CN113480627B - An antibacterial peptide and its application - Google Patents

Abstract

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.

Description

An antibacterial peptide and its application

technical field

The invention relates to the field of microbial infectious diseases and medicine, in particular to an antibacterial peptide and its application.

Background technique

The increasing prevalence of bacterial resistance to antibiotics is a serious public health problem. Infections caused by antibiotic-resistant bacteria are associated with significant morbidity and mortality worldwide. Many previous efforts against multidrug-resistant (MDR) bacteria have focused on methicillin-resistant Staphylococcus aureus (MRSA). Several new treatment options for MRSA have emerged in recent years (David. etl, 2017). Therefore, at present, the main threat of antibiotic-resistant bacteria comes from multidrug-resistant Gram-negative organisms, especially those that develop resistance to carbapenems. Together with carbapenem-resistant Acinetobacter baumannii (CRAB) and carbapenem-resistant Pseudomonas aeruginosa (CRPA), carbapenem-resistant Enterobacteriaceae (CRE) are the greatest threat to human health by WHO One of the top of the list of antibiotic resistance "priority pathogens". Natural antimicrobial peptides limit their further clinical applications due to their unstable metabolism, high production cost, and easy to cause hemolytic side effects. Amino acid substitution to modify polypeptide sequences is the simplest and most commonly used method to improve antibacterial activity and cytotoxicity.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel antibacterial peptide with broad-spectrum antibacterial activity, low toxicity, and no drug resistance induction.

The object of the present invention is achieved through the following technical solutions:

The antibacterial peptide with broad-spectrum antibacterial activity, low toxicity and no induced drug resistance provided by the present invention is based on the band-shaped golden ring snake toxin as a template (GenBank accession number EU753183), selects a section with α-helix characteristics, and then adds It is constructed from a short sequence with a high degree of alpha helix, denoted as L-1, and its amino acid sequence is RLLRKFFRKL. The helical wheel projection of antimicrobial peptide L-1 is shown in Figure 1. In vitro antibacterial experiments, cytotoxicity experiments, hemolytic activity experiments and endotoxin LPS neutralization experiments showed that antimicrobial peptide L-1 has broad-spectrum antibacterial activity, and has good antibacterial activity against carbapenem and colistin-resistant Gram-negative bacteria In addition, antimicrobial peptide L-1 has the characteristics of low toxicity, no hemolytic toxicity, and neutralization of endotoxin LPS. Animal experiments show that antimicrobial peptide L-1 has a good in vivo protective effect on carbapenem-resistant Escherichia coli, which is better than commonly used clinical drugs.

The invention provides the following applications of the antimicrobial peptide L-1: the application of the antimicrobial peptide L-1 in antibacterial, the application is for the purpose of non-disease treatment; the application of the antimicrobial peptide L-1 in the preparation of antibacterial agents; the antimicrobial peptide L-1 Use in the preparation of medicines for preventing or treating bacterial infections.

An antibacterial agent, comprising the antibacterial peptide L-1.

A medicine for preventing or treating bacterial infection, comprising the antibacterial peptide L-1.

Described bacteria include Gram-positive bacteria and Gram-negative bacteria, described Gram-positive bacteria include Staphylococcus aureus, Streptococcus suis, and described Gram-negative bacteria include Escherichia coli, Klebsiella pneumoniae primary bacteria.

The bacteria are drug-resistant bacteria, including drug-resistant Staphylococcus aureus, Streptococcus suis, Escherichia coli, and Klebsiella pneumoniae.

The present invention has the following advantages and beneficial effects:

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

(2) The amino acid sequence of the antimicrobial peptide L-1 of the present invention is greatly shortened, which greatly reduces the production cost.

(3) The antimicrobial peptide L-1 of the present invention has no cross-resistance with commonly used antibiotics.

(4) The antibacterial peptide L-1 of the present invention is expected to be a candidate drug of a new type of antibiotic, and has a good application prospect in clinical antibacterial drugs.

Description of drawings

Figure 1 is a helical wheel projection diagram of antimicrobial peptide L-1, showing that it has good amphiphilicity.

FIG. 2 is a graph showing the results of detecting the toxicity of L-1 to vero cells using the WST-1 method in Example 2. FIG.

FIG. 3 is a graph showing the results of the erythrocyte hemolysis experiment in Example 3. FIG.

4 is a graph showing the results of the interaction between LPS and antimicrobial peptide L-1 measured by ITC in Example 4. FIG.

FIG. 5 is a graph showing the results of the survival rate of mice in Example 5. FIG.

Fig. 6 is a graph showing the detection results of blood inflammatory cytokines in the mice of Example 6, ***: P<0.001.

Detailed ways

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. 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 entrusted to GenScript Corporation for synthesis by Fmoc solid-phase synthesis scheme, and its C-terminus was modified by amidation.

Example 1

According to the operation standard of NCCLS antimicrobial susceptibility test, the classical micro serial double dilution method was used to determine the effect of antimicrobial peptide L-1 on multi-drug resistant Gram-positive bacteria (Staphylococcus aureus, Streptococcus suis) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae) minimum inhibitory concentration, the experiment was repeated 3 times in parallel, the results are shown in Table 1.

Table 1 The minimum inhibitory concentration of antimicrobial peptide L-1 against common multidrug-resistant strains

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

Example 2

Monkey-derived vero cells were used to detect the toxicity of antimicrobial peptide L-1 to cells by WST-1 method.

Vero cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% _CO2 . Vero cells were cultured at 70-80% confluency in 96-well plates at a volume of 100 μL/well of medium. Serially diluted antimicrobial peptides were incubated with cells for 24 h at 37°C in 5% CO ₂ , while a control well of pure cells without drug was set. After 20 h of incubation, 10 μL of WST-1 solution was added to each well, and after 4 h, the reduction of WST-1 was detected by a microplate reader using the absorbance at 490 nm. Cell viability was calculated by calculating the percent fluorescence of drug wells relative to untreated wells, ie, drug well _OD490 /control well _OD490 . The results are shown in Figure 2. L-1 has no obvious inhibition on cell growth within the concentration range of the MIC value.

Example 3

In a 96-well plate, 50 μL of 2% sheep erythrocytes suspended in PBS were added to 50 μL of serially diluted antimicrobial peptide L-1 in PBS, and incubated at 37°C for 1 hour, where the L-1 concentration was 1 , 2, 4, 8, 16, 32, 64, 128 μg/mL, 2.5% TritonX-100 as a positive control. The plate was then centrifuged at 500 g for 5 minutes and 50 μL 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 Figure 3. L-1 did not cause hemolysis of red blood cells at the maximum test concentration of 128 μg/mL, indicating that L-1 does not physically damage mammalian cell membranes.

Example 4

The interaction of E. coli LPS with antimicrobial peptide L-1 was determined in vitro by isothermal calorimetry (ITC). The purified LPS and L-1 were dissolved in PBS at pH 7.4 to make the final concentrations 0.05 mmol/L and 0.5 mmol/L, respectively. L-1 (total volume of 50 μL) was injected into the sample cell containing 300 μL of purified LPS, 2 μL per injection, the injection was repeated 25 times, the injection interval was 200 s, and the experiment was performed at 25 °C. Equilibrium dissociation constant (Kd), stoichiometry (n), enthalpy (ΔH) and entropy (ΔS) were calculated by NanoAnalyzer software. The results are shown in Figure 4, Kd=1.119×10 ^-6 mol/L, n=0.589, ΔH=214.7, ΔS=833.4J/mol/K, indicating that L-1 has a strong binding force to LPS.

Example 5

Forty 6-week-old BALB/c mice were randomly divided into L-1 treatment group, MER treatment group, untreated group and blank control group, with 10 mice in each group. The mice in the L-1 treatment group, the MER treatment group and the untreated group were challenged by intraperitoneal injection of 5×10 ⁸ CFU of carbapenem-resistant Escherichia coli, and the blank control group was injected with the same volume of PBS; the administration group L-1 or MRE were injected once at a dose of 5 mg/kg at 1 h after the challenge, and then administered by intraperitoneal injection every 12 h (dose 5 mg/kg). The untreated group and the blank control group were given the same volume of PBS. After continuous administration for 3 days, the mice were observed for 7 days, and the survival rate was counted. The results are shown in Figure 5. All 10 mice in the untreated group died, the survival rate of the L-1 treatment group was 70%, the survival rate of the MER treatment group was 30%, and the blank control group had no death, indicating that L-1 was resistant to carbapenems. Escherichia coli has a good in vivo protective effect.

Example 6

MSD)检测炎症细胞因子。结果如图6所示，相对于临床常用药物MER，L-1可以明显抑制感染小鼠IL-6和TNF-α的产生、降低炎症反应。Twenty 6-week-old BALB/c mice were randomly divided into L-1 treatment group, MER treatment group, untreated group and blank control group, with 5 mice in each group. The mice in the L-1 treatment group, the MER treatment group and the untreated group were challenged by intraperitoneal injection of 1×10 ⁸ CFU of carbapenem-resistant E. coli E2, and the blank control group was injected with the same volume of PBS; The groups were injected with L-1 or MRE at a dose of 5 mg/kg at 1 h after the challenge, and the untreated group and the blank control group were given the same volume of PBS. After 12 hours of treatment, mice were sacrificed and blood was collected. Using an electrochemiluminescence platform (Quickplex, Meso-

MSD) to detect inflammatory cytokines. The results are shown in Figure 6. Compared with the commonly used clinical drug MER, L-1 can significantly inhibit the production of IL-6 and TNF-α and reduce the inflammatory response in infected mice.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> Huazhong Agricultural University

<120> An antimicrobial peptide and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 1

Arg Leu Leu Arg Lys Phe Phe Arg Lys Leu

1 5 10

Claims (7)

1. an antimicrobial peptide is characterized in that: amino acid sequence is RLLRKFFRKL. 2. the application of the antibacterial peptide according to claim 1 in antibacterial, it is characterized in that: described application is the purpose of non-disease treatment; Described bacteria comprise staphylococcus aureus, streptococcus suis, Escherichia coli, pneumoniae Lebsiella. 3. The application of the antibacterial peptide according to claim 1 in the preparation of an antibacterial agent, wherein the bacteria comprise Staphylococcus aureus, Streptococcus suis, Escherichia coli, and Klebsiella pneumoniae. 4. The application of the antibacterial peptide according to claim 1 in the preparation of a medicine for preventing or treating bacterial infection, wherein the bacteria include Staphylococcus aureus, Streptococcus suis, Escherichia coli, and Klebsiella pneumoniae. 5. An antibacterial agent, characterized in that: comprising the antibacterial peptide of claim 1; and the bacteria include Staphylococcus aureus, Streptococcus suis, Escherichia coli, and Klebsiella pneumoniae. 6. A medicine for preventing or treating bacterial infection, characterized in that: comprising the antibacterial peptide of claim 1; and the bacteria include Staphylococcus aureus, Streptococcus suis, Escherichia coli, and Klebsiella pneumoniae. 7. The application according to any one of claims 2-4, the antibacterial agent according to claim 5 or the medicine according to claim 6, wherein the bacteria are drug-resistant Staphylococcus aureus, Streptococcus suis, Escherichia coli and Klebsiella pneumoniae.

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