WO2023029001A1 - 抗菌肽或肽衍生物、替换体以及其组合物、制备方法和应用 - Google Patents

抗菌肽或肽衍生物、替换体以及其组合物、制备方法和应用 Download PDF

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WO2023029001A1
WO2023029001A1 PCT/CN2021/116530 CN2021116530W WO2023029001A1 WO 2023029001 A1 WO2023029001 A1 WO 2023029001A1 CN 2021116530 W CN2021116530 W CN 2021116530W WO 2023029001 A1 WO2023029001 A1 WO 2023029001A1
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peptide
antimicrobial
amino acid
antimicrobial peptide
peptide derivative
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PCT/CN2021/116530
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English (en)
French (fr)
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温瞳
温雯
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深圳千越生物科技有限公司
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Priority to PCT/CN2021/116530 priority Critical patent/WO2023029001A1/zh
Priority to CN202180056357.XA priority patent/CN116134045A/zh
Priority to JP2023514140A priority patent/JP2023542830A/ja
Publication of WO2023029001A1 publication Critical patent/WO2023029001A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application belongs to the technical field of peptides or peptide derivatives, and in particular relates to an antimicrobial peptide or peptide derivatives, replacement body, composition, preparation method and application thereof.
  • antimicrobial peptides widely exist in organisms such as bacteria, viruses, fungi, insects, amphibians, animals and plants, and antimicrobial peptides have a very significant effect on killing bacteria, fungi, parasites and viruses.
  • organisms such as bacteria, viruses, fungi, insects, amphibians, animals and plants
  • antimicrobial peptides have a very significant effect on killing bacteria, fungi, parasites and viruses.
  • microorganisms have become resistant to drugs, which has promoted and driven the large-scale research and development of antimicrobial peptides by researchers.
  • antimicrobial peptides such as defensins (defensins) were discovered and approved by the FDA (Food and Drug Administration) as drugs for anti-infection, anti-inflammation and other injuries, people have further discovered that antimicrobial peptides have the ability to stimulate the body's innate After the efficacy of acquired and adaptive immunity, antimicrobial peptides increasingly show potential therapeutic advantages against some diseases.
  • antimicrobial peptides are also widely used in food preservation, poultry and animal husbandry, aquaculture and fishery, and daily medical care, hygiene and beauty, cleaning and disinfection, and agricultural pest control.
  • Propionibacterium acnes is caused by the overgrowth of Gram-positive bacteria, a normal human skin microflora, in the pilosebaceous follicles.
  • Propionibacterium acnes is one of the most common diseases affecting human skin, affecting approximately one million Chinese.
  • Acne caused by Propionibacterium has different disease manifestations, such as comedones, papules, tuberculosis, cysts, and pilosebaceous gland inflammation.
  • the traditional treatment of acne caused by Propionibacterium is mostly antibiotics, such as oral oxytetracycline, topical erythromycin, clindamycin, etc., but this treatment will soon lead to antibiotic resistance.
  • benzoyl peroxide (Benzoyl Peroxide) and 5% dapsone gel are also used in clinical treatment of acne caused by propionibacterium. toxic side effect.
  • Pseudomonas aeruginosa is caused by the invasion and infection of Gram-negative bacteria on human and animal bodies. It can infect the respiratory system, urinary system, gastrointestinal system, central nervous system, and blood of organisms. , Heart And Skeletal System. When Pseudomonas aeruginosa infects the eyes and ears, it often causes bacterial keratitis, scleral abscess, endophthalmitis, neonatal ophthalmia and otitis media in children.
  • Pseudomonas aeruginosa/bacterial keratitis in particular, has increased the rate of infection with pupillary lens use, with approximately 25,000 pupillary lens wearers in the United States being infected with this bacteria each year.
  • Pseudomonas aeruginosa can be treated, with the use of antibiotics, the resistance of this microorganism increases, which quickly reduces the efficacy of the treatment, and the combination of drugs and the treatment plan have to be changed.
  • Pseudomonas aeruginosa isolates isolated from intensive care units are resistant to ciprofloxacin, and 31.4% are resistant to piperacillin/tazobactam. Up to 38% for imipenem, up to 23.6% for ceftazidime.
  • Pseudomonas aeruginosa isolates isolated in European intensive care units can be 37-70% resistant to aminoglycosides, 57% resistant to ceftazidime, and 57% resistant to piperacillin/tazobactam.
  • the drug resistance reaches 53%
  • the drug resistance to ciprofloxacin reaches 56%
  • the drug resistance to imipenem reaches 52%. It is very important.
  • the typical antimicrobial peptides currently reported generally consist of 10-100 amino acid residues, contain many basic amino acids and are generally amphipathic.
  • antimicrobial peptides isolated from animals and plants are diverse and widely distributed, and are found in organisms such as insects, fish, mammals, amphibians, and plants.
  • the applicability of antimicrobial peptides from different sources has not been systematically studied.
  • phytoantimicrobial peptides are unstable and easily hydrolyzed by proteolytic enzymes, thereby greatly reducing their antibacterial activity.
  • the purpose of this application is to provide an antimicrobial peptide or a peptide derivative, a synthesis method, a composition and an application, aiming at solving the problems in the prior art that antimicrobial peptides have weak antibacterial activity, are unstable, and do not have broad-spectrum antibacterial activity.
  • the present application provides an antimicrobial peptide or peptide derivative, which includes at least one of the following amino acid sequences:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • X a1 , B a1 , U 1 , 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 , and C a2 are each independently selected from amino acids.
  • the antimicrobial peptides or peptide derivatives provided in the present application can specifically bind to the lipid structure of cell walls/membranes, destroy microorganism walls, and then kill microorganisms and cancer cells.
  • a substitute for the antimicrobial peptide or peptide derivative in which at least one amino acid of the amino acid sequence I or/and amino acid sequence II contained in the antimicrobial peptide or peptide derivative of the present application is replaced.
  • the replacement body provided by this application can ensure the activity of antimicrobial peptides or peptide derivatives, and is also included in the content of this application.
  • the antibacterial activity of antimicrobial peptides or peptide derivatives can be improved. , increasing its broad-spectrum antibacterial activity.
  • the present application provides an antimicrobial peptide or peptide derivative composition, comprising at least one antimicrobial peptide or peptide derivative described in any of the above paragraphs and at least one pharmaceutically acceptable carrier.
  • an antibacterial composition provided in the third aspect of the present application can be mixed with most solvents to form a composition.
  • the present application provides a method for preparing an antibacterial composition, comprising the following steps: mixing the antibacterial peptide or peptide derivative and a solvent.
  • the antimicrobial peptide or peptide derivative in the preparation method of the antibacterial composition of the present application, can be stably present in most solvents, and the antibacterial activity of the antimicrobial peptide or peptide derivative can be preserved by mixing the solvent and the antimicrobial peptide or peptide derivative. On the one hand, the stability of the antimicrobial peptide or peptide derivative against peptidase or protein degradation is ensured.
  • the application provides the application of the antimicrobial peptide or peptide derivative composition of the application in packaging, food processing terminal, clothing, medical supplies, medical instruments, personal hygiene products, disinfectants, cleaning agents, anti-infective drugs, anti-inflammatory drugs , Inhibition of the application of infinite cell proliferation drugs.
  • the application of the antimicrobial peptide or peptide derivative composition of this application is precisely because the antimicrobial peptide or peptide derivative can kill microorganisms, kill tumor cells, anticoagulation and anti-inflammation. Therefore, it has a wide range of uses and mainly has four points advantage:
  • Antimicrobial peptides or peptide organisms can be combined or mixed with packaging, clothing, medical supplies, medical instruments, and personal hygiene products to make them antibacterial, or as preservatives for other materials that are easily degraded by microorganisms;
  • Antimicrobial peptides or peptide organisms can be made into disinfectants, cleaners, anti-infective drugs, and anti-inflammatory drugs to play the role of sterilization, anti-infection, and anti-inflammatory;
  • the antimicrobial peptide or peptide biological composition is made into a coating and applied to the food processing and final terminal, which can reduce the microorganisms on the final surface of food processing and processing, and reduce the risk of people's food poisoning;
  • Fig. 1 statistical histogram of antimicrobial peptides or peptide derivatives to Escherichia coli inhibition test results in the examples of the present application;
  • Figure 2 is a diagram of the inhibition results of antimicrobial peptides or peptide derivatives on Escherichia coli in the examples of the present application;
  • Figure 3 is a diagram of the results of the antimicrobial peptide or peptide derivatives inhibition experiment on wild Staphylococcus aureus in the examples of the present application;
  • Figure 4 is a diagram of the results of the MIC inhibition experiment of the antimicrobial peptide or peptide derivatives against Pseudomonas aeruginosa in the examples of the present application;
  • Figure 5 is a diagram of the experimental results of antibacterial peptides or peptide derivatives killing RNA viruses in the examples of the present application;
  • Figure 6 is a diagram of the experimental results of antibacterial peptides or peptide derivatives antibacterial peptides killing leukemia lymphocytes (Jurkat cell line) in the examples of the present application;
  • Figure 7 is a diagram of the experimental results of antibacterial peptides or peptide derivatives killing leukemia cells (K562 cell line) in the examples of the present application;
  • Figure 8 is a diagram of the experimental results of antimicrobial peptides or peptide derivatives killing liver cancer cells in the examples of the present application.
  • Fig. 9 is a graph showing the experimental results of antimicrobial peptides or peptide derivatives on red blood cells in the examples of the present application.
  • the term "and/or” describes the association relationship of associated objects, indicating that there may be three relationships, for example, A and/or B may mean: A exists alone, A and B exist simultaneously, and B exists alone Condition. Among them, A and B can be singular or plural.
  • the character "/" generally indicates that the contextual objects are an "or" relationship.
  • At least one means one or more, and “multiple” 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 plural items.
  • sequence numbers of the above-mentioned processes do not mean the order of execution, and some or all steps may be executed in parallel or sequentially, and the execution order of each process shall be based on its functions and The internal logic is determined, and should not constitute any limitation on the implementation process of the implementation regulations of this application.
  • the weight of the relevant components mentioned in the description of the embodiments of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the various components.
  • the scaling up or down of the content of the fraction is within the scope disclosed in the description of the embodiments of the present application.
  • the mass described in the description of the embodiments of the present application may be ⁇ g, mg, g, kg and other well-known mass units in the chemical industry.
  • first and second are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features.
  • first XX can also be called the second XX
  • second XX can also be called the first XX.
  • a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features.
  • the embodiment of the present application provides an antimicrobial peptide or peptide derivative, whose amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • X a1 , B a1 , U 1 , 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 , and C a2 are each independently selected from amino acids, wherein a ring bond is formed between Ca 1 and Ca 2 in the amino acid sequence II, and the amino acids include natural amino acids or/and unnatural amino acids, wherein , the antimicrobial peptides or peptide derivatives provided in this application can combine with the lipid structure of the cell wall/membrane to destroy the microbial wall or cell membrane, thereby killing microorganisms and tumor cells.
  • X a1 , X a2 , X a3 , X b1 , X b2 , and X b3 are each independently selected from positively charged or/and B a1 , B a2 , B a3 , Ba4 , B b1 , B b2 , B b3 , B b4 are each independently selected from residues with hydrophobic side chains; or/and U 1 selects One of Gly (G-glycine), Pro (P-proline), Cys (C-cysteine), Cys (R), wherein R represents the protecting group of Cys disulfide bond; or /and Z a1 , Z a2 , Z b1 , Z b2 are each independently selected from non-polar residues; or/and C a1 , C a2 are each independently selected from
  • the antimicrobial peptides and peptide derivatives of the present application contain residues with hydrophobic side chains, positively charged residues, and non-polar residues. Therefore, the antibacterial peptides or peptide derivatives chains provided in this example A typical amphipathic helical structure can be formed.
  • the charge and amphoteric helical structure of the antimicrobial peptide or peptide derivative enable it to combine with the lipid structure of the cell wall/membrane, destroy the microbial wall or cell membrane, and then kill microorganisms and tumor cells.
  • amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • B a1 , U 1 , Z a1 , B a2 , B a3 , Z a2 , B a4 , B b1 , C a1 , Z b1 , B b2 , B b3 , Z b2 , B b4 , and C a2 are independently selected from From the amino acids, X a1 , X a2 , X a3 , X b1 , X b2 , and X b3 are each independently selected from one of the positively charged residues, and the peptide chain with anodic charge is easy to combine with the negatively charged cell membrane .
  • amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • 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 , and C a2 are each independently selected from amino acids
  • X a1 , X a2 , X a3 , X b1 , X b2 , X b3 are each independently selected from at least one of positively charged amino acid residues
  • B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , B b3 , B b3 are each independently selected from at least one of amino acid residues with hydrophobic side chains
  • U 1 is selected from at least one of Gly, Pro, Cys, Cys (R), with The anodically charged peptide chain is easy to combine with
  • amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • C a1 , C a2 are each independently selected from amino acids
  • X a1 , X a2 , X a3 , X b1 , X b2 , and X b3 are each independently selected from one of positively charged residues
  • B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , and B b3 are each independently selected from at least one amino acid residue with a hydrophobic side chain
  • U 1 is selected from Gly, Pro, Cys, Cys (R ), wherein R represents the protecting group of the Cys disulfide bond
  • Z a1 , Z a2 , Z b1 , and Z b2 are each independently selected from at least one of non-polar amino acid residues, with Anodically charged peptide chains are easy to combine with negatively charged cell membranes, non-polar residues and residues of hydrophobic side chains to ensure the balance of water so
  • amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • X a1 , X a2 , X a3 , X b1 , X b2 , and X b3 are each independently selected from positively charged residues;
  • B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , B b3 and B b4 are each independently selected from at least one amino acid residue with a hydrophobic side chain;
  • U 1 is selected from Gly (G-glycine), Pro (P-proline), Cys (C-semi Cystine), Cys (R), wherein, R represents the Cys disulfide bond protecting group;
  • Z a1 , Z a2 , Z b1 , Z b2 are each independently selected from non-polar amino acid residues
  • C a1 and C a2 are each independently selected from at least one of Cys (C-cysteine) and Cys (R), and in amino acid sequence I,
  • the antimicrobial peptides and peptide derivatives of the present application contain residues with hydrophobic side chains, positively charged residues, and nonpolar residues
  • the antimicrobial peptides or peptide derivatives chains provided in this example can be Forming a typical amphipathic helical structure
  • the charge and amphoteric helical structure of the antimicrobial peptide or peptide derivative enable it to combine with the lipid structure of the cell wall/membrane, destroy the microbial wall or cell membrane, and then kill microorganisms and tumor cells.
  • X a1 , X a2 , X a3 , X b1 , X b2 , X b3 , and B b4 are each independently selected from Arg (R-arginine), His(H-histidine), Lys(K-lysine), Orn(ornithine), Har(homoarginine), Dab(2,4 diaminopropionic acid ), one, two or three of unnatural amino acids, on the one hand, Arg (R-arginine), His (H-histidine), Lys (K-lysine), Orn (ornithine acid), Har (homoarginine), Dab (2,4 diaminopropionic acid), and unnatural amino acids are positively charged residues.
  • the number of positively charged residues is one, two or three, or/and B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , and B b3 are each independently selected from Ala (A-alanine), Val (V-valine), Ile (I-isoleucine), Leu (L-leucine acid), Met (M-methionine), two, three, four of unnatural amino acids, Ala (A-alanine), Val (V-valine), Ile (I-iso Leucine), Leu (L-leucine), Met (M-methionine), unnatural amino acids are hydrophobic side chains, and the hydrophobic side chains can balance the water solubility and charge of the peptide chain, that is, If the hydrophobic side chain residues in the peptide chain or peptide derivative are too long, the
  • Z a1 , Z a2 , Z b1 , Z b2 are each independently selected from Asn (N-aspartic acid), Gln (Q-glutamine Amide), Ser (S-serine), Thr (T-threonine) unnatural amino acids, selected from non-polar residues, can balance the water solubility and charge of the peptide chain, that is, If the non-polar residues in the peptide chain or peptide derivative are too long, the peptide chain will easily become unstable.
  • amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • Z a1 , Z a2 , Z b1 , and Z b2 are each independently selected from amino acids, X a1 , X a2 , X a3 , X b1 , X b2 , X b2 , and X b3 are each independently selected from positively charged One, two or three of Arg, His, Lys, Orn, Har, Dab, unnatural amino acids; each of B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , B b3 , B b4 Independently selected from one of the residues with hydrophobic side chains; and/or U is selected from one of Gly, Pro, Cys, Cys (R), wherein R represents the protecting group of the Cys disulfide bond ; C a1 and C a2 are each independently selected from one of Cys and Cys (R), and positively charged residues can form amphipathic
  • amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • X a1 , X a2 , X a3 , X b1 , X b2 , X b2 , and X b3 are each independently selected from one of positively charged Arg, His, Lys, Orn, Har, Dab , and unnatural amino acids.
  • B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , B b3 , B b4 are each independently selected from Ala, Val, Ile, Leu, Two, three or four of Met, unnatural amino acids; or/and U1 is selected from one of Gly, Pro, Cys, Cys (R), wherein R represents the protecting group of the Cys disulfide bond ; Z a1 , Z a2 , Z b1 , Z b2 are each independently selected from at least one of non-polar residues; or/and C a1, C a2 are each independently selected from one of Cys, Cys (R) , positively charged residues can form an amphipathic helical structure in coordination with other residues, increasing the antibacterial activity of antimicrobial peptides or peptide derivatives.
  • the positively charged residues in the antimicrobial peptide chain or peptide derivatives are too long, The charge of antimicrobial peptides or peptide derivatives will be unbalanced and easily become unstable, therefore, in order to ensure the stability of peptides or peptide derivatives against peptidase or protein degradation, the number of positively charged residues is one, two Or three, the hydrophobic side chain can balance the water solubility and charge of the peptide chain, that is to say, if the hydrophobic side chain residue in the peptide chain or peptide derivative is too long, the peptide chain will easily become unstable. Therefore, for 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.
  • amino acid sequence is generally expressed as follows:
  • Amino acid sequence I X a1 B a1 U 1 Z 1 B a2 X a2 B a3 Z a2 B a4 X a3 ;
  • X a1 , X a2 , X a3 , X b1 , X b2 , X b2 , and X b3 are each independently selected from one of positively charged Arg, His, Lys, Orn, Har, Dab , and unnatural amino acids.
  • B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , B b32 , B b4 are each independently selected from Ala, Val, Ile, Leu, Two, three or four of Met and unnatural amino acids;
  • Z a1 , Z a2 , Z b1 , and Z b2 are each independently selected from one of non-polar Asn, Gln, Ser, Thr, and unnatural amino acids Kind;
  • U1 is selected from one of Gly, Pro, Cys, Cys (R), wherein, R represents the protecting group of Cys disulfide bond;
  • C a1, C a2 are independently selected from Cys, Cys (R) One of them, positively charged residues, can cooperate with other residues to form an amphipathic helical structure, increasing the antibacterial activity of antimicrobial peptides or peptide derivatives.
  • the number of positively charged residues is one, Two or three, the hydrophobic side chain can balance the water solubility and charge of the peptide chain, that is, if the hydrophobic side chain residues in the peptide chain or peptide derivative are too long, the peptide chain will easily become unstable, therefore,
  • the number of hydrophobic side chain residues is guaranteed to be one of two, three, or four, which can balance the water solubility and charge of the peptide chain, and also That is to say, if the non-polar residues in the peptide chain or peptide derivative are too long, the peptide chain
  • the Cys disulfide bond includes acetamidomethyl (Acm) , methyl methane thiosulfonate (methyl methane thiosulfonate) or other Cys protecting groups, Cys (R) means containing Cys disulfide The protective group of the bond, wherein, C a2 is the end group of the antimicrobial peptide or peptide derivative, and C a1 is located in the middle position of the antimicrobial peptide or peptide derivative, so 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 sterilization time of the antimicrobial peptide, and make the antimicrobial peptide or peptide derivative have stronger antibacterial activity.
  • At least one amino acid on the amino acid sequence I and/or II has been modified.
  • the stability and antibacterial activity of antimicrobial peptides or peptide derivatives are very important.
  • By modifying the Amino acids on antimicrobial peptides or peptide derivatives can change the performance of the peptide chain, improve the spectrum of antimicrobial peptides or peptide derivatives, improve the stability of antimicrobial peptides or peptide derivatives against peptidase or protein degradation, and increase their broad-spectrum antibacterial activity.
  • the modification includes phosphorylation, halogenation, acetylation, cyclization, and terminal capping reaction, wherein the cyclization includes cyclization formed by disulfide bonds, head-to-tail cyclization, and cyclization of side groups in the internal structure.
  • At least one of the amino acid terminal capping (capping) reaction includes at least one of C-terminal amidation, N-terminal acetylation and N-terminal phospholipidation
  • modification of amino acid such as cyclization can increase the peptide chain Stability, such as phosphorylation of amino acids, can increase the antibacterial activity of antimicrobial peptides or peptide derivatives.
  • the amino acid sequence I and/or II includes at least one motif (motif), usually connected to the predetermined amino acid terminal sequence (N-terminal and/or C-terminal), or directly inserted into the middle of the predetermined amino acid sequence , generally not independently as the amino acid sequence endpoint, the connection between the motif and the peptide chain or the insertion of amino acids, the insertion of the motif in the antimicrobial peptide or peptide derivative can change the performance of the peptide chain, increase the antibacterial activity of the peptide chain, antibacterial peptide or Stability of peptide derivatives against peptidase or protein degradation.
  • motif usually connected to the predetermined amino acid terminal sequence (N-terminal and/or C-terminal)
  • the antimicrobial peptide or peptide derivative is a multimer formed by amino acid sequence I and/or amino acid sequence II, wherein at least one of dimer (Dimer) and tetramer (Tetramer) is relatively common a multibody state.
  • Antimicrobial peptide monomers and/or multimers are conjugated with at least one drug or antibody to form antibacterial drugs, mainly including small molecule chemically synthesized drugs, macromolecular antibodies, and macromolecular drugs synthesized by biorecombination technology.
  • Antimicrobial peptides or peptide derivatives are conjugated with drugs and antibody molecules to form antibacterial drugs, which can be used to modify drugs or antibodies.
  • the antimicrobial peptide or peptide derivative comprises the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 39.
  • a nanostructure of an antimicrobial peptide or peptide derivative, a peptide chain and its peptide derivative or multiple or multimeric peptide chains and peptide derivatives can form a nanostructure, wherein the common nanostructure At least one selected from micelles, vesicles, nanotubes, and nanobelts.
  • the peptide chain and its peptide derivatives form a nanostructure, which is conducive to the stable existence of the peptide chain and its peptide derivatives in the solution, and the transmission in the solution.
  • the present application provides a substitute for an antimicrobial peptide or peptide derivative, which is obtained by replacing any amino acid in the amino acid sequence I or II of the above paragraph, which is also included in the content of the present application, such as L-form Amino acids are replaced with corresponding D-type amino acids or unnatural amino acids.
  • an antimicrobial peptide or peptide derivative obtained by replacing any amino acid in the amino acid sequence I or II of the above paragraph, which is also included in the content of the present application, such as L-form Amino acids are replaced with corresponding D-type amino acids or unnatural amino acids.
  • the application provides an antimicrobial peptide or peptide derivative composition, an antimicrobial peptide or peptide derivative or/and replacement body and at least one pharmaceutically acceptable carrier and/or solvent, the antibacterial peptide provided by the application
  • the peptide or peptide derivative 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.
  • it also includes at least one of excipients, isotonic agents, absorption delaying agents or substrate polymers, mixed with these substances, antimicrobial peptides or peptide derivatives can be made into various antibacterial drugs or Antimicrobial solvent.
  • the excipient is selected from at least one of water, salt, phosphate, glucose, glycerol, and ethanol, which are all commonly used medical reagents, and antimicrobial peptides or derivatives can be mixed with these reagents, which is beneficial to This application applies to the promotion and use of antibacterial peptides and derivatives.
  • the isotonic agent is selected from sugar, polyol and sodium chloride, the isotonic agent can maintain the activity of the peptide, and the antimicrobial peptide and derivatives are mixed with the isotonic agent to make the antimicrobial peptide or peptide derivative more easily with biological Body combination.
  • the polyol is selected from mannitol, sorbitol, and a mixture of polyols and antimicrobial peptides or peptide derivatives.
  • the polyols can maintain the activity of the antimicrobial peptides or derivatives and make the antimicrobial peptides or derivatives easy to combine with organisms.
  • the pharmaceutically acceptable carrier is selected from the minimum amount of auxiliary substances, the pharmaceutically acceptable carrier is selected from wetting agents, emulsifiers, preservatives, buffers, pharmaceutically acceptable carriers and antibacterial peptides or peptide derivatives
  • Forming a composition can maintain the activity of the antimicrobial peptide or derivative, and make the antimicrobial peptide or derivative easy to combine with organisms.
  • the antimicrobial peptide or peptide derivative is combined with the substrate polymer to form an aqueous or non-aqueous solution, and these aqueous or non-aqueous solutions can be applied on the surface of an object to form an antibacterial protective layer.
  • the above-mentioned substances may be composed of various forms or components. Including but not limited to extracts, films, films, laminates, knits, wovens, non-wovens, fibers, filaments, yarns, particles, coatings, and/or foams.
  • This application also includes various models made of the above 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, etc. .
  • the present 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.
  • the mixing temperature is controlled within the range of -10 to 150° C.
  • the mixing time is controlled within the range of 0.1 to 5760 min.
  • the mixing temperature is in the range of 25 to 80° C.
  • the mixing time is in the range of 1 to 1440 minutes, which has a better mixing effect.
  • pretreatment of pharmaceutically acceptable carriers, excipients, solvents, dispersion media, isotonic agents, absorption delaying agents or substrate polymers, wherein pretreatment includes but not limited to oxidation, At least one of reduction, hydrolysis, plasma or radiation, the pretreatment can improve the environment of the antimicrobial peptide or peptide derivative, and then improve the stability of the antimicrobial peptide or peptide derivative against peptidase or protein degradation.
  • the effective concentration range of the antimicrobial peptide or peptide derivative mixed with the above substances is between 1 ug to 20 gwt%, or between 5 pmol to 2 mol concentration.
  • the temperature needs to be controlled within the range of -20 to 25° C. to ensure the antibacterial activity of the antimicrobial peptide or peptide derivative.
  • the temperature is controlled between -4 and 4°C, and the biological activity of the antimicrobial peptide can be ensured at a lower temperature.
  • the application provides the application of any of the following antimicrobial peptides or peptide derivative compositions:
  • Antimicrobial peptides or peptide derivative compositions can be widely used as drugs or non-drugs in humans, animals (including wild animals, livestock, companion animals), plants (including crops), aquatic and aquaculture animals, poultry animals, etc.
  • the concept definition of human beings, animals and plants referred to below refers to the above-mentioned human beings, animals (including wild animals, livestock, companion animals), plants (including crops), aquatic and aquaculture animals, poultry farm animals, etc. The definition of the concept, the following is equivalent.
  • the application of the composition described in the above paragraphs in packaging, antimicrobial peptides or peptide bios can be combined or incorporated into packaging materials, or used as preservatives for other materials that are easily degraded by microorganisms, wherein the packaging components Including but not limited to packaging films, liners, absorbent pads, trays, container assembly elements, caps, lids, adhesives, applicators, etc. for meat products.
  • packaging may be in any form suitable for a particular application, such as aluminum cans, boxes, bottles, glass jars, bags, cosmetic packages, closed tubes, etc.
  • the above packaging includes but is not limited to packaging models made by injection molding, extrusion molding, blow molding, thermoforming, solution coating, blown film and other processes.
  • the packaging is also suitable for the packaging of prescription/non-prescription drugs and health care products, such as capsules, tablets, solutions, emulsifiers, detergents, powders, shampoos, hair conditioners, deodorants, antiperspirants, etc.
  • This applicator is used on different parts and surfaces of the living body. This application greatly reduces or eliminates the growth of bacteria on the surface of the human body.
  • the package also includes other forms of packaging components, such as drinking water for liquids, solutions or suspensions Bottlenecks, replaceable caps, non-replaceable caps, food or medicine dispensing systems; food and beverage delivery systems; baby bottles, caps, pacifiers, etc.
  • the package also contains a droplet dispersing agent or a liquid droplet propellant.
  • the composition described in the above paragraphs is used in food processing and final application, or as a coating for temporary or permanent preparation of food surfaces, such as food additives to replace antibiotics; food processing equipment, food conveyor belts Assemblies and parts of different instruments for mixing, grinding, crushing, rolling, granulating, extruding food; Assemblies and parts of instruments for cutting and slicing food.
  • the surface of the above-mentioned instrument is a metal substance
  • the metal surface must be coated with a layer of functional polymer, which is the polymer containing antimicrobial peptides or peptide derivatives described above in this application.
  • Antimicrobial peptides or peptide biological compositions are made into coatings and applied to food processing and final terminals, which can reduce microorganisms on the final surface of food processing and processing, and reduce the risk of food poisoning for people.
  • this clothing has the function of bactericidal and antibacterial, such as swimwear, underwear, shoe components (such as woven or non-woven inner lining or insole) sports Protective pads, children's clothing, etc.
  • This application also includes protective medical clothing or isolation products, such as protective clothing, masks, gloves, slippers, boots, head coverings or drapes, etc.
  • antimicrobial peptides or peptide organisms can be combined or incorporated into medical supplies and medical materials, or a layer of antimicrobial peptides or peptides can be coated on the surface of medical instruments Peptide derivative coating, or made into a spray to sterilize and disinfect medical supplies and instruments, including medical supplies and instruments including human and animal implants, such as bandages, adhesives, gauze strips, gauze pads, and syringe holders , peripheral or central venous catheters, which may be composed of polyurethane or silicone; urinary catheters, ostomy ports, orthopedic orthotics, orthopedic needles, pacemaker leads, defibrillator leads, ear canal shunts , vascular stents, orthopedic implants, ENT implants, implantable pumps, hernia patches and related thereto plates, screws, blood bags, external blood pumps, infusion systems, cardiopulmonary instruments, dialysis instruments, artificial Skin, Artificial Heart, Ventricular As
  • the application of the composition described in the above paragraphs in personal hygiene products, antimicrobial peptides or peptide organisms can be combined or mixed with personal hygiene products materials, and then personal hygiene products have a bactericidal and antibacterial effect, such as diapers, Inconvenience pads, sanitary napkins, sports pads, tampons, and accessories for their application; in terms of health care products, antimicrobial wipes, baby wipes, personal wipes, cosmetic wipes, diapers, medical wipes (such as wet wipes or cotton pads containing antibiotics, acne medicine, hemorrhoid medicine, anti-itch medicine, anti-inflammatory medicine and preservatives, etc.).
  • antimicrobial peptides or peptide organisms are non-toxic to the human body and can kill viruses, so they can be directly in contact with the oral cavity to achieve the effect of oral cavity sterilization and antibacterial Purpose, among them, items that are in direct contact with the oral cavity, such as baby bottles, pacifiers, dental instruments, elastic bands, dentures, cups, water cups, toothpaste and teething toys, etc., to prevent skin infections in infants and young children.
  • Applications where the primary purpose of protecting children's items from microbial contamination are also included in this protection, such as baby bottles, children's books, plastic clippers, toys, diaper pails, and containers for clean wipes.
  • antimicrobial peptides or peptide organisms can be combined or incorporated into household product materials, wherein household products include telephones, mobile phones, Fiberfill, bedding, window treatments, carpet floor cleaning treatments, foam pads on the back of floor mats or carpet pads, upholstery items such as foam pads, non-woven dryer sheets, paper including softener, car wipes, Household cleaning wipes, countertop wipes, shower curtains, shower curtains, towels, face towels, rags, mops, tablecloths, walls, and counter tops, etc.
  • the antimicrobial peptide or peptide derivative of the present application can be made into disinfectant.
  • the environment where the disinfectant is used includes closed spaces, airplanes, etc. , trains, cinemas, theaters and other air disinfection.
  • Antimicrobial peptides or antimicrobial peptide derivatives can reduce or prevent the formation of biofilms on the surface of independent membranes, such as pervaporation membranes, dialysis membranes, reverse osmosis membranes, ultrafiltration membranes, microfiltration membranes, etc.
  • the composition described in the above paragraphs is used in a cleaning agent.
  • This cleaning agent has a bactericidal effect and needs to be washed with water after use. It is best to clean with deionized water.
  • Washed items can be dried selectively, and the drying methods can be selected from ambient air drying, oven drying, and forced air drying.
  • the drying temperature is between 50°C and 120°C, preferably the drying temperature is between 50°C and 100°C, and the drying time is about 15 minutes to 24 hours.
  • the treatment of the antimicrobial peptide or antimicrobial peptide composition on all the above items includes the whole process before industrial production, after industrial production, and during industrial production.
  • the antimicrobial peptide is first combined with the base polymer, and then the shower curtain is treated with the mixture.
  • the production process includes but not limited to at least one of injection molding, extrusion molding, blow molding, thermoforming, solution coating, and blown film.
  • the application of the composition described in the above paragraphs in anti-infective medicines and anti-inflammatory medicines sterilizes and prevents infections, and infections include bacteria, viruses, fungi, for example; including yeast, pathogens, Gram-negative bacteria and/or Caused by gram-positive bacteria, unicellular or multicellular organisms, symbiotic or non-symbiotic, pathogenic organisms, colonized or non-colonized bacteria.
  • antimicrobial peptides or antimicrobial peptide derivatives can treat or reduce bacterial, viral, and fungal infections on the skin, which can be any part related to the human or animal integumentary system, such as epidermis, dermis, subcutaneous tissue, a Or several hair follicles, one or several sebaceous glands, or other parts associated with the skin.
  • the above-mentioned infection is related to the skin.
  • the infection can also be related to a part of vision or a part of hearing, such as a part of the ear, such as the tympanic cavity or a part of the hearing system including the tympanic cavity.
  • anti-infective drugs can be used to treat human or animal infections caused by any one or several bacteria, and antimicrobial peptides can treat excessive growth of one or more commensal organisms, even if the commensal organisms are non-pathogenic or Symbiotic organisms that are beneficial to humans or animals.
  • Some of these commensal organisms may be from the following species: Staphylococcus, Mycobacterium, and Propionibacterium. Additionally other bacterial infections may be from known pathogens such as Pseudomonas spp.
  • anti-infective drugs may be used to treat infections caused by non-bacterial microorganisms, such as to treat infections caused by one or more fungi (eg, yeast).
  • fungi eg, yeast
  • antimicrobial peptides can treat infections caused by Malassezia furfural.
  • Malassezia furfural is a symbiotic organism. Its excessive growth can cause dandruff, seborrheic dermatitis, pityriasis versicolor and folliculitis, etc. Its treatment often takes control of growth, elimination of related inflammation and control of the first Secondary infection etc. Due to the stubborn growth of Malassezia furfural, it is not easy to treat, and the treatment cycle is long, requiring many drugs to work together. The drugs currently on the market are not only toxic and side effects, but also extremely expensive.
  • the antimicrobial peptide of the present application can treat infections caused by fungi such as Malassezia furfur within the effective therapeutic concentration range.
  • anti-inflammatory drugs can be used to treat inflammation including infection, non-infection and even physical trauma.
  • Antimicrobial peptides or antimicrobial peptide derivatives have been shown to reduce, relieve and treat inflammation in experiments.
  • the above combined peptides have been shown to reduce the release of inflammatory factors such as cytokinesis, chemokines and the like.
  • this kind of inflammation is a physiologically defined inflammatory response, including redness, swelling, induction of one or more pro-inflammatory cytokines at the level of cell molecular transcription and translation; one or more cells associated with inflammation Induced responses of signaling pathways; induced responses of receptors present in cell membranes; presence of cellular infiltration in vascularized tissues, and other manifestations associated with inflammation within the skill of the art.
  • non-immortal cells include human constructs cells in a disease state, such as cancer cells, etc.; such non-immortal cells also include primary cultured cells, such as keratinocytes, microvascular endothelial cells, corneal epithelial cells, and dermal fibroblasts.
  • the antimicrobial peptide composition causes reduction of cell membrane receptors-pattern-recognition receptors specifically bound by bacteria.
  • the animal and a part of the animal body show obvious inflammation-relieving effects, such as reduction or disappearance of tissue redness, decrease or disappearance of cell infiltration, and the like.
  • the above-mentioned pro-inflammatory cytokines include cytokines related to inflammation, including but not limited to tumor necrosis factor, interleukin-8, interleukin-1 and interleukin-6.
  • intracellular signaling pathways include signaling pathways related to inflammation, including but not limited to NFkB and AP-1.
  • Membrane-associated receptors include, but are not limited to, pattern recognition receptors, the TLR receptor family, (including TLR-2 and TLR4).
  • the therapeutic concentration and treatment time of the antimicrobial peptide depend on many factors, such as disease state, age, gender, body weight and personal physical condition, and the antimicrobial peptide is used for stem cell therapy, and its effective concentration is greater than 0.01ug/ml.
  • the effective concentration is above 0.2ug/ml.
  • the anti-infective medicine, anti-inflammatory medicine and medicine for inhibiting cell immortality include at least one of liquid, semi-solidified liquid, creamy solid, ointment, and gel.
  • the external dosage form can be used on skin, hair, And other parts for external use, further speaking, under certain conditions, the external use dosage form can be used in one eye or both eyes, and can also be made into eye drops.
  • the antimicrobial peptide or antimicrobial peptide derivative can be made into an intravenous dosage form, or an intratympanic/transtympanic administration dosage form, and the solvent can be a sterile isotonic aqueous solution.
  • the dosage form of antimicrobial peptides used in medicine should be consistent with the route of administration, which includes gastrointestinal administration, intravenous injection, subcutaneous, intradermal, oral, intranasal (inhalation), vaginal, anal, epidermal, and transmucosal administration , transtympanic, intratympanic, rectal, and other acceptable modes of administration.
  • Antimicrobial peptide preparations should match the route of administration that matches the pharmaceutical composition, such as intravenous injection, subcutaneous, intramuscular, transganglionic, oral, nasal, intraaural, intraaural and subcutaneous administration to humans and/or animals , such as livestock, and companion animals, can also be applied to aquatic species, poultry, etc.
  • Amino acid (Chinese name) Amino acid (English name) three letter abbreviation a letter abbreviation Alanine Alanine Ala A arginine Arginine Arg R Asparagine Asparagine Asn N aspartic acid Aspartic Acid Asp D. cysteine Cysteine Cys C Glutamine Glutamine Gln Q glutamic acid Glutamic acid Glu E.
  • Examples 1 to 39 were synthesized by standard internationally known polypeptide solid-phase synthesis (solid-phase synthesis), liquid-phase synthesis (solution-phase synthesis), or biological combination synthesis (recombinant biosynthesis), and the synthesis results are shown in Table 2 amino acid sequence listing.
  • the 39 kinds of antimicrobial peptides in the polypeptide synthesis examples 1 to 39 were carried out the coliform inhibition experiment, took the same amount of coliform liquid 30ul, added various 30ul antimicrobial peptides (concentration 200ug/ml), the PBS isotonic solution of 30ul was used as the control tube, room temperature React for 15 minutes, then smear the agarose plate, spread it evenly, and incubate at 37°C for 12 hours, count and record the number of coliform bacteria on each plate.
  • Figure 1 for the experimental results.
  • the experimental results show that the antimicrobial peptides SEQ ID NO:1 to SEQ ID NO:39 all have inhibitory effects on Escherichia coli, and the effect of SEQ ID NO:8 is the most obvious.
  • A is the control tube, 251 coliform clones; B is SEQ ID NO: 5, 236 coliform clones; D is SEQ ID NO:6, 226 coliform clones; F is SEQ ID NO:7, 216 coliform clones; C is SEQ ID NO:8, 1 coliform clone; E is 95 SEQ ID NO:35 , indicating that multiple polypeptides have the effect of killing Gram-negative bacteria.
  • SEQ ID NO: 8 is 1, indicating that the optimal amino acid sequence of antimicrobial peptides is SEQ ID NO: 8, and the MIC of coliform bacteria is 32ug/ml .
  • Staphylococcus aureus is a Gram-positive bacterium
  • SEQ ID NO: 8 antimicrobial peptides concentration are 1/4: 375ug/ml; 1/16: 93.7ug/ml; 1/64: 23.34ug/ml
  • concentration are 1/4: 375ug/ml; 1/16: 93.7ug/ml; 1/64: 23.34ug/ml
  • the experimental results are, 1/4: the bacteriostatic zone is 3.95cm; 1/16: the bacteriostatic zone is 3.5cm; 1/64: the bacteriostatic zone is 3.1cm. Please refer to Figure 3.
  • Candida albicans and anaerobic bacteria grew on the control plate, a small amount of bacteria grew on the B: 2ul antimicrobial peptide plate, C: no oral bacteria grew on the 10ul antibacterial peptide, and D: 20ul bacteria also did not see the growth of oral bacteria.
  • 293T was transfected with plasmids pNL4.3 ⁇ , pVSV-G, p-enhancer and lipo2000. After two days of culture at 37°C, the culture supernatant was collected and centrifuged to remove organelle impurities. After 500ul of the cell supernatant was mixed with an equal amount of 200ug/ml SEQ ID NO:8 for 15min, the control group replaced SEQ ID NO:8 with an equal amount of PBS. Filter centrifugation (Millipore Amicon Ultra-15) to remove antimicrobial peptides to eliminate the killing effect of antimicrobial peptides on cells.
  • the control group and the experimental group have the same experimental environment, different What is added is the SEQ ID NO:8 sequence amino acid of the application in the experimental group.
  • K562 cells Cultivate K562 cells, react equal amounts of K562 cells with 10ng, 20ng, and 40ng of SEQ ID NO: 8 respectively, and measure apoptotic cells with flow cytometry PI kit. It is found that cell death is directly proportional to the content of antimicrobial peptides, indicating that Antimicrobial peptides can effectively kill K562 cells.
  • Jurkat cell line leukemia lymphocytes, SEQ ID NO: 1ug/m20ul of peptide No. 10 reacted with 10 5 cells for 10 minutes, centrifuged at 1000rpm for 3 minutes, please refer to the picture 6, the experimental results show that leukemia lymphocytes aggregate into groups , accompanied by cell fragmentation.
  • K562 cell line leukemia cells, SEQ ID NO: 17 antimicrobial peptide 1ug/ml 20ul reacted with 10 5 cells for 10min, centrifuged at 1000rpm for 3min, please refer to Figure 7, the results show that the leukemia cells aggregated into agglomerates.
  • HypG2 cell line liver cancer cells, SEQ ID NO: 15 polypeptide 1ug/ml 20ul reacted with 10 5 cells for 10 minutes, centrifuged at 1000rpm for 3 minutes, please refer to Figure 8, the results show that the liver cancer cells aggregated into groups, and were broken and deformed.

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Abstract

本申请提供了一种抗菌肽或肽衍生物、替换体以及其组合物、制备方法和应用，其包括如下氨基酸序列I和II中的至少一种：氨基酸序列I：Xa1Ba1U1Z1Ba2Xa2Ba3Za2Ba4Xa3；氨基酸序列II如图；其中Xa1、Ba1、U1、Za1、Ba2、Xa2、Ba3、Za2、Ba4、Xa3、Xb1、Bb1、Ca1、Zb1、Bb2、Xb2、Bb3、Zb2、Bb4、Xb3、Ca2各自独立地选自天然氨基酸或/和非天然氨基酸。其中，本申请提供的抗菌肽或肽衍生物可以与细胞壁/膜的脂质结构结合，损坏其理化学性质，破坏微生物壁，进而杀死微生物和肿瘤细胞，另外，也具有外伤消毒、抗感染的功效。

Description

抗菌肽或肽衍生物、替换体以及其组合物、制备方法和应用 技术领域
本申请属于肽或肽衍生物技术领域，尤其涉及一种抗菌肽或肽衍生物、替换体以及其组合物、制备方法和应用。
背景技术
在自然界中，抗菌肽广泛存在于细菌，病毒，真菌，昆虫，两栖类，动物及植物等生物体内，抗菌肽对杀伤细菌、真菌、寄生虫和病毒具有非常显著的作用。随着抗生素大量的使用，造成了微生物具有了耐药性，促进并带动了研发人员对抗菌肽的大规模研发。自从人体内源性抗菌肽如defensins(防御素)被发现，并被FDA(食品药品监督管理局)批准作为抗感染，抗炎症及其他受伤治疗的药物后，人们进一步发现抗菌肽具有激发人体先天获得性及适应性免疫功效以后，抗菌肽越来越显示出针对一些疾病的潜在治疗优势。除了在医用药物上，抗菌肽在食品防腐，禽畜牧业，水产渔业，及日常医用保健，卫生美容，清洁消毒，农业病虫害防治等很多方面也有广泛应用。
例如：痤疮丙酸杆菌是由革兰氏阳性菌，一种正常人类皮肤上的微生物菌群在毛发皮脂腺毛囊中过度生长造成的。痤疮丙酸杆菌是影响人类皮肤的最常见的疾病之一，有大约上百万中国人受此病影响。由丙酸杆菌引起的痤疮有不同的病情表现，如粉刺、丘疹、结核、囊肿、和毛囊皮脂腺炎症等。传统的治疗由丙酸杆菌引起的痤疮多为抗菌素，如口服土霉素，外用红霉素，克林霉素等，但是这种治疗很快就会引起抗生素耐药。另外由丙酸杆菌引起的痤疮治疗临床上还采用过氧化苯甲酰(Benzoyl Peroxide)，5％氨苯砜凝胶，此两种药物低浓度下药效很小，高浓度时有很大的毒副作用。
又如：假单袍体绿脓杆菌是由革兰氏阴性菌对人体及动物体侵入感染造成的，它可以感染到生物体的呼吸系统、泌尿系统、胃肠系统、中枢神经系统，以及血液、心脏和骨骼系统。当假单袍体绿脓杆菌感染眼睛和耳朵时，常常造成成人的细菌角膜炎，巩膜脓肿，眼内炎，新生儿眼炎及小儿中耳炎。特别是绿脓杆菌/细菌角膜炎增加了使用瞳孔眼镜的感染率，美国每年有约两万五千名瞳孔眼镜佩戴者遭遇过此细菌造成的传染。另外，据美国传染病防治中心统计，每1000个入院病人有4个遭遇过假单袍体绿脓杆菌感染。虽然假单袍体绿脓杆菌是可以治疗的，但是随着抗生素的使用，此微生物的抗药性增强，很快就降低了治疗的药效，不得不改变用药组合及治疗方案。有数据显示，在美国从重症监护室单离出的假单袍体绿脓杆菌对环丙沙星的抗药性达51.6％，对哌拉西林/他挫巴坦的耐药性达31.4％，对亚胺培南达38％，对头孢他啶达23.6％。而在欧洲重症监护室单离出的假单袍体绿脓杆菌对氨基糖苷类的耐药性可达37-70％，对头孢他啶耐药达57％，对哌拉西林/他挫巴坦的耐药性达53％，对环丙沙星的抗药性达56％，对亚胺培南耐药可达52％，因此治疗及控制由假单袍体绿脓杆菌造成严重的耐药感染变得至关重要。
目前报道的典型抗菌肽一般由10-100个氨基酸残基组成，含有很多碱性氨基酸并且一般都是两亲性的。
有研究已报道从蛙科类分泌物中提取出来的宿主防御肽PGLa对幽门螺杆菌(革兰氏阴性菌)具有很好的杀伤力。也有从鱼体内分离出来的天然抗菌肽TP4，还有人体中分离出来的天然抗菌肽LL-37都对幽门螺杆菌具有很好的杀伤力。这些发现表明抗菌肽对幽门螺杆菌的治疗效果尤佳，还可作为抑菌添加剂添加到日常生活用品里，从而帮助我们更好的远离幽门螺杆菌。但是，现有抗菌肽存在抗菌活性不强，并且很多不具有广谱抗菌活性的缺陷。
另外，从动植物分离得到的抗菌肽种类多样，分布广泛，在昆虫、鱼类、哺乳动物、两栖动物和植物等生物中均有发现。对于不同来源的抗菌肽的应用性尚未有系统的研究。特别是植物抗菌肽，尤其不稳定，易被蛋白水解酶水解，从而大大降低其抗菌活性。
发明内容
本申请的目的在于提供一种抗菌肽或肽衍生物、合成方法、组合物以及应用，旨在解决现有技术中抗菌肽抗菌活性不强、不稳定、不具有广谱抗菌活性的问题。
为实现上述申请目的，本申请采用的技术方案如下：
第一方面，本申请提供一种抗菌肽或肽衍生物，其包括如下氨基酸序列中的至少一种：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000001
其中，X a1、B a1、U 1、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各自独立地选自氨基酸。
本申请提供的抗菌肽或肽衍生物可以与细胞壁/膜的脂质结构特异性结合，破坏微生物壁，进而杀死微生物和癌细胞。
第二方面，提供一种抗菌肽或肽衍生物的替换体，由本申请抗菌肽或肽衍生物所含氨基酸序列I或/和氨基酸序列II的至少一个氨基酸被替代。
本申请提供的替换体可以确保抗菌肽或肽衍生物的活性，也包含在本申请的内容中，通过对抗菌肽或肽衍生物上氨基酸的替换，可以改善抗菌肽或肽衍生物的抗菌活性，增加其广谱抗菌活性。
第三方面，本申请提供了一种抗菌肽或肽衍生物组合物，至少包括一种上述段落任一所述抗菌肽或肽衍生物和至少一种药剂上可以接受的载体。
本申请第三方面提供的一种抗菌组合物，本申请提供的抗菌肽或肽衍生物可以和大多数溶剂混合，制成组合物。
第四方面，本申请提供了一种抗菌组合物的制备方法，包括如下步骤：将抗菌肽或肽衍生物和溶剂混合处理。
本申请抗菌组合物的制备方法，一方面，抗菌肽或肽衍生物可以稳定存在于大部分溶剂中，将溶剂和抗菌肽或肽衍生物混合可以保存抗菌肽或肽衍生物的抗菌活性，另一方面，确保抗菌肽或肽衍生物对抗肽酶或蛋白质降解的稳定性。
第五方面，本申请提供了本申请抗菌肽或肽衍生物组合物在包装、食品加工最终端、服饰、医疗用品、医疗仪器、个人卫生用品、消毒剂、清洁剂、抗感染药品、消炎药品、抑制细胞无限增殖药品中的 应用。
本申请抗菌肽或肽衍生物组合物的应用，正是由于抗菌肽或肽衍生物可杀死微生物、杀死肿瘤细胞、抗凝血和消炎的功能，因此，其用途广泛，主要有四点优点：
一、抗菌肽或肽生物可与包装、服饰、医疗用品、医疗仪器、个人卫生用品材料结合或掺入其中，使其具有抗菌功能，或作为易被微生物降解的其他材料的保存剂；
二、抗菌肽或肽生物可以被制成消毒剂、清洁剂、抗感染药品、消炎药品，起到杀菌、抗感染、消炎的作用；
三、抗菌肽或肽生物组合物被制成涂层，涂抹在食品处理加工以及最终端中，可以减少食品处理加工最终端表面的微生物，降低人们食品中毒的风险；
四、可以杀死细胞，限制增殖，用于抗肿瘤治疗，局部美容去疣去斑等使用。
附图说明
图1本申请实施例中抗菌肽或肽衍生物对大肠杆菌抑制实验结果统计直方图；
图2本申请实施例中抗菌肽或肽衍生物对大肠杆菌抑制结果图；
图3本申请实施例中抗菌肽或肽衍生物对野生金黄色葡萄球菌抑制实验结果图；
图4本申请实施例中抗菌肽或肽衍生物对假单袍体绿脓杆菌的MIC抑制实验结果图；
图5本申请实施例中抗菌肽或肽衍生物杀死RNA病毒实验结果图；
图6本申请实施例中抗菌肽或肽衍生物抗菌肽杀死白血病淋巴细胞(Jurkat cell line)实验结果图；
图7本申请实施例中抗菌肽或肽衍生物杀死白血病细胞(K562cell line)实验结果图；
图8本申请实施例中抗菌肽或肽衍生物杀死肝癌细胞实验结果图；
图9本申请实施例中抗菌肽或肽衍生物对血红细胞的效果实验结果图。
具体实施方式
为了使本申请要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请，并不用于限定本申请。
本申请中，术语“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B的情况。其中A，B可以是单数或者复数。字符“/”一般表示前后关联对象是一种“或”的关系。
本申请中，“至少一个”是指一个或者多个，“多个”是指两个或两个以上。“以下至少一项(个)”或其类似表达，是指的这些项中的任意组合，包括单项(个)或复数项(个)的任意组合。例如，“a,b，或c中得至少一项(个)”，或，“a,b，和c中的至少一项(个)”，均可以表示：a,b,c,a-b(即a和b),a-c,b-c,或a-b-c，其中a,b,c分别可以是单个，也可以是多个。
应理解，在本申请的各种实施例中，上述各过程的序号的大小并不意味着执行顺序的先后，部分或全部步骤可以并行执行或先后执行，各过程的执行顺序应以其功能和内在逻辑确定，而不应对本申请实施条例的实施过程构成任何限定。
在本申请实施例中使用的术语是仅仅出于描述特定实施条例的目的，而非旨在限制本申请。在本申请实施条例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。
本申请实施例说明书中所提到的相关成分的重量不仅仅可以指代各组分的具体含量，也可以表示各组分间重量的比例关系，因此，只要是按照本申请实施例说明书相关组分的含量按比例放大或缩小均在本申请实施例说明书公开的范围之内。具体地，本申请实施例说明书中所述的质量可以是μg、mg、g、kg等化工领域公知的质量单位。
术语第一、“第二”仅用于描述目的，用来将目的如物质彼此区分开，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。例如，在不脱离本申请实施条例范围的情况下，第一XX也可以被称为第二XX，类似地，第二XX也可以被称为第一XX。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。
第一方面，本申请实施例提供一种抗菌肽或肽衍生物，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000002
其中，X a1、B a1、U 1、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各自独立地选自氨基酸，其中，氨基酸序列II中Ca 1和Ca 2之间形成环键，氨基酸包括天然氨基酸或/和非天然氨基酸，其中，本申请提供的抗菌肽或肽衍生物可以与细胞壁/膜的脂质结构结合，破坏微生物壁或细胞膜，进而杀死微生物和肿瘤细胞。
在实施例中，在上述实施例基础上，为提高抗菌肽或肽衍生物的抗菌活性，X a1、X a2、X a3、X b1、X b2、X b3各自独立地选自带有阳性电荷的残基；或/和B a1、B a2、B a3、B a4、B b1、B b2、B b3、B b4各自独立地选自带有疏水侧链的残基；或/和U 1选自Gly(G－甘氨酸)、Pro(P－脯氨酸)、Cys(C－半胱氨酸)、Cys(R)中的一种，其中，R代表Cys双硫键的保护基团；或/和Z a1、Z a2、Z b1、Z b2各自独立地选自非极性的残基；或/和C a1、C a2各自独自选自Cys(C－半胱氨酸)、Cys(R)中的一种，在氨基酸序列I中，X a1或X a3代表N端，X a1或X a3代表C端，在氨基酸序列II中，X b1和/或B 2代表N端，C a2代表C端。正是由于本申请的抗菌肽和肽衍生物上含有疏水侧链的残基、含有阳性电荷的残基、含有非极性的残基，因此，本实施例提供的抗菌肽或肽衍生物链可以形成典型的两性螺旋结构，该抗菌肽或肽衍生物的荷电性及两性螺旋结构使其能够与细胞壁/膜的脂质结构结合，破坏微生物壁或细胞膜，进而杀死微生物和肿瘤细胞。
实施例中，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000003
其中，B a1、U 1、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各自独立地选自氨基酸，X a1、X a2、X a3、X b1、X b2、X b3各自独立地选自带有阳性电荷的残基中的一个，带有阳极电荷的肽链易于与带负电的细胞膜结合。
实施例中，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000004
其中，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各自独立地选自氨基酸，X a1、X a2、X a3、X b1、X b2、X b3各自独立地选自带有阳性电荷的氨基酸残基中的至少一种，B a1、B a2、B a3、B a4、B b1、B b2、B b3、B b3各自独立地选自带有疏水侧链的氨基酸残基中的至少一种，U 1选自Gly、Pro、Cys、Cys(R)中的至少一种，带有阳极电荷的肽链易于与带负电的细胞膜结合，疏水侧链的残基，以确保平衡抗菌肽或肽衍生物的水溶性和电荷性，进而确保抗菌肽或肽衍生物抗菌活性。
实施例中，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000005
其中，C a1、C a2各自独立地选自氨基酸，X a1、X a2、X a3、X b1、X b2、X b3各自独立地选自带有阳性电荷的残基中的一个；B a1、B a2、B a3、B a4、B b1、B b2、B b3各自独立地选自带有疏水侧链的氨基酸残基中的至少一种；U 1选自Gly、Pro、Cys、Cys(R)中的一种，其中，R代表Cys双硫键的保护基团；Z a1、Z a2、Z b1、Z b2各自独立地选自非极性的氨基酸残基中的至少一种，带有阳极电荷的肽链易于与带负电的细胞膜结合，非极性的残基和疏水侧链的残基以确保平衡抗菌肽或肽衍生物的水溶性和电荷性，进而确保抗菌肽或肽衍生物抗菌活性。
实施例中，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000006
其中，X a1、X a2、X a3、X b1、X b2、X b3各自独立地选自带有阳性电荷的残基；B a1、B a2、B a3、B a4、B b1、B b2、B b3、B b4各自独立地选自带有疏水侧链的氨基酸残基中的至少一种；U 1选自Gly(G－甘氨酸)、Pro(P－脯氨酸)、Cys(C－半胱氨酸)、Cys(R)中的至少一种，其中，R代表Cys双硫键的保护基团；Z a1、Z a2、Z b1、Z b2各自独立地选自非极性的氨基酸残基中的至少一种；C a1、C a2各自独自选自Cys(C－半胱氨酸)、Cys(R)中的至少一个，在氨基酸序列I中，X 1和/或B 1代表N端，B 1和/或X 1代表C端，在氨基酸序列II中，X 1和/或B 2代表N端，Cys代表C端。正是由于本申请的抗菌肽和肽衍生物上含有疏水侧链的残基、含有阳性电荷残基、含有非极性的残基，因此，本实施例提供的抗菌肽或肽衍生物链可以形成典型的两性螺旋结构，该抗菌肽或肽衍生物的荷电性及两性螺旋结构使其能够与细胞壁/膜的脂质结构结合，破坏微生物壁或细胞膜，进而杀死微生物和肿瘤细胞。
在实施例中，在上述实施例基础上，为改善抗菌肽或肽衍生物的稳定性，X a1、X a2、X a3、X b1、X b2、X b3、B b4各自独立地选自Arg(R－精氨酸)、His(H－组氨酸)、Lys(K－赖氨酸)、Orn(鸟氨酸)、Har(高精氨酸)、Dab(2,4二氨基丙酸)、非天然氨基酸中的一种、两种或三种，一方面，Arg(R－精氨酸)、His(H－组氨酸)、Lys(K－赖氨酸)、Orn(鸟氨酸)、Har(高精氨酸)、Dab(2,4二氨基丙酸)、非天然氨基酸为阳性电荷的残基，从整体结构上面来看，可以和其他残基配合可以形成两性螺旋结构，增加抗菌肽或肽衍生物的抗菌活性，另一方面，抗菌肽链或肽衍生物的中的阳性电荷的残基过长，抗菌肽或肽衍生物的电荷将会失衡，容易变得不稳定，因此，为了确保肽或肽衍生物对抗肽酶或蛋白质 降解的稳定性，阳性电荷的残基的数目为一种、两种或三种，或/和B a1、B a2、B a3、B a4、B b1、B b2、B b3各自独立地选自Ala(A－丙氨酸)、Val(V－缬氨酸)、Ile(I－异亮氨酸)、Leu(L－亮氨酸)、Met(M－甲硫氨酸)、非天然氨基酸中的两种、三种、四种，Ala(A－丙氨酸)、Val(V－缬氨酸)、Ile(I－异亮氨酸)、Leu(L－亮氨酸)、Met(M－甲硫氨酸)、非天然氨基酸为疏水侧链，疏水侧链可以平衡肽链的水溶性和电荷性，也就是说，如果肽链或肽衍生物的中的疏水侧链残基过长肽链容易变得不稳定，因此，为了确保肽链对抗肽酶或蛋白质降解的稳定性，疏水侧链残基的个数保证在两种、三种、四种中的一种情况，或/和Z a1、Z a2、Z b1、Z b2各自独立地选自Asn(N－天冬氨酸)、Gln(Q－谷氨酰胺)、Ser(S－丝氨酸)、Thr(T－苏氨酸)非天然氨基酸中的一种，选自非极性的残基，可以平衡肽链的水溶性和电荷性，也就是说，如果肽链或肽衍生物的中的非极性的残基过长肽链容易变得不稳定，因此，为了确保抗菌肽或肽衍生物对抗肽酶或蛋白质降解的稳定性，非极性的残基的个数保证在两种、三种、四种中的一种情况，或/和X a1和B a1、X a2和B a2、X a3和B a3、X b1和B b1、X b2和B b2、X b3和B b3、X b4和B b4残基数目之和为3、4、5、6或7，优选为3或4，针对本申请的抗菌肽或肽衍生物得活性而言，肽链长度越长越好，但考虑到合成的成本和造价，以及为确保肽链的稳定性，X a1和B a1、X a2和B a2、X a3和B a3、X b1和B b1、X b2和B b2、X b3和B b3、X b4和B b4残基数目之和优选个数为3和4。本申请的抗菌肽的氨基酸序列和/或部分氨基酸序列来源于病毒的8至25个氨基酸。
实施例中，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000007
其中，Z a1、Z a2、Z b1、Z b2各自独立地选自氨基酸，X a1、X a2、X a3、X b1、X b2、X b2、X b3各自独立地选自带有阳性电荷的Arg、His、Lys、Orn、Har、Dab、非天然氨基酸中的一种、两种或三种；B a1、B a2、B a3、B a4、B b1、B b2、B b3、B b4各自独立地选自带有疏水侧链的残基中的一个；和/或U 1选自Gly、Pro、Cys、Cys(R)中的一种，其中，R代表Cys双硫键的保护基团；C a1、C a2各自独自选自Cys、Cys(R)中的一个，阳性电荷的残基，可以和其他残基配合可以形成两性螺旋结构，增加抗菌肽或肽衍生物的抗菌活性，另外，抗菌肽链或肽衍生物的中的阳性电荷的残基过长，抗菌肽或肽衍生物的电荷将会失衡，容易变得不稳定，因此，为了确保肽或肽衍生物对抗肽酶或蛋白质降解的稳定性，阳性电荷的残基的数目为一种、两种或三种。
实施例中，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000008
其中，X a1、X a2、X a3、X b1、X b2、X b2、X b3各自独立地选自带有阳性电荷的Arg、His、Lys、Orn、Har、Dab 、非天然氨基酸中的一种、两种或三种；B a1、B a2、B a3、B a4、B b1、B b2、B b3、B b4各自独立地选自带有疏水侧链的Ala、Val、Ile、Leu、Met、非天然氨基酸中的两种、三种或四种；或/和U 1选自Gly、Pro、Cys、Cys(R)中的一种，其中，R代表Cys双硫键的保护基团；Z a1、Z a2、Z b1、Z b2各自独立地选自非极性残基中的至少一种；或/和C a1、C a2各自独自选自Cys、Cys(R)中的一种，阳性电荷的残基，可以和其他残基配合可以形成两性螺旋结构，增加抗菌肽或肽衍生物的抗菌活性，另外，抗菌肽链或肽衍生物 的中的阳性电荷的残基过长，抗菌肽或肽衍生物的电荷将会失衡，容易变得不稳定，因此，为了确保肽或肽衍生物对抗肽酶或蛋白质降解的稳定性，阳性电荷的残基的数目为一种、两种或三种，疏水侧链可以平衡肽链的水溶性和电荷性，也就是说，如果肽链或肽衍生物的中的疏水侧链残基过长肽链容易变得不稳定，因此，为了确保肽链对抗肽酶或蛋白质降解的稳定性，疏水侧链残基的个数保证在两种、三种、四种中的一种情况。
实施例中，其氨基酸序列通用表达如下：
氨基酸序列I：X a1B a1U 1Z 1B a2X a2B a3Z a2B a4X a3；
氨基酸序列II：
Figure PCTCN2021116530-appb-000009
其中，X a1、X a2、X a3、X b1、X b2、X b2、X b3各自独立地选自带有阳性电荷的Arg、His、Lys、Orn、Har、Dab 、非天然氨基酸中的一种、两种或三种；B a1、B a2、B a3、B a4、B b1、B b2、B b32、B b4各自独立地选自带有疏水侧链的Ala、Val、Ile、Leu、Met、非天然氨基酸中的两种、三种或四种；Z a1、Z a2、Z b1、Z b2各自独立地选自非极性的Asn、Gln、Ser、Thr、非天然氨基酸中的一种；U 1选自Gly、Pro、Cys、Cys(R)中的一种，其中，R代表Cys双硫键的保护基团；C a1、C a2各自独自选自Cys、Cys(R)中的一个，阳性电荷的残基，可以和其他残基配合可以形成两性螺旋结构，增加抗菌肽或肽衍生物的抗菌活性，另外，抗菌肽链或肽衍生物的中的阳性电荷的残基过长，抗菌肽或肽衍生物的电荷将会失衡，容易变得不稳定，因此，为了确保肽或肽衍生物对抗肽酶或蛋白质降解的稳定性，阳性电荷的残基的数目为一种、两种或三种，疏水侧链可以平衡肽链的水溶性和电荷性，也就是说，如果肽链或肽衍生物中的疏水侧链残基过长肽链容易变得不稳定，因此，为了确保肽链对抗肽酶或蛋白质降解的稳定性，疏水侧链残基的个数保证在两种、三种、四种中的一种情况，可以平衡肽链的水溶性和电荷性，也就是说，如果肽链或肽衍生物的中的非极性的残基过长肽链容易变得不稳定，因此，为了确保抗菌肽或肽衍生物对抗肽酶或蛋白质降解的稳定性，非极性的残基的个数保证在两种、三种、四种中的一种情况。
在一些实施例中，Cys双硫键包括乙酰氨基甲基(Acetamidomethyl(Acm))、甲硫代磺酸甲酯(methyl methane thiosulfonate)或其他Cys保护基团，Cys(R)表示含有Cys双硫键的保护基团，其中，C a2为抗菌肽或肽衍生物的端基，C a1位于抗菌肽或肽衍生物的中间位置，因此在抗菌肽或肽衍生物分子内部可以形成环链结构，这样可以增强抗菌肽的稳定性，延长抗菌肽杀菌时间，使抗菌肽或肽衍生物具有更强的抗菌活性。
在一些实施例中，氨基酸序列I和/或II上至少包括一个氨基酸被修饰处理过，在实际应用中，抗菌肽或肽衍生物的稳定性和抗菌活性是非常重要的，通过修饰本申请的抗菌肽或肽衍生物上氨基酸可以改变肽链的性能，提高抗菌肽或肽衍生物的光谱性，改善抗菌肽或肽衍生物对抗肽酶或蛋白质降解的稳定性，增加其广谱抗菌活性。
实施例中，修饰包括磷酸化、卤化、乙酰化、环化、端点的加帽反应，其中，环化包含由双硫键形成的环化、首尾环化、内部结构中侧基的环化中的至少一种，氨基酸端点的加帽(capping)反应包括C端酰胺化、N端乙酰化和N端磷脂质化中的至少一种，对氨基酸进行修饰处理例如环化，可以增加肽链的稳定性，又如对氨基酸进行磷酸化处理，可以增加抗菌肽或肽衍生物的抗菌活性。
在一些实施例中，氨基酸序列I和/或II上至少包括一个模体(motif)，常常与既定的氨基酸端点序 列(N端和/或C端)相连，也可以直接插入既定的氨基酸序列中间，一般不独立作为氨基酸序列端点，模体与肽链的连接或插入氨基酸之间，在抗菌肽或肽衍生物中插入模体可以改变肽链的性能，增加肽链的抗菌活性，抗菌肽或肽衍生物对抗肽酶或蛋白质降解的稳定性。
在一些实施例中，抗菌肽或肽衍生物为包括氨基酸序列I和/或氨基酸序列II形成的多体，其中，双体(Dimer)、四体(Tetramer)中的至少一种，是比较常见的一种多体状态。抗菌肽单体和/或多体与至少一种药物、抗体共轭结合形成抗菌药品，主要是包含小分子化学合成药物、大分子的抗体、生物组换技术合成的大分子药物，本申请的抗菌肽或肽衍生物形成的多体和药物、抗体分子共轭结合形成抗菌药品，可以用于对药品或抗体的改性。
在一些实施例中，抗菌肽或肽衍生物包括SEQ ID NO:1至SEQ ID NO:39所示氨基酸序列。
在一些实施例中，一种抗菌肽或肽衍生物的纳米结构，一条肽链及其肽衍生物或复数或多聚的肽链和肽衍生物都可以形成纳米结构，其中，常见的纳米结构选自胶束、囊泡、纳米管、纳米带中的至少一种。肽链及其肽衍生物形成纳米结构，有利于肽链及其肽衍生物在溶液中稳定存在，并在溶液中传送。
第二方面，本申请提供了一种抗菌肽或肽衍生物的替换体，替换上述段落任一氨基酸序列I或II中的氨基酸得到替换体，也包含在本申请的内容中，如将L型氨基酸替换成对应的D型氨基酸或非自然氨基酸，通过对抗菌肽或肽衍生物上氨基酸的替换，可以改善抗菌肽或肽衍生物的抗菌活性，增加其广谱抗菌活性。
第三方面，本申请提供了一种抗菌肽或肽衍生物组合物，抗菌肽或肽衍生物或/和替换体以及至少一种药剂上可以接受的载体和/或溶剂，本申请提供的抗菌肽或肽衍生物可以和大多数药剂上可以接受的载体结合形成药组合物，同样也可以和大部分溶剂混合，制成组合物。
在一些实施例中，还包括赋形剂、等渗剂、吸收延迟剂或基底多聚物中的至少一种，和这些物质混合，可以将抗菌肽或肽衍生物制成各种抗菌药品或者抗菌溶剂。
在一些实施例中，赋形剂选自水、盐、磷酸盐、葡萄糖、甘油、乙醇中的至少一种，这些都是常用的医学试剂，抗菌肽或衍生物可以和这些试剂混合，有利于本申请抗菌肽和衍生物的推广使用。
在一些实施例中，等渗剂选自糖、多醇及氯化钠，等渗剂可保持肽活性，抗菌肽和衍生物和等渗剂混合，使抗菌肽或肽衍生物更容易与生物体结合。
在一些实施例中，多醇选自甘露醇，山梨糖醇，多醇和抗菌肽或肽衍生物混合，多醇可保持抗菌肽或衍生物活性，使抗菌肽或衍生物易于与生物体结合。
其中，药学上可以接受的载体选自最小量的辅助物质，药学上可以接受的载体选自润湿剂、乳化剂、防腐剂、缓冲液，药学上可以接受的载体和抗菌肽或肽衍生物形成组合物，可保持抗菌肽或衍生物活性，使抗菌肽或衍生物易于与生物体结合。
实施例中，抗菌肽或肽衍生物与基底多聚物结合制成水相或非水相溶液，这些水相或非水相溶液可以涂抹在物体表面形成抗菌保护层。
另外，上述物质可以以多种形式或由多种成分而构成。包括但不限制于抽提物、胶片、膜、层压板、针织面料、编织面料、非编织面料、纤维、细丝、纱线、颗粒、涂层、和/或泡沫。本申请也包含由上述物质与抗菌肽制成的各种模型，包括但不限制于注塑成型、挤压成型、吹塑成型、热成型、溶液涂层、 吹膜、针织、编织及纺织制品等。
第四方面，本申请提供了一种抗菌肽或肽衍生物组合物的制备方法，包括如下步骤：将抗菌肽或肽衍生物和所述溶剂混合处理。
实施例中，抗菌肽或肽衍生物与基底多聚物在制成混合物时，为了确保充分混合，混合温度控制在-10至150℃范围之间，混合时间控制在0.1至5760min范围之间。其中，混合温度在25至80℃范围之间，混合时间在1至1440min范围之间，具有更好的混合效果。
在一些实施例中，还包括对药剂上可以接受的载体、赋形剂、溶剂、分散介质、等渗剂、吸收延迟剂或基底多聚物进行预处理，其中预处理包括但不限于氧化、还原、水解、等离子体或辐射中的至少一种，预处理可以改善抗菌肽或肽衍生物的所述环境，进而提高抗菌肽或肽衍生物对抗肽酶或蛋白质降解的稳定性。
在一些实施例中，抗菌肽或肽衍生物与上述物质混合的有效浓度范围介于1ug至20gwt％之间，或介于5pmol至2mol浓度之间。为保存抗菌肽或肽衍生物组合物，需控制温度在-20至25℃范围之间，以确保抗菌肽或肽衍生物的抗菌活性。另外，控制温度在-4至4℃范围之间，较低的温度下才能确保抗菌肽的生物学活性特性。
第五方面，本申请提供了下述任一抗菌肽或肽衍生物组合物的应用：
在包装、食品加工最终端、服饰、医疗用品、医疗仪器、个人卫生用品、消毒剂、清洁剂、抗感染药品、消炎药品、抑制细胞无限增殖药品中。抗菌肽或肽衍生物组合物作为药品或非药品可以广泛使用在人类，动物(包括野生动物、牲畜、伴侣动物)、植物(包括农作物)、水生及水产养殖动物，禽类养殖动物等。在下文中称之为人类及动植物的概念定义，即为上述提到的包含人类、动物(包括野生动物、牲畜、伴侣动物)、植物(包括农作物)，水生及水产养殖动物、禽类养殖动物等的概念定义，以下等同。
在一些实施例中，上述段落所述组合物在包装中的应用，抗菌肽或肽生物可与包装材料结合或掺入其中，或作为易被微生物降解的其他材料的保存剂，其中，包装成分包括但不限制于肉制品的包装膜、衬垫、吸水垫、托盘、容器组合原件、帽、盖子、黏合剂、涂抹器等。此种包装可以是适合于特殊应用的任何包装形式，如铝制罐、盒子、瓶子、玻璃罐、袋子、化妆品包装、封闭式管子等。上述包装包括但不限制于由注塑成型、挤压成型、吹塑成型、热成型、溶液涂层、吹膜等工艺制成的包装模型。
其中，包装品还适用于处方/非处方药及保健卫生用品的包装，如对胶囊、片剂、溶液、乳化剂、清洁剂、粉末、洗发剂、护发剂、除臭剂、止汗剂等制成的瓶子、尖端物、涂抹器、盖子等的包装；包装品还适用于涂抹器的包装，例如口红、唇膏、上光油等，眼部化妆品包装、如睫毛膏、眼线、眼影、喷粉、洗澡粉、腮红、粉底物及乳液的包装。此涂抹器用于生体的不同部位及表面，该种应用极大地减少或消灭了细菌在人体表面的生长，另外，还包含其他形式的包装组成，如承装液体、溶液或悬浊液的喝水瓶颈、可替换盖子、不可替换盖子、食物或药品的分装系统；食物及饮料的输送系统；婴儿奶瓶、盖子、奶嘴等。包装也包含做成胶滴离散剂或者液滴喷射剂。
在一些实施例中，上述段落所述组合物在食品处理加工以及最终端中的应用，或者用作临时或永久准备食品表面的涂层，如替代抗生素的食品添加物；食品加工仪器、食品传送带组装及部件，用来混合、 研磨、压碎、滚动、造粒、挤压食物的不同仪器的组装及部件；用来切断、切片食物的仪器组装及部件。如果上述仪器的表面为金属物质，该金属表面须涂抹一层具有功能性的多聚物，该多聚物即为本申请在上述阐述的含有抗菌肽或肽衍生物的多聚物。抗菌肽或肽生物组合物被制成涂层，涂抹在食品处理加工以及最终端中，可以减少食品处理加工最终端表面的微生物，降低人们食品中毒的风险。
在一些实施例中，上述段落所述组合物在服饰中的应用，这种服饰具有杀菌抑菌的功能，如游泳衣、内衣、鞋组成部分(如编织或非编织的内衬或鞋垫)运动防护垫、儿童服装等。本申请也包含防护性医疗服装或隔离用品，如防护服、口罩、手套、拖鞋、靴子、头盖物或垂帘等。
在一些实施例中，上述段落所述组合物在医疗用品、仪器中的应用，抗菌肽或肽生物可与医疗用品、医疗材料结合或掺入其中，或在医疗仪器表面涂一层抗菌肽或肽衍生物涂层，或制成喷雾对医疗用品、仪器进行杀毒和消毒，其中，医疗用品、仪器包括人体及动物植入物上、如绷带、粘结剂、纱布条、纱布垫、注射器支架、周围静脉或中央静脉插管，该插管可以是由聚氨酯或硅胶组成的；尿插管造口术端口、骨科矫形器、矫形针、起搏器导线、除颤器导线、耳道分流器、血管支架、整形植入物、耳鼻喉植入物、可植入泵、疝气贴片，以及与此相关的盘子、螺丝、血液袋、外用血泵、输液系统、心肺仪器、透析仪器、人工皮肤、人工心脏、心室辅助装置、助听器、血管移植物、起搏器组成元件、髋关节植入物、膝盖植入物、齿科植入物等。
在一些实施例中，上述段落所述组合物在个人卫生用品中的应用，抗菌肽或肽生物可与个人卫生用品材料结合或掺入，进而个人卫生用品具有杀菌抑菌的作用，如尿布、不便垫、卫生巾、运动垫、卫生棉条以及应用它们的附属器械；在卫生保健物方面，抗微生物湿巾、婴儿湿巾、个人擦拭湿巾、化妆湿巾、尿布、医疗用湿巾(如含有抗生素的湿巾或棉垫、治疗痤疮药物、治疗痔疮药物、止痒药物、抗炎症药物及防腐剂等)。
在一些实施例中，上述段落所述组合物在与口腔直接接触的物品中的应用，抗菌肽或肽生物对人体无毒，并可以杀毒，因此可以直接和口腔接触，达到口腔杀菌抑菌的目的，其中，口腔直接接触的物品，防止婴幼儿皮肤感染等，如婴儿奶瓶、奶嘴、齿科器械、松紧带、假牙、杯子、水杯、牙膏及出牙玩具等。以小儿物品防止微生物感染为主要目的的应用也在此保护中，如婴儿瓶、小儿书籍、塑料剪子、玩具、尿布桶、以及装干净湿巾的容器。
在一些实施例中，上述段落所述组合物在家庭用品中的使用，为防止菌从口入，抗菌肽或肽生物可与家庭用品材料结合或掺入，其中，家庭用品包括电话、手机、纤维充填物、床上用品、窗户处理、地毯地面清洁处理、脚垫或地毯垫背面的泡沫垫、室内装潢物品(如泡沫垫)、非编织干燥纸、包括柔软剂的纸张、汽车擦拭湿巾、家庭用清洁湿巾、台面湿巾、浴帘、浴帘布、毛巾、面巾、抹布、拖布、桌布、墙、以及柜台面等。
在一些实施例中，上述段落所述组合物在消毒剂中的应用，为避免交叉感染，可本申请的抗菌肽或肽衍生物制成消毒剂，消毒剂所使用的环境包括封闭空间、飞机、火车、影院、剧场等空气消毒。例如：医用内窥镜等消毒。抗菌肽或抗菌肽衍生物可以减少或防止在独立膜表面形成生物膜(biofilm),这些独立膜包括渗透蒸发膜、透析膜、反渗透膜、超滤膜、微滤膜等。
在一些实施例中，上述段落所述组合物在清洁剂中的应用，此种清洁剂具有杀菌的作用，使用后需 要用水清洗。最好使用去离子水清洗。可以选择性地干燥清洗过的物品，干燥方法可以选择环境空气干燥，烘箱干燥，和空气强制干燥。干燥温度介于50℃至120℃，优选干燥温度为50℃至100℃，干燥时间约15min至24h。
另外，抗菌肽或抗菌肽组合物对上述所有物品的处理包括在工业化生产之前，工业化生产之后，及正在工业化生产之中的全过程。例如制作抗菌浴帘，首先将抗菌肽与基底多聚物结合，然后用混合物处理浴帘。生产工艺包括但不限制于注塑成型、挤压成型、吹塑成型、热成型、溶液涂层、吹膜中的至少一种。
在一些实施例中，上述段落所述组合物在抗感染药品、消炎药品中的应用，杀菌防止感染，感染包括细菌、病毒、真菌，例如；包括酵母、病原体、革兰氏阴性菌和/或革兰氏阳性菌、单细胞或多细胞生物体引起的，也可以是共生生物或非共生生物、病原体生物、聚落性地或非聚落性的细菌引起的。具体地，抗菌肽或抗菌肽衍生物可以治疗或减少细菌、病毒、真菌在皮肤上的感染，这种感染可以是与人类或动物外皮系统相关的任何部分，如表皮，真皮，皮下组织，一个或数个毛发毛囊，一个或数个皮脂腺，或与皮肤相关联的其他部位。换言之，上述的感染是与皮肤相关的，进一步讲，感染也可以是与视力的一部分相关的或与听力的一部分相关的，如耳朵的一部分，如鼓室或包括鼓室在内的听力系统的一部分。
实施例中，抗感染药品可以被用于治疗人类或动物由任何一种或数种细菌造成的感染，抗菌肽可以治疗一种或多种共生生物过度增长，即使这种共生生物是非病原体或是对人体或动物有益的共生生物。其中一些共生生物可能来自以下种属：葡萄球菌，分枝杆菌，和丙酸杆菌。此外其他细菌感染可能来自于已知的病原体，如假单袍菌属。
实施例中，抗感染药品可以用来治疗由非细菌微生物造成的感染，如治疗由一种或多种真菌(如酵母)引起的感染。例如抗菌肽可以治疗由糠醛马拉色菌引起的感染。糠醛马拉色菌是一种共生生物，它的过度增长会造成头皮屑，脂溢性皮炎，糠疹花斑癣及毛囊炎等，对它的治疗常采取控制生长，消除相关炎症及控制第二次感染等。由于糠醛马拉色菌生长顽固不容易治疗，加之治疗周期长，需要很多药物共同作用，现行市场上使用的药物不仅毒副作用大而且极为昂贵。本申请的抗菌肽在有效治疗浓度范围内可以治疗由真菌如糠醛马拉色菌引起的感染。
实施例中，消炎药品可以被来治疗包括感染及非感染甚至物理创伤引起的炎症，抗菌肽或抗菌肽衍生物在实验中上显示具有减少，缓解和治疗炎症的作用。如在细胞、细菌及动物中，上述组合肽显示了减少炎症因子，如胞质分裂(cytokinesis)，趋化因子(chemokines)及其相似物的释放。
另外，此种炎症为生理定义的炎症反应，既包括发红，肿胀，在细胞分子转写及翻译水平上的一个或多个促炎症细胞因子的诱导反应；与炎症相关的一个或多个细胞信号传导路径的诱导反应；细胞膜存在的受体诱导反应；在血管化组织内存在的细胞浸润，及在本领域技术范畴内与炎症相关的其它表现症状。
在一些实施例中，上述段落所述组合物在抑制细胞无限增殖药品中的应用，抗肿瘤的作用，此处的非无限增殖的细胞(细胞系内In Vitro及生体内In vivo)包括人类构建的细胞，疾病状态的细胞，例如癌细胞等；此种非永生细胞也包括原代培养细胞，如角质细胞，微血管内皮细胞，角膜上皮细胞及真皮成纤维细胞等。抗菌肽组合物造成了细胞膜受体－细菌特异性结合的模型识别受体(pattern-recognition  receptors)的减少。作为上述反应的结果，动物及动物体一部分显示了明显的炎症缓解作用，如组织红肿减少或消失，细胞渗透减少或消失等。
在一些实施例中，上述的促炎症细胞因子包括与炎症相关的细胞因子，包括但不限制于肿瘤坏死因子，白介素8，白介素1和白介素6。另外，细胞内信号传导经路包括与炎症相关的传导信号经路，包括但不限制于NFkB，AP-1。“细胞膜相关受体”包括但不限制于模式识别受体，TLR受体家族，(包括TLR-2和TLR4)。
实施例中，抗菌肽的治疗浓度及治疗时间取决于很多因子，如病情状态，年龄，性别，体重及个人身体状况，抗菌肽应用干细胞治疗，其有效浓度大于0.01ug/ml。
实施例中，有效浓度为0.2ug/ml以上。
实施例中，抗感染药品、消炎药品和抑制细胞无限增殖药品包括液体、半固体化液体、奶油状固体、软膏、凝胶中的至少一种，例如，外用药剂型可以用在皮肤，头发，及其他外用部分，进一步讲，在一定条件下，外用剂型可以用在单眼或双眼，也可以做成滴眼液。
实施例中，抗菌肽或抗菌肽衍生物可以做成静注剂型，或者是鼓室内/跨鼓室投药剂型，溶剂可以是灭菌的等渗水溶液。
另外，医药上使用的抗菌肽剂型要与投药路径相一致，该投药路径包括胃肠投药，静注，皮下，皮内，口腔，鼻内(吸入)，阴道、肛门、表皮，经黏膜给药，跨鼓室，鼓室内，直肠给药及其他可以接受的投药方式。抗菌肽制剂要与药学组成成分相吻合的投药路径相匹配，如以静注，皮下，肌肉内，跨神经节，口服，鼻、内，耳内及表皮下等投药方式给予人类和/或动物，如牲畜，及陪伴动物等，也可以应用给水生物种，禽类等。
另外，申请中一个字母代表氨基酸的定义与国际氨基酸编码及代表序列标准一致(IUPAC-IYUB)，请参考表1所示。
表1
氨基酸(中文名) 氨基酸(英文名) 三个字母缩写 一个字母缩写
丙氨酸 Alanine Ala A
精氨酸 Arginine Arg R
天冬酰胺 Asparagine Asn N
天冬氨酸 Aspartic Acid Asp D
半胱氨酸 Cysteine Cys C
谷氨酰胺 Glutamine Gln Q
谷氨酸 Glutamic acid Glu E
甘氨酸 Glycine Gly G
组氨酸 Histidine His H
异亮氨酸 Isoleucine Ile I
亮氨酸 Leucine Leu L
赖氨酸 Lysine Lys K
甲硫氨酸 Methionine Met M
苯丙氨酸 Phenylalanine Phe F
脯氨酸 Proline Pro P
丝氨酸 Serine Ser S
苏氨酸 Threonine Thr T
色氨酸 Tryptophan Trp W
络氨酸 Tyrosine Tyr Y
缬氨酸 Valine Val V
鸟氨酸 Ornithine Orn  
高精氨酸 Homoarginine Har  
2,4二氨基丙酸 2,4-diaminobutyric acid Dab  
下面结合具体实施例进行说明。
实施例1至39
实施例1至39通过标准的国际公知的多肽固相合成(solid-phase synthesis),液相合成(solution–phase synthesis),或者生物组换合成(recombinant biosynthesis)合成，合成结果如表2所示的氨基酸序列表。
表2
Figure PCTCN2021116530-appb-000010
Figure PCTCN2021116530-appb-000011
Figure PCTCN2021116530-appb-000012
性能测试及结果分析
(1)大肠杆菌抑制实验
对多肽合成实施例1至39中39种抗菌肽进行大肠菌抑制实验，取等量大肠菌液体30ul，加入各种30ul抗菌肽(浓度200ug/ml)，对照管用30ul的PBS等渗液，室温下反应15min，然后涂抹琼脂糖平板，涂均匀后在温度37℃下，培养12h，统计和记录各平板的大肠菌个数。实验结果，请参照图1。实验结果表明，抗菌肽SEQ ID NO:1至SEQ ID NO:39对大肠杆菌均具有抑制效果，其中SEQ ID NO:8的效果最为明显。
为了使实验结果更加清楚，选取6个实验样本进行对照分析，请参照图2所示，A为对照管，251个大肠菌克隆；B为SEQ ID NO:5，236个大肠菌克隆；D为SEQ ID NO:6，226个大肠菌克隆；F为SEQ ID NO:7，216个大肠菌克隆；C为SEQ ID NO:8，1个大肠菌克隆；E为SEQ ID NO:35为95个，表明复数多肽有杀死革兰氏阴性菌的作用，另外，SEQ ID NO:8为1个，表明，最优的抗菌肽氨基酸序列为SEQ ID NO:8，大肠菌的MIC为32ug/ml。
(2)野生金黄色葡萄球菌抑制环实验
取临床分离的野生金黄葡萄球菌(金黄色葡萄球菌为革兰氏阳性菌)液体涂满琼脂糖平板，放入接种用牛津杯，加入SEQ ID NO:8抗菌肽(浓度各为1/4：375ug/ml；1/16:93.7ug/ml；1/64：23.34ug/ml)200ul，在37℃，培养18h，测定抑菌环大小。实验结果为，1/4：抑菌环为3.95cm；1/16:抑菌环为3.5cm；1/64:抑菌环为3.1cm。请参照图3所示。实验结果表明，随着抗菌肽的浓度减少，抑菌环变小，另外，和大肠菌相比，抗菌肽对金黄色葡萄球菌更为敏感，本申请的抗菌肽具有很好的抗革兰氏阳性菌性。
(3)铜绿假单胞菌(绿脓杆菌ATCC27853)的MIC测定
取浓度为1024ug/ml，512ug/ml，256ug/ml，128ug/ml，64ug/ml，32ug/ml，16ug/ml，8ug/ml，4ug/ml，2ug/m的SEQ ID NO:8抗菌肽，并设置阳性对照，阴性对照，分别和标准绿脓杆菌混合后，分别加入培养板12个孔中，并对培养板12个孔进行编号，其中，请参照图4所示，1对应1024ug/ml，2对应512ug/ml，3对应256ug/ml，4对应128ug/ml，5对应64ug/ml，6对应32ug/ml，7对应16ug/ml，8对应8ug/ml，9对应4ug/ml，10对应2ug/m，11对应阳性对照，12对应阴性对照，在37℃下，培养2天后，观察培养板中的浑浊程度。实验结果，请参照图4所示。实验结果表明，抗菌肽对绿脓杆菌具有很好的抑制效果，对绿脓杆菌的MIC为64ug/ml。
(4)口腔细菌的抑制作用
取老人漱口水，分成等量的4份，并分别与不同量的2mg/mlSEQ ID NO:8混合30min后均匀涂抹到含有琼脂糖羊血的平板中，其中，A:PBS(对照)，B:2ul、C:10ul、D:20ul混合。在37℃下，厌氧培养4天后，观察平板菌落数。实验结果，请参照图5所示。
具体地，发现对照平板有白色念珠菌及厌氧菌生长，B:2ul抗菌肽平板有少量菌生长，C:10ul抗菌肽未见任何口腔细菌生长，D:20ul菌同样未见口腔细菌生长。
(5)抗菌肽杀死RNA病毒实验
用质粒pNL4.3Δ、pVSV-G，p-enhancer与lipo2000转染293T，在37℃下，培养两天后，取培养上清，离心去除细胞器杂质。将细胞上清液500ul与等量的200ug/ml SEQ ID NO:8混合反应15min后，对照组用等量的PBS替代SEQ ID NO:8。过滤离心法(Millipore Amicon Ultra-15)，来除去抗菌肽，以剔除抗菌肽本身对细胞的杀伤作用。用待测定样品感染293Tcell line，在37℃下，培养12h，回收293T cell line，采用RT-PCR法测定可感染细胞的活病毒差异，其中，对照组和实验组的各种实验环境相同，不同的是实验组中加入本申请的SEQ ID NO:8序列氨基酸。
结果表明：对照组的活性病毒基因拷贝为1.7×10 7；实验组的抗菌肽组活性病毒拷贝为5×10 5，表明本申请的抗菌肽有明显的杀死病毒的作用。
(6)抗菌肽杀死肿瘤细胞实验
培养K562细胞，将等量的K562细胞分别与10ng、20ng、40ng的SEQ ID NO:8反应后，用流式细胞仪PI试剂盒测定凋亡细胞，发现细胞死亡与抗菌肽含量成正比，说明抗菌肽可以有效地杀死K562细胞。
(7)抗菌肽杀死白血病淋巴细胞实验
Jurkat cell line:白血病淋巴细胞，SEQ ID NO:10号多肽1ug/m20ul与10 5cell反应10min，在1000rpm下，离心3min后，请参照图片6所示，实验结果表明，白血病淋巴细胞凝聚成团，并伴有细胞破碎。
(8)抗菌肽杀死白血病细胞实验
K562 cell line:白血病细胞，SEQ ID NO:17抗菌肽1ug/ml 20ul与10 5cell反应10min，1000rpm离心3min后，请参照图7所示，结果表明，显示白血病细胞凝聚成团。
(9)抗菌肽杀死肝癌细胞实验
HypG2 cell line:肝癌细胞，SEQ ID NO:15号多肽1ug/ml 20ul与10 5cell反应10min，1000rpm离心3min后，请参照图8所示，结果表明，肝癌细胞凝聚成团，并破碎变形。
(10)抗菌肽对血红细胞的效果实验
人血红细胞，SEQ ID NO:27抗菌肽1ug/ml 20ul与2％RBC反应10min，1000rpm离心3min后，实验结果，请参照图9所示，结果表明，人血红细胞凝聚成团，未见明显的溶血。
以上所述仅为本申请的较佳实施例而已，并不用以限制本申请，凡在本申请的精神和原则之内所做的任何修改、等同替换和改进等，均应包含在本申请的保护范围之内。

Claims (16)

  1. 一种抗菌肽或肽衍生物，其特征在于，其包括如下氨基酸序列中的至少一种：
    氨基酸序列I：X a1B a1U 1Z a1B a2X a2B a3Z a2B a4X a3；
    氨基酸序列II：
    Figure PCTCN2021116530-appb-100001
    其中，X a1、B a1、U 1、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各自独立地选自氨基酸。
  2. 如权利要求1所述的抗菌肽或肽衍生物，其特征在于，
    所述X a1、X a2、X a3、X b1、X b2、X b3各自独立地选自带有阳性电荷的残基中的至少一种；和/或
    所述B a1、B a2、B a3、B a4、B b1、B b2、B b3、B b4各自独立地选自带有疏水侧链的残基中的至少一种；和/或
    所述U 1选自Gly、Pro、Cys、Cys(R)中的一种，其中，所述(R)代表Cys双硫键的保护基团；和/或
    所述Z a1、Z a2、Z b1、Z b2各自独立地选自非极性残基中的至少一种；和/或
    所述C a1、C a2各自独自选自Cys、Cys(R)中的一种。
  3. 如权利要求2所述的抗菌肽或肽衍生物，其特征在于，
    所述X a1、X a2、X a3、X b1、X b2、X b2、X b3各自独立地选自Arg、His、Lys、Orn、Har、Dab、非天然氨基酸中的一种、两种或三种；或/和
    B a1、B a2、B a3、B a4、B b1、B b2、B b3、B b4各自独立地选自Ala、Val、Ile、Leu、Met、非天然氨基酸中的两种、三种或四种；或/和
    所述Z a1、Z a2、Z b1、Z b2各自独立地选自Asn、Gln、Ser、Thr、非天然氨基酸中的一种。
  4. 如权利要求2所述的抗菌肽或肽衍生物，其特征在于，所述Cys双硫键选自乙酰氨基甲基、甲硫代磺酸甲酯或其他Cys保护基团中的一种。
  5. 如权利要求1-4任一项所述的抗菌肽或肽衍生物，其特征在于，所述氨基酸序列I和/或氨基酸序列II上至少包括一个氨基酸被修饰处理过。
  6. 如权利要求5所述的抗菌肽或肽衍生物，其特征在于，所述修饰处理包括磷酸化、卤化、乙酰化、环化、端点的加帽反应中的至少一种。
  7. 如权利要求1所述的抗菌肽或肽衍生物，其特征在于，所述氨基酸序列I和/或氨基酸序列II上至少包括一个模体。
  8. 如权利要求1-4、6-7任一项所述的抗菌肽或肽衍生物，其特征在于，所述抗菌肽或肽衍生物为包括氨基酸序列I和/或氨基酸序列II形成的多体。
  9. 如权利要求1-4、6-7任一项所述的抗菌肽或肽衍生物，其特征在于，所述抗菌肽或肽衍生物包括SEQ ID NO:1至SEQ ID NO:39所示氨基酸序列。
  10. 如权利要求1-4、6-7任一项所述的抗菌肽或肽衍生物，其特征在于，至少一条所述抗菌肽或肽衍生物形成纳米结构。
  11. 如权利要求10所述的抗菌肽或肽衍生物，其特征在于，所述纳米结构包括胶束、囊泡、纳米管、 纳米带中的至少一种。
  12. 一种抗菌肽或肽衍生物的替换体，其特征在于，由权利要求1-11任一所述抗菌肽或肽衍生物所含氨基酸序列I或/和氨基酸序列II中的至少一个氨基酸被替代。
  13. 一种抗菌肽或肽衍生物组合物，其特征在于，包括权利要求1-11任一所述抗菌肽或肽衍生物或/和权利要求12所述替换体以及至少一种药剂上可以接受的载体和/或溶剂。
  14. 根据权利要求13所述的抗菌肽或肽衍生物组合物，其特征在于，还包括赋形剂、等渗剂、吸收延迟剂或基底多聚物中的至少一种。
  15. 一种根据权利要求13所述抗菌肽或肽衍生物组合物的制备方法，其特征在于，包括如下步骤：
    将抗菌肽或肽衍生物和所述溶剂混合处理。
  16. 根据权利要求14所述抗菌肽或肽衍生物组合物在包装、食品加工最终端、服饰、医疗用品、医疗仪器、个人卫生用品、消毒剂、清洁剂、抗感染药品、消炎药品、抑制细胞无限增殖药品中的应用。
PCT/CN2021/116530 2021-09-03 2021-09-03 抗菌肽或肽衍生物、替换体以及其组合物、制备方法和应用 WO2023029001A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1363558A (zh) * 2001-05-10 2002-08-14 上海华谊生物技术有限公司 蛙皮抗菌肽衍生物
CN101570569A (zh) * 2008-04-29 2009-11-04 昆山博青生物科技有限公司 合成抗菌肽、其制备方法及应用
CN101914565A (zh) * 2010-07-27 2010-12-15 中国农业科学院饲料研究所 一种在毕赤酵母中有效的表达阳离子抗菌肽的方法
CN103484466A (zh) * 2013-09-11 2014-01-01 华南农业大学 一种小菜蛾抗菌肽moricin及其制备方法与应用
CN104672307A (zh) * 2015-03-23 2015-06-03 吉林大学 一种提高阳离子短肽抗菌性和稳定性的方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1363558A (zh) * 2001-05-10 2002-08-14 上海华谊生物技术有限公司 蛙皮抗菌肽衍生物
CN101570569A (zh) * 2008-04-29 2009-11-04 昆山博青生物科技有限公司 合成抗菌肽、其制备方法及应用
CN101914565A (zh) * 2010-07-27 2010-12-15 中国农业科学院饲料研究所 一种在毕赤酵母中有效的表达阳离子抗菌肽的方法
CN103484466A (zh) * 2013-09-11 2014-01-01 华南农业大学 一种小菜蛾抗菌肽moricin及其制备方法与应用
CN104672307A (zh) * 2015-03-23 2015-06-03 吉林大学 一种提高阳离子短肽抗菌性和稳定性的方法

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