Antimicrobial peptides or peptide derivatives, alternatives and compositions, methods of preparation and use thereof

Abstract

This application provides an antimicrobial peptide or peptide derivative, substitute, and compositions thereof, preparation methods, and applications thereof, comprising at least one of the following amino acid sequences I and II: Amino acid sequence I: X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 Amino acid sequence II: where X a1 B a1 U1, Z a1 B a2 X a2 B a3 Z a2 B a4 X a3 X b1 B b1 C a1 Z b1 B b2 X b2 B b3 Z b2 B b4 X b3 C a2 Each peptide is independently selected from natural amino acids and / or non-natural amino acids. Among them, the antimicrobial peptides or peptide derivatives provided in this application can bind to the lipid structure of cell walls / membranes, damage their physicochemical properties, destroy microbial walls, and thus kill microorganisms and tumor cells. In addition, they also have the effects of external wound disinfection and anti-infection.

Description

Technical Field

[0001] This application belongs to the field of peptide or peptide derivative technology, and particularly relates to an antimicrobial peptide or peptide derivative, a substitute, a composition thereof, a preparation method thereof, and an application thereof. Background Technology

[0002] In nature, antimicrobial peptides are widely found in organisms such as bacteria, viruses, fungi, insects, amphibians, animals, and plants. They exhibit significant effects in killing bacteria, fungi, parasites, and viruses. The widespread use of antibiotics has led to drug resistance in microorganisms, spurring large-scale research into antimicrobial peptides. Since the discovery of endogenous antimicrobial peptides such as defensins and their approval by the FDA (Food and Drug Administration) for anti-infective, anti-inflammatory, and other wound treatment purposes, the ability of antimicrobial peptides to stimulate both acquired and adaptive immunity has been further discovered, demonstrating their potential therapeutic advantages against various diseases. Beyond pharmaceutical applications, antimicrobial peptides also find wide application in food preservation, livestock farming, aquaculture, daily medical and health care, hygiene and beauty, cleaning and disinfection, and agricultural pest and disease control.

[0003] For example, Propionibacterium acnes is a Gram-positive bacterium, a normal human skin microbiome, that overgrows in the hair follicles of the sebaceous glands. Propionibacterium acnes is one of the most common skin diseases affecting humans, impacting approximately one million Chinese people. Acne caused by Propionibacterium acnes presents with various symptoms, such as comedones, papules, nodules, cysts, and folliculitis. Traditional treatments for Propionibacterium acnes often involve antibiotics, such as oral oxytetracycline and topical erythromycin and clindamycin. However, this treatment quickly leads to antibiotic resistance. Additionally, benzoyl peroxide and 5% dapsone gel are also used clinically to treat Propionibacterium acnes. These two drugs have little efficacy at low concentrations and significant toxic side effects at high concentrations.

[0004] For example, *Pseudomonas aeruginosa* is a Gram-negative bacterium that infects humans and animals. It can infect the respiratory, urinary, gastrointestinal, central nervous, blood, cardiovascular, and skeletal systems. When *Pseudomonas aeruginosa* infects the eyes and ears, it often causes bacterial keratitis, scleral abscess, endophthalmitis in adults, neonatal conjunctivitis, and otitis media in children. In particular, *Pseudomonas aeruginosa* / bacterial keratitis increases the infection rate among iris-wearing eyeglasses; approximately 25,000 iris-wearing eyeglasses users in the United States are infected with this bacterium annually. Furthermore, according to the U.S. Centers for Disease Control and Prevention, 4 out of every 1,000 hospitalized patients have been infected with *Pseudomonas aeruginosa*. Although *Pseudomonas aeruginosa* is treatable, with the use of antibiotics, this microorganism has developed increased resistance, quickly reducing the effectiveness of treatments and necessitating changes in medication combinations and treatment regimens. Data shows that in the United States, *Pseudomonas aeruginosa* isolated from intensive care units exhibits resistance rates of 51.6% to ciprofloxacin, 31.4% to piperacillin / tazobactam, 38% to imipenem, and 23.6% to ceftazidime. In Europe, *Pseudomonas aeruginosa* isolated from intensive care units shows resistance rates of 37-70% to aminoglycosides, 57% to ceftazidime, 53% to piperacillin / tazobactam, 56% to ciprofloxacin, and 52% to imipenem. Therefore, the treatment and control of severe drug-resistant infections caused by *Pseudomonas aeruginosa* are crucial.

[0005] Currently reported typical antimicrobial peptides generally consist of 10-100 amino acid residues, contain many basic amino acids, and are generally amphiphilic.

[0006] Studies have reported that the host defense peptide PGLa, extracted from the secretions of frogs, has excellent cytotoxic activity against Helicobacter pylori (a Gram-negative bacterium). Similarly, the natural antimicrobial peptide TP4 isolated from fish and the natural antimicrobial peptide LL-37 isolated from humans also exhibit good cytotoxic activity against Helicobacter pylori. These findings suggest that antimicrobial peptides are particularly effective in treating Helicobacter pylori and can be added to everyday products as antibacterial additives to help us better combat the bacteria. However, existing antimicrobial peptides suffer from drawbacks such as weak antimicrobial activity and the lack of broad-spectrum antimicrobial activity.

[0007] Furthermore, antimicrobial peptides isolated from plants and animals are diverse and widely distributed, found in insects, fish, mammals, amphibians, and plants. However, the applicability of antimicrobial peptides from different sources has not yet been systematically studied. Plant antimicrobial peptides, in particular, are unstable and easily hydrolyzed by proteolytic enzymes, thus significantly reducing their antimicrobial activity. Summary of the Invention

[0008] The purpose of this application is to provide an antimicrobial peptide or peptide derivative, a synthesis method, a composition, and an application, aiming to solve the problems of weak, unstable, and lack of broad-spectrum antimicrobial activity of existing antimicrobial peptides.

[0009] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0010] In a first aspect, this application provides an antimicrobial peptide or peptide derivative comprising at least one of the following amino acid sequences:

[0011] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0012] Amino acid sequence II:

[0013] Among them, X a1 B a1 U1, Z a1 B a2 X a2 B a3 Z a2 B a4 X a3 X b1 B b1 C a1 Z b1 B b2 X b2 B b3 Z b2 B b4 X b3 C a2 Each is independently selected from amino acids.

[0014] The antimicrobial peptides or peptide derivatives provided in this application can specifically bind to the lipid structure of the cell wall / membrane, disrupting the microbial wall and thereby killing microorganisms and cancer cells.

[0015] In a second aspect, an alternative to an antimicrobial peptide or peptide derivative is provided, wherein at least one amino acid of amino acid sequence I and / or amino acid sequence II contained in the antimicrobial peptide or peptide derivative of this application is replaced.

[0016] The substitutes provided in this application can ensure the activity of antimicrobial peptides or peptide derivatives, and are also included in the content of this application. By replacing the amino acids on the antimicrobial peptides or peptide derivatives, the antimicrobial activity of the antimicrobial peptides or peptide derivatives can be improved and their broad-spectrum antimicrobial activity can be increased.

[0017] Thirdly, this application provides an antimicrobial peptide or peptide derivative composition comprising at least one antimicrobial peptide or peptide derivative described in any of the preceding paragraphs and at least one pharmaceutically acceptable carrier.

[0018] The third aspect of this application provides an antibacterial composition, wherein the antibacterial peptide or peptide derivative provided in this application can be mixed with most solvents to form a composition.

[0019] Fourthly, this application provides a method for preparing an antibacterial composition, comprising the following steps: mixing an antibacterial peptide or peptide derivative with a solvent.

[0020] The method for preparing the antimicrobial composition of this application, on the one hand, allows the antimicrobial peptide or peptide derivative to exist stably in most solvents, and mixing the solvent and the antimicrobial peptide or peptide derivative can preserve the antimicrobial activity of the antimicrobial peptide or peptide derivative. On the other hand, it ensures the stability of the antimicrobial peptide or peptide derivative against peptidase or protein degradation.

[0021] Fifthly, this application provides the use of the antimicrobial peptide or peptide derivative composition of this application in packaging, food processing end products, clothing, medical supplies, medical instruments, personal hygiene products, disinfectants, cleaning agents, anti-infective drugs, anti-inflammatory drugs, and drugs that inhibit unlimited cell proliferation.

[0022] The application of the antimicrobial peptide or peptide derivative composition of this application is due to the fact that antimicrobial peptides or peptide derivatives can kill microorganisms, kill tumor cells, and have anticoagulant and anti-inflammatory functions. Therefore, they have a wide range of uses and mainly have four advantages:

[0023] I. Antimicrobial peptides or peptide organisms can be combined with or incorporated into packaging, clothing, medical supplies, medical instruments, and personal hygiene products to give them antimicrobial functions, or serve as preservatives for other materials that are easily degraded by microorganisms.

[0024] II. Antimicrobial peptides or peptide organisms can be made into disinfectants, cleaning agents, anti-infective drugs, and anti-inflammatory drugs, which play a role in sterilization, anti-infection, and anti-inflammation.

[0025] 3. Antimicrobial peptides or peptide biocomposites are made into coatings and applied to food processing and final stages to reduce microorganisms on the surface of food processing final stages and reduce the risk of food poisoning.

[0026] Fourth, it can kill cells and limit their proliferation, and can be used for anti-tumor treatment, local cosmetic treatment such as wart removal and spot removal. Attached Figure Description

[0027] Figure 1 Statistical histogram of experimental results on the inhibition of Escherichia coli by antimicrobial peptides or peptide derivatives in the embodiments of this application;

[0028] Figure 2 The results of the inhibition of Escherichia coli by antimicrobial peptides or peptide derivatives in the embodiments of this application are shown in the figure.

[0029] Figure 3 Figure 1 shows the experimental results of the inhibition of wild Staphylococcus aureus by antimicrobial peptides or peptide derivatives in the embodiments of this application.

[0030] Figure 4 The MIC inhibition experiment results of antimicrobial peptides or peptide derivatives against Pseudomonas aeruginosa in the embodiments of this application are shown in the figure.

[0031] Figure 5 Figure showing experimental results of antimicrobial peptides or peptide derivatives killing RNA viruses in the embodiments of this application;

[0032] Figure 6 The experimental results of the antimicrobial peptide or peptide derivative killing leukemia lymphocytes (Jurkat cellline) in the embodiments of this application are shown in the figure.

[0033] Figure 7 The experimental results of the antimicrobial peptide or peptide derivative killing leukemia cells (K562 cell line) in the embodiments of this application are shown in the figure.

[0034] Figure 8 Figure showing experimental results of antimicrobial peptides or peptide derivatives killing liver cancer cells in the embodiments of this application;

[0035] Figure 9 The experimental results of the effect of antimicrobial peptides or peptide derivatives on red blood cells in the embodiments of this application are shown in the figure. Detailed Implementation

[0036] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0037] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0038] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0039] It should be understood that, in the various embodiments of this application, the sequence number of each process does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the implementation regulations of this application.

[0040] The terminology used in the embodiments of this application is for the purpose of describing particular implementations only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the implementations of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0041] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0042] The terms "first" and "second" are used only to describe the purpose of distinguishing objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the provisions of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

[0043] In a first aspect, embodiments of this application provide an antimicrobial peptide or peptide derivative, the amino acid sequence of which is generally expressed as follows:

[0044] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 Ba4 X a3 ;

[0045] Amino acid sequence II:

[0046] Among them, X a1 B a1 U1, Z a1 B a2 X a2 B a3 Z a2 B a4 X a3 X b1 B b1 C a1 Z b1 B b2 X b2 B b3 Z b2 B b4 X b3 C a2 Each amino acid is independently selected, wherein a ring bond is formed between Ca1 and Ca2 in amino acid sequence II, and the amino acids include natural amino acids and / or non-natural amino acids. The antimicrobial peptides or peptide derivatives provided in this application can bind to the lipid structure of the cell wall / membrane, destroy the microbial wall or cell membrane, and thereby kill microorganisms and tumor cells.

[0047] In this embodiment, based on the above embodiments, to improve the antibacterial activity of the antimicrobial peptide or peptide derivative, X a1 X a2 X a3 X b1 X b2 X b3 Each is independently selected from residues carrying a positive charge; or / and B a1 B a2 B a3 B a4 B b1 B b2 B b3 B b4 Each residue is independently selected from residues with hydrophobic side chains; or / and U1 is selected from one of Gly (G-glycine), Pro (P-proline), Cys (C-cysteine), and Cys(R), wherein R represents a protecting group of the Cys disulfide bond; or / and Z a1 Z a2 Z b1 Z b2 Each is independently selected from nonpolar residues; or / and C a1 C a2Each is individually selected from Cys(C-cysteine) and Cys(R), and in amino acid sequence I, X a1 or X a3 Representing the N-terminal, X a1 or X a3 Representing the C-terminus, in amino acid sequence II, X b1 And / or B2 represents the N-terminus, C a2 Representing the C-terminus. Because the antimicrobial peptides and peptide derivatives of this application contain hydrophobic side chain residues, positively charged residues, and nonpolar residues, the antimicrobial peptides or peptide derivative chains provided in this embodiment can form a typical amphoteric helical structure. The charge and amphoteric helical structure of these antimicrobial peptides or peptide derivatives enable them to bind to the lipid structure of the cell wall / membrane, disrupting the microbial wall or cell membrane, thereby killing microorganisms and tumor cells.

[0048] In the examples, the amino acid sequences are generally expressed as follows:

[0049] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0050] Amino acid sequence II:

[0051] Among them, B a1 U1, Z a1 B a2 B a3 Z a2 B a4 B b1 C a1 Z b1 B b2 B b3 Z b2 B b4 C a2 Each is independently selected from amino acids, X a1 X a2 X a3 X b1 X b2 X b3 Each peptide is independently selected from one of the positively charged residues, and the anoly charged peptide chains readily bind to the negatively charged cell membrane.

[0052] In the examples, the amino acid sequences are generally expressed as follows:

[0053] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0054] Amino acid sequence II:

[0055] Among them, B a1 Z a1 B a2 B a3 Z a2 B a4 B b1 C a1 Z b1 B b2 B b3 Z b2 B b4 C a2 Each is independently selected from amino acids, X a1 X a2 X a3 X b1 X b2 X b3 Each is independently selected from at least one of the positively charged amino acid residues, B a1 B a2 B a3 B a4 B b1 B b2 B b3 B b3 Each is independently selected from at least one of amino acid residues with hydrophobic side chains, and U1 is selected from at least one of Gly, Pro, Cys, and Cys(R). The peptide chain with an anodic charge is easy to bind to the negatively charged cell membrane. The hydrophobic side chain residues ensure a balance between the water solubility and charge of the antimicrobial peptide or peptide derivative, thereby ensuring the antimicrobial activity of the antimicrobial peptide or peptide derivative.

[0056] In the examples, the amino acid sequences are generally expressed as follows:

[0057] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0058] Amino acid sequence II:

[0059] Among them, C a1 C a2 Each is independently selected from amino acids, X a1 X a2 X a3 X b1 X b2 X b3 Each is independently selected from one of the positively charged residues; B a1 B a2 B a3 B a4 B b1 B b2 B b3 Each is independently selected from at least one amino acid residue with a hydrophobic side chain; U1 is selected from Gly, Pro, Cys, and Cys(R), wherein R represents a protecting group of the Cys disulfide bond; Z a1 Z a2 Z b1 Z b2 Each peptide is independently selected from at least one nonpolar amino acid residue. The anolyl peptide chain readily binds to the negatively charged cell membrane. The nonpolar residues and hydrophobic side chain residues ensure a balance between the water solubility and charge of the antimicrobial peptide or peptide derivative, thereby ensuring the antimicrobial activity of the antimicrobial peptide or peptide derivative.

[0060] In the examples, the amino acid sequences are generally expressed as follows:

[0061] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0062] Amino acid sequence II:

[0063] Among them, X a1 X a2 X a3 X b1 X b2 X b3 Each is independently selected from residues carrying a positive charge; B a1 B a2 B a3 B a4 B b1 B b2 Bb3 B b4 Each residue is independently selected from at least one amino acid residue with a hydrophobic side chain; U1 is selected from at least one of Gly (G-glycine), Pro (P-proline), Cys (C-cysteine), and Cys(R), wherein R represents a protecting group of the Cys disulfide bond; Z a1 Z a2 Z b1 Z b2 Each is independently selected from at least one nonpolar amino acid residue; C a1 C a2 Each peptide is individually selected from at least one of Cys(C-cysteine) and Cys(R). In amino acid sequence I, X1 and / or B1 represent the N-terminus, and B1 and / or X1 represent the C-terminus. In amino acid sequence II, X1 and / or B2 represent the N-terminus, and Cys represents the C-terminus. Because the antimicrobial peptides and peptide derivatives of this application contain hydrophobic side chain residues, positively charged residues, and nonpolar residues, the antimicrobial peptides or peptide derivative chains provided in this embodiment can form a typical amphipathic helical structure. The charge and amphipathic helical structure of these antimicrobial peptides or peptide derivatives enable them to bind to the lipid structure of the cell wall / membrane, disrupting the microbial wall or cell membrane, thereby killing microorganisms and tumor cells.

[0064] In this embodiment, based on the above embodiments, to improve the stability of the antimicrobial peptide or peptide derivative, X a1 X a2 X a3 X b1 X b2 X b3 B b4 Each residue is independently selected from one, two, or three of the following: Arg (R-arginine), His (H-histidine), Lys (K-lysine), Orn (ornithine), Har (homogeneous arginine), Dab (2,4-diaminopropionic acid), and non-natural amino acids. On the one hand, Arg (R-arginine), His (H-histidine), Lys (K-lysine), Orn (ornithine), Har (homogeneous arginine), and Dab (2,4-diaminopropionic acid) are positively charged residues. From an overall structural perspective, these residues can combine with other residues to form an amphoteric helix structure, increasing the antimicrobial activity of the antimicrobial peptide or peptide derivative. On the other hand, if the positively charged residues in the antimicrobial peptide chain or peptide derivative are too long, the charge of the antimicrobial peptide or peptide derivative will become unbalanced and unstable. Therefore, to ensure the stability of the peptide or peptide derivative against peptidase or protein degradation, the number of positively charged residues is one, two, or three, and / or B... a1 B a2 B a3 Ba4 B b1 B b2 B b3 Each amino acid is independently selected from two, three, or four non-natural amino acids: Ala (A-alanine), Val (V-valine), Ile (I-isoleucine), Leu (L-leucine), and Met (M-methionine). These non-natural amino acids serve as hydrophobic side chains. Hydrophobic side chains can balance the water solubility and charge of the peptide chain. In other words, if the hydrophobic side chain residues in the peptide chain or peptide derivative are too long, the peptide chain is prone to instability. Therefore, to ensure the stability of the peptide chain against peptidase or protein degradation, the number of hydrophobic side chain residues is guaranteed to be one of two, three, or four, or / and Z. a1 Z a2 Z b1 Z b2 Each residue is independently selected from one of the non-natural amino acids Asn (N-aspartic acid), Gln (Q-glutamine), Ser (S-serine), and Thr (T-threonine). The selection of nonpolar residues helps balance the water solubility and charge of the peptide chain. In other words, if the nonpolar residues in the peptide chain or peptide derivative are too long, the peptide chain becomes unstable. Therefore, to ensure the stability of antimicrobial peptides or peptide derivatives against peptidase or protein degradation, the number of nonpolar residues is guaranteed to be one of two, three, or four, or / and X. a1 and B a1 X a2 and B a2 X a3 and B a3 X b1 and B b1 X b2 and B b2 X b3 and B b3 X b4 and B b4 The sum of the residue numbers is 3, 4, 5, 6, or 7, preferably 3 or 4. For the activity of the antimicrobial peptide or peptide derivative of this application, a longer peptide chain is better. However, considering the cost and expense of synthesis, and to ensure the stability of the peptide chain, X a1 and B a1 X a2 and B a2 X a3 and B a3 X b1 and B b1 X b2 and B b2 Xb3 and B b3 X b4 and B b4 The sum of the residues is preferably 3 or 4. The amino acid sequence and / or part of the amino acid sequence of the antimicrobial peptide of this application are derived from 8 to 25 amino acids of a virus.

[0065] In the examples, the amino acid sequences are generally expressed as follows:

[0066] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0067] Amino acid sequence II:

[0068] Among them, Z a1 Z a2 Z b1 Z b2 Each is independently selected from amino acids, X a1 X a2 X a3 X b1 X b2 X b2 X b3 Each is independently selected from one, two, or three of the positively charged Arg, His, Lys, Orn, Har, Dab, and non-natural amino acids; B a1 B a2 B a3 B a4 B b1 B b2 B b3 B b4 Each residue is independently selected from one of the residues with a hydrophobic side chain; and / or U1 is selected from one of Gly, Pro, Cys, and Cys(R), wherein R represents a protecting group of the Cys disulfide bond; C a1 C a2 Each residue is individually selected from one of Cys or Cys(R). These positively charged residues can combine with other residues to form amphoteric helical structures, increasing the antimicrobial activity of the antimicrobial peptide or peptide derivative. In addition, if the positively charged residues in the antimicrobial peptide chain or peptide derivative are too long, the charge of the antimicrobial peptide or peptide derivative will become unbalanced and easily become unstable. Therefore, in order to ensure the stability of the peptide or peptide derivative against peptidase or protein degradation, the number of positively charged residues is one, two, or three.

[0069] In the examples, the amino acid sequences are generally expressed as follows:

[0070] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0071] Amino acid sequence II:

[0072] Among them, X a1 X a2 X a3 X b1 X b2 X b2 X b3 Each is independently selected from one, two, or three of the positively charged Arg, His, Lys, Orn, Har, Dab, and non-natural amino acids; B a1 B a2 B a3 B a4 B b1 B b2 B b3 B b4 Each amino acid is independently selected from two, three, or four of the following non-natural amino acids with hydrophobic side chains: Ala, Val, Ile, Leu, Met; or / and U1 is selected from one of Gly, Pro, Cys, and Cys(R), where R represents the protecting group of the Cys disulfide bond; Z a1 Z a2 Z b1 Z b2 Each is independently selected from at least one of nonpolar residues; or / and C a1 C a2Each residue is individually selected from Cys or Cys(R), and is positively charged. It can combine with other residues to form an amphoteric helical structure, increasing the antimicrobial activity of the antimicrobial peptide or peptide derivative. However, if the positively charged residues in the antimicrobial peptide chain or peptide derivative are too long, the charge of the antimicrobial peptide or peptide derivative will become unbalanced and unstable. Therefore, in order to ensure the stability of the peptide or peptide derivative against peptidase or protein degradation, the number of positively charged residues is one, two, or three. Hydrophobic side chains can balance the water solubility and charge of the peptide chain. That is to say, if the hydrophobic side chain residues in the peptide chain or peptide derivative are too long, the peptide chain is prone to instability. Therefore, in order to ensure the stability of the peptide chain against peptidase or protein degradation, the number of hydrophobic side chain residues is guaranteed to be one of two, three, or four.

[0073] In the examples, the amino acid sequences are generally expressed as follows:

[0074] Amino acid sequence I:X a1 B a1 U1Z1B a2 X a2 B a3 Z a2 B a4 X a3 ;

[0075] Amino acid sequence II:

[0076] Among them, X a1 X a2 X a3 X b1 X b2 X b2 X b3 Each is independently selected from one, two, or three of the positively charged Arg, His, Lys, Orn, Har, Dab, and non-natural amino acids; B a1 B a2 B a3 B a4 B b1 B b2 B b32 B b4 Each amino acid is independently selected from two, three, or four non-natural amino acids, including Ala, Val, Ile, Leu, Met, and those with hydrophobic side chains; Z a1 Z a2 Z b1 Z b2Each is independently selected from one of the nonpolar Asn, Gln, Ser, Thr, or non-natural amino acids; U1 is selected from one of Gly, Pro, Cys, or Cys(R), where R represents the protecting group of the Cys disulfide bond; C a1 C a2 Each residue is individually selected from one of Cys or Cys(R). These positively charged residues can combine with other residues to form an amphoteric helical structure, increasing the antimicrobial activity of the antimicrobial peptide or peptide derivative. However, if the positively charged residues in the antimicrobial peptide chain or derivative are too long, the charge of the peptide or derivative will become unbalanced and unstable. Therefore, to ensure the stability of the peptide or peptide derivative against peptidase or protein degradation, the number of positively charged residues should be one, two, or three. Hydrophobic side chains can balance the water solubility and charge of the peptide chain. If the hydrophobic side chain residues in the peptide chain or peptide derivative are too long, the peptide chain is prone to instability. Therefore, in order to ensure the stability of the peptide chain against peptidase or protein degradation, the number of hydrophobic side chain residues should be guaranteed to be one of two, three, or four types to balance the water solubility and charge of the peptide chain. In other words, if the nonpolar residues in the peptide chain or peptide derivative are too long, the peptide chain is prone to instability. Therefore, in order to ensure the stability of antimicrobial peptides or peptide derivatives against peptidase or protein degradation, the number of nonpolar residues should be guaranteed to be one of two, three, or four types.

[0077] In some embodiments, the Cys disulfide bond includes acetamidomethyl (Acm), methyl methane thiosulfonate, or other Cys protecting groups, where Cys(R) represents a protecting group containing a Cys disulfide bond, wherein C a2 For the terminal group of antimicrobial peptides or peptide derivatives, C a1 Located in the middle position of antimicrobial peptides or peptide derivatives, a ring chain structure can be formed inside the antimicrobial peptide or peptide derivative molecule. This can enhance the stability of the antimicrobial peptide, prolong the bactericidal time of the antimicrobial peptide, and make the antimicrobial peptide or peptide derivative have stronger antimicrobial activity.

[0078] In some embodiments, at least one amino acid in amino acid sequence I and / or II is modified. In practical applications, the stability and antimicrobial activity of antimicrobial peptides or peptide derivatives are very important. By modifying the amino acids on the antimicrobial peptides or peptide derivatives of this application, the performance of the peptide chain can be changed, the spectral properties of the antimicrobial peptides or peptide derivatives can be improved, the stability of the antimicrobial peptides or peptide derivatives against peptidase or protein degradation can be improved, and their broad-spectrum antimicrobial activity can be increased.

[0079] In the embodiments, the modifications include phosphorylation, halogenation, acetylation, cyclization, and capping reactions at the ends. Cyclation includes at least one of cyclization formed by disulfide bonds, head-to-tail cyclization, and cyclization of side groups in the internal structure. Capping reactions at the ends of amino acids include at least one of C-terminal amidation, N-terminal acetylation, and N-terminal phospholipidation. Modifying amino acids, such as cyclization, can increase the stability of the peptide chain. For example, phosphorylation of amino acids can increase the antimicrobial activity of antimicrobial peptides or peptide derivatives.

[0080] In some embodiments, amino acid sequence I and / or II includes at least one motif, often linked to a predetermined amino acid endpoint sequence (N-terminus and / or C-terminus), or directly inserted into the middle of a predetermined amino acid sequence. Generally, it does not stand alone as an amino acid sequence endpoint. The motif is linked to the peptide chain or inserted between amino acids. Inserting a motif into an antimicrobial peptide or peptide derivative can alter the properties of the peptide chain, increase the antimicrobial activity of the peptide chain, and improve the stability of the antimicrobial peptide or peptide derivative against peptidase or protein degradation.

[0081] In some embodiments, the antimicrobial peptide or peptide derivative is a polymorph comprising amino acid sequence I and / or amino acid sequence II, wherein at least one of dimer and tetramer is a relatively common polymorphic state. Antimicrobial peptide monomers and / or polymorphs are conjugated with at least one drug or antibody to form antimicrobial drugs, mainly including small molecule chemically synthesized drugs, large molecule antibodies, and large molecule drugs synthesized using biotransformation technology. The polymorphs of the antimicrobial peptides or peptide derivatives of this application, conjugated with drug or antibody molecules to form antimicrobial drugs, can be used for the modification of drugs or antibodies.

[0082] In some embodiments, the antimicrobial peptide or peptide derivative includes the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:39.

[0083] In some embodiments, a nanostructure of an antimicrobial peptide or peptide derivative can be formed by a single peptide chain and its peptide derivatives, or by multiple or polymeric peptide chains and peptide derivatives. Common nanostructures are selected from at least one of micelles, vesicles, nanotubes, and nanoribbons. The formation of nanostructures by peptide chains and their derivatives facilitates the stable existence and transport of peptide chains and their derivatives in solution.

[0084] Secondly, this application provides a substitute for an antimicrobial peptide or peptide derivative, which is obtained by replacing any amino acid in amino acid sequence I or II of the above paragraph. This is also included in the content of this application. For example, replacing L-type amino acids with corresponding D-type amino acids or non-natural amino acids can improve the antimicrobial activity of the antimicrobial peptide or peptide derivative and increase its broad-spectrum antimicrobial activity by replacing the amino acids on the antimicrobial peptide or peptide derivative.

[0085] Thirdly, this application provides an antimicrobial peptide or peptide derivative composition, an antimicrobial peptide or peptide derivative and / or a substitute, and at least one pharmaceutically acceptable carrier and / or solvent. The antimicrobial peptide or peptide derivative provided in this application can be combined with most pharmaceutically acceptable carriers to form a pharmaceutical composition, and can also be mixed with most solvents to form a composition.

[0086] In some embodiments, at least one of excipients, isotonic agents, absorption delay agents, or base polymers is also included. When mixed with these substances, the antimicrobial peptides or peptide derivatives can be formulated into various antimicrobial drugs or antimicrobial solvents.

[0087] In some embodiments, the excipient is selected from at least one of water, salt, phosphate, glucose, glycerol, and ethanol, which are all commonly used medical reagents. The antimicrobial peptide or its derivatives can be mixed with these reagents, which is beneficial for the promotion and use of the antimicrobial peptides and their derivatives in this application.

[0088] In some embodiments, the isotonic agent is selected from sugars, polyols, and sodium chloride. The isotonic agent can maintain peptide activity. The antimicrobial peptides and derivatives are mixed with the isotonic agent to make the antimicrobial peptides or peptide derivatives more easily bind to organisms.

[0089] In some embodiments, the polyol is selected from mannitol, sorbitol, and a mixture of polyols and antimicrobial peptides or peptide derivatives, wherein the polyol can maintain the activity of the antimicrobial peptides or derivatives and make the antimicrobial peptides or derivatives readily bind to organisms.

[0090] Among them, pharmaceutically acceptable carriers are selected from the minimum amount of auxiliary substances, and pharmaceutically acceptable carriers are selected from wetting agents, emulsifiers, preservatives, and buffers. Pharmaceutically acceptable carriers and antimicrobial peptides or peptide derivatives form a composition that can maintain the activity of antimicrobial peptides or derivatives and make antimicrobial peptides or derivatives easy to bind to organisms.

[0091] In the embodiments, antimicrobial peptides or peptide derivatives are combined with a substrate polymer to form an aqueous or non-aqueous solution, which can be applied to the surface of an object to form an antimicrobial protective layer.

[0092] Furthermore, the aforementioned substances can be in various forms or composed of various components. These include, but are not limited to, extracts, films, membranes, laminates, knitted fabrics, woven fabrics, non-woven fabrics, fibers, filaments, yarns, particles, coatings, and / or foams. This application also includes various models made from the aforementioned substances and antimicrobial peptides, including, but not limited to, injection molding, extrusion molding, blow molding, thermoforming, solution coating, blown film, knitting, weaving, and textile products.

[0093] Fourthly, this application provides a method for preparing an antimicrobial peptide or peptide derivative composition, comprising the following steps: mixing the antimicrobial peptide or peptide derivative with the solvent.

[0094] In the embodiments, when the antimicrobial peptide or peptide derivative is mixed with the base polymer, the mixing temperature is controlled between -10 and 150°C, and the mixing time is controlled between 0.1 and 5760 min to ensure thorough mixing. A mixing temperature between 25 and 80°C and a mixing time between 1 and 1440 min results in better mixing performance.

[0095] In some embodiments, the method further includes pretreatment of a pharmaceutically acceptable carrier, excipient, solvent, dispersion medium, isotonic agent, absorption delay agent, or substrate polymer, wherein the pretreatment includes, but is not limited to, at least one of oxidation, reduction, hydrolysis, plasma, or radiation, which may improve the environment of the antimicrobial peptide or peptide derivative, thereby enhancing the stability of the antimicrobial peptide or peptide derivative against peptidase or protein degradation.

[0096] In some embodiments, the effective concentration range of the antimicrobial peptide or peptide derivative mixed with the above-mentioned substances is between 1 μg and 20 g wt%, or between 5 pmol and 2 mol. To preserve the antimicrobial peptide or peptide derivative composition, the temperature needs to be controlled between -20 and 25°C to ensure the antimicrobial activity of the antimicrobial peptide or peptide derivative. Additionally, the temperature should be controlled between -4 and 4°C, as lower temperatures are necessary to ensure the biological activity properties of the antimicrobial peptide.

[0097] Fifthly, this application provides for the use of any of the following antimicrobial peptide or peptide derivative compositions:

[0098] Antimicrobial peptides or peptide derivative compositions can be widely used as pharmaceuticals or non-pharmaceuticals in packaging, food processing, clothing, medical supplies, medical instruments, personal hygiene products, disinfectants, cleaning agents, anti-infective drugs, anti-inflammatory drugs, and drugs that inhibit unlimited cell proliferation. They are used in packaging, food processing, clothing, medical supplies, medical instruments, personal hygiene products, disinfectants, cleaning agents, anti-infective drugs, anti-inflammatory drugs, and drugs that inhibit unlimited cell proliferation. The following definition of "human" and "animals and plants" refers to the aforementioned definition encompassing humans, animals (including wild animals, livestock, and companion animals), plants (including crops), aquatic and aquatic animals, and farmed poultry, and is equivalent to these definitions below.

[0099] In some embodiments, the application of the compositions described in the foregoing paragraphs in packaging involves the combination or incorporation of antimicrobial peptides or peptide biomolecules with or as preservatives for other materials that are easily degraded by microorganisms. The packaging components include, but are not limited to, packaging films, liners, absorbent pads, trays, container assemblies, caps, lids, adhesives, applicators, etc., for meat products. Such packaging can be any form of packaging suitable for a particular application, such as aluminum cans, boxes, bottles, glass jars, bags, cosmetic packaging, closed tubes, etc. The aforementioned packaging includes, but is not limited to, packaging models manufactured by processes such as injection molding, extrusion molding, blow molding, thermoforming, solution coating, and blown film.

[0100] Packaging materials are also suitable for packaging prescription / non-prescription drugs and health and hygiene products, such as bottles, tips, applicators, and caps for capsules, tablets, solutions, emulsifiers, detergents, powders, shampoos, conditioners, deodorants, antiperspirants, etc. Packaging materials are also suitable for applicator packaging, such as lipsticks, lip glosses, etc., and eye cosmetics packaging, such as mascara, eyeliner, eyeshadow, powder, bath powder, blush, foundation, and lotions. These applicators are used on different parts and surfaces of the body, and this application greatly reduces or eliminates bacterial growth on the human body surface. In addition, it includes other forms of packaging components, such as drinking bottle necks, replaceable caps, non-replaceable caps, food or medicine dispensing systems, food and beverage delivery systems, baby bottles, caps, nipples, etc. Packaging also includes products made into droplet dispersants or droplet atomizers.

[0101] In some embodiments, the compositions described in the foregoing paragraphs are used in food processing and final stages, or as temporary or permanent coatings for preparing food surfaces, such as food additives as antibiotic alternatives; food processing instruments, food conveyor belt assemblies and components; assemblies and components of various instruments used for mixing, grinding, crushing, rolling, granulating, and extruding food; and assemblies and components of instruments used for cutting and slicing food. If the surface of the aforementioned instruments is metallic, the metallic surface must be coated with a functional polymer, which is the polymer containing antimicrobial peptides or peptide derivatives described above in this application. Antimicrobial peptides or peptide biocompositions are formulated into coatings and applied to food processing and final stages to reduce microorganisms on the surface of the food processing final stage, thereby reducing the risk of food poisoning.

[0102] In some embodiments, the composition described in the preceding paragraphs is used in clothing that has antibacterial and bacteriostatic functions, such as swimwear, underwear, shoe components (such as woven or non-woven linings or insoles), sports protective pads, children's clothing, etc. This application also includes protective medical clothing or isolation supplies, such as protective suits, masks, gloves, slippers, boots, head coverings, or curtains.

[0103] In some embodiments, the application of the compositions described in the preceding paragraphs in medical supplies and instruments involves combining or incorporating antimicrobial peptides or peptide biopeptides with or into medical supplies and medical materials, or coating the surface of medical instruments with an antimicrobial peptide or peptide derivative coating, or preparing a spray to disinfect and sterilize medical supplies and instruments. These medical supplies and instruments include implants in humans and animals, such as bandages, adhesives, gauze strips, gauze pads, syringe holders, peripheral or central venous cannulas (which may be made of polyurethane or silicone); urinary catheterization ports; orthopedic orthopedic devices, orthopedic pins, pacemaker leads, defibrillator leads, ear canal shunts, vascular stents, orthopedic implants, ear, nose and throat implants, implantable pumps, hernia patches, and related plates, screws, blood bags, external blood pumps, infusion systems, cardiopulmonary instruments, dialysis instruments, artificial skin, artificial hearts, ventricular assist devices, hearing aids, vascular grafts, pacemaker components, hip implants, knee implants, dental implants, etc.

[0104] In some embodiments, the use of the compositions described in the preceding paragraphs in personal hygiene products involves the combination or incorporation of antimicrobial peptides or peptide organisms with or into the materials of the personal hygiene products, thereby giving the personal hygiene products a bactericidal and bacteriostatic effect, such as diapers, pads, sanitary napkins, sports mats, tampons, and related devices for their use; in terms of hygiene and health care products, antimicrobial wipes, baby wipes, personal wipes, cosmetic wipes, diapers, medical wipes (such as wipes or pads containing antibiotics, acne medications, hemorrhoid medications, antipruritic medications, anti-inflammatory medications, and preservatives, etc.).

[0105] In some embodiments, the application of the compositions described in the preceding paragraphs in articles that come into direct contact with the oral cavity utilizes antimicrobial peptides or peptide organisms that are non-toxic to humans and capable of killing viruses. Therefore, they can directly contact the oral cavity to achieve the purpose of oral sterilization and bacteriostasis. These articles, which come into direct contact with the oral cavity, prevent skin infections in infants and young children, such as baby bottles, nipples, dental instruments, elastic bands, dentures, cups, water cups, toothpaste, and teething toys. Applications primarily aimed at preventing microbial infections in children's items are also included in this protection, such as baby bottles, children's books, plastic scissors, toys, diaper containers, and containers for clean wipes.

[0106] In some embodiments, the use of the compositions described in the preceding paragraphs in household products, to prevent bacteria from entering through the mouth, may involve the combination or incorporation of antimicrobial peptides or peptide biopeptides with household product materials, wherein household products include telephones, mobile phones, fiber fillings, bedding, window treatments, carpet floor cleaning treatments, foam pads on the back of floor mats or carpet mats, upholstery items (such as foam pads), nonwoven dry paper, paper containing softeners, car wipes, household cleaning wipes, countertop wipes, shower curtains, shower curtain fabrics, towels, face towels, rags, mops, tablecloths, walls, and countertops, etc.

[0107] In some embodiments, to avoid cross-infection, the composition described in the foregoing paragraphs can be used in disinfectants formulated with the antimicrobial peptides or peptide derivatives of this application. The environments in which the disinfectants are used include enclosed spaces, airplanes, trains, cinemas, theaters, and other air disinfection environments. For example, it can be used for disinfection of medical endoscopes. Antimicrobial peptides or antimicrobial peptide derivatives can reduce or prevent the formation of biofilms on the surface of independent membranes, including pervaporation membranes, dialysis membranes, reverse osmosis membranes, ultrafiltration membranes, microfiltration membranes, etc.

[0108] In some embodiments, the composition described in the preceding paragraphs is used in a cleaning agent that has a bactericidal effect and requires rinsing with water after use. Deionized water is preferred. The cleaned items can be selectively dried using methods such as ambient air drying, oven drying, and forced air drying. The drying temperature is between 50°C and 120°C, preferably between 50°C and 100°C, and the drying time is approximately 15 minutes to 24 hours.

[0109] Furthermore, the treatment of all the aforementioned articles by antimicrobial peptides or antimicrobial peptide compositions includes the entire process before, after, and during industrial production. For example, in the manufacture of antimicrobial shower curtains, the antimicrobial peptides are first combined with a base polymer, and then the shower curtain is treated with the mixture. Manufacturing processes include, but are not limited to, at least one of injection molding, extrusion molding, blow molding, thermoforming, solution coating, and blown film.

[0110] In some embodiments, the application of the compositions described in the foregoing paragraphs in anti-infective or anti-inflammatory drugs kills bacteria and prevents infection. Infections include bacteria, viruses, and fungi, such as yeast, pathogens, Gram-negative and / or Gram-positive bacteria, caused by single-celled or multicellular organisms, and can also be caused by symbiotic or non-symbiotic organisms, pathogenic organisms, or colony-forming or non-colonial bacteria. Specifically, antimicrobial peptides or antimicrobial peptide derivatives can treat or reduce bacterial, viral, or fungal infections on the skin. Such infections can be any part of the human or animal exodermal system, such as the epidermis, dermis, subcutaneous tissue, one or more hair follicles, one or more sebaceous glands, or other sites associated with the skin. In other words, the aforementioned infections are skin-related. Further, the infections can also be related to a part of vision or hearing, such as a part of the ear, such as the tympanic cavity, or a part of the auditory system including the tympanic cavity.

[0111] In the embodiments, anti-infective drugs can be used to treat infections in humans or animals caused by any one or more types of bacteria, and antimicrobial peptides can treat the overgrowth of one or more symbiotic organisms, even if such symbiotic organisms are non-pathogenic or beneficial to humans or animals. Some of these symbiotic organisms may originate from species such as Staphylococcus, Mycobacterium, and Propionibacterium. Other bacterial infections may also originate from known pathogens, such as Pseudomonas.

[0112] In the embodiments, anti-infective drugs can be used to treat infections caused by non-bacterial microorganisms, such as infections caused by one or more fungi (e.g., yeast). For example, antimicrobial peptides can treat infections caused by *Malassezia furfur*. *Malassezia furfur* is a symbiotic organism whose overgrowth can cause dandruff, seborrheic dermatitis, pityriasis versicolor, and folliculitis. Treatment often involves controlling its growth, eliminating related inflammation, and preventing secondary infections. Because *Malassezia furfur* is stubborn and difficult to treat, and the treatment cycle is long, requiring the combined action of many drugs, currently available drugs are not only highly toxic but also extremely expensive. The antimicrobial peptides of this application can treat infections caused by fungi such as *Malassezia furfur* within an effective therapeutic concentration range.

[0113] In the embodiments, anti-inflammatory drugs can be used to treat inflammation caused by infection, non-infection, and even physical trauma. Antimicrobial peptides or their derivatives have been shown in experiments to reduce, alleviate, and treat inflammation. For example, in cells, bacteria, and animals, the aforementioned combination peptides have shown to reduce the release of inflammatory factors such as cytokinesis, chemokines, and their analogues.

[0114] In addition, this inflammation is a physiologically defined inflammatory response, including redness, swelling, induction of one or more pro-inflammatory cytokines at the cellular molecular transcription and translation level; induction of one or more cell signaling pathways associated with inflammation; receptor-induced responses present in the cell membrane; cellular infiltration in vascularized tissues; and other manifestations of inflammation within the scope of the art.

[0115] In some embodiments, the use of the composition described in the preceding paragraphs in drugs that inhibit unlimited cell proliferation, and their antitumor effects, refers to the use of non-unlimited proliferating cells (in vivo cells in cell lines and in vivo cells in living organisms) including human-constructed cells and cells in disease states, such as cancer cells; such non-immortalized cells also include primary cultured cells, such as keratinocytes, microvascular endothelial cells, corneal epithelial cells, and dermal fibroblasts. The antimicrobial peptide composition results in a reduction of pattern-recognition receptors—cell membrane receptors that specifically bind to bacteria. As a result of the above-mentioned response, animals and parts of animals exhibit significant anti-inflammatory effects, such as reduced or absent tissue redness and swelling, and reduced or absent cell permeability.

[0116] In some embodiments, the pro-inflammatory cytokines described above include inflammation-related cytokines, including but not limited to tumor necrosis factor, interleukin-8, interleukin-1, and interleukin-6. Additionally, intracellular signaling pathways include inflammation-related signaling pathways, including but not limited to NF-κB and AP-1. "Cell membrane-associated receptors" include, but are not limited to, pattern recognition receptors and the TLR receptor family (including TLR-2 and TLR4).

[0117] In the embodiments, the therapeutic concentration and treatment time of the antimicrobial peptide depend on many factors, such as disease status, age, gender, weight and individual physical condition. When the antimicrobial peptide is used for stem cell therapy, its effective concentration is greater than 0.01 ug / ml.

[0118] In the examples, the effective concentration was above 0.2 ug / ml.

[0119] In the embodiments, the anti-infective drugs, anti-inflammatory drugs, and drugs that inhibit unlimited cell proliferation include at least one of liquids, semi-solid liquids, creamy solids, ointments, and gels. For example, the topical dosage form can be used on the skin, hair, and other external parts. Furthermore, under certain conditions, the topical dosage form can be used on one or both eyes, or it can be made into eye drops.

[0120] In the embodiments, the antimicrobial peptide or antimicrobial peptide derivative can be formulated as an intravenous injection or an intratympanic / transtympanic administration formulation, and the solvent can be a sterile isotonic aqueous solution.

[0121] Furthermore, the formulation of antimicrobial peptides used in pharmaceuticals must be consistent with the route of administration, which includes gastrointestinal, intravenous, subcutaneous, intradermal, oral, intranasal (inhalation), vaginal, anal, epidermal, mucosal, transtympanic, intratympanic, rectal, and other acceptable methods of administration. Antimicrobial peptide formulations must be matched with a route of administration consistent with their pharmaceutical composition, such as intravenous, subcutaneous, intramuscular, transganglionic, oral, nasal, intraoral, intraauricular, and subcutaneous administration to humans and / or animals, such as livestock and companion animals, and may also be used in aquatic organisms and poultry.

[0122] In addition, the definition of one letter representing an amino acid in the application is consistent with the International Standard for Amino Acid Coding and Representative Sequence (IUPAC-IYUB), please refer to Table 1.

[0123] Table 1

[0124]

[0125]

[0126] The following description is based on specific embodiments.

[0127] Examples 1 to 39

[0128] Examples 1 to 39 were synthesized using standard, internationally recognized solid-phase synthesis, solution-phase synthesis, or recombinant biosynthesis of polypeptides. The results are shown in Table 2, which contains the amino acid sequence.

[0129] Table 2

[0130]

[0131]

[0132]

[0133] Performance testing and results analysis

[0134] (1) Escherichia coli inhibition experiment

[0135] Escherichia coli inhibition experiments were conducted on 39 antimicrobial peptides from Examples 1 to 39 of peptide synthesis. Equal volumes of E. coli liquid (30 μL) were taken, and 30 μL of each antimicrobial peptide (concentration 200 μg / mL) were added. 30 μL of isotonic PBS was used as a control. The mixture was reacted at room temperature for 15 min, then spread evenly onto agarose plates and incubated at 37°C for 12 h. The number of E. coli on each plate was counted and recorded. For experimental results, please refer to [reference needed]. Figure 1 Experimental results showed that antimicrobial peptides SEQ ID NO:1 to SEQ ID NO:39 all had inhibitory effects on Escherichia coli, with SEQ ID NO:8 showing the most significant effect.

[0136] To make the experimental results clearer, six experimental samples were selected for comparative analysis. Please refer to [the provided text]. Figure 2 As shown, A is the control tube with 251 E. coli clones; B is SEQ ID NO:5 with 236 E. coli clones; D is SEQ ID NO:6 with 226 E. coli clones; F is SEQ ID NO:7 with 216 E. coli clones; C is SEQ ID NO:8 with 1 E. coli clone; E is SEQ ID NO:35 with 95 clones, indicating that the complex peptide has the effect of killing Gram-negative bacteria. In addition, SEQ ID NO:8 has 1 clone, indicating that the optimal amino acid sequence of the antimicrobial peptide is SEQ ID NO:8, and the MIC of E. coli is 32ug / ml.

[0137] (2) Wild Staphylococcus aureus inhibition loop experiment

[0138] Spread clinically isolated wild-type Staphylococcus aureus (Staphylococcus aureus is a Gram-positive bacterium) liquid onto an agarose plate, place it in an Oxford inoculation cup, and add 200 μL of SEQ ID NO:8 antimicrobial peptide (concentrations of 1 / 4: 375 ug / ml; 1 / 16: 93.7 ug / ml; 1 / 64: 23.34 ug / ml). Incubate at 37°C for 18 h, and determine the size of the inhibition zone. The results were: 1 / 4: inhibition zone 3.95 cm; 1 / 16: inhibition zone 3.5 cm; 1 / 64: inhibition zone 3.1 cm. Please refer to... Figure 3 As shown in the figure. Experimental results show that as the concentration of the antimicrobial peptide decreases, the inhibition zone becomes smaller. In addition, compared with Escherichia coli, the antimicrobial peptide is more sensitive to Staphylococcus aureus. The antimicrobial peptide of this application has good anti-Gram-positive bacteria activity.

[0139] (3) MIC determination of Pseudomonas aeruginosa (ATCC27853)

[0140] SEQ ID NO:8 antimicrobial peptide at concentrations of 1024 ug / ml, 512 ug / ml, 256 ug / ml, 128 ug / ml, 64 ug / ml, 32 ug / ml, 16 ug / ml, 8 ug / ml, 4 ug / ml, and 2 ug / ml were prepared, along with positive and negative controls. These were mixed with standard Pseudomonas aeruginosa and added to 12 wells of a culture plate, with each well numbered. Please refer to [reference needed]. Figure 4As shown, 1 corresponds to 1024 ug / ml, 2 to 512 ug / ml, 3 to 256 ug / ml, 4 to 128 ug / ml, 5 to 64 ug / ml, 6 to 32 ug / ml, 7 to 16 ug / ml, 8 to 8 ug / ml, 9 to 4 ug / ml, 10 to 2 ug / ml, 11 to the positive control, and 12 to the negative control. After incubation at 37℃ for 2 days, the degree of turbidity in the culture plates was observed. For experimental results, please refer to... Figure 4 As shown in the figure. Experimental results indicate that the antimicrobial peptide has a good inhibitory effect on Pseudomonas aeruginosa, with a MIC of 64 ug / ml.

[0141] (4) Inhibition of oral bacteria

[0142] Take the elderly person's mouthwash, divide it into 4 equal portions, and mix each portion with a different amount of 2 mg / ml SEQ ID NO:8. After 30 min, evenly spread the mixture onto agarose-containing sheep blood plates. A: PBS (control), B: 2 μL, C: 10 μL, D: 20 μL. Incubate anaerobically at 37°C for 4 days, then observe the colony count. For experimental results, please refer to [reference needed]. Figure 5 As shown.

[0143] Specifically, Candida albicans and anaerobic bacteria were found to grow on the control plate, a small amount of bacteria grew on the B: 2ul antimicrobial peptide plate, no oral bacteria were observed to grow on the C: 10ul antimicrobial peptide plate, and no oral bacteria were observed to grow on the D: 20ul antimicrobial peptide plate.

[0144] (5) Experiment on the killing of RNA viruses by antimicrobial peptides

[0145] 293T cells were transfected with plasmids pNL4.3Δ, pVSV-G, p-enhancer, and lipo2000. After two days of incubation at 37°C, the culture supernatant was collected and centrifuged to remove organelles and impurities. 500 μL of the cell supernatant was mixed with an equal volume of 200 μg / ml SEQ ID NO:8 and reacted for 15 min. The control group was treated with an equal volume of PBS instead of SEQ ID NO:8. The antimicrobial peptides were removed by filtration centrifugation (Millipore Amicon Ultra-15) to eliminate their cytotoxic effects. The 293T cell line was infected with the test sample and incubated at 37°C for 12 h. The 293T cell line was then recovered, and the difference in viable virus in infectable cells was determined by RT-PCR. The experimental and control groups had identical experimental environments; the difference was that the experimental group contained the amino acid sequence of SEQ ID NO:8 from this application.

[0146] The results showed that the number of active viral gene copies in the control group was 1.7 × 10⁻⁶. 7The experimental group had 5 × 10⁻⁶ viral copies of antimicrobial peptides. 5 This indicates that the antimicrobial peptides in this application have a significant virus-killing effect.

[0147] (6) Experiment on the killing of tumor cells by antimicrobial peptides

[0148] K562 cells were cultured, and equal amounts of K562 cells were reacted with 10 ng, 20 ng, and 40 ng of SEQ ID NO:8, respectively. Apoptotic cells were measured using a flow cytometry PI kit. It was found that cell death was directly proportional to the content of antimicrobial peptides, indicating that antimicrobial peptides can effectively kill K562 cells.

[0149] (7) Experiment on the killing of leukemia lymphocytes by antimicrobial peptides

[0150] Jurkat cell line: Leukemic lymphocytes, SEQ ID NO: 10 polypeptide 1ug / m20ul and 10 5 After reacting with the cell for 10 minutes and centrifuging at 1000 rpm for 3 minutes, as shown in Figure 6, the experimental results show that leukemia lymphocytes clump together and are accompanied by cell breakage.

[0151] (8) Experiment on the killing of leukemia cells by antimicrobial peptides

[0152] K562 cell line: Leukemia cells, SEQ ID NO:17 antimicrobial peptide 1ug / ml 20ul and 10 5 After reacting in the cell for 10 minutes and centrifuging at 1000 rpm for 3 minutes, please refer to... Figure 7 As shown, the results indicate that leukemia cells clump together.

[0153] (9) Experiment on the killing of liver cancer cells by antimicrobial peptides

[0154] HypG2 cell line: liver cancer cells, SEQ ID NO: 15 peptide 1ug / ml 20ul and 10 5 After reacting in the cell for 10 minutes and centrifuging at 1000 rpm for 3 minutes, please refer to... Figure 8 As shown, the results indicate that liver cancer cells clump together and break down and deform.

[0155] (10) Experiment on the effect of antimicrobial peptides on hemoglobin cells

[0156] Human red blood cells, SEQ ID NO:27 antimicrobial peptide 1ug / ml 20ul reacted with 2% RBC for 10min, centrifuged at 1000rpm for 3min. For experimental results, please refer to [reference needed]. Figure 9 As shown, the results indicate that human red blood cells clump together, with no obvious hemolysis observed.

[0157] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. sequence list SEQUENCE LISTING <110> Shenzhen Qianyue Biotechnology Co., Ltd. <120> Antimicrobial peptides or peptide derivatives, substitutes, and compositions thereof, preparation methods and applications <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> PRT <213> Artificial sequence <400> 1 His Ala Val Gly Asn Ile Met His Ile Ala Ser Ala Val Leu Val 1 5 10 15 Arg His <210> 2 <211> 18 <212> PRT <213> Artificial sequence <400> 2 His Lys Ala Val Gly Gln Ile Met His Ile Ala Ser Ala Val Leu 1 5 10 15 Val Arg His <210> 3 <211> 19 <212> PRT <213> Artificial sequence <400> 3 His Arg Ala Met Pro Asn Met Leu Lys Ile Met Ala Ser Leu Val 1 5 10 15 Leu Ala Lys Arg <210> 4 <211> 19 <212> PRT <213> Artificial sequence <400> 4 His Ala Met Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val Leu 1 5 10 15 Ala Arg His Lys <210> 5 <211> 18 <212> PRT <213> Artificial sequence <400> 5 His Lys Ala Val Pro Asn Met Leu His Ile Ala Ser Ala Val Leu 1 5 10 15 Val Arg His <210> 6 <211> 18 <212> PRT <213> Artificial sequence <400> 6 His Lys Ala Val Gly Asn Met Leu Lys Ile Ala Ser Ala Val Leu 1 5 10 15 Val His Lys <210> 7 <211> 18 <212> PRT <213> Artificial sequence <400> 7 His Ala Met Pro Asn Met Leu Arg Ile Met Ala Ser Ala Val Leu 1 5 10 15 Val Lys Arg <210> 8 <211> 18 <212> PRT <213> Artificial sequence <400> 8 His Ala Met Gly Asn Met Leu Lys Ile Met Ala Ser Ala Val Leu 1 5 10 15 Val Arg His <210> 9 <211> 20 <212> PRT <213> Artificial sequence <400> 9 His Arg Ala Met Gly Gln Ile Val Lys Met Ile Ala Thr Leu Val 1 5 10 15 Leu Ala Arg Lys His 20 <210> 10 <211> twenty one <212> PRT <213> Artificial sequence <400> 10 His Lys Arg Ala Met Pro Ser Ile Val Lys Met Ile Ala Thr Leu 1 5 10 15 Val Leu Ala Arg Lys His 20 <210> 11 <211> 19 <212> PRT <213> Artificial sequence <400> 11 Arg Lys Ala Met Pro Asn Met Leu Arg Ile Met Ala Ser Ala Val 1 5 10 15 Leu Val His Lys <210> 12 <211> 20 <212> PRT <213> Artificial sequence <400> 12 Arg His Ala Met Gly Asn Met Leu His Ile Met Ala Ser Leu Val 1 5 10 15 Leu Ala Arg Lys His 20 <210> 13 <211> 20 <212> PRT <213> Synthetic Sequence <400> 13 Arg His Ala Val Pro Asn Met Leu Arg Ile Met Ala Ser Lys Val 1 5 10 15 Lys Ala His Lys Arg 20 <210> 14 <211> 20 <212> PRT <213> Synthetic Sequence <400> 14 Arg Ala Met Pro Asn Met Leu Lys Ile Met Ala Ser Ala Val Leu 1 5 10 15 Val His Lys <210> 15 <211> 19 <212> PRT <213> Synthetic Sequence <400> 15 Arg Ala Met Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val Leu 1 5 10 15 Ala Arg Lys His <210> 16 <211> 20 <212> PRT <213> Synthetic Sequence <400> 16 Arg Lys Ala Val Pro Asn Met Leu Arg Ile Met Ala Ser Ala Val 1 5 10 15 Leu Val His Lys Arg 20 <210> 17 <211> 20 <212> PRT <213> artificial sequence <400> 17 Arg His Ala Met Gly Gln Met Leu Arg Ile Met Ala Ser Leu Val 1 5 10 15 Leu Ala Arg Lys <210> 18 <211> 21 <212> PRT <213> artificial sequence <400> 18 Arg Lys His Ala Met Pro Ser Ile Val Lys Met Ile Ala Thr Leu 1 5 10 15 Val Leu Ala Arg Lys His 20 <210> 19 <211> 20 <212> PRT <213> artificial sequence <400> 19 Arg Lys His Ala Met Gly Gln Ile Val Lys Met Ile Ala Thr Leu 1 5 10 15 Val Leu Ala His Arg 20 <210> 20 <211> 20 <212> PRT <213> artificial sequence <400> 20 Arg His Ala Val Pro Asn Met Leu Arg Ile Met Ala Ser Ala Val 1 5 10 15 Leo Val Lys His Arg 20 <210> 21 <211> 18 <212> PRT <213> artificial sequence <400> 21 Lys Ala Val Pro Asn Ile Met His Ile Met Ala Ser Ala Val Leu 1 5 10 15 Val Arg His <210> 21 <211> 19 <212> PRT <213> artificial sequence <400> 21 Lys Arg Ala Met Pro Asn Ile Val Arg Ile Met Ala Ser Ala Val 1 5 10 15 Leo Val Arg His <210> 22 <211> 19 <212> PRT <213> artificial sequence <400> 22 Lys His Ala Met Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val 1 5 10 15 Lion Wing Arg His <210> 23 <211> 19 <212> PRT <213> artificial sequence <400> 23 Lys Ala Val Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val Leu 1 5 10 15 Ala Arg Lys His <210> 24 <211> 20 <212> PRT <213> artificial sequence <400> 24 Lys His Ala Val Gly Gln Ile Val Lys Met Ile Ala Thr Leu Val 1 5 10 15 Leu Ala Lys His Arg 20 <210> 25 <211> 20 <212> PRT <213> artificial sequence <400> 25 Lys Arg Ala Met Pro Ser Ile Val Lys Met Ile Ala Thr Ala Val 1 5 10 15 Leu Val Arg Lys His 20 <210> 26 <211> 18 <212> PRT <213> artificial sequence <400> 26 Lys Ala Val Pro Asn Met Lys Lys Ile Met Ala Ser Leu Val Leu 1 5 10 15 Ala Arg His <210> 27 <211> 20 <212> PRT <213> artificial sequence <400> 27 Lys His Ala Val Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val 1 5 10 15 Leu Ala His Lys Arg 20 <210> 28 <211> 20 <212> PRT <213> Artificial sequence <400> 28 Lys His Arg Ala Val Gly Asn Met Leu Lys Ile Ala Ser Leu Val 1 5 10 15 Leu Ala Arg His Lys 20 <210> 29 <211> 19 <212> PRT <213> Artificial sequence <400> 29 Lys Ala Met Pro Asn Ile Val Arg Ile Met Ala Ser Leu Val Leu 1 5 10 15 Ala Arg His Lys <210> 30 <211> twenty one <212> PRT <213> Artificial sequence <400> 30 His Arg Ala Met Cys Pro Asn Met Leu Lys Ile Met Ala Ser Leu 1 5 10 15 Val Leu Ala Lys Arg Cys 20 <210> 31 <211> twenty one <212> PRT <213> Artificial sequence <400> 31 His Ala Met Cys Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val 1 5 10 15 Leu Ala Arg His Lys Cys 20 <210> 32 <211> 21 <212> PRT <213> Artificial sequence <400> 32 His Arg Ala Met Cys Pro Asn Met Leu Lys Ile Met Ala Ser Leu 1 5 10 15 Val Leu Ala Lys Arg Cys 20 <210> 33 <211> 21 <212> PRT <213> Artificial sequence <400> 33 His Ala Met Cys Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val 1 5 10 15 Leu Ala Arg His Lys Cys 20 <210> 34 <211> 23 <212> PRT <213> Artificial sequence <400> 34 His Lys Arg Ala Met Cys Pro Ser Ile Val Lys Met Ile Ala Thr 1 5 10 15 Leu Val Leu Ala Arg Lys His Cys 20 <210> 35 [[ID=6O]]<211> 21 <212> PRT <213> Artificial sequence <400> 35 Arg Lys Ala Met Cys Pro Asn Met Leu Arg Ile Met Ala Ser Ala 1 5 10 15 Val Leu Val His Lys Cys 20 <210> 36 <211> 21 <212> PRT <213> Synthetic sequence <400> 36 Arg Ala Met Cys Pro Asn Met Leu Arg Ile Met Ala Ser Leu Val 1 5 10 15 Leu Ala Arg Lys His Cys 20 <210> 37 <211> 21 <212> PRT <213> Synthetic sequence <400> 37 Arg His Ala Met Cys Gly Gln Met Leu Arg Ile Met Ala Ser Leu 1 5 10 15 Val Leu Ala Arg Lys Cys 20 <210> 38 <211> 21 <212> PRT <213> Synthetic sequence <400> 38 Lys Arg Ala Met Cys Pro Asn Ile Val Arg Ile Met Ala Ser Ala 1 5 10 15 Val Leu Val Arg His Cys 20 <210> 39 <211> 21 <212> PRT <213> Synthetic sequence <400> 39 Lys His Ala Met Cys Pro Asn Met Leu Arg Ile Met Ala Ser Leu 1 5 10 15 Val Leu Ala Arg His Cys 20

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide consists of the amino acid sequence SEQ ID NO:

8.

2. The application of the antimicrobial peptide according to claim 1 in the preparation of products that inhibit Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa, wherein the product is any one of disinfectant, cleaning agent, or anti-infective drug.

3. The use of the antimicrobial peptide according to claim 1 in the preparation of drugs for treating leukemia or liver cancer.

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