阅读说明：本技术 抗菌肽dn6nh2及其应用 (Antibacterial peptide DN6NH2And uses thereof ) 是由 王建华 王秀敏 王振龙 李婷 韩卉卉 毛若雨 杨娜 郝娅 马炫炫 于 2020-04-16 设计创作，主要内容包括：本发明涉及新型的抗菌肽DN6NH<Sub>2</Sub>及其应用。抗菌肽DN6NH<Sub>2</Sub>是在抗菌肽N6的C末端羧基经酰胺化修饰得到的,且除了甘氨酸之外,其余氨基酸均为D型氨基酸。抗菌肽DN6NH<Sub>2</Sub>可显著提高母体肽N6NH<Sub>2</Sub>对蛋白酶和血清的稳定性,对多重耐药维氏气单胞菌具有快速杀菌、无反弹的杀菌特点,同时兼具低细胞毒性和低溶血性。DN6NH<Sub>2</Sub>在体外能够抑制维氏气单胞菌生物被膜的形成；在体内它能够显著改善小鼠因感染维氏气单胞菌生物膜而引发的背部皮肤脓肿坏死现象,其效果优于母体肽和环丙沙星。实验结果表明,DN6NH<Sub>2</Sub>明显改善了N6NH<Sub>2</Sub>的酶解稳定性和生物活性。抗菌肽DN6NH<Sub>2</Sub>是极具应用价值的小分子多肽,具有广阔的应用前景。(The invention relates to a novel antibacterial peptide DN6NH 2 And applications thereof. Antibacterial peptide DN6NH 2 Is obtained by amidation modification of carboxyl at the C terminal of antibacterial peptide N6, and the other amino acids except glycine are D-type amino acids. Antibacterial peptide DN6NH 2 Can obviously improve the parent peptide N6NH 2 Has the characteristics of quick sterilization and no rebound sterilization on multiple drug-resistant Aeromonas veronii, and has low cytotoxicity and low hemolysis. DN6NH 2 Can inhibit the formation of aeromonas veronii biofilm in vitro; in vivo, the composition can obviously improve the phenomenon of back skin abscess necrosis of mice caused by infection of Aeromonas veronii biomembranes, and the effect is superior to that of parent peptide and ciprofloxacin. The experimental result shows that DN6NH 2 Obviously improves N6NH 2 Stabilization of enzymatic hydrolysisSex and biological activity. Antibacterial peptide DN6NH 2 Is a micromolecular polypeptide with great application value and has wide application prospect.)

1. Antibacterial peptide DN6NH₂Characterized in that the antibacterial peptide DN6NH₂Is obtained by amidation modification of carboxyl at the C terminal of antibacterial peptide N6, and the other amino acids except glycine are D-type amino acids.

2. The antibacterial peptide DN6NH according to claim 1₂Characterized in that the antibacterial peptide DN6NH₂The structure of (A) is as follows:

3. DN6NH containing the antibacterial peptide of claim 1 or 2₂The broad spectrum antibacterial drug or composition of (1).

4. DN6NH containing the antibacterial peptide of claim 1 or 2₂The preservative or the bactericide of (1).

5. The antibacterial peptide DN6NH of claim 1 or 2₂Any of the following uses:

1) for preparing broad-spectrum antibacterial drugs or compositions;

2) for the preparation of preservatives;

3) is used for preparing bactericide.

6. Use according to claim 5, wherein the bacteria comprise gram-positive and gram-negative bacteria.

7. Use according to claim 6, characterized in that said bacteria comprise bacteria of the genera Staphylococcus (Staphylococcus), Escherichia (Escherichia), Salmonella (Salmonella), Pseudomonas (Pseudomonas), Aeromonas (Aeromonas).

8. The use according to claim 7, wherein the bacteria comprise Staphylococcus suis (Staphyloccocus suicus), Staphylococcus aureus (Staphyloccocus aureus), Escherichia coli (Escherichia coli), Salmonella typhimurium (Salmonella typhimurium), Salmonella pullorum (Salmonella pullorum), Salmonella enteritidis (Salmonella enteritidis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Aeromonas veronii (Aeromonas veronii).

9. Antibacterial peptide DN6NH₂The application in preparing biological products for treating or preventing infection caused by Aeromonas veronii and biomembrane thereof and related diseases caused by the infection.

10. The use according to claim 8, wherein the disease comprises subcutaneous abscesses caused by biofilms produced by aeromonas veronii.

Technical Field

The invention relates to the field of biological medicine, in particular to antibacterial peptide DN6NH₂And applications thereof.

Background

Antimicrobial peptides (AMPs) are mostly cationic amphiphilic molecules of linear or cyclic structure, generally less than 100 amino acids in length, present in almost all organisms and being part of the host innate immune system. The number of synthetic peptide mimetics using natural antimicrobial peptides as templates has reached thousands. They have been shown to possess a wide range of biological activities (antibacterial, fungal, protozoan, viral, parasitic, and tumor, etc.). Compared with the traditional antibiotics, the antibacterial peptide has the characteristics of multiple action targets, low drug resistance and the like on pathogenic bacteria, and is considered to be one of ideal substitutes of the antibiotics. The antibacterial peptide has an antibacterial function, and can inhibit the formation of bacterial biofilms and neutralize bacterial endotoxins; meanwhile, the traditional Chinese medicine composition has the functions of immunoregulation, cell apoptosis inhibition, angiogenesis promotion, wound healing and the like, and plays an important role in the health of human and animals.

Aeromonas veronii is a common pathogenic bacterium of human, livestock and fish. Relevant research reports indicate that Aeromonas veronii is one of the important pathogenic sources causing the fish infection. It can infect various fishes, and the clinical symptoms are mainly manifested by blackening the whole body of the fish, severe bleeding on the body surface and fin rays and forming an ulcer focus; the gill filaments of seriously ill fishes are seriously necrotic, are grey brown and rot, and have a large amount of light yellow ascites in abdominal cavities. In recent years, the drug resistance of Aeromonas veronii is continuously enhanced due to the abuse of antibiotics, which poses a potential threat to national health and aquaculture in China. Therefore, the development of novel antibacterial agents is urgently required.

Disclosure of Invention

The invention aims to provide a novel antibacterial peptide DN6NH₂And applications thereof.

In order to achieve the aim, the invention provides an antibacterial peptide DN6NH in a first aspect₂Antibacterial peptide DN6NH₂Is obtained by amidation modification of carboxyl at the C terminal of antibacterial peptide N6, and the other amino acids except glycine are D-type amino acids.

Antibacterial peptide DN6NH₂The structure of (A) is as follows:

the antibacterial peptide DN6NH of the invention₂An active polypeptide designed to be synthesized artificially, comprising 21 amino acid residues. Antibacterial peptide DN6NH₂The molecular weight of (A) is 2474.88Da, and the amino acid sequence is shown in SEQ ID NO. 1.

In a second aspect, the invention provides a peptide containing the antibacterial peptide DN6NH₂The broad spectrum antibacterial drug or composition of (1).

In a third aspect, the invention provides a peptide containing antibacterial DN6NH₂The preservative or the bactericide of (1).

In a fourth aspect, the invention provides the antimicrobial peptide DN6NH₂Any of the following uses:

1) for preparing broad-spectrum antibacterial drugs or compositions;

2) for the preparation of preservatives;

3) is used for preparing bactericide.

The bacteria include gram positive bacteria and gram negative bacteria.

Such bacteria include, but are not limited to, Staphylococcus (Staphylococcus), Escherichia (Escherichia), Salmonella (Salmonella), Pseudomonas (Pseudomonas), Aeromonas (Aeromonas) bacteria.

Preferably, the bacteria include Staphylococcus suis (Staphylococcus hyicus), Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Salmonella typhimurium (Salmonella typhimurium), Salmonella pullorum (Salmonella pullorum), Salmonella enteritidis (Salmonella enteritidis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Aeromonas veronii (Aeromonas veronii), and the like.

In a fifth aspect, the invention provides the antimicrobial peptide DN6NH₂The application in preparing biological products for treating or preventing infection caused by Aeromonas veronii and biomembrane thereof and related diseases caused by the infection.

The disease includes subcutaneous abscesses caused by biofilms produced by aeromonas veronii.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the antibacterial peptide DN6NH provided by the invention₂Can obviously improve the parent peptide N6NH₂(the C-terminal carboxyl group of the antimicrobial peptide N6 was amidated and modified, GFAWNVCVYRNGVRVCHRRAN-NH)₂) Has the characteristics of quick sterilization and no rebound sterilization on multiple drug-resistant Aeromonas veronii, and has low cytotoxicity and low hemolysis. DN6NH₂Can inhibit the formation of aeromonas veronii biofilm in vitro; in vivo, the composition can obviously improve the phenomenon of abscess necrosis of back skin of a mouse caused by infection of an aeromonas veronii biofilm, and the effect is superior to that of parent peptide and ciprofloxacin. The experimental result shows that DN6NH₂Obviously improves N6NH₂The enzymatic stability and the biological activity of the compound. DN6NH₂The molecular weight is small, the artificial synthesis is easy, the polypeptide is a small molecular polypeptide with great application value, and the polypeptide can be used for preparing novel antibacterial agents and has wide application prospect.

Drawings

FIG. 1 shows the antibacterial peptide DN6NH in example 1 of the present invention₂Mass spectrum of (2).

FIG. 2 shows the antibacterial peptide DN6NH in example 3 of the invention₂The bactericidal power curve for the clinical isolate strain Aeromonas veronii L1.

FIG. 3 shows the antibacterial peptide DN6NH in example 5 of the invention₂The result of the serum stability test.

FIG. 4 shows the antibacterial peptide DN6NH in example 6 of the invention₂The result of the cytotoxicity assay of (1).

FIG. 5 shows the antibacterial peptide DN6NH in example 7 of the invention₂The result of the hemolytic assay.

FIG. 6 shows the antibacterial peptide DN6NH in example 8 of the invention₂Influence on primary membranes of Aeromonas veronii.

FIG. 7 shows the antibacterial peptide DN6NH in example 9 of the invention₂Influence on the mature membrane of Aeromonas veronii.

FIG. 8 shows the antibacterial peptide DN6NH in example 10 of the present invention₂Influence on the number of Aeromonas veronii in mice.

FIG. 9 shows the antibacterial peptide DN6NH in example 11 of the invention₂The curative effect on the biomembrane in the body of the mouse.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.