Novel antibacterial peptide with broad-spectrum antibacterial property and application thereof

Abstract

The invention discloses a novel antibacterial peptide with broad-spectrum antibacterial performance and application thereof, and belongs to the technical field of biomedicine. According to the novel antibacterial peptide disclosed by the invention, the amino acid sequence of the novel antibacterial peptide is H2N-Lys-Trp-Lys-Trp-Ala-Lys-Arg-Trp-Trp-Ile-COOH. The antibacterial peptide is short in sequence chain, high in yield and good in stability, and has broad-spectrum antibacterial activity on common strains and multi-drug-resistant bacteria. The antibacterial peptide can be used for preparing antibacterial dressings and antibacterial drugs. The antibacterial peptide disclosed by the invention has relatively outstanding antibacterial effects on common strains (staphylococcus aureus, escherichia coli, tritirachium album, staphylococcus epidermidis, bacillus subtilis and pseudomonas aeruginosa) and superbacteria (multi-drug-resistant acinetobacter baumannii, multi-drug-resistant klebsiella pneumoniae and multi-drug-resistant escherichia coli) which have generated multiple drug resistance. The antibacterial peptide disclosed by the invention is easy to chemically synthesize, has relatively good biological safety, has excellent performance of resisting drug-resistant bacteria, and has a wide application prospect in the fields of preparation of antibacterial drugs, dressings and the like.

Description

Technical field:

[0001] The present invention belongs to the field of biomedical technology, and more specifically relates to a novel antimicrobial peptide with broad-spectrum antimicrobial performance and application thereof. Background technology:

[0002] Since the discovery of antibiotics, they have been widely used by human society. However, with the increasingly frequent and irrational use of antibiotics, some bacteria have developed multidrug resistance, making it increasingly difficult for antibiotics to kill these bacteria, which in turn leads to a surge in the morbidity and mortality of bacterial infections. These bacteria are called superbugs. Superbugs are microorganisms that are both highly toxic and resistant to antibiotics. The rampant spread of superbugs is an increasingly serious global threat, especially in the field of healthcare. At present, more and more patients in intensive care units (ICUs) are exposed to multidrug-resistant microbial infections. The mortality rate of patients infected with superbugs is very high, and they themselves become a source of infection for superbugs. At present, because microbial resistance causes millions of deaths every year, humans have spent a lot of manpower, material resources and financial resources to fight against resistant microorganisms. Despite this, the spread of resistant bacteria has not been effectively curbed, and the development of new antibacterial agents that can fight resistant bacteria has become very urgent.

[0003] Antimicrobial peptides (AMPs) are short peptides produced by organisms, usually composed of 6-60 amino acids. Antimicrobial peptides, also known as host defense peptides (HDPs), represent one of the oldest innate defense mechanisms in organisms. Currently, more than 3,000 AMPs have been reported in the AMP database. Compared with traditional antibiotics, antimicrobial peptides have obvious advantages, including slower emergence of resistance, broad-spectrum antimicrobial membrane activity, and the ability to modulate host immune responses. Therefore, antimicrobial peptides are widely considered to be one of the potential alternatives to antibiotics.

[0004] However, among the antimicrobial peptides currently developed, the antimicrobial properties of short-chain antimicrobial peptides are somewhat biased and cannot achieve true broad-spectrum antibacterial properties. They also have disadvantages such as poor antimicrobial activity against drug-resistant bacteria, short shelf life, low yield, poor in vivo activity, and insufficient stability, which bring inconvenience to the preparation of antimicrobial products. Artificial synthesis can make up for the above shortcomings to a certain extent, enhancing the feasibility of the application of antimicrobial peptides. Summary of the invention:

[0005] Therefore, the technical problem to be solved by the present invention is that the existing antimicrobial peptides have poor antimicrobial activity, insufficient antimicrobial range, poor stability and other problems.

[0006] In order to address the above problems, the present invention provides the following technical solutions:

[0007] A novel antimicrobial peptide with broad-spectrum antimicrobial properties and its application, comprising the following steps:

[0008] Preparation of antimicrobial peptides: Using solid phase synthesis, natural or synthetic amino acid matrices containing carboxyl groups and amino groups are mainly polycondensed through amidation reactions. In addition to being used directly, these synthetic antimicrobial peptides can also be designed to have other functional peptides according to different occasions. Alternatively, reactive functional groups such as carboxyl or hydroxyl groups can be introduced into the peptide molecule through partial bonding of ester bonds and amide bonds.

[0009] Preparation of antimicrobial peptide ointment: The antimicrobial peptide is mixed with polyethylene glycol 2000 (PEG2000), polyethylene glycol 200 (PEG200), and glycerol, and the mixture is heated and cooled to prepare the antimicrobial peptide ointment.

[0010] Compared with the prior art, the advantages of the present invention are:

[0011] (1) The antimicrobial peptide prepared by the present invention has a short amino acid sequence, low synthesis cost, high yield and good reproducibility;

[0012] (2) The antimicrobial peptide prepared by the present invention has high antibacterial activity and broad-spectrum antibacterial property, can inhibit Gram-negative bacteria, Gram-positive bacteria, and fungi, and has good inhibitory activity against a variety of multidrug-resistant bacteria. The minimum inhibitory concentration (MIC) of the antimicrobial peptide to Staphylococcus aureus (S.aureus) is 15 μg / mL, the MIC to Escherichia coli (E.coli) is 40 μg / mL, the MIC to Candida albicans (C.albicans) is 30 μg / mL, the MIC to Staphylococcus epidermidis (S.epidermidis) is 10 μg / mL, the MIC to Bacillus subtilis (B.subtilis) is 20 μg / mL, the MIC to Pseudomonas aeruginosa (P.aeruginosa) is 20 μg / mL, the MIC to multidrug-resistant Acinetobacter baumannii (MDR-A.baumannii) is 30 μg / mL, the MIC to multidrug-resistant Klebsiella pneumoniae (MDR-K.pneumoniae) is 250 μg / mL, and the MIC to multidrug-resistant Escherichia coli (MDR-E.coli) is 150 μg / mL.

[0013] (3) The antibacterial ointment prepared by the present invention has a simple preparation process, excellent antibacterial effect, is easy to produce on a large scale, and is more conducive to clinical application. Description of the drawings:

[0014] Figure 1 This is a diagram showing the inhibition zone effect of the antimicrobial peptide obtained in Example 1 on Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Candida albicans (C. albicans);

[0015] Figure 2This is a test graph of the minimum inhibitory concentration (MIC) of the antimicrobial peptide obtained in Example 1 against Staphylococcus aureus (S. aureus);

[0016] Figure 3 This is a graph showing the MIC test of the antimicrobial peptide obtained in Example 1 against Escherichia coli (E.coli);

[0017] Figure 4 This is the MIC test graph of the antimicrobial peptide obtained in Example 1 against Candida albicans (C.albicans);

[0018] Figure 5 This is a graph showing the MIC test of the antimicrobial peptide obtained in Example 1 against Staphylococcus epidermidis (S. epidermidis);

[0019] Figure 6 This is a graph showing the MIC test of the antimicrobial peptide obtained in Example 1 against Bacillus subtilis;

[0020] Figure 7 This is a graph showing the MIC test of the antimicrobial peptide obtained in Example 1 against Pseudomonas aeruginosa (P. aeruginosa);

[0021] Figure 8 This is a MIC test graph of the antimicrobial peptide obtained in Example 1 against multidrug-resistant Acinetobacter baumannii (MDR-A.baumannii);

[0022] Fig. 9 This is a MIC test graph of the antimicrobial peptide obtained in Example 1 against multidrug-resistant Klebsiella pneumoniae (MDR-K.pneumoniae);

[0023] Fig.10 This is a MIC test graph of the antimicrobial peptide obtained in Example 1 against multidrug-resistant Escherichia coli (MDR-E.coli);

[0024] Fig.11 The stability tests of the antimicrobial peptide obtained in Example 1 are as follows: (a) thermal stability, (b) UV irradiation stability, and (c) pH stability;

[0025] Fig.12 is a graph showing the hemolysis rate of the antimicrobial peptide obtained in Example 1;

[0026] Fig.13 is a graph showing the survival rate of L929 cells treated with the antimicrobial peptide obtained in Example 1;

[0027] Fig.14 These are pictures of blank ointment and antimicrobial peptide ointment;

[0028] Fig.15This is the antibacterial zone effect diagram of the antimicrobial peptide ointment on Staphylococcus aureus, Escherichia coli, and Candida albicans;

[0029] Fig.16 This is the recovery of superficial epidermal wounds on the back of mice infected with Candida albicans under different treatments;

[0030] Fig.17 These are pictures of colony smears on the superficial epidermal injury wounds on the backs of mice infected with Candida albicans under different treatments. Specific implementation method:

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. The present invention can be implemented in many different forms and should not be understood as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0032] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0033] Example 1: Preparation of antimicrobial peptides

[0034] The peptides were synthesized on solid phase using amino acids with Fmoc protecting groups.

[0035] (1) First, 0.25 g of dichlorotrityl chloride resin was swollen with 5 mL of N,N-dimethylformamide (DMF) for 2 h.

[0036] (2) Add 0.32 g of isoleucine and 150 μL of N,N-diisopropylethylamine (DIEA) and react for 2 h, then add 5 mL of methanol and 150 μL of DIEA to block the unreacted linker.

[0037] (3) Use 5 mL of 20% piperidine solution to remove the Fmoc protecting group. Then, add 0.38 g of tryptophan,

[0038] 0.4601 g PyBoP, 0.1195 g HoBT and 150 μL DIEA were reacted for 2 h.

[0039] (4) Repeat step (3) and add amino acids in sequence. The amount of each Fmoc-protected amino acid is: isoleucine 0.32 g, tryptophan 0.38 g, arginine 0.35 g, lysine 0.42 g, and alanine 0.28 g.

[0040] (5) After deprotecting the last amino acid, use 5 mL of cleavage agent (trifluoroacetic acid: water: triisopropylsilane = 95:2.5:2.5)

[0041] After cutting for 2 h, the peptide was precipitated in 20 mL of ice ether and finally freeze-dried in a freeze dryer for later use.

[0042] Example 2: Antimicrobial peptide antibacterial effect test

[0043] The antimicrobial peptide prepared in this example was lyophilized and dissolved in PBS buffer to conduct the inhibition zone experiment. Figure 1 As shown, the antimicrobial peptide has good antibacterial activity at 1 mg / mL.

[0044] Example 3: Antimicrobial peptide minimum inhibitory concentration test

[0045] First, different concentrations of antimicrobial peptide solutions were prepared in centrifuge tubes using PBS as a solvent. Bacteria in the logarithmic growth phase were added to each tube to make the bacterial concentration 1×10 7 CFU / mL. After mixing, place in a bacterial incubator and culture for 2 hours. Spread 100 μL of the mixture evenly on LB culture medium and observe the number of colonies after incubation at 37°C in a bacterial incubator for 24 hours. Figure 2 , 3 , 4, 5, 6, 7, 8, 9, and 10 show that the antimicrobial peptide has good antimicrobial activity against different bacterial species and has a broad-spectrum antimicrobial activity. As shown in the figure, the minimum inhibitory concentration of the antimicrobial peptide against Staphylococcus aureus is 15μg / mL, the minimum inhibitory concentration against Escherichia coli is 40μg / mL, the minimum inhibitory concentration against Candida albicans is 30μg / mL, the minimum inhibitory concentration against Staphylococcus epidermidis is 10μg / mL, the minimum inhibitory concentration against Bacillus subtilis is 20μg / mL, the minimum inhibitory concentration against Pseudomonas aeruginosa is 20μg / mL, the minimum inhibitory concentration against multidrug-resistant Acinetobacter baumannii is 30μg / mL, the minimum inhibitory concentration against multidrug-resistant Klebsiella pneumoniae is 250μg / mL, and the minimum inhibitory concentration against multidrug-resistant Escherichia coli is 150μg / mL.

[0046] Example 4: Thermal stability test of antimicrobial peptides

[0047] Prepare several 1.5mL centrifuge tubes, weigh equal masses of antimicrobial peptides into the centrifuge tubes, place the centrifuge tubes in a water bath, and heat at 100°C for 30min, 60min, 90min, and 120min gradient times as the experimental group, and use the 0min heating group as the blank control group. Then add 1mL of ultrapure water to each small centrifuge tube to dissolve the antimicrobial peptide. After that, use a high performance liquid chromatograph to detect, observe the changes in the peak area of ​​the antimicrobial peptide, and perform normalized plots to show the thermal stability of the antimicrobial peptide. The results are as follows: Fig.11 As shown in (a), the antimicrobial peptide can still stably exist at more than 70% under heating conditions at 100°C for 120 minutes, and has good thermal stability.

[0048] Example 5: UV irradiation stability test of antimicrobial peptides

[0049] Prepare several 1.5mL centrifuge tubes, weigh equal masses of antimicrobial peptides into the centrifuge tubes, and dissolve them with 1mL of ultrapure water. Place the centrifuge tubes under ultraviolet light for 0min, 10min, 20min, 30min, 40min, 50min, and 60min, and then perform HPLC detection to observe and record the changes in the peak area of ​​the antimicrobial peptides. Take the group with ultraviolet irradiation time of 0min as the 100% blank control group for normalization plotting to evaluate the ultraviolet irradiation stability of the antimicrobial peptides. The results are shown in Figure 2. Fig.11 As shown in (b), the antimicrobial peptide can still stably exist at more than 80% under ultraviolet light irradiation conditions for 60 minutes; the antimicrobial peptide has good ultraviolet irradiation stability.

[0050] Example 6: pH stability test of antimicrobial peptides

[0051] Prepare several 1.5mL centrifuge tubes, weigh equal amounts of antimicrobial peptides in the centrifuge tubes, add 1mL of buffer solution with pH=2, 4, 6, 7, 8, 10, 12 to the small centrifuge tubes to dissolve the antimicrobial peptides, let stand for 4 hours, and then use high performance liquid chromatography to detect the changes in the peak area of ​​the antimicrobial peptides. The group with pH=7 was used as the 100% blank control group for normalization plotting to evaluate the pH stability of the antimicrobial peptides. Fig.11 As shown in (c), the antimicrobial peptide can still stably exist at more than 70% after 4 hours in an environment with a pH value of 2-12, and the antimicrobial peptide has good pH stability.

[0052] Example 7: Hemolytic activity test of antimicrobial peptides

[0053] Different concentrations of antimicrobial peptide solutions dissolved in PBS buffer were incubated with fresh blood, and the supernatant was centrifuged and tested for hemolysis rate using an ELISA instrument. Fig.12 As shown, the antimicrobial peptide had no obvious hemolytic activity at concentrations below 300 μg / mL.

[0054] Example 8: Cytotoxicity test of antimicrobial peptides

[0055] Different concentrations of antimicrobial peptide solutions dissolved in cell culture medium were incubated with L929 cells, and the cell survival rate was tested using an ELISA instrument. Fig.13 As shown, the antimicrobial peptide had no obvious cytotoxicity at concentrations below 500 μg / mL, indicating that it had good biocompatibility.

[0056] Example 9: Preparation of antimicrobial peptide ointment

[0057] Two polyethylene glycols (PEG2000 and PEG200) with different molecular weights and glycerol are used as raw materials to prepare ointments, and antimicrobial peptides are incorporated therein. The specific preparation method is as follows: weigh PEG2000 in a glass bottle, stir and heat at 50-60°C in a water bath for 1 hour to melt it, then take an equal amount of glycerol and four times the mass of PEG200 in the bottle and continue to stir and heat for 0.5 hour to mix evenly, then add antimicrobial peptide powder accounting for about 2% of the total mass of the antimicrobial ointment, continue to heat and stir for 0.5 hour, and then let the glass bottle stand at room temperature to obtain a slightly light yellow antimicrobial peptide ointment. Fig.14 The following are pictures of blank ointment and antimicrobial peptide ointment.

[0058] Example 10: In vitro antibacterial function test of antimicrobial peptide ointment

[0059] The antibacterial effect of the ointment in vitro was tested using the inhibition zone method. 100 μL of the ointment was used for the inhibition zone experiment to observe whether there was an obvious inhibition zone. The selected bacteria were Staphylococcus aureus, Escherichia coli, and Candida albicans. The results are as follows Fig.15 As shown, the antibacterial peptide ointment has obvious inhibition zones against these three bacteria and possesses excellent in vitro antibacterial ability.

[0060] Example 11: In vivo antibacterial function test of antimicrobial peptide ointment

[0061] In order to verify the in vivo antibacterial effect of antimicrobial peptide ointment, a mouse superficial epidermal Candida albicans infection model was established. The mice were divided into four groups: no treatment group, blank ointment treatment group, antimicrobial peptide ointment treatment group, and commercial miconazole nitrate cream treatment group. There were three mice in each group. The back hair of each group of mice was shaved clean under anesthesia, and the back skin of the mice was rubbed slowly and gently with a sterile steel wool until the skin was shiny and red but without obvious bleeding. A 2×2 cm sterile gauze was soaked in a solution with a bacterial concentration of 1×10 8 CFU / mL of Candida albicans liquid for 2h, take out the gauze and stick it on the back skin of the mouse after friction, then fix it with tape and remove it after a period of time. Then treat it with the above different treatment methods every day, apply twice a day, 50mg each time. Take pictures every day to record the changes in the back wound of the mouse, then wipe the back wound of the mouse with a wet cotton swab, fold the cotton swab head down and soak it in 5mL of sterilized PBS solution for 2h, take 100μL of the soaked PBS buffer for flat coating to record the changes in the back colony of the mouse. Fig.16 This is the recovery of superficial epidermal injury wounds on the back of mice infected with Candida albicans under different treatments. Fig.17The following are colony smears of superficial epidermal wounds on the back of mice infected with Candida albicans under different treatments. The results show that the antimicrobial peptide ointment has a good killing effect on Candida albicans in vivo and promotes the repair of infected wounds.

[0062] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A novel antimicrobial peptide with broad-spectrum antimicrobial properties, characterized in that: The antimicrobial peptide sequence is H2N-Lys-Trp-Lys-Trp-Ala-Lys-Arg-Trp-Trp-Ile-COOH, and the molecular weight is 1487.79 Da.

2. Use of the novel antimicrobial peptide with broad-spectrum antimicrobial properties as claimed in claim 1 in the preparation of antimicrobial and antibacterial products for bacterial / fungal infections.

3. The use according to claim 2, characterized in that: The antimicrobial peptide is used for preparing antimicrobial drugs / carriers / dressings for treating bacterial / fungal infections.

4. The use according to claim 2, characterized in that: The bacteria include common bacteria and multi-drug resistant super bacteria.

5. The use according to claim 2, characterized in that: The fungus is Candida albicans (C.albicans).

6. The use according to claim 4, characterized in that: The common bacteria include Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Staphylococcus epidermidis (S. epidermidis), Bacillus subtilis (B. subtilis), and Pseudomonas aeruginosa (P. aeruginosa).

7. The use according to claim 4, characterized in that: The super bacteria include multidrug-resistant Acinetobacter baumannii (MDR-A.baumannii), multidrug-resistant Klebsiella pneumoniae (MDR-K.pneumoniae), and multidrug-resistant Escherichia coli (MDR-E.coli).

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