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Pyronema omphalodes natural antibacterial 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, good bactericidal effect, broad application value and market prospect.

Active Publication Date: 2018-06-12
北京生泰云科技有限公司
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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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  • Pyronema omphalodes natural antibacterial peptides and application thereof
  • Pyronema omphalodes natural antibacterial peptides and application thereof
  • Pyronema omphalodes natural antibacterial 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, four polypeptide sequences (U4LUG1, U4LWE8, U4LNY7, U4LND7) derived from Pyronema omphalodes were found from the fungal proteome data of the UniProt database (http: / / www.uniprot.org / ). The group of polypeptide sequences and their functional regions are respectively shown in SEQ ID NO: 1-4 and SEQ ID NO: 5-8.

[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) on the NCBI website, and the results showed that, The encoded product of this gene may be the precursor of fungal defensin family antimicrobial peptide. The sequence of the polypeptide precursor coded by this group of genes was further compared with the protein blast software (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TY PE=BlastSe...

Embodiment 2

[0040] Embodiment 2 antimicrobial peptide P1-P4 gene synthesis

[0041] Antimicrobial peptide P1-P4 gene sequences (SEQ ID NO:9-12) 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 vectors pPICP1-pPICP4

[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 pPICP1-pPICP4;

[0044] Detailed construction process of vectors pPICP1~pPICP4: use restriction endonucleases 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 Tiangen B...

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Abstract

The invention discloses a group of pyronema omphalodes natural antibacterial peptides, wherein the amino acid sequences of the pyronema omphalodes natural antibacterial peptides are shown by SEQ ID NO: 5 to 8. A fungus protein database is retrieved so as to obtain the mature peptide sequences of the antibacterial peptides are obtained by retrieving a fungus protein database and screening and analyzing; through a comparative analysis, the group of natural antibacterial peptides has an obvious difference from the amino acid sequences of all antibacterial peptides known at present, and belongs toa novel type of antibacterial peptides. The group of antibacterial peptides is optimized by a codon to realize recombination expression in pichia pastoris. The result of an antibacterial experiment indicates that the antibacterial peptide has a remarkable bacteriostatic activity against gram-positive bacteria, particularly against staphylococcus aureus ATCC43300 and streptococcus suis CVCC 3928 and CVCC 606, and can be applied to the fields such as antibacterial agents, food additives, cosmetics, feed additives and preservatives, and has a great application value and a broad market prospect.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the natural antibacterial peptide of Phylothrix terrestris and its application. 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): C07K14/37C12N15/31C12N1/19A23L33/18A23K20/147A61K38/16A61P31/04A61K8/64A61Q19/00C12R1/84
CPCA23K20/147A23L33/18A61K8/64A61K38/00A61K2800/10A61Q19/00C07K14/37
Inventor 王建华杨娜毛若雨王秀敏滕达郝娅王潇
Owner 北京生泰云科技有限公司
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