LL-37 derived antibacterial peptide and application thereof

Abstract

The invention belongs to the technical field of antibacterial peptides, and particularly relates to an LL-37 derived antibacterial peptide and application thereof. According to the invention, 17-29aa of the LL-37 antibacterial peptide is used as a core fragment and is connected with an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 through an amido bond, the antibacterial activity of the obtained antibacterial peptide is obviously higher than that of the LL-37 antibacterial peptide, and the antibacterial peptide shows an obvious bactericidal effect on clinical drug-resistant bacteria such as acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli, staphylococcus aureus, klebsiella pneumoniae and the like; meanwhile, the cytotoxicity and hemolytic activity are weak, drug resistance is not prone to being generated, bacteria can be rapidly killed in vivo through multiple mechanisms, no obvious cytotoxicity exists, the bacterium load in tissue is remarkably reduced, and the lifetime of a bacterium-infected mouse is prolonged.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an LL-37-derived antimicrobial peptide and its applications. Background Technology

[0002] Antimicrobial peptide LL-37 is the only member of the cathelicidin family in the human innate immune system. It not only possesses broad-spectrum antimicrobial activity but also regulates the immune system, promotes damage repair and angiogenesis, and treats tumors. LL-37 is the C-terminal cleavage product of the cathelicidin peptide Hcap-18, and is named LL-37 because it contains 37 amino acids. However, the antimicrobial activity of LL-37 itself is not strong; therefore, optimizing the sequence of LL-37 antimicrobial peptide to enhance its antimicrobial activity is crucial. Summary of the Invention

[0003] The purpose of this invention is to optimize the sequence of the LL-37 antimicrobial peptide, so that the obtained antimicrobial peptide has better antimicrobial activity, weaker cytotoxicity and hemolytic activity, and is less likely to induce drug resistance.

[0004] To achieve the above objectives, the present invention provides an antimicrobial peptide, which includes a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are linked by an amide bond; The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; The second polypeptide comprises the amino acid sequence shown in SEQ ID NO:3.

[0005] Preferably, the amino acid sequence of the antimicrobial peptide is as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0006] The present invention also provides the application of the antimicrobial peptides described in the above technical solutions in bactericides or bacteriostatic agents.

[0007] Preferably, the bactericide or bacteriostatic agent targets one or more of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0008] Preferably, the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus; and the Klebsiella pneumoniae includes drug-resistant Klebsiella pneumoniae.

[0009] This invention provides a microbial agent, which is a bactericide or bacteriostatic agent, and the active ingredient of the microbial agent includes the antimicrobial peptide described in the above technical solution.

[0010] This invention provides the application of the antimicrobial peptide or the bacterial agent described in the above-mentioned technical solutions in the preparation of anti-infective products.

[0011] Preferably, the anti-infective product includes an antibacterial product; the bacteria include one or more of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0012] Preferably, the product includes a drug.

[0013] The present invention provides an anti-infective drug, wherein the active ingredient of the anti-infective drug includes the antimicrobial peptide or the bacterial agent described in the above-mentioned technical solution.

[0014] Beneficial effects: This invention provides an antimicrobial peptide comprising a first polypeptide and a second polypeptide linked by an amide bond. The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:3. The amino acid sequence shown in SEQ ID NO:3 corresponds to amino acids 17-29a of the LL-37 antimicrobial peptide. This invention selects this as the core fragment and links it to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 via an amide bond to optimize and modify the LL-37 antimicrobial peptide. The resulting antimicrobial peptide exhibits significantly higher antimicrobial activity than the LL-37 antimicrobial peptide, demonstrating significant bactericidal effects against Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. Simultaneously, it exhibits weak cytotoxicity and hemolytic activity, is less prone to inducing drug resistance, and can rapidly kill bacteria in vivo through multiple mechanisms. It has no significant cytotoxicity, significantly reduces bacterial load in tissues, and prolongs the survival time of mice infected with bacteria. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 Diagram of the KF-22's helical wheel; Figure 2 The circular dichroism chromatogram of KF-22; Figure 3 The image shows the results of the hemolytic activity assay for KF-22. Figure 4 The image shows the cytotoxicity test results for KF-22 and LL-37; where A represents HEK 293T cells and B represents RAW264.7 cells. Figure 5 The results of scanning electron microscopy and transmission electron microscopy on the effects of KF-22 on bacterial morphology are shown; where A is Acinetobacter baumannii ATCC 19606 and B is Staphylococcus aureus ATCC 25923. Figure 6 The results are for bactericidal kinetics testing; where A is Acinetobacter baumannii ATCC 19606 and B is Staphylococcus aureus ATCC 25923. Figure 7 The results represent the induction of drug resistance; where A is Acinetobacter baumannii and B is Staphylococcus aureus. Figure 8 The effects of KF-22 on biofilm and its ability to eliminate persistent bacteria are investigated. A represents the effect on bacterial biofilm formation; B represents the ability to eliminate bacterial biofilm; and C represents the ability to eliminate persistent bacteria. Figure 9 The results represent in vivo antibacterial activity; where A represents the bacterial load in the kidney, lung, liver, and spleen tissues; and B represents the survival time of the mice. Figure 8 and Figure 9 In the middle, there was no significant difference in the expression of ns. express P <0.05, express P <0.01, express P <0.001. Detailed Implementation

[0017] The present invention provides an antimicrobial peptide comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are linked by an amide bond; the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:3.

[0018] As one embodiment, the amino acid sequence of the antimicrobial peptide of the present invention is shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0019] The present invention does not impose strict requirements on the preparation method of the antimicrobial peptide; it can be prepared using conventional methods in the art, such as artificial synthesis.

[0020] The present invention also provides the application of the antimicrobial peptides described in the above technical solutions in bactericides or bacteriostatic agents.

[0021] In one embodiment, the bactericide or bacteriostatic agent of the present invention targets one or more of the following bacteria: Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In one embodiment, the Staphylococcus aureus of the present invention includes methicillin-resistant Staphylococcus aureus (MRSA); in another embodiment, the MRSA is MRSA4. In one embodiment, the Klebsiella pneumoniae of the present invention includes drug-resistant Klebsiella pneumoniae; in another embodiment, the drug-resistant Klebsiella pneumoniae is Klebsiella pneumoniae. K. pneumoniae 6183.

[0022] This invention provides a microbial agent, which is a bactericide or bacteriostatic agent, and the active ingredient of the microbial agent includes the antimicrobial peptide described in the above technical solution.

[0023] This invention provides the application of the antimicrobial peptide or the bacterial agent described in the above-mentioned technical solutions in the preparation of anti-infective products.

[0024] In one embodiment, the anti-infective product of the present invention includes an antibacterial product. In one embodiment, the bacteria of the present invention include one or more of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In one embodiment, the product of the present invention includes a drug.

[0025] The present invention provides an anti-infective drug, wherein the active ingredient of the anti-infective drug includes the antimicrobial peptide or the bacterial agent described in the above-mentioned technical solution.

[0026] In one embodiment, the anti-infective drug of the present invention further includes pharmaceutically acceptable excipients. The present invention does not have strict requirements on the type of pharmaceutically acceptable excipients; they can be conventionally selected according to the drug dosage form requirements. The present invention also does not have strict requirements on the dosage form of the anti-infective drug; conventional drug dosage forms in the art can be used.

[0027] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an LL-37-derived antimicrobial peptide and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 1. The sequence of the cathelicidin BF antimicrobial peptide (KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF, SEQ ID NO:8) was split into three fragments: cathelicidin-BF (1-9), cathelicidin-BF (10-20), and cathelicidin-BF (21-30). These fragments were then randomly combined with the three fragments LL-37 (1-16), LL-37 (17-29), and LL-37 (30-37) from the sequence of the LL-37 antimicrobial peptide (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, SEQ ID NO:9) to obtain a series of hybrid peptides. The activity of these peptides was predicted using an antimicrobial peptide prediction website (http: / / www.camp3.bicnirrh.res.in / predict / ), and the results are shown in Table 1.

[0029] Table 1. Predicted antibacterial activities of different peptides

[0030] Note: Support Vector Machine (SVM); Random Forest (RF); Discriminant Analysis (DA).

[0031] As shown in Table 1, among the obtained heterozygous peptides, KF-22, VK-24, FK-22 and FK-24 all showed high probability in the prediction results of the three antimicrobial peptides, with KF-22 showing the best prediction effect.

[0032] 2. Physiological and biochemical tests Based on the conclusions of step 1, the physiological and biochemical characteristics of peptides Cathelicidin-BF, LL-37, KF-9, VK-11, FK-13, KF-22, VK-24, FK-22, and FK-24 were detected using an online website (https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py). The results are shown in Table 2.

[0033] Table 2. Physiological and biochemical detection results of different peptides

[0034] As can be seen from Table 2, the peptides Cathelicidin-BF, LL-37, KF-9, VK-11, FK-13, KF-22, VK-24, FK-22 and FK-24 are all cationic peptides, and all except VK-11 have a certain degree of hydrophobicity.

[0035] Example 2 1. Cathelicidin-BF, LL-37, KF-9, VK-11, FK-13, KF-22, VK-24, FK-22 and FK-24 in Example 1 were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0036] 2. Use the website https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py to generate the helical wheel diagram of KF-22. The result is as follows: Figure 1 As shown.

[0037] 3. Determination of the circular dichroism chromatogram of KF-22: The secondary structure of KF-22 in ultrapure water, sodium dodecyl sulfate (SDS), and trifluoroethanol (TFE) buffer was determined using circular dichroism spectroscopy. In short, KF-22 was dissolved in ultrapure water, 20 mM SDS solution, and 50% TFE solution, respectively. 200 μL of each mixture was added to a quartz cuvette with a path length of 0.1 cm, and the spectra were scanned and recorded in the wavelength range of 190-250 nm. The results are shown below. Figure 2 As shown. By Figure 2 As shown, the secondary structure of KF-22 is an α-helix. The molecular weight of KF-22 is 2882.58 d Daltons, and its isoelectric point is 12.02.

[0038] 4. Determination of the minimum inhibitory concentration (MIC) 4.1. Strains used: Standard strain of Staphylococcus aureus S.aureus (ATCC 25923), Staphylococcus aureus standard strain S.aureus (ATCC 6538) and methicillin-resistant Staphylococcus aureus MRSA4; Escherichia coli standard strain E. coli (ATCC 8739); Acinetobacter baumannii standard strain A. baumannii (ATCC 19606); Pseudomonas aeruginosa P. aeruginosa (ATCC 27853); Drug-resistant Klebsiella pneumoniae K. pneumoniae 6183. Methicillin-resistant Staphylococcus aureus (MRSA4) and drug-resistant Klebsiella pneumoniae. K. pneumoniae6183 are disclosed in the reference: Integrating a cationic backbone with a hydrophobic core: A structure-function strategy for designing self-assembling antimicrobial peptides with enhanced activity.

[0039] 4.2. Culture conditions of the strain: cultured in Luria-Bertani (LB) medium at 37°C with shaking.

[0040] 4.3. After the strain reached the exponential growth phase, vancomycin (Vanco) and colistin (Coli) were used as positive controls, and sterile 0.9% NaCl was used as a negative control. KF-22 was diluted to various concentrations in sterile physiological saline. (1 OD = 1 × 10⁻⁶) 9 CFU / mL, dilute the above bacterial culture with physiological saline to 2×10⁻⁶. 5 CFU / mL. 100 µL of physiological saline was added to a sterile 96-well plate beforehand, and 200 µL of the test sample was added to the first row of wells. The test sample was then serially diluted twofold, with 100 µL of a 2×10⁻⁶ concentration added to each well. 5 CFU / mL bacterial suspension was cultured at 37℃ for different times. The absorbance of the bacterial suspension at 600 nm was measured using a microplate reader, and the wells where no microbial growth was detected were taken as the MIC values. The experimental results are shown in Table 3.

[0041] Table 3. Antibacterial activity of different peptides (MIC, μg / mL)

[0042] As shown in Table 3, the experimental results indicate that KF-9, VK-11, FK-13, KF-22, VK-24, FK-22, and FK-24 all exhibited good antibacterial activity against both standard strains and clinically resistant strains. KF-22, VK-24, FK-22, and FK-24 showed relatively better antibacterial effects, with MICs ranging from 1.25 to 40 μg / ml. KF-22 exhibited the most significant antibacterial activity.

[0043] Example 3 Hemolytic activity assay Blood was collected from the eyes of specific pathogen-free (SPF-grade) female C57BL / 6 mice (6-8 weeks old) into anticoagulant tubes and centrifuged at 3500 rpm for 10 minutes at 4°C. The supernatant was discarded, and the red blood cells were washed twice with PBS to prepare an 8% red blood cell suspension. In 96-well plates, the peptides were serially diluted with PBS (concentration range 0.625-320 µg / mL), and an equal volume of red blood cell suspension was added to each well. PBS solution containing 1% Triton X-100 and PBS were used as positive and negative controls, respectively. After incubation at 37°C for 1 hour, the plates were centrifuged at 3500 rpm for 5 minutes at 4°C. 100 µl of the supernatant from each well was transferred to a new 96-well plate, and the absorbance was measured at 540 nm using a microplate reader to calculate the hemolysis rate. The formula for calculating the hemolysis rate is as follows: Hemolysis rate (%) = [(Sample A540 - Negative control A540) / (Positive control A540 - Negative control A540)] × 100. The results are as follows... Figure 3 As shown. According to Figure 3 It can be seen that KF-22 does not have obvious hemolytic activity.

[0044] Example 4 Cytotoxicity assay The cytotoxicity of the peptides was detected using a cell proliferation and toxicity assay kit (CCK-8). Human embryonic kidney 293T (HEK293T) cells and mouse macrophage RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin at 37°C in a 5% CO2 incubator. Cells were sporadically grown at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours. The original culture medium was discarded and replaced with fresh medium containing different concentrations of peptides (0-240 µg / mL), and cultured for another 24 hours. 10 µl of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 4 hours. Wells containing only cells were designated as positive controls, and wells containing only DMEM medium were designated as blank controls. Absorbance was measured at 450 nm using a Tecan microplate reader, and cell viability was calculated using the formula: Cell viability (%) = [(A450 of drug-treated group - A450 of blank group) / (A450 of positive control group - A450 of blank group)] × 100. Results are as follows: Figure 4 As shown. According to Figure 4 It can be seen that KF-22 has certain cytotoxicity against HEK293T at high concentrations (120 and 240 μg / m), while LL-37 is more toxic than KF-22; neither KF-22 nor LL-37 has obvious cytotoxicity against RAW264.7 cells.

[0045] Example 5 Effects of polypeptides on bacterial morphology Logarithmic growth phase bacteria (Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923) were cultured in LB medium and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL. The peptide was added to the bacterial suspension at final concentrations of 5-fold and 10-fold MIC, and incubated at 37°C with shaking for 2 hours. After incubation, all samples were centrifuged at 5400 rpm for 5 minutes and the bacterial cells were resuspended in PBS. The bacterial samples were then fixed overnight at 4°C with 2.5% glutaraldehyde. Ultrastructural changes in the bacteria were observed using transmission electron microscopy, and bacterial surface morphology was observed using scanning electron microscopy. Results are as follows: Figure 5 As shown. According to Figure 5 It can be seen that KF-22 at 5 times and 10 times the MIC can cause the cell membrane rupture of Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923, thereby inducing bacterial death.

[0046] Example 6 KF-22 bactericidal kinetics Overnight cultures of Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923 were diluted 1:100 with PBS and adjusted to a concentration of approximately 1×10⁻⁶. 5 CFU / mL. The bacterial suspensions were then treated with PBS, KF-22, polymyxin, or vancomycin, at concentrations set at 1, 5, and 10 times the minimum inhibitory concentration (MIC). All cultures were incubated at 37°C with shaking at 180 rpm. Samples of 10 µL were taken at 0, 30, 60, 120, 180, and 240 minutes, diluted 100-fold, and then plated into 100 µL plates. Colony counts were performed after 24 hours of incubation at 37°C. Results are as follows: Figure 6 As shown. According to Figure 6 It can be seen that KF-22 exhibits rapid bactericidal effects, which are faster than those of clinical antibiotics such as polymyxin and vancomycin, and therefore it is not easy to induce drug resistance.

[0047] Example 7 KF-22 induces resistance to Acinetobacter baumannii and Staphylococcus aureus. Cultured Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923 were continuously treated with sub-inhibitory concentrations of KF-22, polymyxin, or vancomycin. The simplified procedure is as follows: First, the treatment concentrations of KF-22, polymyxin, and vancomycin were determined according to the minimum inhibitory concentration (MIC) determination protocol. Then, the bacterial suspensions from the wells treated with the sub-inhibitory concentrations of KF-22, polymyxin, and vancomycin were diluted to 1×10⁻⁶ using RPMI 1640 medium containing 10% fetal bovine serum. 5 CFU / mL, then proceed to the next round of MIC determination. Repeat this process for at least 20 days. Results are as follows: Figure 7 As shown. According to Figure 7 It can be seen that, compared to polymyxin and vancomycin, which rapidly induce resistance with sub-inhibitory concentrations, KF-22 treatment is less likely to induce resistance. Therefore, KF-22 is less likely to induce resistance than polymyxin and vancomycin and has better safety.

[0048] Example 8 The effects of KF-22 on biofilm and its bactericidal effect on resident bacteria 1. The inhibitory effect of KF-22 on biofilm formation of Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923 was investigated using the following method: Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923 were cultured overnight at 37°C in LB medium. The bacterial suspension was then diluted to 1×10⁻⁶ with RPMI 1640 medium containing 10% fetal bovine serum. 8 CFU / mL was used as the working bacterial culture. KF-22 was serially diluted in RPMI 1640 medium containing 10% fetal bovine serum in 96-well plates to a final concentration ranging from 0.5 to 8 times the minimum inhibitory concentration (MIC), with an equal volume of working bacterial culture added to each well. Untreated bacteria in RPMI medium containing 10% fetal bovine serum served as a control. After incubating the 96-well plates at 37°C for 24 hours, the culture medium was removed, and the plates were washed three times with PBS. Then, each well was fixed with 95% methanol for 15 minutes. After the biofilm air-drying, it was stained with 0.1% crystal violet solution for 15 minutes, followed by rinsing with PBS. Finally, the crystal violet-stained biofilm was dissolved in 100 µL of 95% ethanol and incubated at 37°C for 30 minutes. The absorbance was measured at 600 nm using a microplate reader to assess the biofilm quantity. Results are as follows. Figure 8 As shown in Figure A. According to... Figure 8 As can be seen from Figure A, KF-22 can significantly inhibit biofilm formation in Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923.

[0049] 2. The effect of KF-22 on the removal of biofilms formed by Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923sy was determined by the following method: Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923 were diluted to 1×10⁻⁶ during the logarithmic growth phase. 8 After achieving a CFU / mL concentration, 100 μL of bacterial culture was added to each well of a 96-well plate and cultured for 24 hours to form a biofilm. The supernatant was then removed, and KF-22 peptide was added to RPMI medium containing 10% fetal bovine serum at concentrations ranging from 1 to 8 times the minimum inhibitory concentration (MIC). The biofilm was then treated at 37°C for another 24 hours. Wells containing only 100 μL of fresh RPMI medium (containing 10% fetal bovine serum) served as blank controls, while culture media without the peptide served as positive controls. Biofilm staining was performed using crystal violet as described above, and quantitative analysis was performed at 600 nm using a microplate reader. Results are as follows: Figure 8 As shown in Figure B. According to... Figure 8 As can be seen from Figure B, KF-22 can effectively remove the biofilms already formed by Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923.

[0050] 3. The bactericidal effect of KF-22 on persistent Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923 was determined by the following method: Mid-log growth broths of multidrug-resistant Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923 were diluted to 1×10⁻⁶ with fresh LB medium. 8 CFU / mL. 100 μL of the bacterial suspension was added to a 96-well plate and incubated at 37°C for 24 hours to induce biofilm formation and retain bacteria. After incubation, the supernatant was removed by washing three times with sterile PBS. Then, 100 μL of RPMI medium containing 10% fetal bovine serum and 50 times the minimum inhibitory concentration (MIC) of polymyxin or vancomycin were added to each well, and the plate was incubated at 37°C for another 24 hours. After washing three times with PBS again, the adhered retaining bacteria were detached by sonication (100 μL fresh PBS, 5 minutes). KF-22 was serially diluted (0.5–8 times the MIC) and added to the retaining bacterial suspension, incubated at 37°C for 12 hours, and the number of viable bacteria was determined using microbiological methods. Results are as follows: Figure 8 As shown in C. According to Figure 8 As can be seen from Figure C, KF-22 also has a good ability to eliminate persistent bacteria such as Acinetobacter baumannii ATCC 19606 and Staphylococcus aureus ATCC 25923.

[0051] Example 9 KF-22's in vivo antibacterial activity 1. The in vivo antibacterial activity of KF-22 was determined using a mouse model of intraperitoneal systemic infection: Female C57BL / 6J mice (purchased from Charles River Pharmaceuticals) aged 6-8 weeks and weighing 18±2 g were selected. Neutropenia was induced in mice by intraperitoneal injection of cyclophosphamide on day 1 (150 mg / kg) and day 4 (100 mg / kg). On day 5, Acinetobacter baumannii ATCC 19606 bacterial suspension (1×10⁻⁶) was injected intraperitoneally. 8 An infection model was established using CFU / mL. One hour after infection, mice were treated with KF-22 (2, 4, 8 mg / kg) and polymyxin (2, 4 mg / kg), respectively, while the control group received an equal volume of PBS. Twelve hours after infection, mice were euthanized by cervical dislocation, and lung, liver, spleen, and kidney tissues were collected for bacterial load determination. Results are as follows: Figure 9 As shown in Figure A. According to... Figure 9 As can be seen from Figure A, KF-22 concentration-dependently inhibited the load of Acinetobacter baumannii ATCC 19606 in the kidneys, lungs, liver, and spleen, with effects comparable to those of the clinical antibiotic polymyxin.

[0052] 2. Based on step 1, the effect of KF-22 on the survival rate of mice infected with Acinetobacter baumannii ATCC 19606g was determined. The specific procedure is as follows: Female C57BL / 6J mice aged 6-8 weeks and weighing approximately 18±2 g were selected and intraperitoneally injected with logarithmic growth phase Acinetobacter baumannii ATCC 19606 bacterial suspension (concentration of 1×10⁻⁶). 7 An infection model was established using CFU / mL. One hour after infection, mice were treated with PBS, polymyxin (5 mg / kg), and KF-22 (5 and 10 mg / kg) via intraperitoneal injection, once daily for three consecutive days. The survival of mice was continuously observed and recorded over 7 days. Results are as follows: Figure 9 As shown in Figure B. According to... Figure 9 As can be seen from Figure B, F-22 and polymyxin significantly prolonged the survival rate of mice infected with Acinetobacter baumannii ATCC 19606g.

[0053] As can be seen from the above, the LL-37-derived antimicrobial peptide provided by this invention has strong antimicrobial activity, weak cytotoxicity and hemolytic activity, and is not prone to inducing drug resistance.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antibacterial peptide, characterized in that, The antibacterial peptide comprises a first polypeptide and a second polypeptide, and the first polypeptide and the second polypeptide are connected by an amide bond. The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2. The second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:

3.

2. The antimicrobial peptide according to claim 1, characterized in that, The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:

7.

3. The antibacterial peptide of claim 1 or 2 is used in a bactericidal or bacteriostatic agent.

4. Use according to claim 3, characterized in that, The bacteria against which the bactericidal or bacteriostatic agent is directed include one or more of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae.

5. Use according to claim 4, characterized in that, The Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus; the Klebsiella pneumoniae includes drug-resistant Klebsiella pneumoniae.

6. A microbial agent, which is a fungicide or a bacteriostat, characterized in that, The active ingredient of the bactericidal agent includes the antibacterial peptide of claim 1 or 2.

7. The antibacterial peptide of claim 1 or 2 or the bactericidal agent of claim 6 is used in the preparation of an anti-infection product.

8. Use according to claim 7, characterized in that, The anti-infection product includes an anti-bacterial infection product; the bacteria include one or more of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae.

9. Use according to claim 7 or 8, characterized in that, The product includes a drug.

10. An anti-infective medicament comprising, as an active ingredient, a compound or a salt thereof according to claim 1. The active ingredient of the anti-infection drug includes the antibacterial peptide of claim 1 or 2 or the bactericidal agent of claim 6.

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