Novel antimicrobial peptides and their pharmaceutical compositions

Abstract

The present invention provides an antimicrobial peptide and its pharmaceutical composition that is highly stable, safe, has good antimicrobial activity, and has a shorter sequence, and can treat and alleviate rosacea, adolescent acne, atopic dermatitis, genital warts and vulvar intraepithelial neoplasia, inflammatory reactions, inflammatory response syndromes, etc., and the antimicrobial polypeptide contains the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and specifically to antibacterial peptides and their applications.

Background Art

[0002] Antimicrobial peptides (AMPs) are immune-activating molecules produced by living organisms during long-term evolution to adapt to the environment. They possess antibacterial, antiviral, antifungal, and antiparasitic functions and play an important role in the body's natural immune defense system, and are also called "host defense peptides" (HDPs). Antimicrobial peptides typically consist of 12-50 amino acid residues, and most are cationic short peptides. Their charge levels range from +2 to +9, with antimicrobial peptides carrying +2 to +4 charge being the most abundant (Rodriguez AA, Otero-Gonzalez A, Ghattas M, Standker L. Discovery, optimization, and clinical application of natural antimicrobial peptides. Biomedicines, 2021, 9(10):1381. Yount NY, Bayer AS, Xiong YQ, Yeaman MR. Advances in antimicrobial peptide immunobiology. Biopolymers, 2006, 84(5):435-458). The large positive charge on the surface of antimicrobial peptides is due to the fact that the initial interaction between antimicrobial peptides and bacterial cell membranes is primarily achieved through electrostatic interaction between cationic residues on the surface of the antimicrobial peptide and anions on the bacterial cell membrane. Furthermore, while small amounts of anionic antimicrobial peptides are also present, their antimicrobial activity is far lower than that of cationic antimicrobial peptides, and their role in innate immunity is not yet fully understood. Therefore, research on antimicrobial peptides has mainly focused on cationic antimicrobial peptides (Teixeira V, Feio MJ, Bastos M. Role of lipids in the interaction of antimicrobial peptides with membranes. Progress in Lipid Research, 2012, 51(2):149-177).Structurally, antimicrobial peptides have three configurations: α-helical, β-folding, and elongated / random curl. These structures are amphiphilic or capable of converting to amphiphilicity, and by dissolving antimicrobial peptides in a water- and lipid-rich environment (Lazzaro BP, Zasloff M, Rolff J. Antimicrobial peptides: application informed by evolution. Science, 2020, 368(6490):eaau5480.), they can adhere to the surface of bacterial cell membranes and exert membrane-lysis effects. Antimicrobial peptides also have relatively small molecular weights, usually less than 10 kDa. This small molecular weight helps antimicrobial peptides to rapidly diffuse and be secreted in host cells, stimulating immediate defense against pathogenic microorganisms. To date, the Antimicrobial Peptide Database (APD) contains more than 3000 AMPs, covering antibacterial, antifungal, antiviral, antiparasitic, and other functions.

[0003] In recent years, the overuse of antibiotics has led to a public health crisis due to bacterial resistance. According to the latest research by the Center for Disease Control and Prevention (CDC), antibiotic resistance causes millions of different diseases worldwide every year, and it is estimated that by 2050, the number of deaths due to antibiotic resistance will reach tens of millions. Compared to conventional antibiotics, the application advantages of antimicrobial peptides mainly include: (1) selective toxicity. Differences in cell membrane composition between pathogens and hosts are the basis of antimicrobial peptide target specificity, allowing antimicrobial peptides to specifically enter pathogens (Zhang QY, Yan ZB, Meng YM, Hong XY, Shao G, Ma JJ, Cheng XR, Liu J, Kang J, Fu CY. Antimicrobial peptides: mechanism of action, activity and clinical potential. Military Medical Research, 2021, 8(1):48.), (2) rapid action. The time required for antimicrobial peptides to kill target bacteria is much shorter than the doubling time of the target bacteria (Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology, 2005, 3(3):238-250.), (3) Effectively kills retainer bacteria. Retainer bacteria are a subgroup of bacteria within a bacterial population that can survive lethal doses of antibiotics and other stressful environments, and are bacterial cells that do not undergo genetic mutations, have reduced metabolic activity, and are not killed by antimicrobial drugs (Liu XL, Yang WX, Ma YL, Wang D, Chen H. Research progress in the formation mechanism and treatment of persister. Chinese Journal of Infection Control, 2020, 19(2):184-188. (in Chinese)).The target of antimicrobial peptides is usually located on the cell membrane and is independent of cellular metabolic activity, making them advantageous for killing swarming bacteria (Defraine V, Schuermans J, Grymonprez B, Govers SK, Aertsen A, Fauvart M, Michiels J, Lavigne R, Briers Y. Efficacy of artilysin art-175 against resistant and persistent Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy, 2016, 60(6):3480-3488.). (4) Broad-spectrum. In addition to commonly seen Gram-negative and Gram-positive bacteria, antimicrobial peptides are also effective against viruses, protozoa, and fungi (Yeung ATY, Gellatly SL, Hancock REW. Multifunctional cationic host defence peptides and their clinical applications. Cellular and Molecular Life Sciences: CMLS, 2011, 68(13):2161-2176). (5) Less likely to induce chemical resistance. Antimicrobial peptides act on very conservative cell membranes and are rarely modified by bacteria to induce chemical resistance. Secondly, antimicrobial peptides have a short half-life, making it difficult for them to accumulate in the environment and induce bacterial chemical resistance. Furthermore, the duration of action of antimicrobial peptides is short, and compared to antibiotics, the intermediate therapeutic concentration range of antimicrobial peptides is smaller. The smaller the intermediate therapeutic concentration range, the less likely it is to induce the production of chemical resistance. Currently, antimicrobial peptides have some applications in fields such as food, agriculture, and medicine, and have shown good results.Furthermore, antimicrobial peptides have a certain degree of clinical application. For example, omiganan, a synthetic analog of indolicidin, has great potential in treating fungal infections, including rosacea, adolescent acne, atopic dermatitis, genital warts, and vulvar intraepithelial neoplasia (Zyrek D, Wajda A, Czechowicz P, Nowica J, Jaskiewicz M, Neubauer D, Kamysz W. The antimicrobial activity of omiganan alone and in combination against Candida isolated from vulvovaginal candidiasis and bloodstream infections. Antibiotics:Basel, Switzerland, 2021, 10(8):1001.).

[0004] While the application prospects for antimicrobial peptides are promising, there are many problems, mainly manifesting in three areas: (1) Pharmacokinetics: Antimicrobial peptides have problems such as hemolytic activity of red blood cells, low oral bioavailability, and sensitivity to gastrointestinal protease degradation, making most antimicrobial peptides unsuitable for oral administration. On the other hand, with systemic administration methods such as intravenous injection, plasma protein hydrolases rapidly degrade antimicrobial peptides, and the rapid removal action of the liver and kidneys shortens the half-life of antimicrobial peptides, thus limiting their use to topical administration. (2) Antimicrobial activity: Although antimicrobial peptides have broad-spectrum antimicrobial activity, their antimicrobial activity is relatively low compared to conventional antibiotics. In other words, to achieve the same antibacterial effect, the concentration of antimicrobial peptides used may be higher (Chen X, Zhang M, Zhou CH, Kallenbach NR, Ren DC. Control of bacterial persister cells by Trp / Arg-containing antimicrobial peptides. Applied and Environmental Microbiology, 2011, 77(14):4878-4885.), but high concentrations of antimicrobial peptides increase the hemolysis of host red blood cells. (3) Drug production and processing aspects: Compared to conventional small molecule therapeutics, antimicrobial peptide sequences are relatively long, and production and manufacturing costs are often relatively high. In particular, disulfide-rich antimicrobial peptides have, to some extent, limited the clinical production and use of antimicrobial peptides.

[0005] Currently, many solutions have been proposed to address the problems associated with antimicrobial peptides, including chemical modifications to enhance stability, substitution of antimicrobial peptide amino acid sequences to increase antimicrobial activity, fusion of antimicrobial peptides with endocrines to reduce cytotoxicity, and designing short-linear antimicrobial peptides to lower costs. However, based on the above antimicrobial peptide optimization policies, these improvements cannot simultaneously achieve desirable effects. In later stages, an artificial intelligence research and development method was proposed, which involves creating predictive models to estimate the molecular properties of antimicrobial peptides and using them for candidate selection. Typically, predictive models are constructed using manually selected or automatically learned sets of components, structures, and physicochemical features. Candidate antimicrobial peptides are then usually obtained by combining and counting reasonable subsequences, and then randomly selecting or modifying them from a conventional molecular base. Simultaneously, by representing antimicrobial peptide amino acid residues as strings and performing instrumental learning / deep learning training and analysis on an antimicrobial peptide amino acid sequence dataset, novel antimicrobial peptide amino acid sequences can be efficiently identified.

[0006] Simultaneously, many researchers have integrated their knowledge of the relationship between microorganisms and protein polypeptides, and have established numerous policies for designing antimicrobial agents based on natural template peptides. Compared to natural AMP, modified and synthesized AMP has shown superior antimicrobial efficacy and biocompatibility. Specifically, first, after previous researchers summarized the properties of natural AMP, antimicrobial activity is enhanced by increasing the ratio of positively charged and hydrophobic amino acids according to the purpose. Next, the secondary structure of the polypeptide is adjusted. Many AMPs have a disordered structure in aqueous solution, and after entering the membrane environment, they interact with phospholipid biomolecules to form a very stable secondary structure, which is of crucial importance for antimicrobial activity. Therefore, the secondary structure is a fundamental element of the interaction between AMP and the membrane, and folding into a regular secondary structure helps in the insertion of AMP into the bacterial membrane. Thirdly, the amphiphilicity of AMP is adjusted. Amphiphilicity is the degree of spatial separation of hydrophobic and hydrophilic residues on opposite sides of the molecular backbone. Typically, amphiphilicity is quantified by the hydrophobic moment, which is the vector sum of individual hydrophobic amino acids in a normalized helix. The amphiphilic α-helical is the most common conformation, where the main chain of the peptide ascends in a regular helical pattern around a central axis, helicing once every 3-4 amino acid residues. The hydrophilic surface of the amphiphilic α-helical peptide and negatively charged bacterial membrane components exert an electrostatic effect, increasing membrane permeability by inserting the peptide's hydrophobic surface into the membrane. Usually, AMP can be modified to form a perfect amphiphilic structure by deleting, substituting, or adding amino acids to the sequence. However, studies have shown that AMP with a perfect amphiphilic structure, despite having optimal activity, also exhibits increased cytotoxicity, suggesting that this perfect amphiphilic structure non-selectively increases membrane disruption.

[0007] While various polypeptide design optimization techniques have yielded some results, AMP itself still suffers from drawbacks such as high toxicity, unclear toxicology, and immature clinical trial stability, which remain the biggest obstacles to its application. Furthermore, most previous studies have focused on simply cutting or modifying natural AMP to obtain stronger performance, often ignoring the inherent structure and performance of the peptide sequence itself. As a result, the poor performance of most AMPs remains unresolved, and there are very few antimicrobial templates suitable for practical use.

[0008] Previous studies have shown that after cleaving the amino and carboxyl ends of zebrafish yolk hyperphosphoprotein (Phosvitin, Pv), the 194 amino acids at the carboxyl end still retain antimicrobial activity, but the antimicrobial activity of the 55-amino acid sequence at the amino end (Pt6) completely disappears. By proportionally shortening the amino acids at the carboxyl end, it was ultimately discovered that the 55 amino acids at the carboxyl end still retain antimicrobial activity, and this was named yolk hyperphosphoprotein-inducing polypeptide (Pt5) (Wang SHWang YMa J, Ding YCZhang SC: Phosvitin Plays a Critical Role in the Immunity of Zebrafish Embryos via Acting as a Pattern Recognition Receptor and an Antimicrobial Effector Journal of Biological Chemistry 2011 286(25):22653-22664). Since yolk hyperphosphoprotein-derived polypeptides (Pt5) are derived from yolk hyperphosphoprotein (Phosvitin, Pv) in vitellogenin (Vg), many of these antimicrobial proteins possess the molecular characteristics of both parents, have a general net positive charge between 2 and 9, and have a very high proportion of hydrophobic amino acids (Manchekar MR. Richardson PE, Forte TM., Datta GSegrest JPDashti N: Apolipoprotein b containing lipoprotein particle assembly - Lipid capacity of the nascent lipoprotein particle Journal of Biological Chemistry 2004 279(38):39757-39766).Subsequent studies have shown that Pt5 not only has antibacterial properties but also exhibits some protective effects against zebrafish infected with aerobic monoseptic bacteria (Ding YCLiu XMBu LZLi HYZhang SC: Antimicrobial-immunomodulatory activities Of zebrafish phosvitin-derived peptide Pt5. Peptides 201237(2):309-313).

[0009] From the above, it can be seen that most natural AMPs have a series of problems, such as weak biological activity, high toxicity, and low stability, which are due to the limitations of their own mechanism of action, and therefore most AMPs cannot be directly produced and applied. Based on prior art and prior research, it has been found that the antimicrobial peptide obtained by selecting and detecting the active site of an antimicrobial peptide and then modifying its activity using a point sequence as a template creatively solves the challenges of practical application of antimicrobial peptides. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] This invention involves a method of amino acid shortening expression, in which the carboxyl and amino ends of egg yolk high-phosphoprotein Pv are removed, respectively, to obtain an antimicrobial peptide having an active site. The selected and detected active site sequence is then used as a template for design modification, and finally, selection is performed to obtain an antimicrobial peptide with high stability, good safety, good antimicrobial activity, and a shorter sequence. This completes the following invention. [Means for solving the problem]

[0011] According to a first aspect, the present invention provides a novel antimicrobial polypeptide comprising the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2.

[0012] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 allows for the substitution of one and / or several amino acids, and the antibacterial activity of the substituted amino acid sequence is not significantly reduced, retains considerable antibacterial activity, or exhibits significantly enhanced antibacterial activity. Moreover, the substituted amino acids may consist of 1 to 5 amino acids, preferably 1 to 3, and more preferably 1 amino acid. The substituted amino acids may be sequential substitutions and / or interval substitutions.

[0013] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2, when one and / or several amino acids are added to the carboxyl group and / or amino group, exhibits either no significant decrease in antibacterial activity, a substantial increase in antibacterial activity, or a significant enhancement of antibacterial activity. Moreover, the number of added amino acids can range from 1 to 30, preferably 1 to 20, and more preferably 1 to 10. The added amino acids may be concentrated at the carboxyl group, concentrated at the amino group, or simultaneously at both the carboxyl and amino groups.

[0014] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 has an increase or decrease of 1 to 5 amino acids, preferably an increase and / or decrease of 1 to 3 amino acids, and more preferably an increase and / or decrease of 1 amino acid, and the increased or decreased amino acids can be concentrated or dispersed at any position in the amino acid sequence, and the antibacterial activity of the modified amino acid sequence is not significantly reduced, retains considerable antibacterial activity, or has significantly enhanced antibacterial activity.

[0015] Furthermore, the spatial arrangement of the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 or its variant sequence (the amino acid sequence after the above-mentioned amino acid substitution, addition, or deletion) is such as α-helical, β-folding, and / or elongation / random curl.

[0016] According to a second aspect, the present invention provides an antimicrobial polypeptide comprising an amino acid sequence having 90-100% homology to the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2, referred to as the homologous sequence of Ac-SRMKKWAKIIEKWRKWH-NH2, wherein the spatial arrangement of the homologous sequence is such as α-helical, β-folding and / or elongated / random curl.

[0017] According to a third aspect, the present invention provides an antimicrobial pharmaceutical composition comprising an antimicrobial polypeptide of the first aspect of the present invention and / or an antimicrobial polypeptide of the second aspect of the present invention and a pharmaceutically acceptable adjuvant.

[0018] In specific embodiments, the antimicrobial pharmaceutical composition of the present invention contains only the antimicrobial polypeptide active component of the first embodiment of the present invention, wherein the antimicrobial polypeptide comprises the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2.

[0019] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 allows for the substitution of one and / or several amino acids, and the antibacterial activity of the substituted amino acid sequence is not significantly reduced, retains considerable antibacterial activity, or exhibits significantly enhanced antibacterial activity. Moreover, the substituted amino acids may consist of 1 to 5 amino acids, preferably 1 to 3, and more preferably 1 amino acid. The substituted amino acids may be sequential substitutions and / or interval substitutions.

[0020] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2, when one and / or several amino acids are added to the carboxyl group and / or amino group, exhibits either no significant decrease in antibacterial activity, a substantial increase in antibacterial activity, or a significant enhancement of antibacterial activity. Moreover, the number of added amino acids can range from 1 to 30, preferably 1 to 20, and more preferably 1 to 10. The added amino acids may be concentrated at the carboxyl group, concentrated at the amino group, or simultaneously at both the carboxyl and amino groups.

[0021] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 has an increase or decrease of 1 to 5 amino acids, preferably an increase and / or decrease of 1 to 3 amino acids, and more preferably an increase and / or decrease of 1 amino acid, and the increased or decreased amino acids can be concentrated or dispersed at any position in the amino acid sequence, and the antibacterial activity of the modified amino acid sequence is not significantly reduced, retains considerable antibacterial activity, or has significantly enhanced antibacterial activity.

[0022] Furthermore, the spatial arrangement of the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 or its variant sequence (the amino acid sequence after the above-mentioned amino acid substitution, addition, or deletion) is such as α-helical, β-folding, and / or elongation / random curl.

[0023] In another specific embodiment, the antimicrobial pharmaceutical composition of the present invention comprises only the antimicrobial polypeptide active component of the second embodiment of the present invention, wherein the antimicrobial polypeptide comprises an amino acid sequence having 90-100% homology to the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2, and is referred to as the homologous sequence of Ac-SRMKKWAKIIEKWRKWH-NH2, and the spatial arrangement of the homologous sequence is such as α-helical, β-folding and / or elongated / random curl.

[0024] In another specific embodiment, the antimicrobial pharmaceutical composition of the present invention simultaneously contains the antimicrobial polypeptide described in the first aspect of the present invention and the antimicrobial polypeptide described in the second aspect of the present invention as active ingredients.

[0025] In another specific embodiment, the antimicrobial pharmaceutical composition of the present invention further comprises other active ingredients different from the antimicrobial polypeptide described in the first aspect of the present invention and the antimicrobial polypeptide described in the second aspect, the other active ingredients may be antibiotics, immunomodulators, minerals, trace elements and / or vitamins, etc.

[0026] Furthermore, the antibacterial pharmaceutical composition of the present invention can be manufactured into dosage forms such as injections, oral preparations, freeze-dried powder injections, and the like.

[0027] According to a fourth aspect, the present invention provides a nucleic acid molecule, and the nucleic acid molecule encodes an antibacterial polypeptide containing the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2.

[0028] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 can have one and / or several amino acid substitutions, and the antibacterial activity of the amino acid sequence after substitution is not obviously decreased, retains considerable antibacterial activity, or has obviously enhanced antibacterial activity. Furthermore, there are 1-5, preferably 1-3, more preferably 1 amino acid substitutions. The substituted amino acids may be continuous substitutions and / or spaced substitutions.

[0029] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 has one and / or several amino acids added to the carboxyl group end and / or amino group end, and the antibacterial activity of the increased amino acid sequence is not obviously decreased, retains considerable antibacterial activity, or has obviously enhanced antibacterial activity. Furthermore, the increased amino acids are 1-30, preferably 1-20, more preferably 1-10 amino acids. The increased amino acids may concentrate at the carboxyl group end, concentrate at the amino group end, or increase simultaneously at the carboxyl group end and the amino group end.

[0030] Furthermore, the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 has an increase or decrease of 1-5 amino acids, preferably an increase and / or decrease of 1-3 amino acids, more preferably an increase and / or decrease of 1 amino acid, and the increased or decreased amino acids can concentrate or disperse at any position of the amino acid sequence, and the antibacterial activity of the amino acid sequence after modification is not obviously decreased, retains considerable antibacterial activity, or has obviously enhanced antibacterial activity.

[0031] Furthermore, the spatial arrangement of the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2 or its variant sequence (the amino acid sequence after the above-mentioned amino acid substitution, addition, or deletion) is such as α-helical, β-folding, and / or elongation / random curl.

[0032] Furthermore, the nucleic acid molecule encodes an antimicrobial polypeptide containing an amino acid sequence having 90-100% homology to the amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH2.

[0033] According to a fifth aspect, the present invention provides a host cell that can express the antimicrobial polypeptide described in the present invention.

[0034] According to a sixth aspect, the present invention provides uses for antimicrobial polypeptides used to treat bacterial infectious diseases, viral infectious diseases, chlamydia infectious diseases, fungal infectious diseases, mycoplasma infectious diseases, rickettsial infectious diseases, such as rosacea, adolescent acne, atopic dermatitis, genital warts and vulvar intraepithelial neoplasia, inflammatory reactions, and inflammatory response syndromes. [Brief explanation of the drawing]

[0035] [Figure 1] E. coli antibacterial activity detection [Figure 2] Detection of antimicrobial activity in S. aureus [Figure 3] Toxicity experiments of AC-SH-17 on in vitro cultured mammalian cells [Figure 4] Detection of inflammatory responses in cells [Figure 5] The effect of Ac-SH-17 on the survival rate of mice with LPS-induced pustules. [Modes for carrying out the invention]

[0036] Definitions and general technologies Unless otherwise defined herein, scientific and technical terms used in connection with the present invention should have the meanings commonly known to those skilled in the art. Generally, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0037] The term "polypeptide" refers to a molecule consisting of monomers (amino acids) that surround a single "polypeptide" and multiple "polypeptides" and are linearly linked via amide bonds (also called peptide bonds). The term "polypeptide" means any one or more chains containing two or more amino acids, and does not mean a product of a specific length. The polypeptides of the present invention may have an amino acid size of about 10-30 or 10-20, and the polypeptide may have a limited spatial structure, but is not required to have such a structure. In one embodiment of the present invention, the antimicrobial peptide is obtained by artificial chemical synthesis and its carboxyl group terminus is amidated.

[0038] Viterogenin (Vg) is a protein found in the blood of non-mammalian mature oviparous animals. It belongs to a large superfamily of transporters and is present in almost all oviparous animals, including fish, amphibians, reptiles, birds, and many invertebrates, as a precursor to oviparous proteins. Viterogenin is generally synthesized exogenously and transported to the ovaries via blood circulation.

[0039] Phosvitin (Pv), a high-phosphoprotein in egg yolks, is formed by the hydrolysis of Vg by cathepsin D, a member of the aspartic acid protease family, in oocytes. Pv possesses not only a conservative serine structural region but also a relatively high phosphorus content, making it one of the most phosphorus-containing natural proteins known to date. In chickens, African clawed frogs, and bony fish, Pv can contain up to 50% serine residues, which can covalently bind to phosphates and ionically bind to calcium ions, giving Pv the ability to transport phosphates and calcium ions.

[0040] As used herein, “pharmaceutical composition” is prepared by mixing the antimicrobial polypeptide of the present invention with a pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carrier is typically nontoxic to the recipient at the dosage and concentration used and is prepared in liquid, solid, aerosol, or other oral ingestion form. In a sense, the pharmaceutical compositions provided in the present invention can be used in a method of treating a subject’s disease, comprising administering an antimicrobial polypeptide, a site-mutated antimicrobial polypeptide, a homologous sequence antimicrobial polypeptide, a polypeptide derivative, or a pharmaceutical composition thereof to a subject.

[0041] The subjects of the pharmaceutical compositions of the present invention include humans and non-human animals, such as mammals including mice, rats, guinea pigs, dogs, cats, rabbits, cattle, horses, sheep, goats, pigs, birds, fish, reptiles, and amphibians. The terms further include cells and microorganisms. In some embodiments, the subjects are selected from cells, specifically from mouse fibroblasts (L929), frozen cells, and human erythrocytes (RBCs).

[0042] In this specification, "administration" as it may be used interchangeably means an amount and method of administration sufficient to improve one or more symptoms of a target disease, using the antimicrobial polypeptide, site-mutated antimicrobial polypeptide, homologous sequence antimicrobial polypeptide, polypeptide derivative, or pharmaceutical composition disclosed herein.

[0043] Antimicrobial activity refers to the ability of an antimicrobial agent to suppress or kill pathogenic microorganisms. The minimum concentration at which bacterial growth in a culture medium can be suppressed is called the minimum inhibitory concentration (MIC). When the evaluation criterion is to kill bacteria, a reduction of 99% or 99.5% or more in the total number of viable bacteria is called the minimum bactericidal concentration (MBC). In the examples of the present invention, the antimicrobial activity of the antimicrobial peptide Ac-SH-17 is detected, and the minimum inhibitory concentration is adopted.

[0044] As used herein, the term “symptom or disease” includes, but is not limited to, rosacea, adolescent acne, atopic dermatitis, genital warts and vulvar intraepithelial neoplasia, and inflammatory response syndromes.

[0045] The Gram-negative and Gram-positive bacteria used in the examples of the present invention are the Gram-negative Escherichia coli strain 25922 (ATCC 25922, E. coli 25922) and the Gram-positive Staphylococcus aureus strain 25923 (ATCC 25923, S. aureus 25923).

[0046] Gram-positive bacterial antimicrobial peptides are obtained by artificial chemical synthesis, and their carboxyl group ends are amidated. The purity of the synthesized product is greater than 95%. After the synthesized modified antimicrobial peptides pass quality inspection, they are dissolved in 1% (v / v) trifluoroethanol (2,2,2-trifluoroethanol, TFE) to prepare a mother liquor with a final concentration of 1 mg / mL, which is then dispensed and stored in a -20°C refrigerator.

[0047] The reagents used in the examples of the present invention are LB nutritional meat soup, DMSO (solarbio brand), DMEM nutritional solution (GIBCO), fetal bovine serum (Hyclone), 1.8 ml cryopreservation tube, 0.25% trypsin (GIBCO), PBS, 10% fetal bovine serum DMEM nutritional solution, DMSO, MTT, and trypsin.

[0048] The equipment required for the embodiments of the present invention includes a 4 ml sterile centrifuge tube, a shaker (culture conditions 37°C, 150 r / min), a water bath, a centrifuge, an inverted microscope, a CO2 incubator, a clean bench, a 96-orifice plate, a water bath constant temperature oscillator, a plate reader, and the like.

[0049] Example 1: Synthesis of Ac-SH-17 polypeptide 1. An amino group resin with a substitution degree of approximately 0.5 mmol was selected, the resin was placed in a reaction tube, DCM (15 ml / g) was added, and the tube was shaken for 30 minutes.

[0050] 2. Following the first amino acid: The solvent was filtered by suction using a sand core, the amino acid was added in a 3x molar excess, dissolved with DMF, then DIEA was added in a 10x molar excess, and the mixture was shaken for 60 minutes. The mixture was sealed with methanol.

[0051] 3. Deprotection: Remove the DMF, add 20% piperidine DMF solution (15 ml / g), leave for 5 minutes, remove, add another 20% piperidine DMF solution (15 ml / g), and leave for 15 minutes.

[0052] 4. Detection: Remove the piperidine solution, take a dozen or so resin particles, wash them three times with ethanol, add the detection reagent and perform detection. Heat at 105°C-110°C for 5 minutes, and a positive reaction was indicated when it turned dark blue.

[0053] 5. Washing: DMF (10ml / g) twice, DCM (10ml / g) twice, DMF (10ml / g) twice 6. Condensation: Protect the amino acids with a 3x excess and HBTU with a 3x excess, dissolve both in as little DMF as possible, place in a reaction tube, immediately add a 10x excess of DIEA, and react for 30 minutes.

[0054] The process was repeated, and the amino acids in the Ac-SRMKKWAKIIEKWRKWH-NH2 sequence were sequentially linked from right to left, until the last amino acid, the Fmoc protecting group, was removed.

[0055] 7. Cutting polypeptides from resin: Prepare a cutting solution (10 ml / g) of 95% TFA, 1% water, 2% EDT, and 2% TIS, and cut for 120 min.

[0056] 8. Drying and washing: The cleavage fluid was dried as much as possible with nitrogen, washed six times with ether, and then dried by shaking at room temperature.

[0057] 9. Analysis and purification: The crude product was purified by high-performance liquid chromatography.

[0058] 10. Freeze-drying: The target polypeptide solution was collected, concentrated in a freeze-dryer, and freeze-dried into a powder.

[0059] Example 2: Detection of antimicrobial activity of modified peptide Ac-SH-17 2.1 Qualitative analysis of the antibacterial activity of modified peptides using a liquid antimicrobial method Eight 4 ml sterile centrifuge tubes were prepared and numbered 1-8. 1.5 ml of sterile nutrient-rich meat broth was placed in sterile centrifuge tubes 2-8. The prepared samples were then placed in centrifuge tubes 1 and 2. After mixing centrifuge tube 2, 1.5 ml was aspirated from centrifuge tube 2 into centrifuge tube 3. After mixing centrifuge tube 3, 1.5 ml was aspirated from centrifuge tube 3 into centrifuge tube 4, and so on. Finally, 1.5 ml of liquid was aspirated from centrifuge tube 7 and discarded. In this way, centrifuge tubes 1-7 contained samples with double the mass concentration, tube 8 was designated as the negative control group, and tube 1 as the positive control group. Finally, 1.5 ml of reserve bacterial suspension was placed in each centrifuge tube. Three parallel samples were prepared for each sample. Immediately, 100 μl was placed in a plate reader and its OD540 was measured and recorded as OD 0h. Subsequently, the samples were cultured in a 37°C constant temperature incubator, and 100 μl was taken every hour. The OD540 of each centrifuge tube was measured. The change in absorbance indicated the bacterial growth status, and the antimicrobial activity of the polypeptide and the minimum inhibitory concentration (MIC) were determined.

[0060] 2.2 Quantitative Analysis of Antimicrobial Activity of Modified Peptides by Plate Reader Turbidimetric Method 1. Preparation of bacterial solution The bacterial solution was diluted with LB nutritional meat broth, and the bacterial solution was adjusted to a concentration of 104 CFU / mL.

[0061] 2. Polypeptide resilience The polypeptide was dissolved in LB nutritional meat broth, and then restored with 1% trifluoroethanol for 0.5 hours.

[0062] 3. Incubation culture Eight 4 ml sterile centrifuge tubes were prepared and numbered 1-8. 1.5 ml of sterile nutrient-rich meat broth was placed in sterile centrifuge tubes 2-8. The prepared samples were then placed in centrifuge tubes 1 and 2. After mixing centrifuge tube 2, 1.5 ml was aspirated from centrifuge tube 2 into centrifuge tube 3. After mixing centrifuge tube 3, 1.5 ml was aspirated from centrifuge tube 3 into centrifuge tube 4. Finally, 1.5 ml of liquid was aspirated from centrifuge tube 7 and discarded. In this way, centrifuge tubes 1-7 contained samples with double the mass concentration, tube 8 was designated as the negative control group, and tube 1 as the positive control group. Finally, 1.5 ml of reserve bacterial suspension was placed in each centrifuge tube. Three parallel samples were prepared for each sample. Immediately, 100 μl was placed in a plate reader and its OD540 was measured and recorded as OD 0h. Subsequently, the samples were incubated in a 37°C constant temperature incubator, and 100 μl was taken every hour. The OD540 of each centrifuge tube was measured once.

[0063] Example 3: Cytotoxicity and hemolytic analysis of Ac-SH-17 3.1 Cytotoxicity Experiments 1. Prepare the solution (5 mg / ml MTT): Dissolve 15 mg of MTT in 3 ml of PBS, filter it through a 0.22 μm filter in a clean bench, and store it away from light at 4°C (wrapped in tin foil).

[0064] 2. Each material was weighed to 20 mg, sterilized by UV light for 24 hours, and dissolved in DMEM culture medium containing 10% fetal bovine serum. Culture media containing five concentration gradients of sample concentrations were prepared: 5 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, and 0.25 mg / ml.

[0065] 3. Inoculated cells: L929 cells that were growing vigorously were digested, and the cell suspension was adjusted to 104 cells / ml. 100 μl of the cell suspension was placed in each well of a cell drop plate and three 96 orifice plates, and the cells were cultured in a CO2 incubator for 24 hours to allow the cells to form walls.

[0066] 4. Add the sample. Blank control group: PBS, no cells Sample control group: Cultured cells in culture medium containing the material. Negative control group: Cultured cells in culture medium without material. Positive control group: Culture medium containing 0.5% phenol Set up 3-5 parallel samples for each group. 5. The original culture medium in the 96-orifice plates was discarded and placed in each experimental group. Each group was incubated with 100 μl / well of a blank orifice, 3-5 parallel samples per group, and 24, 48, and 72 hours on three plates in a CO2 incubator.

[0067] 6. Observation of cell morphology and measurement of OD values ​​using the MTT method. Cell plates were removed at 48h, 72h, and 96h, respectively. Cell morphology was observed and recorded under an inverted microscope, and photographs were taken. Under low light conditions, the stock solution was discarded into each well, 100 μl of culture medium and 20 μl of MTT were added, and the plates were incubated at 37°C in a CO2 incubator for 4-5 hours. The stock solution was then discarded, 150 μl of DMSO was added, and the mixture was mixed for 8 minutes on a shaker. The OD value was measured with a 490 nm plate reader, the relative value added (RGR) of the cells was calculated, and cytotoxicity was evaluated. RGR (%) = (Experimental group OD - Blank group OD) / (Normal group OD - Blank group OD) * 100% 3.2 Hemolysis Experiment The hemolytic activity of Ac-SH-17 was detected using human erythrocytes (RBCs). All subjects were provided with written informed consent. Healthy human blood was collected in EDTA anticoagulant tubes. Equal portions of 200 μl of erythrocyte suspension were mixed with 200 μl of Ac-SH-17 solution at different concentrations (12.5, 25, 50, 100, and 200 μg / ml). After incubation at 37°C, the mixtures were centrifuged for 1 hour, and the supernatant was collected and added to a 96-orifice plate. Erythrocytes incubated with PBS, BSA solution (100 μg / ml), or 0.1% Triton X-100 solution were used as blank, negative, and positive controls, respectively. Absorbance was measured at 540 nm under a microplate reader. Three parallel samples were set up for each experimental group, and the experiment was repeated at least three times. Significant difference analysis was performed on the results using the two-way ANOVA method, with p < 0.05 being considered statistically significant.

[0068] Table 3.1 Hemolytic analysis Hemolytic activity of polypeptide [Table 1] Table 3.1 shows that Ac-SH-17 does not exhibit significant hemolytic activity compared to the positive control group. Therefore, it is considered that Ac-SH-17 does not have hemolytic activity against mammalian blood cells.

[0069] Example 4: Antimicrobial peptide anti-inflammatory experiment NO is a diffusible, low-membrane-permeable molecule obtained when NOS oxidizes L-arginine. In vivo, NO is involved in many physiological reactions. At high concentrations, NO is cytotoxic and has antibacterial and insecticidal effects in mammalian cellular immunity. However, at low concentrations, it can play a signaling role, positively and negatively regulating and controlling innate and acquired immunity. Therefore, detecting the concentration of NO released from cells can detect cellular inflammatory responses.

[0070] RAW264.7 mouse macrophage cell lines were used as experimental material and cultured in DMEM hypersaccharide medium containing FBS (10%) and penicillin streptomycin secondary antibody (100 U / ml). Cells were used in experiments when they were stable, well-formed, and had clear edges.

[0071] 4.1 Cell Processing 1. Remove the culture medium from the cells and rinse twice with room temperature PBS to remove excess medium. Then, add 0.25% trypsin and digest for 3 minutes. Gently tap the culture bottle to dislodge the cells, then add the culture medium to complete the digestion process. Tap the bottle repeatedly to homogenize the cells.

[0072] 2. Take a brand new, sterile 6-orifice plate (Corning, cargo number 3516), place 2 ml of cell suspension in each well, and gently shake the 6-orifice plate to evenly distribute the cells. Then, incubate the 6-orifice plate in a 37°C incubator for 4 hours, ensuring that the cells adhere well to the walls and reach a density of 1.5 × 10⁻⁶. 6 Set to cells / ml

[0073] After 3-4 hours, the 6-orifice plate was removed, the culture medium was aspirated, and the plate was washed twice with PBS solution. Opti-MEM medium was then added and the plate was incubated for 6 hours to eliminate the effect of specific components in the serum on LPS.

[0074] 4. The cells were randomly divided into eight groups: (a) control group: normal cultured cells; (b) LPS-treated group: cells treated with LPS at a final concentration of 1 μg / ml; (c) BSA group: cells treated with 10 M BSA; (d) polypeptide group: cells treated with different concentrations of antimicrobial peptides; (e) LPS and BSA group: cells treated with LPS at a final concentration of 1 μg / ml and 10 M BSA; (f) LPS and polypeptide group: cells treated with LPS at a final concentration of 1 μg / ml and different concentrations of antimicrobial peptides.

[0075] 5. 4 μL LPS was added to the LPS-containing treatment group to bring the final systemic LPS concentration to 1 μg / ml. PBS and BSA were added to the corresponding cell population, and the cells were returned to the incubator and cultured for 24 hours to induce an inflammatory response. During this time, the state of the cells was observed. If tentacles appeared on the cells, activation was possible. In this case, the secretion of cellular inflammatory factors was relatively complex, making it unsuitable to continue the experiment.

[0076] 6. Remove the treated cells from the incubator, transfer the supernatant to a new sterile test tube, and store it in a refrigerator at -20°C for subsequent experiments.

[0077] 4.2 This experiment detected and improved NO using the method provided in the total nitric oxide content measurement kit. The specific method is as follows: 1. Sample preparation: Each supernatant was centrifuged at room temperature at 14000g in an ultrafiltration tube to remove proteins for 5 minutes, and the lower layer of clear liquid was retained for subsequent measurements.

[0078] 2. Dilution of the standard: 1 ml of sterile ddH2O was added to the KNO2 dry powder to prepare a 10 mM solution, which was then mixed uniformly for preparatory purposes and diluted to 2, 5, 10, 20, and 50 μmol / L standard solutions.

[0079] 3. Prepare the reagents: A. Add approximately 1 ml of sterile ddH2O to 5 mg of NADPH, mix by inverting the mixture, then add ddH2O to a final volume of 3 ml to prepare 2 mM NADPH. Except for the portion to be used immediately, the remaining NADPH solution must be dispensed immediately and stored at -80°C.

[0080] B.FAD, LDH, and Nitratereductase were dispensed separately and then stored at -80°C for reserve.

[0081] C. Nitratereductase was removed immediately before use, and the remaining reagents were placed in an ice bath after dissolution. GriessReagentI and GriessReagentII need to be balanced to room temperature before use.

[0082] 4. Detection of NO: A. 30 μl of sample or standard was collected in a 96-orifice plate, and the process was repeated three times for each sample.

[0083] B. 2.5 μl of NADPH, 5 μl of FAD, and 2.5 μl of nitratereductase were added to each sample and mixed thoroughly to ensure homogeneity. Care was taken to avoid hair aspiration during the sample addition process. The samples were incubated at 37°C for 15 minutes, or at room temperature (20-30°C) for 40 minutes.

[0084] C. Add 5 μl LDA Buffer and 5 μl LDH to each sample and mix thoroughly. Incubate at 37°C for 5 minutes, or at room temperature (20-30°C) for 20 minutes.

[0085] D. 25 μl each of GriessReagent I and GriessReagent II were added in order, mixed uniformly, and incubated at room temperature (20-30°C) for 10 minutes. Then, the absorbance at 540 nm was measured and the reading was recorded.

[0086] 5. A standard curve was created, and the concentration of nitric oxide in the sample was calculated.

[0087] Example 5: Antimicrobial peptide anti-pusodermemia experiment Pseudomycin (sepsis) is a systemic inflammatory response syndrome caused by an infectious agent. A rat model of enteric pyoderma was constructed by injecting rat fecal homogenate into the peritoneal cavity of model rats. Antimicrobial peptides were then injected into the peritoneal cavity, and the survival rate of mice within 10-48 hours was observed to evaluate the therapeutic effect of antimicrobial peptides on mouse pyoderma.

[0088] 5.1 The experimental animals were grouped together.

[0089] Sixty SD rats were randomly divided into six groups: Model Group 1, Model Group 2, Model Group 3, Model Group 4, Model Group 5, and a blank group, with 10 rats in each group. These six groups of rats were sequentially weighted and numbered.

[0090] 5.2 We performed duplication on a rat model of endotoxemia. 5.3 The supernatant of fecal homogenate was prepared.

[0091] After 60 SD rats were adapted to a 1-week rearing regimen, freshly formed feces were collected. After weighing the rat feces, an appropriate amount of saline solution was added, and the mixture was homogenized and thoroughly polished to produce a 10% rat fecal homogenate. The rat fecal homogenate was centrifuged for 5 minutes at a rotation speed of 3000 rpm to obtain the supernatant, and an appropriate amount of the supernatant was aspirated with a syringe for reserve.

[0092] 5.4 Manufacture a rat model.

[0093] Before constructing the model, 1-5 groups of model rats were fasted for 12 hours, and then injected intraperitoneally with 10% rat fecal homogenate supernatant at a dose of 1 ml / 200g. After the injection was completed, the vital signs and findings of these five groups of rats were observed. If these five groups of rats exhibited vital signs such as shriveling, reduced movement, lethargy, sparse hair, shivering, and obvious bloodstains around the nose, it indicated that the model had been successfully constructed. The rats that successfully formed the model were used as the subjects of this study.

[0094] Model mice were randomly divided into two groups: a model group and a treatment group. After successful model construction, the treatment group received 0.5 ml of antimicrobial peptide (80 μM) intraperitoneally. Both groups were given the same amount of water and feed and housed in separate cages. The activity and feeding status of all mice were observed.

[0095] Example 6: Cell system and culture thereof In this study, mouse epithelioid fibrillary cells L929 were used as experimental material and cultured in DMEM medium containing 10% FBS at a culture environment of 37°C and 4% CO2.

[0096] 6.1 Cell resuscitation 1. Remove the cryopreservation tubes from the liquid nitrogen tank and immediately dissolve them in a 37°C water bath (1-2 min).

[0097] 2. Add the culture medium (1 ml of cryopreservation solution + 4 ml of fresh culture medium), transfer to a culture bottle, and incubate at constant temperature. 3. The suspension of black dots was observed using an inverted microscope. 6.2 Cell Passaging 1. Culture medium (including suspension cells): The culture medium was drawn up with a pipette and transferred to a sterile centrifuge tube.

[0098] 2. Wall-covered portion: The cells were washed with 1 ml of PBS, the PBS was aspirated, and the culture medium from Step 1 was placed in a centrifuge tube to collect the cells. 3. Add 1 ml of trypsin to the culture bottle and allow it to digest in an incubator.

[0099] 4. Digestion time varies depending on the characteristics of the cells. Digestion can be completed when the cells are clearly contracted and curled up under a microscope, and the cells can slide out of the culture bottle. The culture bottle should not be tapped at any point during the process.

[0100] 5. Complete digestion with 3 ml of culture medium containing serum, blow off the digested cells from the cell suspension, and collect it in the centrifuge tube from step 1.

[0101] 6. Centrifuge the centrifuge tube at 1200 rpm (approximately 250 g) for 3 minutes. After centrifugation, discard the supernatant, add fresh culture medium, and resuspend. Divide the mixture into individual bottles as needed and incubate in an incubator. 6.3 Cryopreservation of Cells 1. Sterilize in a clean bench using UV sterilization for 30 minutes, ventilate for 10 minutes, preheat the culture medium, trypsin, and PBS to 37°C, and sterilize the equipment and cryopreservation tubes under high temperature and pressure. 2. Elute the cells. The liquid was aspirated from the cell culture vial, washed once with 3 ml of PBS, aspirated again, digested with 3 ml of 0.25% trypsin for 3 minutes, finished digestion with 2 ml of digestant, eluted the cells by repeatedly tapping, centrifuged at 1000 g for 3 minutes, allowed the cells to suspend and settle in 1.2 ml of cryopreservation solution, placed in a cryopreservation tube, sealed, refrigerated at 4°C for 30 minutes, refrigerated at 20°C for 1 hour, spent the night in an 80°C refrigerator, and stored in a liquid nitrogen tank. Example 7: Experimental Results As shown in Figures 1 and 2, the modified antimicrobial peptide Ac-SH-17 exhibited antimicrobial activity against two types of drug-sensitive bacterial strains. Furthermore, as shown in Figure 3, no significant toxic or damaging effect was observed on polypeptide AC-SH-17 at a concentration of 20 μM, suggesting that AC-SH-17 does not exhibit significant toxicity to normal mammalian tissue cells cultured in vitro.

[0102] In Figure 4, after stimulating and inducing a cellular inflammatory response with 1 μg / ml LPS, the NO release concentration reached 12 μM (M). Later, when different concentrations of antimicrobial peptides were added, NO release was significantly suppressed when the antimicrobial peptide concentration reached 80 μM, and the NO release concentration was found to be almost the same as that of the blank control. Therefore, it was concluded that antimicrobial peptides can effectively suppress cellular inflammatory responses under certain concentration conditions.

[0103] Figure 5 shows that after housing mice under the same conditions for 48 hours, the administered group significantly improved the survival rate of pyodermaceutical mice within 24 hours compared to the model group. Therefore, it is considered that the antimicrobial peptide Ac-SH-17 has good therapeutic and inhibitory properties against pyodermaceuticals.

Claims

1. Amino acid sequence Ac-SRMKKWAKIEKWRKWH-NH ２ A new antibacterial polypeptide containing

2. The aforementioned amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH ２ The novel antimicrobial polypeptide according to claim 1, wherein one and / or several amino acids can be substituted, and the antimicrobial activity of the substituted amino acid sequence is not significantly reduced, retains considerable antimicrobial activity, or has significantly enhanced antimicrobial activity.

3. The aforementioned amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH ２ The novel antimicrobial polypeptide according to claim 1, wherein one and / or several amino acids are added to the carboxyl group and / or amino group, and the antimicrobial activity of the increased amino acid sequence is not significantly reduced, retains a considerable amount of antimicrobial activity, or has significantly enhanced antimicrobial activity.

4. The aforementioned amino acid sequence Ac-SRMKKWAKIIEKWRKWH-NH ２ The novel antimicrobial polypeptide according to claim 1, wherein there is an increase or decrease of 1 to 5 amino acids, the increased or decreased amino acids can be concentrated or dispersed at any position in the amino acid sequence, and the antimicrobial activity of the modified amino acid sequence is not significantly reduced, retains a considerable amount of antimicrobial activity, or has significantly enhanced antimicrobial activity.

5. Amino acid sequence Ac-SRMKKWAKIEKWRKWH-NH ２ A novel antimicrobial polypeptide containing an amino acid sequence with 90-100% homology.

6. An antimicrobial pharmaceutical composition comprising an antimicrobial polypeptide according to any one or more of claims 1 to 5 and a pharmaceutically acceptable adjuvant.

7. An antimicrobial pharmaceutical composition further comprising other active ingredients different from the antimicrobial polypeptides described in any one or more of claims 1 to 5, wherein the other active ingredients may be antibiotics, immunomodulators, minerals, trace elements and / or vitamins.

8. Amino acid sequence Ac-SRMKKWAKIEKWRKWH-NH ２ A nucleic acid molecule encoding an antimicrobial polypeptide containing the above.

9. A host cell capable of expressing an antimicrobial polypeptide according to any one or more of claims 1 to 5.

10. Use of an antimicrobial polypeptide according to any one of claims 1 to 5 in the manufacture of a drug for treating a microbial infectious disease, including bacterial infectious diseases, viral infectious diseases, chlamydia infectious diseases, fungal infectious diseases, mycoplasma infectious diseases and / or rickettsial infectious diseases.

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