Broad-spectrum antibacterial peptide as well as screening method and application thereof

Abstract

The invention discloses a broad-spectrum antibacterial peptide as well as a screening method and application thereof, and belongs to the technical field of antibacterial peptides, a bioinformatics method is mainly adopted for searching a new antibacterial peptide, then amino acid sequence optimization is carried out, and finally the antibacterial peptide with a better antibacterial effect is obtained. Sterilization activity evaluation is carried out by using various known strains with drug resistance, and the result shows that the antibacterial peptide provided by the invention has broad-spectrum antibacterial activity on drug-resistant bacteria, more comprehensive antibacterial effect and wider indications; the compound has the advantages of no hemolysis, low cytotoxicity, efficient in-vivo antibacterial activity and high stability, and has application safety; the compound has a wide application prospect of being developed into a human body antibacterial drug.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and particularly relates to a broad-spectrum antimicrobial peptide, a screening method thereof, and an application thereof. Background Art

[0002] Since the successful industrialization of penicillin in 1938, the research and development of antibiotics have entered a period of rapid development. Antibiotics have treated countless patients with bacterial or fungal infections and made great contributions to human health. However, due to the inappropriate use of antibiotics and the lack of management of antibiotics, many bacteria have developed resistance to antibiotics, resulting in the ineffectiveness of antibiotic treatment. Bacterial resistance has seriously threatened human health. In recent years, the emergence of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii) and Gram-positive bacteria (Staphylococcus aureus) has been an increasingly serious problem and challenge worldwide. In the face of the increasingly serious resistance problem, however, not enough drugs have been developed to solve this problem so far. Polymyxins (such as colistin) have become the last effective means for the treatment of drug-resistant bacterial infections. Therefore, it is urgent to develop antibacterial agents with different action mechanisms and targets from traditional antibiotics in order to reduce the probability of severe infections caused by drug-resistant bacteria.

[0003] Antimicrobial peptides (AMPs) are short polypeptides that are mainly positively charged and have a length of 10 - 80 amino acid residues. Antimicrobial peptides are an important part of the innate immune system, have biological activities against a variety of bacteria, viruses, and fungi, and have the characteristics of rapid bactericidal action, low host cell toxicity, and low bacterial resistance. Some antimicrobial peptides can also activate the immune system and have important functions in cell signal transduction, cell proliferation regulation, and anti-tumor, anti-virus, and anti-parasite aspects.

[0004] As promising antimicrobial drugs against multi-drug resistant bacteria, antimicrobial peptides have shown unprecedented advantages. Different from antibiotics that interfere with the metabolic processes of pathogenic microorganisms, antimicrobial peptides usually produce antimicrobial effects by physically destroying the lipids of the microbial cell membrane and inducing the leakage of cell contents. Therefore, they have little impact on the probability of the evolution of bacterial resistance. Currently, due to their strong antimicrobial potential and unique action mechanism, antimicrobial peptides are considered ideal candidates to replace antibiotics. Research and development of new antimicrobial peptides as antibiotic alternatives have become an important topic that needs to be studied urgently.

[0005] To find antimicrobial peptides from polypeptides, people initially studied polypeptides through experiments and identified them by observing whether they had antimicrobial activity. In this way, although it was possible to accurately determine whether a polypeptide had an antimicrobial effect, the experimental process was cumbersome, time-consuming, required a large amount of funds, and could not predict the activity of antimicrobial peptides. With the development of high-throughput proteomics, the number of protein and polypeptide sequences has increased rapidly. It is difficult to identify effective antimicrobial peptides from a large number of peptide samples and predict their antimicrobial activity through experiments. Therefore, we need to find other methods to identify and predict effective antimicrobial peptides. Summary of the Invention

[0006] To solve the deficiencies in the prior art, the present invention provides a broad-spectrum antimicrobial peptide, as well as a screening method and application thereof. The purpose of this study is to screen a peptide compound with strong antimicrobial activity, in order to solve the increasingly serious problem of bacterial resistance to existing market antibiotics and the pain caused by common hospital bacterial infections to the majority of patients.

[0007] The specific technical solution is as follows:

[0008] The present invention provides a broad-spectrum antimicrobial peptide, selected from the following polypeptides or combinations thereof:

[0009] (1) The polypeptides shown in SEQ ID NO.1 to SEQ ID NO.5;

[0010] (2) Truncated peptides of the polypeptides shown in SEQ ID NO.1 to SEQ ID NO.5;

[0011] (3) Derived peptides of the polypeptides described in (1) or (2), or modified forms and polypeptides having the same function as the polypeptides described in (1) or (2), and the polypeptides are cyclic peptides or linear peptides.

[0012] Wherein: the truncated peptide is a polypeptide obtained by truncating one or more amino acids from the polypeptides described in SEQ ID NO.1 to SEQ ID NO.5, and the polypeptide is a cyclic peptide or a linear peptide. Each of the above truncated peptides represents an independent embodiment of the present invention.

[0013] The sequences of SEQ ID NO.1 to SEQ ID NO.5 are:

[0014] HHHHGVRGTCKLTIMPKPLKRRGRRSRSMAIAHLVRSRHMKRWRWGLRKRISTVLPPWRYITRIISAVSVTGRSSYCFHH (SEQ ID NO.1);

[0015] HHHHKKQQKIFKFVFLHRRGSRQPWRPARMAWKPHFHKDWQLARGHMVQPAGPKIRRCKAGQAKVPHIALHTALGYCFHH(SEQ ID NO.2);

[0016] HHHHRKWKKKSHHVLRKFTNLCWAAFKTFMGHMWPVGYRFNKLALNHHCLVQTSADHCGMNRMQPSSLLRPNMCAYCFHH(SEQ ID NO.3);

[0017] HHHHRQRCTAGRRRRSSTNINRCLCQSARFARRWKNNWRCAGGWACVTVHIPWRNWRRHLPKVRRCVFPAFRIRNYCFHH(SEQ ID NO.4);

[0018] HHHHLKGKGASVSAPYWNRGRLLPVVMTKLSTKPVMRRSAHRSSSRWWRNAKRIIKSRAGNRKCLPDARSWRCKWYCFHHH(SEQ ID NO.5).

[0019] The configuration of each amino acid is independently selected from D-configuration or L-configuration.

[0020] Based on the above technical solution, further, the truncated peptide has a length of 10 to 50 amino acids, and the truncated peptide is a cyclic peptide or a linear peptide.

[0021] Based on the above technical solution, further, the truncated peptide has a length of 10 to 30 amino acids, and the truncated peptide is a cyclic peptide or a linear peptide.

[0022] Based on the above technical solution, further, the truncated peptide has a length of 15 to 30 amino acids, and the truncated peptide is a cyclic peptide or a linear peptide.

[0023] Based on the above technical solution, further, the derivative peptide is a polypeptide having the same function obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence of the polypeptide of (1) or (2).

[0024] Based on the above technical solution, further, the derivative peptide is a polypeptide having the same function obtained by substituting, deleting, and / or adding one, two, three, or four amino acids to the amino acid sequence of the polypeptide of (1) or (2).

[0025] Optimized derivative peptides are obtained by truncating the sequence and replacing amino acids. The derivative peptides are placed on the public website heliquest (https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py) to predict their hydrophobic moment, hydrophobic surface, positive charge, etc., and polypeptides are synthesized based on this.

[0026] Based on the above technical solution, further, the sites modified by the modification form include but are not limited to N-terminal modification, C-terminal modification, backbone modification, side-chain modification, and amino acid modification.

[0027] Specifically, the modification is selected from the following forms or combinations thereof:

[0028] (1) Cyclic peptide synthesis: head-to-tail cyclization, side-chain cyclization (lactone, lactam, ether bond, etc.), multiple disulfide bonds, monothioether cyclization, etc.;

[0029] (2) Isotope labeling: 13 C, 15 N, 18 Isotope labeling such as O;

[0030] (3) Polyethylene glycol (PEG) modification: Modification with PEG2, PEG4, PEG8, PEG12, PEG24, PEG36, PEG2000, PEG5000, PEG3400, PEG20K, PEG40K, etc.;

[0031] (4) Phosphorylation modification: Phosphorylation modification of L-type or D-type amino acids (such as threonine T, serine S), single or multiple amino acid phosphorylation modifications;

[0032] (5) Polypeptide-protein conjugation: KLH, BSA, OVA, etc.;

[0033] (6) Modification of N-terminal or side-chain amino acids: Acetylation, formylation, biotinylation, trifluoroacetylation, benzoylation, 2-aminobenzoylation, maleimidation, chloroacetylation, bromoacetylation, succinylation, palmitoylation, malic acidification, fatty acidification, formaldehydeation, chelation reaction (such as Hynic, DTPA, DOTA, NOTA), chlorination, fluorination, bromination, nitro or methoxy substitution, fluorescence labeling (such as Cy series, Texas series, Alexa series, rhodamine, Bodipy, Rox, FAM, FITC, MCA, TAMRA, Dnp), etc.;

[0034] (7) C-terminal modification: Amidation, esterification, aldehydeation, alcoholization, succinylation, fluorescence labeling (such as Cy series, rhodamine, AMC, AFC, PNA, CMK, FMK), etc.;

[0035] (8) Alkylation modification: N-methylation, side chain methylation, N-ethylation, N-phenylpropylatation, N-allylation, etc.;

[0036] (9) Other special modifications: glycopeptide, sulfonation, MAPS, etc.

[0037] In the process of polypeptide structure optimization, unnatural modification strategies can also be adopted to improve the activity, stability and selectivity of polypeptides. By replacing natural amino acids in the sequence with unnatural amino acids with similar physicochemical properties, the stability of polypeptides can be improved.

[0038] Based on the above technical solution, further, the broad-spectrum antibacterial peptide includes at least one of the following listed amino acid sequences, or a combination thereof,

[0039] (I) KRWRW;

[0040] (II) ISTVL;

[0041] (III) GSRQP;

[0042] (IV) RKWKKK;

[0043] (V) WRNWRRHL;

[0044] (VI) WWRNAKRIIK.

[0045] Based on the above technical solution, further, the broad-spectrum antibacterial peptide includes at least one of the following listed amino acid sequences, or a combination thereof,

[0046] (I) KRWRWGLR;

[0047] (II) ISTVLPPW;

[0048] (III) HRRGSRQP;

[0049] (IV) RKWKKKWHH;

[0050] (V) WRNWRRHLPKV;

[0051] (VI) RWWRNAKRIIKS.

[0052] Further, the length of the broad-spectrum antibacterial peptide does not exceed 50 amino acids.

[0053] More preferably, the length of the broad-spectrum antibacterial peptide does not exceed 35 amino acids.

[0054] On the other hand, the present invention provides a broad-spectrum antibacterial peptide, and the broad-spectrum antibacterial peptide has an amino acid sequence shown in any one of SEQ ID No: 6 to SEQ ID No: 152 in Table 1 below:

[0055] Table 1 Partial polypeptide sequences of the present invention

[0056]

[0057]

[0058]

[0059]

[0060] On the other hand, the present invention provides an antibacterial peptide having an amino acid sequence with at least 90%, at least 95%, or at least 99% identity to any one of the amino acid sequences of SEQ ID No: 1 to SEQ ID No: 152.

[0061] On the other hand, the present invention provides a pharmaceutical composition comprising an effective amount of the above-mentioned broad-spectrum antibacterial peptide.

[0062] Based on the above technical solution, further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient, and the pharmaceutically acceptable carrier and / or excipient includes a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, and a flavoring agent.

[0063] On the other hand, the present invention provides the application of the above-mentioned broad-spectrum antibacterial peptide and pharmaceutical composition in the preparation of a drug for killing or inhibiting pathogenic bacteria;

[0064] In some embodiments, the pathogenic bacteria include bacteria.

[0065] On the other hand, the present invention provides any one of the following applications of the above-mentioned broad-spectrum antibacterial peptide and pharmaceutical composition:

[0066] A1) Destroy the integrity of the bacterial cell membrane;

[0067] A2) Prepare a product for destroying the integrity of the bacterial cell membrane;

[0068] A3) Reduce the permeability of the outer membrane of bacteria;

[0069] A4) Prepare a product for reducing the permeability of the outer membrane of bacteria;

[0070] A5) Destroy the membrane potential of bacteria;

[0071] A6) Prepare a product for destroying the membrane potential of bacteria;

[0072] A7) Destroy the integrity of the bacterial cell wall;

[0073] A8) Preparing a product that disrupts the integrity of the bacterial cell wall;

[0074] A9) Inhibiting the activity of bacteria;

[0075] A10) Preparing a drug that inhibits the activity of bacteria.

[0076] Based on the above technical solution, further, the bacteria are Gram-positive bacteria and / or Gram-negative bacteria.

[0077] Based on the above technical solution, further, the Gram-negative bacteria include Escherichia coli, Salmonella typhimurium, Salmonella pullorum, Salmonella spp., Pseudomonas aeruginosa, Acinetobacter baumannii, Shigella spp., and Enterobacter sakazakii, and the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Listeria monocytogenes, and Bacillus cereus.

[0078] Based on the above technical solution, further, the bacteria include one or more of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0079] Based on the above technical solution, further, the Pseudomonas aeruginosa is a drug-resistant Pseudomonas aeruginosa, and the Staphylococcus aureus is a drug-resistant Staphylococcus aureus.

[0080] Based on the above technical solution, further, the drug-resistant Pseudomonas aeruginosa is a carbapenem-resistant Pseudomonas aeruginosa, and the drug-resistant Staphylococcus aureus is a methicillin-resistant Staphylococcus aureus.

[0081] As used herein, the "peptide library" is a peptide library constructed by Hunan Zhongcheng Peptide Biochemical Co., Ltd. using the PICT (Peptide Information Compression Technology) patented technology. This technology compresses peptide information by biological means, integrating the information of multiple peptides into one peptide, thereby achieving a relatively small library capacity containing a large amount of peptide information; a cyclic peptide library containing nearly 73,000 80-amino acid peptides is constructed by the PICT technology. The specific construction method can be referred to in Patent CN201580081102.3 and Patent CN201780089941.9.

[0082] As used herein, the "antibacterial peptide", also referred to as "antibacterial polypeptide", is synonymous with "antibacterial protein" and "antibacterial protein", and is used herein to refer to a polymer of amino acid residues having antibacterial, bacteriostatic, or bactericidal functions. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a specific polypeptide sequence also implicitly encompasses its conservatively modified variants. Description of the Drawings

[0083] Figure 1 It is the detection result of the bacterial survival rate at different time points in Example 5;

[0084] Figure 2 It is the hemolysis rate detection result of polypeptides with different concentrations in Example 6;

[0085] Figure 3 It is the cytotoxicity experiment result of the polypeptide on NHDF cells and HEK293 cells in Example 7;

[0086] Figure 4 It is the result of bacteria taking up NPN after being treated with polypeptides at different concentrations in Example 9;

[0087] Figure 5 It is the change of the absorbance value of bacteria over time after being treated with polypeptides at different concentrations in Example 10;

[0088] Figure 6 It is the ColabFold structure prediction result of some polypeptides of the present invention in Example 11;

[0089] Figure 7 It is the liquid phase detection result of the polypeptide shown in SEQ ID No: 58 in Example 12;

[0090] Figure 8 It is the mass spectrometry detection result of the polypeptide shown in SEQ ID No: 58 in Example 12;

[0091] Figure 9 It is the liquid phase detection result of the polypeptide shown in SEQ ID No: 63 in Example 12;

[0092] Figure 10 It is the mass spectrometry detection result of the polypeptide shown in SEQ ID No: 63 in Example 12;

[0093] Figure 11 It is the liquid phase detection result of the polypeptide shown in SEQ ID No: 79 in Example 12;

[0094] Figure 12 It is the mass spectrometry detection result of the polypeptide shown in SEQ ID No: 79 in Example 12;

[0095] Figure 13 It is the liquid phase detection result of the polypeptide shown in SEQ ID No: 113 in Example 12;

[0096] Figure 14 It is the mass spectrometry detection result of the polypeptide shown in SEQ ID No: 113 in Example 12;

[0097] Figure 15 Liquid detection results of the polypeptide shown in SEQ ID No: 133 of Example 12;

[0098] Figure 16 Mass spectrometry detection results of the polypeptide shown in SEQ ID No: 133 of Example 12;

[0099] Figure 17 Liquid detection results of the polypeptide shown in SEQ ID No: 150 of Example 12;

[0100] Figure 18 Mass spectrometry detection results of the polypeptide shown in SEQ ID No: 150 of Example 12. Detailed implementation manners

[0101] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0102] Example 180 Screening of cyclic peptide antibacterial peptides

[0103] Take out the 96-well deep well plate of 80-cyclic peptides, and dilute the polypeptide to 5 mg / mL (final concentration is 250 μg / mL). Add Escherichia coli ATCC25922 with a bacterial liquid amount of 2×105 / mL to each well (total volume of each well is 100 μL). Incubate statically at 37 °C for 20 h, observe whether bacteria grow, and sequentially judge whether the polypeptide has antibacterial activity.

[0104] Through high-throughput screening, the present invention conducts a primary screening of antibacterial activity against Escherichia coli ATCC25922 (polypeptide primary screening concentration is 250 μg / mL) on the pentapeptide full library of the company, and screens out 80-cyclic peptides with better antibacterial activity from nearly 73,000 80-cyclic peptides, namely SEQ ID No. 1 to SEQ ID No. 5. The antibacterial activity results of SEQ ID No. 1 to SEQ ID No. 5 are shown in Table 2, and the antibacterial activity of the active polypeptides is all below 250 μg / mL.

[0105] Table 2 Antibacterial activity of 80-cyclic peptides of the present invention

[0106]

[0107] Example 2. Design of truncated peptides and derivative polypeptides

[0108] The first round of amino acid decompression was performed on the 80-ring peptides (SEQ ID No.1 to SEQ ID No.5), and multiple linear peptides with amino acid sequences of different lengths were designed. Through the primary screening of the antibacterial activity of the first-round decompressed peptides against Escherichia coli ATCC25922, antibacterial peptides with antibacterial activity below 62.5 μg / mL were obtained. The broad-spectrum antibacterial and bacteriostatic activities of the above antibacterial peptides were screened, and antibacterial peptides with good broad-spectrum antibacterial activities were obtained. The second and third rounds of decompression and optimization were carried out on them. By further means such as truncating and optimizing the sequence, replacing amino acids to increase the positive charge number or hydrophobicity, and using the public website heliquest (https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py) to predict the hydrophobic moment, hydrophobic surface, charge number, etc. of the antibacterial peptides, the effect of optimizing the antibacterial peptides was achieved.

[0109] From the perspective of improving the druggability of peptide molecules, the polypeptide structures obtained by disassembly were modified and transformed. The modification strategies were divided into two categories: one was the modification of the peptide chain backbone, including non-natural amino acid modification, cyclization strategy, terminal structure modification, etc.; the other was to introduce other groups for structural optimization and performance modification on the basis of the unchanged polypeptide backbone, including higher fatty acid modification, polyethylene glycol modification, protein fusion strategy, cholesterol modification, etc. According to the above modification strategies, cyclic peptides or linear peptides with different amino acid sequences were designed. Some of the derivative peptides of the present invention are shown in Table 3.

[0110] Table 3 Some derivative peptides of the present invention

[0111]

[0112]

[0113]

[0114]

[0115] Example 3 Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of polypeptides

[0116] The antibacterial (Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, and Pseudomonas aeruginosa ATCC27853) activities of the truncated peptides and derivative peptides described in Example 2 were detected.

[0117] The broth dilution method was used to determine the minimum inhibitory concentration and minimum bactericidal concentration. The specific steps were as follows:

[0118] (1) Take out the glycerol bacteria from the ultra-low temperature refrigerator at 80 °C. Use a sterile inoculation loop to scrape a little of the frozen bacteria and streak-inoculate it on a TSA solid medium petri dish. Place it in a constant temperature incubator and culture at 37 °C for 16 - 20 hours; (2) Pick a single colony and inoculate it into 10 mL of CAMHB liquid medium. Culture at 37 °C with 250 rpm until the OD600 is about 0.6 - 0.8;

[0119] (3) Dilute the bacterial solution with sterile normal saline to an OD 600 of about 0.15, that is, the colony count is about 10 8 cfu / mL;

[0120] (4) Dilute the bacterial solution:CAMHB medium at a ratio of 1:300. At this time, the colony count of the bacterial solution is about 10 5 cfu / mL;

[0121] (5) Dissolve the polypeptide in sterile water to prepare a stock solution of 5.12 mg / mL. Transfer the corresponding volume of the stock solution to the starting well of the dilution plate, and then perform two-fold serial dilution 9 times. There are a total of 10 different concentrations of the same polypeptide; Dissolve imipenem into 80 μg / mL, transfer the corresponding volume to the starting well of the dilution plate, and then perform two-fold serial dilution 9 times;

[0122] (6) Add 5 μL of the polypeptide in the dilution plate to the corresponding wells of the test plate, and then add 95 μL of the bacterial solution culture to the test plate (except for the SC well). Repeat each test plate 2 times;

[0123] Growth control (GC): Liquid medium containing microbial inoculum and sterile water, without polypeptide. Sterile control (SC): Liquid medium and sterile water, without polypeptide.

[0124] Table 4 Concentration gradient of broth two-fold dilution method

[0125]

[0126] (7) Place the test plate in a constant temperature incubator and culture at 37 °C for 20 h;

[0127] (8) Observe the growth of bacteria in each well. The lowest concentration without bacterial growth is the minimum inhibitory concentration;

[0128] (9) Dip a sterile inoculation loop into the liquid in each well respectively, coat it on a TSA agar plate, wait for the liquid to be completely absorbed, then place it in a 37 °C constant temperature incubator and culture for 20 h. Observe the colonies on each plate and record the minimum bactericidal concentration.

[0129] Six polypeptides with good antibacterial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were screened from the designed polypeptides. The experimental results are shown in Table 5, namely SEQ ID No.58, SEQ ID No.63, SEQ ID No.79, SEQ ID No.113, SEQ ID No.133, and SEQ ID No.150. The geometric mean of their minimum inhibitory concentrations against Escherichia coli ATCC25922 was in the range of 3.78 - 13.24 μg / mL, the geometric mean of their minimum inhibitory concentrations against Staphylococcus aureus ATCC29213 was in the range of 5.77 - 19.13 μg / mL, and the geometric mean of their minimum inhibitory concentrations against Pseudomonas aeruginosa ATCC27853 was in the range of 8 - 20.89 μg / mL. These 6 polypeptides all had broad-spectrum antibacterial activities and could effectively kill bacteria at concentrations below 32 μg / mL.

[0130] Table 5 Determination results of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of polypeptides

[0131]

[0132] a is the mode, GM is the geometric mean

[0133] Antibacterial experiment of Example 4

[0134] The radial diffusion method was used to determine the antibacterial effect of the polypeptide. The specific operation was as follows:

[0135] (1) Take out the glycerol bacteria from the -80 °C ultra-low temperature refrigerator, scrape a little of the frozen bacteria with a sterile inoculation loop and streak inoculate on the TSA solid medium petri dish, and place it in a constant temperature incubator at 37 °C for 16 - 20 hours;

[0136] (2) Pick a single colony and inoculate it into 10 mL of CAMHB liquid medium, and culture it at 37 °C and 250 rpm until the OD600 is about 0.6 - 0.8;

[0137] (3) Dilute the bacterial solution with sterile normal saline to OD 600 about 0.15, that is, the number of colonies is about 10 8 cfu / mL;

[0138] (4) Dilute the polypeptide to 0.8 mg / mL according to the concentration requirements, and dilute imipenem to 250 μg / mL, 5.2 μg / mL, and 1.3 μg / mL, respectively, for the detection of Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25922, and Staphylococcus aureus ATCC29213;

[0139] (5) Prepare the bottom agar: Prepare a bottom agar with a TSB concentration of 0.03% (W / V), a low electroosmotic agarose concentration of 1% (W / V), and a Tween-20 concentration of 0.02% (V / V). After autoclaving, cool it to 42 - 46 °C.

[0140] (6) Add the bacterial solution to the bottom agar at a ratio of 1:300, mix well and pour it into a petri dish (9 cm), about 15 mL, and let it solidify at room temperature.

[0141] (7) Use a gel puncher to punch 9 sample holes with a diameter of 4 mm on the petri dish, add 6 μL of the polypeptide (0.8 mg / mL) to the holes, and incubate at 37 °C for 3 h to allow the sample to fully diffuse into the bottom agar.

[0142] (8) Prepare the overlay agar: Prepare an overlay agar with a TSB concentration of 6% (W / V) and a low electroosmotic agarose concentration of 1% (W / V). After autoclaving, cool it to 42 - 46 °C, add 15 mL of the overlay agar to the above petri dish, and after it fully solidifies, incubate it at 37 °C for about 20 h. Among them, invert the agar containing Pseudomonas aeruginosa into a new petri dish so that the bottom agar containing bacteria contacts the air, thereby allowing the bacteria to grow fully.

[0143] (9) Measure and record the diameter of each inhibition zone.

[0144] The experimental results are shown in Table 6. The inhibition zone diameters of SEQ ID No.58, SEQ ID No.63, SEQ ID No.79, SEQ ID No.133, and SEQ ID No.150 against Escherichia coli ATCC25922 are between 8.5 - 15.5 mm. Among them, the inhibition zone diameters of SEQ ID No.63, SEQ ID No.113, and SEQ ID No.150 are 15, 15.5, and 15 mm respectively; the inhibition zone diameters against Staphylococcus aureus ATCC2921 are between 7 - 13 mm. Among them, the inhibition zone diameters of SEQ ID No.63, SEQ ID No.113, and SEQ ID No.150 are 13, 10.5, and 11 mm respectively; the inhibition zone diameters against Pseudomonas aeruginosa ATCC27853 are between 8.5 - 15.5 mm. Among them, the inhibition zone diameters of SEQ ID No.63, SEQ ID No.113, and SEQ ID No.150 are 13.5, 15.5, and 13.5 mm respectively.

[0145] Table 6 Measurement results of polypeptide inhibition zones

[0146]

[0147] Example 5 Time-kill kinetics experiment of the polypeptide

[0148] The polypeptide has inhibitory effects on Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. In this experiment, the effects of the polypeptide on the growth of the above bacteria were investigated at different times. The specific operations are as follows:

[0149] (1) Take out the glycerol bacteria from the -80°C ultra-low temperature refrigerator. Use a sterile inoculation loop to scrape a little of the frozen bacteria and streak-inoculate it on a TSA solid medium petri dish. Place it in a constant temperature incubator and incubate at 37°C for 16 - 20 hours;

[0150] (2) Pick a single colony and inoculate it into 10 mL of CAMHB liquid medium. Incubate at 37°C and 250 rpm until the OD600 is approximately 0.6 - 0.8;

[0151] (3) Dilute the bacterial solution with sterile normal saline to an OD600 of approximately 0.15, that is, the number of colonies is approximately 10 8 cfu / mL;

[0152] (4) Dilute the bacterial solution:CAMHB medium at a ratio of 1:300. At this time, the number of colonies in the bacterial solution is approximately 10 5 cfu / mL;

[0153] (5) Add the polypeptide to the bacterial solution (the total volume is 1 mL, and the final concentration of the polypeptide is 1×MIC), and incubate at 37°C;

[0154] (6) Take 10 μL of samples at 0, 0.5, 1, 2, 4, and 6 h. After diluting 100 times, take 100 μL and spread it on a plate. Place the plate in a 37°C constant temperature incubator and incubate for 20 h.

[0155] (7) Count the colonies and compare the number of colonies at different time points.

[0156] The experimental results are as Figure 1 shown. When the 6 polypeptides tested (SEQ ID No.58, SEQ ID No.63, SEQ ID No.79, SEQ ID No.113, SEQ ID No.133, SEQ ID No.150) were added to the bacterial solution at a concentration of 1×MIC, the bacteria were basically killed within 2 hours, and most of the polypeptides completed bactericidal action within 30 minutes after being added to the bacterial solution. Therefore, the screened polypeptides have the effect of rapid bactericidal action, which is consistent with the characteristics of cationic antimicrobial peptides acting on the cell membrane.

[0157] Example 6 Hemolysis Experiment

[0158] Use rabbit blood to detect the hemolytic effect of the polypeptide on red blood cells. The specific detection steps are as follows:

[0159] (1) Wash rabbit red blood cells three times with PBS solution and prepare a cell suspension with a concentration of 1% (V / V).

[0160] (2) Dissolve the polypeptide in DMSO to 10 mM, and dilute the polypeptide stock solution with PBS to 250 μM, 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, and 2 μM.

[0161] (3) Mix the polypeptide and blood at a ratio of 4:1 and incubate at 37 °C for 1 h; set positive control: 0.1% Triton X-100; negative control: PBS solution; quality control control: polymyxin B (hemolysis rate < 5%).

[0162] (4) Use a multifunctional microplate reader to detect the absorbance at a wavelength of 540 nm, and calculate the hemolysis rate of the polypeptide using the following formula: Hemolysis rate (%) = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%.

[0163] The results are as Figure 2 shown in Table 7. SEQ ID No.79 and SEQ ID No.113 showed no obvious toxicity to red blood cells within a concentration of 512 μg / mL; SEQ ID No.150 showed no obvious toxicity to red blood cells within a concentration of 285 μg / mL; SEQ ID No.58 was toxic to red blood cells.

[0164] Table 7 Minimum hemolytic concentration of polypeptides

[0165]

[0166] Example 7 Cytotoxicity experiment

[0167] The in vitro cytotoxicity of the polypeptide was detected using human skin fibroblasts (NHDF) and human embryonic kidney cells (HEK293). The specific operation steps are as follows:

[0168] (1) Resuscitate NHDF cells and HEK293 cells. The culture medium is DMEM containing 10% FBS and 1% double antibody (penicillin-streptomycin mixture), and culture them in a constant temperature incubator at 37 °C and 5% CO 2 conditions; the cell growth density reaches 80-90% of the culture flask.

[0169] (2) Rinse the cells with DPBS and then digest the cells with 0.25% trypsin (containing 0.5 mM EDTA).

[0170] (3) Add the corresponding growth medium to terminate digestion, collect the cell suspension into a centrifuge tube, centrifuge at 1000 rpm for 3 min, and remove the supernatant medium.

[0171] (4) Resuspend the cells by adding 3 - 5 mL of fresh growth medium and passage them at a ratio of 1:3 to 1:8. Incubate them in an incubator at 37°C with 5% CO 2 2. Culture. Change the medium or passage the cells every 2 - 3 days after passage;

[0172] (5) Passage and amplify all the above cells to the required cell number respectively. After digesting and resuspending the cells, incubate them in a 96 - well cell culture plate at a cell density of 2000 cells per well and 100 μL of cell suspension per well for 24 h for experimental detection. (6) Dilute the bioactive polypeptides to 1× concentrations of 20 μM, 10 μM, 5 μM, and 1 μM respectively using complete medium.

[0173] (7) Aspirate the culture medium supernatant in the cell plate. Add the bioactive polypeptides to the cell plate according to the plate layout, 80 μL of sample per well, and incubate in an incubator at 37°C with 5% CO 2 2 for 48 h.

[0174] (8) Add 8 μL of CCK - 8 cell viability detection reagent to each well and incubate in an incubator at 37°C with 5% CO 2 2 for 1 h. Detect the OD 450 value using a Cytation5 microplate reader.

[0175] The results are as Figure 3 shown. SEQ ID No.41, SEQ ID No.111, and SEQ ID No.113 have little toxic effect on NHDF cells and HEK293 cells. When 20 μM (56 μg / mL) of the polypeptide is added, the inhibition rate on these two types of cells is less than 20%; SEQ ID No.150 has certain cytotoxicity to NHDF cells and HEK293 cells, but within 1 MIC (8 μg / mL) concentration, the inhibition rate on NHDF cells is less than 20%.

[0176] Example 8 Drug Resistance Experiment

[0177] Long - term use of antibiotics can cause bacteria to develop drug resistance. The minimum inhibitory concentration experiment is used to monitor and culture bacteria to study the drug resistance of bacteria to antimicrobial peptides. The specific operations are as follows:

[0178] (1) Open the freeze - dried bacterial strain tube in a sterile operation manner, add an appropriate amount of culture medium, and aspirate and blow several times to melt and disperse the bacterial strain. Take a test tube containing 5.0 mL - 10.0 mL of the corresponding culture medium, drop a little bacterial strain suspension, and culture at 37°C for 18 h - 24 h. This is the first - generation culture.

[0179] (2) Take the first-generation culture for the minimum inhibitory concentration experiment, and record the MIC of the polypeptide against the bacteria. Add the polypeptide-bacteria mixture at 1 / 2 MIC to 10 mL of CAMHB, and culture it at 37 °C for 3 - 5 h to obtain the second-generation culture.

[0180] (3) Take the second-generation culture for the minimum inhibitory concentration experiment, and record the MIC of the polypeptide against the bacteria. Add the polypeptide-bacteria mixture at 1 / 2 MIC to 10 mL of CAMHB, and culture it at 37 °C for 3 - 5 h to obtain the third-generation culture.

[0181] (4) And so on until the bacteria develop resistance to the polypeptide.

[0182] The experimental results are shown in Table 8. By incubating the polypeptide at 1 / 2 MIC concentration with Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, among the 13 polypeptides selected, when cultured and passaged to the 17th generation with the bacteria, they still had good bactericidal effects on Escherichia coli and Pseudomonas aeruginosa and no drug resistance was generated; while when passaged to the 9th generation, more than half of the polypeptides developed drug resistance to most of the Staphylococcus aureus. Compared with the antibiotic imipenem, Escherichia coli developed drug resistance at the 8th generation, Staphylococcus aureus developed drug resistance at the 3rd generation, and Pseudomonas aeruginosa developed drug resistance at the 8th generation. Therefore, the selected membrane-breaking antibacterial peptides will not generate drug resistance to Gram-negative bacteria for at least 17 generations, and for Gram-positive bacteria, it is also more difficult to develop drug resistance than antibiotics.

[0183] Table 8 Detection results of bacterial drug resistance to polypeptides

[0184]

[0185]

[0186] Example 9 N-Phenyl-1-naphthylamine (NPN) uptake assay

[0187] Determine the ability of the peptide to penetrate the outer membrane through the NPN uptake assay. The specific operation steps are as follows:

[0188] NPN (taking ATCC25922 as an example, applicable to ATCC29213 and ATCC27853);

[0189] (1) Pick a bacterial monoclonal, culture it in MHB at 37 °C until the logarithmic growth phase, and harvest by centrifugation;

[0190] (2) Wash the cells 3 times with 10 mM HEPES buffer (pH 7.4, containing 5 mM glucose);

[0191] (3) Resuspend with 10 mM HEPES buffer (pH 7.4, containing 5 mM glucose), and adjust the OD600 To 0.15, diluted 3-fold;

[0192] (4) Mix 90 μL of bacterial culture and 5 μL of polypeptides at different concentrations with 5 μL of NPN (final concentration 10 μM) and incubate for 1 hour. Use 0.5% NaCl solution as the negative control and add 20 μg / mL polymyxin B as the positive control;

[0193] (5) If the cell membrane is damaged, NPN can enter the membrane phospholipid layer, enhancing the fluorescence intensity. The excitation and emission wavelengths of the resulting blue fluorescent group are 350 nm and 420 nm, respectively;

[0194] (6) Use the following formula to calculate the percentage of NPN uptake:

[0195] NPN uptake (%) = (F obs - F 0 ) / (F 100 - F 0 ) × 100

[0196] where, F obs —— Fluorescence value measured in the presence of polypeptides;

[0197] F 0 —— Initial fluorescence value of the NPN-containing solution;

[0198] F 100 —— Fluorescence value produced by adding 20 μg / mL polymyxin B.

[0199] The experimental results are as Figure 4 shown. The antibacterial peptides screened in this study can disrupt the bacterial membrane, enabling the hydrophobic fluorophore NPN to enter the membrane. Moreover, as the polypeptide concentration increases, the amount of NPN penetrating into the membrane also increases. This proves that the polypeptides screened in this study are membrane-disrupting antibacterial peptides and have the effect of disrupting the membrane and killing bacteria.

[0200] Example 10 2-Nitrophenyl-β-D-galactopyranoside (ONPG) inner membrane penetration experiment

[0201] Evaluate the interaction between antibacterial peptides and bacterial cell membranes by measuring the activity of bacterial β-galactosidase. The specific operation steps are as follows:

[0202] ONPG (taking Escherichia coli ATCC25922 as an example, equally applicable to Pseudomonas aeruginosa ATCC27853):

[0203] (1) Pick a single colony and culture it in MHB medium at 37 °C until the logarithmic growth phase;

[0204] (2) Transfer the bacterial culture to MHB containing 2% lactose and incubate at 37°C (to induce the production of β-galactosidase, and the induction time varies for different bacteria), then harvest by centrifugation;

[0205] (3) After collecting the bacterial culture, wash it 3 times with 10 mM PBS (pH 7.4) and resuspend, and adjust the OD600 to 0.15.

[0206] (4) Mix the bacterial resuspension: 10 mM PBS (pH 7.4): 10 mM PBS (pH 7.4) containing 4.5 mM ONPG at a ratio of 1:1:1 to form a 10 mM PBS (pH 7.4) bacterial suspension containing 1.5 mM ONPG.

[0207] (5) Add 95 μL of the bacterial suspension and 5 μL of the antimicrobial peptide (1×MIC) to a 96-well microplate and mix well by oscillation.

[0208] (6) Use 10 mM PBS (pH 7.4) as the negative control and 1% Triton as the positive control.

[0209] (7) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the change in absorbance at OD 405 nm wavelength over time (OD measurement is carried out at 405 nm from 0 to 10 h, and measured once every 1 min).

[0210] The results are as Figure 5 shown. When the polypeptide screened in this study was added to the bacterial suspension containing ONPG, the bacterial membrane was damaged and intracellular β-galactosidase was released (among the bacteria used in this study, Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 can secrete β-galactosidase, while Staphylococcus aureus ATCC29213 cannot secrete β-galactosidase), causing ONPG to be enzymatically hydrolyzed into o-nitrophenol (yellow). Therefore, it is further proved that the polypeptide screened in this study is a membrane-breaking antimicrobial peptide, which can effectively damage the bacterial membrane and thus achieve a bactericidal effect.

[0211] Example 11 Heliquest Prediction and ColabFold Structure Prediction

[0212] The hydrophobic surface and charge number prediction were performed on SEQ ID No.58, SEQ ID No.63, SEQ ID No.79, SEQ ID No.113, SEQ ID No.133, and SEQ ID No.150 using the website https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py. The tertiary structure prediction was performed on SEQ ID No.58, SEQ ID No.63, SEQ ID No.79, SEQ ID No.113, SEQ ID No.133, and SEQ ID No.150 using the website https: / / github.com / sokrypton / ColabFold.

[0213] Structure Figure 6 As shown, through Heliquest prediction and ColabFold structure prediction, the six peptides in this study all showed a consistent α-helical structure, which is consistent with the membrane-breaking structure of cationic antimicrobial peptides.

[0214] Example 12 Peptide Synthesis and Liquid Phase Mass Spectrometry Detection

[0215] The polypeptide compound and its derivative provided by the present invention adopt a solid phase synthesis method to synthesize its linear precursor, and obtain the target crude peptide after cutting. The synthetic carrier is 2-Chlotrityl Resin resin. In the synthesis process, the 2-Chlotrityl Resin resin is first fully swollen in N, N-dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90: 2.5: 2.5: 5, v: v: v: v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the target polypeptide crude product is obtained after precipitation by frozen methyl tert-butyl ether. The crude polypeptide is purified and separated by a C18 reverse phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile / water to obtain a pure product of the polypeptide and its derivatives.

[0216] Experimental reagents

[0217]

[0218]

[0219] (1) Synthesis of the polypeptide represented by SEQ ID No.58

[0220]

[0221] Step 1: Coupling of the first amino acid Fmoc-Ala-OH

[0222] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Weigh Fmoc-Ala-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol), dissolve them in 5 ml of DCM and add to the resin, and react at room temperature for 2 h. After the reaction is completed, add the capping solution (10 ml) of DCM: methanol: DIEA (85:10:5, v:v:v) and cap at room temperature for 10 min. The capped resin was washed 5 times with DCM and 5 times with DMF.

[0223] Step 2: Synthesis of the linear precursor peptide chain

[0224] The linear precursor peptide chain of SEQ ID No. 58

[0225] K-R-W-R-W-G-L-R-K-R-I-S-T-V-L-P-P-W-R-Y-I-T-R-I-I-S-A.

[0226] The resin obtained in Step 1 was fully swollen in DMF for 1 h, and then synthesized in the order from the second W at the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows:

[0227] · Deprotect Fmoc twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0228] · Rinse the resin 6 - 8 times with DMF until neutral pH.

[0229] · Dissolve 0.5 mmol of Fmoc-AA, 0.5 mmol of 6-chlorobenzotriazol-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 h.

[0230] · Rinse the resin 4 - 6 times with DMF before coupling the next amino acid.

[0231] After the synthesis of the linear polypeptide, the resin was rinsed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0232] Step 3: Cleavage of the linear precursor peptide chain

[0233] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0234] Step 4: Peptide purification

[0235] The crude polypeptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 20-50% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0236] Step 5: Detection and characterization methods

[0237] The purity and molecular weight of the peptide purified in step 4 were determined by analytical HPLC and LC / MS. Figure 7 , 8 shown.

[0238] (2) Synthesis of the polypeptide represented by SEQ ID No.63

[0239]

[0240] Step 1: Coupling of the first amino acid Fmoc-Ile-OH

[0241] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. Fmoc-Ile-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 ml of DCM and added to the resin, and reacted at room temperature for 2 hours. After the reaction was completed, a blocking solution (10 ml) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 minutes for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0242] Step 2: Synthesis of linear precursor peptide chain

[0243] The linear precursor peptide chain of SEQ ID No. 63, L-V-W-S-R-H-M-K-R-W-R-W-G-L-R-K-L-I.

[0244] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then synthesized in the order from the second W at the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows:

[0245] · 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time.

[0246] · The resin was rinsed with DMF 6 - 8 times until neutral pH.

[0247] · 0.5 mmol of Fmoc-AA, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol of 4-methylmorpholine (NMM) were dissolved in DMF, added to the resin, and reacted at room temperature for 1 h.

[0248] · The resin was rinsed with DMF 4 - 6 times before the next amino acid coupling.

[0249] After the synthesis of the linear polypeptide, the resin was rinsed with DMF 5 times and with DCM 5 times. The resin was dried in vacuo.

[0250] Step 3: Cleavage of the linear precursor peptide chain

[0251] Freshly prepared cleavage cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 2, and the reaction was shaken at room temperature for 2 h. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid and combined with the reaction solution. The crude product was precipitated with 4 volumes of cold MTBE. The crude product was washed with MTBE 3 times and dried in vacuo.

[0252] Step 4: Purification and preparation of the polypeptide

[0253] The crude polypeptide was dissolved in 20% aqueous acetonitrile solution, filtered through a 0.45 μm membrane, and then separated by a reverse-phase high performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column was a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 20 - 50% acetonitrile in 40 min. The relevant fractions of the product were collected, and the fractions with a purity > 95% were combined after HPLC identification, freeze-dried to obtain the pure polypeptide.

[0254] Step 5: Detection and characterization methods

[0255] The pure polypeptide from Step 4 was analyzed by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine its purity and the molecular weight of the target substance. The results are shown as Figure 9 and 10 shown.

[0256] Other polypeptides of the present invention can be synthesized with reference to the synthesis methods of SEQ ID No. 58 and SEQ ID No. 63. The analytical results of high performance liquid chromatography and liquid chromatography / mass spectrometry for SEQ ID No. 79, SEQ ID No. 113, SEQ ID No. 133, and SEQ ID No. 150 are shown as Figures 11 to 18 shown.

[0257] The preferred embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent deformations or substitutions without departing from the spirit of the present invention. These equivalent deformations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A broad-spectrum antibacterial peptide, characterized in that it is selected from the following polypeptides or their combinations: (1) The polypeptides shown in SEQ ID NO.1 to SEQ ID NO.5; (2) Truncated peptides of the polypeptides shown in SEQ ID NO.1 to SEQ ID NO.5; (3) Derived peptides of the polypeptides described in (1) or (2), or modified forms of polypeptides having the same function as the polypeptides described in (1) or (2), and the polypeptides are cyclic peptides or linear peptides.

2. The broad-spectrum antibacterial peptide according to claim 1, characterized in that the truncated peptide has a length of 10 to 50 amino acids, and the truncated peptide is a cyclic peptide or a linear peptide.

3. The broad-spectrum antibacterial peptide according to claim 1, characterized in that the derived peptide is a polypeptide having the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence of the polypeptide described in (1) or (2).

4. The broad-spectrum antibacterial peptide according to claim 1, characterized in that the sites modified in the modified form include, but are not limited to, N-terminal modification, C-terminal modification, backbone modification, side chain modification, and amino acid modification.

5. The broad-spectrum antibacterial peptide according to claim 1, characterized in that the broad-spectrum antibacterial peptide includes at least one of the following listed amino acid sequences, or their combination, (I) KRWRW; (II) ISTVL; (III) GSRQP; (IV) RKWKKK; (V) WRNWRRHL; (VI) WWRNAKRIIK.

6. The broad-spectrum antibacterial peptide according to claim 5, characterized in that the broad-spectrum antibacterial peptide includes at least one of the following listed amino acid sequences, or their combination, (I) KRWRWGLR; (II) ISTVLPPW; (III) HRRGSRQP; (IV) RKWKKKWHH; (V) WRNWRRHLPKV; (VI) RWWRNAKRIIKS.

7. A broad-spectrum antibacterial peptide, characterized in that the broad-spectrum antibacterial peptide has an amino acid sequence shown in any one of SEQ ID NO.6 to SEQ ID NO.

152.

8. A pharmaceutical composition, characterized in that the pharmaceutical composition contains an effective amount of the broad-spectrum antibacterial peptide according to any one of claims 1 to 7; further, the pharmaceutical composition also contains a pharmaceutically acceptable carrier and / or excipient, and the pharmaceutically acceptable carrier and / or excipient include fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

9. Use of the broad-spectrum antibacterial peptide according to any one of claims 1 to 7 and the pharmaceutical composition according to claim 8 in the preparation of a drug for killing or inhibiting pathogenic bacteria; preferably, the pathogenic bacteria include bacteria.

10. The use according to claim 9 is any one of the following applications: A1) Destroy the integrity of the bacterial cell membrane; A2) Prepare a product for destroying the integrity of the bacterial cell membrane; A3) Reduce the permeability of the bacterial outer membrane; A4) Prepare a product for reducing the permeability of the bacterial outer membrane; A5) Destroy the bacterial membrane potential; A6) Preparing a product for disrupting the bacterial membrane potential; A7) Disrupting the integrity of the bacterial cell wall; A8) Preparing a product for disrupting the integrity of the bacterial cell wall; A9) Inhibiting the activity of bacteria; A10) Preparing a drug for inhibiting the activity of bacteria; Furthermore, the bacteria are Gram-positive bacteria and / or Gram-negative bacteria; Even further, the Gram-negative bacteria include Escherichia coli, Salmonella typhimurium, Salmonella pullorum, Salmonella spp., Pseudomonas aeruginosa, Acinetobacter baumannii, Shigella spp., and Enterobacter sakazakii; the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Listeria monocytogenes, and Bacillus cereus.

Images