阅读说明：本技术 一种抗菌肽及其应用 (Antibacterial peptide and application thereof ) 是由 谭臣 鲁浩 鲁文嘉 王晨晨 李晓丹 王高岩 于 2021-06-25 设计创作，主要内容包括：本发明公开了一种抗菌肽及其应用,属于微生物传染病及医药领域。本发明抗菌肽的氨基酸序列为RLLRKFFRKL,其具有广谱抗菌活性、低毒性、无溶血毒性、中和外毒素LPS的特点。本发明的抗菌肽可用于制备抗菌剂、制备预防或治疗细菌感染的药物,其对耐药菌(大肠杆菌、肺炎克雷伯菌等)的抗菌效果优于临床常用药物。本发明的抗菌肽L-1的氨基酸序列大大缩短,大幅降低了生产成本,其有望成为新型抗生素的候选药物,在临床抗菌药物中具有良好的应用前景。(The invention discloses an antibacterial peptide and application thereof, belonging to the fields of microbial infectious diseases and medicines. The amino acid sequence of the antibacterial peptide is RLLRKFFRKL, and the antibacterial peptide has the characteristics of broad-spectrum antibacterial activity, low toxicity, no hemolytic toxicity and exotoxin LPS neutralization. The antibacterial peptide can be used for preparing antibacterial agents and medicines for preventing or treating bacterial infection, and has an antibacterial effect on drug-resistant bacteria (escherichia coli, klebsiella pneumoniae and the like) superior to that of clinical common medicines. The amino acid sequence of the antibacterial peptide L-1 is greatly shortened, the production cost is greatly reduced, and the antibacterial peptide L-1 is expected to become a candidate drug of a novel antibiotic and has a good application prospect in clinical antibacterial drugs.)

1. An antimicrobial peptide, characterized by: the amino acid sequence is RLLRKFFRKL.

2. The use of the antimicrobial peptide of claim 1 for antimicrobial applications, wherein: the use is for non-disease treatment purposes.

3. Use of the antimicrobial peptide of claim 1 for the preparation of an antimicrobial agent.

4. Use of the antimicrobial peptide of claim 1 for the preparation of a medicament for the prevention or treatment of a bacterial infection.

5. An antimicrobial agent characterized by: comprising the antimicrobial peptide of claim 1.

6. A medicament for the prevention or treatment of bacterial infections, characterized by: comprises the antibacterial peptide L-1.

7. The use according to any one of claims 2 to 4, the antibacterial agent according to claim 5 or the medicament according to claim 6, wherein: the bacteria include gram-positive bacteria and gram-negative bacteria.

8. The use according to any one of claims 2 to 4, the antibacterial agent according to claim 5 or the medicament according to claim 6, wherein: the bacteria include Staphylococcus aureus, Streptococcus suis, Escherichia coli, and Klebsiella pneumoniae.

9. The use according to any one of claims 2 to 4, the antibacterial agent according to claim 5 or the medicament according to claim 6, wherein: the bacteria are drug-resistant bacteria.

10. The use according to any one of claims 2 to 4, the antibacterial agent according to claim 5 or the medicament according to claim 6, wherein: the bacteria are drug-resistant staphylococcus aureus, streptococcus suis, escherichia coli and klebsiella pneumoniae.

Technical Field

The invention relates to the fields of microbial infectious diseases and medicines, in particular to an antibacterial peptide and application thereof.

Background

Bacterial resistance to antibiotics is becoming increasingly prevalent and is a serious public health problem. Infections caused by antibiotic-resistant bacteria are associated with significant morbidity and mortality worldwide. Previous efforts against multidrug resistant (MDR) bacteria have focused on methicillin-resistant staphylococcus aureus (MRSA). In recent years, several new treatment options have emerged for MRSA (david. etl, 2017). Thus, at present, the major threat to antibiotic-resistant bacteria comes from multi-drug resistant gram-negative organisms, particularly those that develop resistance to carbapenems. Along with carbapenem-resistant acinetobacter baumannii (CRAB) and carbapenem-resistant pseudomonas aeruginosa (CRPA), carbapenem-resistant enterobacteriaceae (CRE) are one of the top levels of the WHO list of antibiotic-resistant "priority pathogens" that pose the greatest threat to human health. The natural antibacterial peptides limit further clinical application due to the defects of unstable metabolism, high production cost, easy hemolysis side effect and the like. Amino acid substitutions have been the simplest and most common method for modifying polypeptide sequences to improve antibacterial activity and cytotoxicity.

Disclosure of Invention

The invention aims to provide a novel antibacterial peptide which has broad-spectrum antibacterial activity, low toxicity and no induced drug resistance.

The purpose of the invention is realized by the following technical scheme:

the antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and no induced drug resistance is prepared by selecting a section with alpha-helix characteristics from banded chrysotoxin as a template (GenBank accession number EU753183) and adding a short sequence with high alpha-helix, and the antibacterial peptide is marked as L-1, and has an amino acid sequence of RLLRKFFRKL. The helical wheel projection of antimicrobial peptide L-1 is shown in FIG. 1. In vitro antibacterial experiments, cytotoxicity experiments, hemolytic activity experiments and endotoxin LPS neutralization experiments show that the antibacterial peptide L-1 has broad-spectrum antibacterial activity and has good inhibition effect on gram-negative bacteria resistant to carbapenem and colistin; in addition, the antibacterial peptide L-1 has the characteristics of low toxicity, no hemolytic toxicity and endotoxin LPS neutralization. Animal experiments show that the antibacterial peptide L-1 has good in-vivo protection effect on carbapenem-resistant escherichia coli, and is superior to clinical common medicines.

The invention provides the following applications of antibacterial peptide L-1: the use of antimicrobial peptide L-1 for antimicrobial applications, which is not a disease treatment; the application of the antibacterial peptide L-1 in preparing antibacterial agents; application of antibacterial peptide L-1 in preparing medicine for preventing or treating bacterial infection is provided.

An antibacterial agent comprising the antibacterial peptide L-1.

A medicament for preventing or treating bacterial infection, which comprises the antibacterial peptide L-1.

The bacteria comprise gram-positive bacteria and gram-negative bacteria, the gram-positive bacteria comprise staphylococcus aureus and streptococcus suis, and the gram-negative bacteria comprise escherichia coli and klebsiella pneumoniae.

The bacteria are drug-resistant bacteria, including drug-resistant staphylococcus aureus, streptococcus suis, escherichia coli and klebsiella pneumoniae.

The invention has the following advantages and beneficial effects:

(1) the antibacterial peptide L-1 has the advantages of broad-spectrum antibacterial activity, low toxicity and no induced drug resistance.

(2) The amino acid sequence of the antibacterial peptide L-1 is greatly shortened, and the production cost is greatly reduced.

(3) The antibacterial peptide L-1 has no cross resistance with common antibiotics.

(4) The antibacterial peptide L-1 is expected to become a candidate drug of novel antibiotics and has good application prospect in clinical antibacterial drugs.

Drawings

FIG. 1 is a perspective view of a helical wheel of antimicrobial peptide L-1, showing its good amphiphilicity.

FIG. 2 is a graph showing the results of example 2 in which L-1 was tested for toxicity to vero cells using the WST-1 method.

FIG. 3 is a graph showing the results of the hemolysis experiment of red blood cells in example 3.

FIG. 4 is a graph showing the results of interaction of LPS with antibacterial peptide L-1 as determined by ITC in example 4.

FIG. 5 is a graph showing the results of the survival rate of the mouse in example 5.

Fig. 6 is a graph of the blood inflammatory cytokine assay results for the mice of example 6: p < 0.001.

Detailed Description

The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The antibacterial peptide L-1 used in the following examples was synthesized by Fmoc solid phase synthesis protocol by Kingsler, and its C-terminal was amidated.

Example 1

According to the NCCLS antibacterial drug sensitivity test operation standard, the minimum inhibitory concentration of the antibacterial peptide L-1 to multi-drug-resistant gram-positive bacteria (staphylococcus aureus, streptococcus suis) and gram-negative bacteria (escherichia coli and klebsiella pneumoniae) is determined by adopting a classical micro continuous two-fold dilution method, the experiment is repeated for 3 times in parallel, and the result is shown in table 1.

TABLE 1 minimum inhibitory concentration of antimicrobial peptide L-1 against common multi-drug resistant strains

Abbreviations: AMP, ampicillin; TET, tetracycline; LEV, levofloxacin; MER, meropenem; COL, colistin.

Example 2

The toxicity of the antibacterial peptide L-1 to the cells is detected by adopting monkey vero cells and using a WST-1 method.

Vero cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂Culturing under the condition. Vero cells were cultured at 70-80% confluence in 96-well plates at a volume of 100 μ L/well medium. Mixing serially diluted antibacterial peptide with cells at 37 deg.C and 5% CO₂Incubated for 24h in the same wayPure cell control wells without drug were set. After incubation for 20h, 10. mu.L of WST-1 solution was added to each well, and after 4h, the reduction of WST-1 was detected by a microplate reader using absorbance at 490 nm. The percent fluorescence, i.e., drug well OD, of the drug wells relative to the non-drug-treated wells was calculated₄₉₀Control well OD₄₉₀And obtaining the cell survival rate. The results are shown in FIG. 2, where L-1 had no significant inhibition of cell growth over the concentration range of MIC values.

Example 3

In a 96-well plate, 50. mu.L of 2% sheep red blood cells suspended in PBS were added to 50. mu.L of antimicrobial peptide L-1 serially diluted in PBS and incubated at 37 ℃ for 1 hour, wherein the L-1 concentration was 1, 2, 4, 8, 16, 32, 64, 128. mu.g/mL, and 2.5% TritonX-100 was used as a positive control. The plates were then centrifuged at 500g for 5 minutes and 50 μ Ι _ of supernatant from each well of the assay plate was transferred to a fresh 96-well plate. Hemolysis was confirmed by visual observation and measurement of absorbance at 543 nm. The results are shown in FIG. 3, where L-1 did not cause hemolysis of red blood cells at the maximum concentration tested of 128. mu.g/mL, indicating that L-1 did not physically damage mammalian cell membranes.

Example 4

The interaction of E.coli LPS with the antimicrobial peptide L-1 was determined in vitro using isothermal calorimetry titration (ITC). The purified LPS and L-1 were dissolved in PBS at pH 7.4 to give final concentrations of 0.05mmol/L and 0.5mmol/L, respectively. L-1 (total volume 50. mu.L) was injected into a cuvette containing 300. mu.L of purified LPS, 2. mu.L each, and the injection was repeated 25 times with 200s intervals, and the experiment was performed at 25 ℃. Equilibrium dissociation constant (Kd), stoichiometry (n), enthalpy (Δ H) and entropy (Δ S) were calculated by NanoAnalyzer software. The results are shown in FIG. 4, with Kd of 1.119 × 10^-6mol/L, n is 0.589, Δ H is 214.7, and Δ S is 833.4J/mol/K, which shows that L-1 has strong binding force with LPS.

Example 5

40 BALB/c mice, 6 weeks old, were randomly divided into L-1 treated, MER treated, untreated, and placebo groups, with 10 mice per group. The mice in the L-1 treatment group, the MER treatment group and the untreated group are 5X 10⁸CFU carbapenem-resistant abdominal cavity injection of E.coliPerforming toxicity attack, and injecting PBS with the same volume into a blank control group; the administration group was administered with L-1 or MRE at a dose of 5mg/kg 1h after challenge, and then administered by intraperitoneal injection every 12h (dose of 5mg/kg), and the untreated group and the blank control group were administered with PBS in the same volume. After 3 consecutive days of administration, mice were observed for 7 days and survival rates were counted. The results are shown in figure 5, the survival rate of 10 mice in the untreated group is 70%, the survival rate of the MER treatment group is 30%, and the survival rate of the blank control group is not dead, which indicates that L-1 has good in vivo protection effect on the carbapenem-resistant Escherichia coli.

Example 6

20 BALB/c mice, 6 weeks old, were randomly divided into L-1 treated, MER treated, untreated, and placebo groups, with 5 mice per group. The mice in the L-1 treatment group, the MER treatment group and the untreated group are 1 × 10⁸C, injecting the CFU carbapenem-resistant escherichia coli E2 into an abdominal cavity for toxicity counteracting, and injecting PBS with the same volume into a blank control group; the administration group was injected with L-1 or MRE at a dose of 5mg/kg 1h after challenge, and the untreated group and the blank control group were given PBS of the same volume. After 12 hours of treatment, mice were sacrificed and blood was collected. Use of an electrochemiluminescence platform (Quickplex, Meso-MSD) to detect inflammatory cytokines. The results are shown in figure 6, compared with the clinical commonly used medicine MER, the L-1 can obviously inhibit the generation of IL-6 and TNF-alpha of infected mice and reduce inflammatory reaction.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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