion on the synthesized gene fragment and pPICZαA respectively, the enzyme digestion system and conditions are as follows:

[0045]

[0046] After adding the above enzyme digestion system, react at 37°C for 3 hours, and detect by 2% agarose gel electrophoresis, electrophoresis conditions: 130V, 30min. After electrophoresis, use a scalpel to cut the electrophoretic bands corresponding to the carrier fragment and the gene fragment under the ultraviolet light, and use the ...

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Abstract

The invention discloses a group of natural antimicrobial peptides derived from fungi. Amino acid sequences of the natural antimicrobial peptides are separately shown in SEQ ID NO: 3-4. Mature peptidesequences of the antimicrobial peptides are obtained through retrieving a fungal protein database and carrying out screening analysis; proven by comparative analysis, the amino acid sequences of the natural antimicrobial peptides are obviously different from those of all currently-known antimicrobial peptides, and the natural antimicrobial peptides belong to novel antimicrobial peptides. The antimicrobial peptides are optimized through codon, and then, the recombinant expression of the antimicrobial peptides can be achieved in pichia pastoris. Proven by antimicrobial experiments, the antimicrobial peptides disclosed by the invention have remarkable bacteriostasis activity to gram-positive bacteria, particularly Staphylococcus aureus ATCC43300, Streptococcus suis CVCC 3928 and CVCC 606, canbe applied to the fields of antibacterials, food additives, cosmetics, feed additives, preservatives and the like and have broad application values and market prospects.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a group of novel natural antibacterial peptides and their applications. Background technique [0002] Antibiotics have played an important role in the treatment of infectious diseases, but due to the abuse of antibiotics, drug-resistant strains continue to increase, and a variety of superbugs have appeared. one of the main threats and challenges. Staphylococcus aureus is an important pathogenic bacterium for humans, which often causes food poisoning and wound infection, and mainly causes mastitis in cattle and sheep in the animal breeding industry. Staphylococcus aureus in raw milk is prone to produce harmful substances such as Staphylococcus aureus enterotoxin and contaminate dairy products ( etc., EnterotoxigenicStaphylococcus aureus in bulk milk in Norway. Journal of Applied Microbiology. 2005, 99: 158-166.), which affects the quality and safety of dairy products and brin...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/81C12N1/19A61K38/16A61K8/64A61P31/04A23K20/147A23L3/3526C12R1/84
CPCA23L3/3526A61K8/64A61K38/00A23K20/147C07K14/37
Inventor 王建华杨娜王潇滕达毛若雨郝娅王秀敏
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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