阅读说明：本技术 一种棕榈酸化抗酶解抗菌肽及其制备方法和应用 (Palmitic acid anti-enzymolysis antibacterial peptide and preparation method and application thereof ) 是由 单安山 来振衡 邵长轩 于 2020-03-30 设计创作，主要内容包括：本发明提供一种棕榈酸化抗酶解抗菌肽及其制备方法和应用。抗菌肽B<Sub>3</Sub>C<Sub>16</Sub>的序列为C<Sub>16</Sub>-GGGK(PRPR)K(PRPR)RPRP,其中C<Sub>16</Sub>为棕榈酸,每个赖氨酸的侧链通过酰胺键各链接一个多肽支链PRPR,形成树枝状结构和肽链之间的空间位阻,进一步的使用棕榈酸作为抗菌肽序列的主要疏水性来源,完美规避了所有蛋白酶对疏水性氨基酸的水解,最后使用柔性氨基酸连接体GGG将棕榈酸与分支肽链接,形成完美两亲结构。该抗菌肽对革兰氏阴性菌和革兰氏阳性菌等标准菌和耐药菌均有高效的抑制作用,同时具有很低的溶血毒性,高浓度的蛋白酶对B<Sub>3</Sub>C<Sub>16</Sub>的水解程度很弱。(The invention provides a palmitic acid anti-enzymolysis antibacterial peptide and a preparation method and application thereof. Antibacterial peptide B 3 C 16 Has the sequence of C 16 GGGK (PRPR) K (PRPR) RPRP, wherein C 16 The side chain of each lysine is respectively linked with a polypeptide branched chain PRPR through an amido bond to form steric hindrance between a dendritic structure and the peptide chain, further, the palmitic acid is used as a main hydrophobic source of an antibacterial peptide sequence, the hydrolysis of hydrophobic amino acids by all proteases is perfectly avoided, and finally, a flexible amino acid connector GGG is used for connecting the palmitic acid and the branched peptide chainThen, a perfect amphiphilic structure is formed. The antibacterial peptide has high-efficiency inhibiting effect on standard bacteria and drug-resistant bacteria such as gram-negative bacteria, gram-positive bacteria and the like, and simultaneously has very low hemolytic toxicity, and high-concentration protease is used for inhibiting B 3 C 16 The degree of hydrolysis of (a) is weak.)

1. Palmitic acid anti-enzymolysis antibacterial peptide B₃C₁₆Characterized in that the antibacterial peptide B₃C₁₆Has the sequence of C₁₆GGGK (PRPR) K (PRPR) RPRP, wherein C₁₆The side chain of each lysine is linked with a polypeptide branched chain PRPR through an amido bond to form steric hindrance between a dendritic structure and the peptide chain.

2. The palmitoylated enzymolysis-resistant antibacterial peptide B as claimed in claim 1₃C₁₆The method of (1) is characterized in that proline Pro is placed at the carboxyl terminal of each arginine Arg to form steric hindrance between amino acids, and lysine L ys is added to the polypeptide backboneAdding two branched peptide chains to form a dendritic structure and steric hindrance between the peptide chains, further using palmitic acid as a hydrophobic source of an antibacterial peptide sequence, finally using a flexible amino acid connector GGG to link the palmitic acid and the branched peptide to form a perfect amphiphilic structure, and naming the branched palmitic acid enzymolysis-resistant antibacterial peptide as B₃C₁₆The sequence of which is C₁₆-GGGK(PRPR)K(PRPR)RPRP。

3. The palmitoylated enzymolysis-resistant antibacterial peptide B as claimed in claim 1₃C₁₆The application in preparing medicine for treating infectious diseases of gram-negative bacteria and gram-positive bacteria.

4. Use according to claim 3, characterized in that: the gram-negative bacteria comprise ciprofloxacin-resistant pseudomonas aeruginosa.

5. Use according to claim 3, characterized in that: the gram-positive bacteria comprise methicillin-resistant staphylococcus aureus.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a palmitic acid anti-enzymolysis antibacterial peptide, and a preparation method and application thereof.

Background

The majority of antibiotics currently in use are extracted from natural microorganisms in the 40 to 60 th century, with a lack of renewal of effectiveness against antibiotics in recent decades the bacteria have gradually increased resistance to existing antibiotics through changes in target sites, increased efflux or decreased influx into cells, drug inactivation, and increased survival tolerance in recent years infection events caused by drug-resistant bacteria have increased worldwide, and are expected to result in 1000 million deaths each year by 2050.

Currently, 3000 or more natural antimicrobial peptides have been extracted from animals, plants and microorganisms, but natural antimicrobial peptides show only limited therapeutic efficacy. And tend to lose activity under physiological conditions after systemic administration. This phenomenon occurs because the antibacterial peptide is easily proteolytically degraded in vivo, and the antibacterial peptide needs to maintain the integrity of the molecule to maintain its biological activity, so the design of the novel antibacterial peptide is imperative.

Disclosure of Invention

Based on the defects, the invention provides the palmitic acid enzymolysis resistant antibacterial peptide B₃C₁₆The antibacterial peptide B₃C₁₆Strong enzymolysis resistance, biological activity and finenessThe cell selectivity is higher.

The technical scheme adopted by the invention is as follows: palmitic acid anti-enzymolysis antibacterial peptide B₃C₁₆Sequence is C₁₆GGGK (PRPR) K (PRPR) RPRP, wherein C₁₆In the case of palmitic acid, a polypeptide branch PRPR is linked to each lysine side chain via an amide bond, forming a steric hindrance between the dendritic structure and the peptide chain.

Another object of the present invention is to provide a palmitoylated enzymolysis-resistant antibacterial peptide B as described above₃C₁₆The preparation method is characterized in that proline Pro is placed at the carboxyl end of each arginine Arg to form steric hindrance between amino acids, two branched peptide chains are added to a polypeptide main chain by utilizing lysine L ys to form steric hindrance between a dendritic structure and the peptide chains, further, palmitic acid is used as a hydrophobic source of an antibacterial peptide sequence, finally, a flexible amino acid connector GGG is used for linking the palmitic acid and the branched peptide to form a perfect amphiphilic structure, and the branched palmitic acid enzymolysis-resistant antibacterial peptide is named as B₃C₁₆Sequence is C₁₆-GGGK(PRPR)K(PRPR)RPRP。

Another object of the present invention is to provide a palmitoylated enzymolysis-resistant antibacterial peptide B as described above₃C₁₆The application in preparing medicine for treating infectious diseases of gram-negative bacteria and gram-positive bacteria.

It is preferably that:

1. the gram-negative bacteria as described above include ciprofloxacin pseudomonas aeruginosa.

2. Gram-positive bacteria as described above include methicillin-resistant staphylococcus aureus.

According to the invention, the palmitic acid is used for replacing hydrophobic amino acids in the traditional antibacterial peptide, and the amino acid sequence is reasonably arranged according to the protease science, so that the enzyme cutting sites are effectively avoided, and further, the branch peptide sequence is added to the polypeptide part, so that the protease is difficult to identify, and the purpose of resisting enzymolysis is achieved. The antibacterial activity, hemolytic activity and protease hydrolysis resistance of the obtained antibacterial peptide are detected, and the palmitic acid enzymolysis resistant antibacterial peptide B is found₃C₁₆The compound has obvious inhibition effect on gram-negative bacteria and gram-positive bacteria such as escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis and the like, has high-efficiency inhibition effect on drug-resistant bacteria such as ciprofloxacin pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus and the like, and has low hemolytic toxicity; and high concentrations of trypsin, chymotrypsin, pepsin, proteinase K, p₃C₁₆Is very weak, and thus, in combination, B₃C₁₆Is an antibacterial peptide with higher practical application potential.

Drawings

FIG. 1 shows palmitoylated enzymolysis-resistant antibacterial peptide B₃C₁₆Hemolytic activity of (2).

FIG. 2 shows palmitoylated enzymolysis-resistant antimicrobial peptide B₃C₁₆Mass spectrum of (2).

FIG. 3 shows palmitoylated enzymolysis-resistant antimicrobial peptide B₃C₁₆The high performance liquid chromatogram of (1).

FIG. 4 shows palmitoylated enzymolysis-resistant antimicrobial peptide B₃C₁₆3D structure diagram of (1).

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.