Antibacterial peptide M-389 and its antibacterial effect
Patent Information
- Application Number
- CN202611350637.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-02
- Publication Date
- 2026-11-03
AI Technical Summary
Existing antibiotics are becoming less effective due to drug resistance, necessitating the development of antimicrobial peptides with broad-spectrum antibacterial activity and low toxicity.
A polypeptide M-389 was designed and synthesized with the amino acid sequence MGILSKIGKSIGKQVLKNSKKKIAKEVIKYLTKKLLK. It was obtained through solid-phase synthesis and biological expression. Its amino acid sequence was optimized to improve antibacterial activity and reduce hemolytic effects. It was prepared into various pharmaceutical salt forms for inhibiting bacteria.
The peptide M-389 exhibits significant antibacterial activity against a variety of bacteria, especially Gram-positive and Gram-negative bacteria, and has low toxicity within the effective concentration range, showing potential as a novel antibacterial drug.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial control, specifically relating to an antimicrobial peptide M-389 and its antimicrobial effects. Background Technology
[0002] Faced with the increasingly serious problem of microbial resistance, the academic and industrial communities have a growing demand for novel antimicrobial active substances. Although traditional antibiotics have made significant contributions to the fight against infectious diseases over the past few decades, the resistance problems resulting from their long-term and extensive use cannot be ignored. Some common clinical strains have shown significant resistance to multiple antibiotics, seriously weakening the effectiveness of existing treatment options.
[0003] Antimicrobial peptides, a class of naturally occurring defensive molecules in the innate immune system of organisms, have attracted much attention due to their broad-spectrum antimicrobial activity. These polypeptide molecules are ubiquitous in various organisms in nature, from lower microorganisms to higher mammals, all of which produce different types of antimicrobial peptides. Their small molecular weight allows them to inhibit or kill various pathogens, including bacteria and fungi. Compared to traditional antibiotics, the action characteristics of antimicrobial peptides make it less likely to induce rapid drug resistance in microorganisms, showing promising potential as novel antimicrobial agents. However, natural antimicrobial peptides still face limitations in practical applications, such as short half-life, insufficient in vivo stability, and reduced activity in high-salt or acidic environments. These limitations require improvement through rational molecular modification, sequence optimization, or advanced delivery strategies to meet the needs of practical applications. Summary of the Invention
[0004] The main problem to be solved by this invention is how to obtain an antimicrobial peptide with broad-spectrum antibacterial activity and low toxicity.
[0005] To address the above problems, the present invention provides a polypeptide.
[0006] The amino acid sequence of the polypeptide provided by the present invention is: MGILSKIGKSIGKQVLKNSKKKIAKEVIKYLTKKLLK (SEQ ID No:1); the polypeptide is an antimicrobial peptide M-389, the molecular weight of the antimicrobial peptide M-389 is 4.16 kDa, the isoelectric point is 10.887, and the average hydrophobicity is -0.251.
[0007] The aforementioned polypeptides can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.
[0008] Those skilled in the art can readily mutate the nucleotide sequence encoding the polypeptide of the present invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the polypeptide isolated according to the present invention, as long as they encode the polypeptide and have the polypeptide function, are derived from and equivalent to the nucleotide sequence of the present invention.
[0009] In this article, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid or nucleotide sequences, then the identity value (%) can be obtained.
[0010] The pharmaceutical salts of the aforementioned polypeptides also fall within the scope of protection claimed in this invention.
[0011] The present invention also provides a medicament for inhibiting bacteria or treating bacterial infectious diseases, said medicament containing the polypeptide or the pharmaceutical salt described above.
[0012] The pharmaceutical salts of the polypeptides of the present invention include acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, and carbonate. rbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate, embonate, estolate, palmitate, esylate, pantothenate, fumarate, phosphate / diphosphate.Gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycolyllarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, triethyliodide, lactate, valerate, etc. Depending on their intended use, pharmaceutical salts can be formed from cations such as sodium, potassium, aluminum, calcium, lithium, manganese, zinc, and bismuth, or from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, and procaine.These salts are formed from diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide, among others. They can be prepared using standard methods, such as the reaction of free acids with organic or inorganic bases. In the presence of a basic group, such as an amino group, acidic salts such as hydrochloride, hydrobromide, acetate, and pamoate can be used as dosage forms; in the presence of an acidic group (such as -COOH) or an alcohol group, pharmaceutically acceptable esters such as acetate, maleate, and pivaloyloxymethyl acetate, as well as esters known in the literature for improving solubility and hydrolysis, can be used as sustained-release and prodrug formulations.
[0013] In addition to the polypeptide of SEQ ID No:1 or its pharmaceutically acceptable salt, the drug described above may also contain a suitable carrier or excipient. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Various dosage forms can be formulated using these materials, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems.
[0014] In this article, the bacteria referred to are either Gram-positive or Gram-negative bacteria.
[0015] Furthermore, the Gram-positive bacterium is *Bacillus stearothermii* (…). Bacillusspizizenii ) and Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp. aureus The Gram-negative bacteria mentioned are Escherichia coli (Escherichia coli). Escherichiacoli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Acinetobacter baumannii ( Acinetobacter baumannii ) and Klebsiella pneumoniae subspecies of pneumonia ( Klebsiella pneumoniae subsp. pneumoniae ).
[0016] Furthermore, the concentration of the polypeptide used in the drug is 8 to 128 μg / mL.
[0017] In one specific embodiment, the antimicrobial peptide M-389 is effective against Escherichia coli (E. coli). Escherichia coli ATCC25922 and Acinetobacter baumannii ( Acinetobacter baumannii The minimum inhibitory concentration (MIC) of ATCC 19606 is 8 μg / mL; The antimicrobial peptide M-389 is effective against Klebsiella pneumoniae subsp. pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae subsp. pneumoniae ATCC 13883 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The minimum inhibitory concentration (MIC) of ATCC 27853 is 16 μg / mL; The antimicrobial peptide M-389 is effective against Bacillus stearothermia ( Bacillus spizizenii ATCC 6633 and Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp. aureus The minimum inhibitory concentration (MIC) of ATCC 25923 is 128 μg / mL.
[0018] The methods for preparing antimicrobial peptides mentioned above include chemical synthesis or biosynthesis. The chemical synthesis method can be a solid-phase synthesis method. The biosynthesis method can include the following: 1) Biosynthesis method: synthesizing antimicrobial peptides through a microbial expression system; 2) DNA synthesis method: such as using a DNA synthesizer to synthesize antimicrobial peptide genes and producing antimicrobial peptides in Escherichia coli through fusion expression; 3) Amino acid residue substitution and chemical modification: improving the antibacterial effect and reducing hemolytic activity of natural antimicrobial peptides by designing and modifying them.
[0019] In one specific embodiment, the antimicrobial peptide is synthesized into a crude polypeptide by solid-phase synthesis; the polypeptide is then purified to obtain a purity greater than 95%.
[0020] This invention also provides the following biomaterials: C1) A nucleic acid molecule encoding the polypeptide described above; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2).
[0021] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.
[0022] The vectors described herein are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.
[0023] The use of the aforementioned polypeptides in the preparation of drugs for inhibiting bacteria or treating bacterial infectious diseases also falls within the scope of protection claimed in this invention.
[0024] The use of the medicinal salts described above in the preparation of drugs for inhibiting bacteria or treating bacterial infectious diseases also falls within the scope of protection claimed by this invention.
[0025] The present invention also provides the use of the foregoing drug in any of the following: 1) Application in the preparation of products for treating bacterial infectious diseases; 2) Application in the preparation of products for the treatment of bacterial infectious diseases; 3) Applications in the preparation of products that inhibit bacterial activity; 4) Application in the preparation of products for inhibiting bacterial activity. Attached Figure Description
[0026] Figure 1 The six-sided diagram is a prediction of the structure of the antimicrobial peptide M-389.
[0027] Figure 2 This is a schematic diagram of the HPLC detection results for the antimicrobial peptide M-389.
[0028] Figure 3 This is a schematic diagram of the MS detection results for the antimicrobial peptide M-389.
[0029] Figure 4 The results show the growth inhibition of various bacterial strains by the antimicrobial peptide M-389 at different concentrations.
[0030] Figure 5 The results of enzyme-linked immunosorbent assay (ELISA) analysis show the growth inhibition rate of antimicrobial peptide M-389 at different concentrations on various bacterial strains.
[0031] Figure 6 The results show the hemolytic effects of antimicrobial peptide M-389 on sheep erythrocytes.
[0032] Figure 7 The results show the hemolytic effects of antimicrobial peptide M-389 on mouse erythrocytes.
[0033] Figure 8 The results show the toxicity of the antimicrobial peptide M-389 to human embryonic kidney cells. Detailed Implementation
[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0036] The following examples use statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, and P < 0.001 (***) indicates a highly significant difference.
[0037] Example 1: Screening and physicochemical property study of antimicrobial peptide M-389 1. Screening of antimicrobial peptide M-389 To obtain high-quality antimicrobial peptides, sheep intestinal metagenomic data were collected from the NCBI database. Bioinformatics tools were used to assemble, bin, and remove redundancy from the data to obtain high-quality metagenome blocks (MAGs). Species annotation was performed using tools such as GTDB-TK, and further, tools such as Prokka were used to predict open reading frames (ORFs) to identify hypothetical proteins with unknown functions, providing foundational data for subsequent antimicrobial peptide screening. Language models such as ESMFold were used to predict the structures of hypothetical proteins, obtaining their PDB structural information. Subsequently, software such as PyMOL was used to convert the structures into six-sided images, which were then used as input data for deep learning models to screen for novel antimicrobial peptides.
[0038] Protein sequences and their three-dimensional structural information from positive and negative samples were collected from the UniProt and PDB databases and converted into six-sided images. Based on this data, a DeepSeek-JanUS antimicrobial peptide screening model was constructed, and the model was trained and fine-tuned by combining image and sequence information to improve its antimicrobial peptide recognition ability.
[0039] The ORFs (and their corresponding image information) mined from the metagenomic data were input into a trained deep learning model for antimicrobial peptide screening. Based on the model's predictions, candidate sequences were scored using evaluation software, and sequence alignment was performed using BLAST. The candidate antimicrobial peptide sequence M-389 (full name: MDINBBMK_01389_hypothetical_protein) with the highest score was selected for subsequent experimental validation. The amino acid sequence of antimicrobial peptide M-389 is: MGILSKIGKSIGKQVLKNSKKKIAKEVIKYLTKKLLK (SEQ ID No:1).
[0040] The sequence alignment results of the antimicrobial peptide are shown in Table 1. These results indicate that the sequence has no highly homologous sequences in known antimicrobial peptide databases (APD3, dbAMP, DRAMP). The model prediction results for antimicrobial peptide M-389 are shown in Table 2. The prediction results of the trained model are consistent with those of the online prediction website, both identifying the sequence as an antimicrobial peptide.
[0041] Table 1. Comparison results of antimicrobial peptide M-389 with antimicrobial peptide databases
[0042] Table 2. Prediction results of antimicrobial peptide M-389 model
[0043] 2. Physicochemical Properties Study of Antimicrobial Peptide M-389 The physicochemical properties of antimicrobial peptide M-389 are as follows: it contains 37 amino acids and has an extinction coefficient of 1490 M. -1 cm -1 The molecular weight is 4.16 kDa, isoelectric point is 10.887, and average hydrophobicity is -0.251. The tertiary structure of the screened antimicrobial peptides was predicted using an online prediction tool, and the results are as follows: Figure 1 As shown, M-389 contains an α-helical structure.
[0044] Example 2: Synthesis and purification of antimicrobial peptide M-389 Based on the designed amino acid sequence SEQ ID No:1, the crude polypeptide was synthesized by Shenyang Shengjing Biological Cell Research and Development Center Co., Ltd. using solid-phase synthesis; the polypeptide was then purified to obtain a purity greater than 95%.
[0045] The purity and molecular weight of the antimicrobial peptide M-389 were identified by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry (MS). HPLC detection was performed using a ChromCore 120 C18 5u column (4.6 × 250 mm) with gradient elution of 0.1% TFA aqueous solution (mobile phase A) and 0.1% TFA acetonitrile solution (mobile phase B) at a flow rate of 1.0 mL / min, a detection wavelength of 220 nm, and an injection volume of 10 μL.
[0046] Test results as follows Figure 2 As shown, the retention time of the target peak is 7.940 min, the peak shape is symmetrical and sharp, the main peak area accounts for 96.5895% of the total peak area, and the impurity peak content is low. Figure 2 This indicates that the purity of the antimicrobial peptide M-389 is greater than 95%, which meets the purity requirements for subsequent experiments.
[0047] Mass spectrometry was performed in ESI positive ion mode, and a series of multicharged protonated molecular ion peaks were acquired. Among them, m / z 520.50, 594.75, 693.65, 832.20, 1039.90 and 1386.25 were attributed to [M+8H]8+. 8+ [M+7H] 7+ [M+6H] 6+ [M+5H] 5+ [M+4H] 4+ and [M+3H] 3+ The molecular weights derived from the peaks of each charged ion state are highly consistent, with an average measured molecular weight of approximately 4156 Da. This is basically consistent with the theoretical molecular weight of the antimicrobial peptide M-389 (Mw = 4156.20 Da), and the error is within the allowable deviation range of the instrument. Figure 3 The combined HPLC and MS results confirm that the synthesized antimicrobial peptide M-389 has the correct molecular weight, correct structure, high purity, and meets the requirements for subsequent experiments.
[0048] Example 3: Antimicrobial activity of antimicrobial peptide M-389 The tested strains included: Escherichia coli ( Escherichia coli ATCC 25922 (bio-52502), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ATCC 27853 (bio-52506), Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp aureus ATCC 25923 (bio-52504), Klebsiella pneumoniae subspecies of pneumonia ( Klebsiella pneumoniae subsp pneumoniaeATCC 13883 (bio-107381), Acinetobacter baumannii ( Acinetobacter baumannii ATCC 19606 (bio-53250), Bacillus stearothermiae ( Bacillus spizizenii ATCC 6633 (bio-52499) was used as the test strain. All strains were purchased from Beijing BioBio Biotechnology Co., Ltd. The test method was the determination of the minimum inhibitory concentration (MIC), and the specific steps are as follows: The glycerol-preserved bacterial strains of the above six species were inoculated into cation-adjusted Mueller-Hinton broth (CaMHB) and cultured overnight at 37 ℃ with shaking at 180 rpm. The CaMHB medium used in the experiment was purchased from Qingdao HopeBio Co., Ltd. (product name: CAMHB broth, catalog number: HB6231-1). The medium was prepared according to the instructions: 22.0 g of medium powder was weighed, added to 1000 mL of distilled water, heated to dissolve, dispensed, and autoclaved at 116-121 ℃ for 10 min. After overnight culture, the bacterial suspension was diluted 1:100 with fresh CaMHB medium and cultured at 37 ℃ with shaking at 180 rpm until the exponential growth phase (OD200). 600 =0.6-0.8). The bacterial concentration was then adjusted to 1×10⁻⁶ using sterile CaMHB medium. 6 CFU / mL, used for subsequent experiments.
[0049] The dilution method for antimicrobial peptide M-389 is the two-fold dilution method. First, 5 mg of lyophilized antimicrobial peptide M-389 is dissolved in 1 mL of sterile enzyme-free water to prepare a 5 mg / mL antimicrobial peptide stock solution. Then, it is serially diluted with sterile enzyme-free water to obtain working solutions of different concentrations.
[0050] MIC determination was performed using the microbroth dilution method in 96-well plates. Well 1 contained 200 μL of sterile, enzyme-free water as a blank control; well 2 contained 200 μL of bacterial culture as a growth control; well 3 contained 100 μL of sterile, enzyme-free water and 100 μL of bacterial culture as a negative control; well 4 contained 100 μL of antibiotic solution and 100 μL of bacterial culture as a positive control, with the antibiotic being either kanamycin (KAN) or tetracycline (TET), both at a final concentration of 128 μg / mL. Wells 5 through 12 contained 100 μL of antimicrobial peptide solution at different concentrations and 100 μL of bacterial culture, respectively. The antimicrobial peptide was serially diluted twofold, with final concentrations of 512, 256, 128, 64, 32, 16, 8, and 4 μg / mL, respectively. The final bacterial concentration in each well was 5 × 10⁻⁶.5 CFU / mL. After incubating 96-well plates at 37 °C for 16-18 h, bacterial growth was observed visually. The lowest concentration of antimicrobial peptide that completely inhibited visible bacterial growth was defined as the minimum inhibitory concentration (MIC). Figure 4 All experiments were performed with three technical replicates.
[0051] Simultaneously, the absorbance at 600 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The growth inhibition rate was calculated using the following formula: Inhibition rate (%) = 1 - [(OD200) / 200] 样品 -OD 空白对照 ) / (OD 阴性对照 -OD 空白对照 Growth inhibition rate ≥80% and the result is statistically significant are judged as having an inhibitory effect.
[0052] Experimental results are as follows Figure 4 and Figure 5 As shown: Antimicrobial peptide M-389 against Escherichia coli ( Escherichia coli ATCC25922 and Acinetobacter baumannii ( Acinetobacter baumannii The minimum inhibitory concentration (MIC) of ATCC 19606 is 8 μg / mL; against Klebsiella pneumoniae subsp. pneumoniae (… Klebsiella pneumoniae subsp pneumoniae ATCC13883 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The minimum inhibitory concentration (MIC) of ATCC 27853 is 16 μg / mL; against Bacillus stearothermia (… Bacillus spizizenii ATCC 6633 and Staphylococcus aureus subsp. aureus ( Staphylococcus aureus subsp aureus The minimum inhibitory concentration (MIC) of ATCC 25923 is 128 μg / mL.
[0053] Example 4: Biosafety of antimicrobial peptide M-389 To evaluate the biosafety of the antimicrobial peptide M-389, its hemolytic activity and mammalian cytotoxicity were detected using erythrocyte hemolysis and human embryonic kidney cell (HEK-293) toxicity assays. The experimental materials were as follows: 4% sheep erythrocytes (Beijing Solarbio Science & Technology Co., Ltd., catalog number: S9450), 4% mouse erythrocytes (Beijing Solarbio Science & Technology Co., Ltd., catalog number: S9551), and human embryonic kidney cells HEK-293 (STR) (Hyfehring Biotechnology Co., Ltd., catalog number: FH0242). The specific experimental procedures are as follows: Fresh 4% sheep red blood cells and 4% mouse red blood cells were collected, washed with sterile PBS, and centrifuged three times at 1000×g at 4 ℃ until the supernatant was clear. The red blood cells were collected and prepared into a 2% red blood cell suspension with PBS. The red blood cell suspension was mixed with an equal volume of antimicrobial peptide M-389 solution at different final concentrations (2, 4, 8, 16, 32, 64, 128, 256 μg / mL). A negative control (equal volume of PBS, 0% hemolysis) and a positive control (equal volume of 2% Triton X-100) were also included. After incubation at 37 ℃ for 1 h, the mixture was centrifuged at 1000×g for 10 min, and the absorbance at 540 nm was measured. Hemolysis rate (%) = (OD 样品 OD 阴性对照 ) / (OD 阳性对照 OD 阴性对照 () × 100%. Each group has 3 replicates.
[0054] The results are as follows: At a concentration of 256 μg / mL, the hemolytic rate of antimicrobial peptide M-389 on sheep erythrocytes was 0.5% ( Figure 6 The hemolysis rate of mouse erythrocytes was 0.7% ( ). Figure 7 The results showed that within the effective antibacterial concentration range, the hemolytic activity of antimicrobial peptide M-389 was low, and its safety to mammalian erythrocytes at a concentration of 256 μg / mL was within acceptable limits.
[0055] The toxicity of antimicrobial peptide M-389 to human embryonic kidney 293 (HEK-293) cells was determined using the CCK-8 assay. HEK-293 cells in logarithmic growth phase were seeded at 5000 cells / well in 96-well plates and cultured at 37 ℃ in a 5% CO2 incubator for 24 h. After cell adhesion, the culture medium was discarded, and culture medium containing different final concentrations (16, 32, 64, 128, 256, 512 μg / mL) of antimicrobial peptide M-389 was added to each well. Control wells without antimicrobial peptide and blank wells without cells were also included. Cells were cultured for another 24 h. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 ℃ in the dark for 2 h. The absorbance at 450 nm was then measured. Cell viability (%) = (OD) / (Cell viability percentage). 样品 -OD 空白 ) / (OD 细胞对照 -OD 空白 () × 100%. Each group has 3 replicates.
[0056] The results are as follows Figure 8As shown, at a concentration of 16 μg / mL, the survival rate of HEK-293 cells remained above 50%. In summary, the results of both hemolysis and cytotoxicity assays indicate that the antimicrobial peptide M-389 exhibits low toxicity / hemolysis at low concentrations, which increases significantly with increasing concentration, thus providing a safe basis for its development as an antimicrobial drug.
[0057] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A polypeptide, characterized in that, The polypeptide is the polypeptide whose amino acid sequence is SEQ ID No:1 in the sequence listing.
2. The pharmaceutical salt of the polypeptide according to claim 1.
3. A drug for inhibiting bacteria or treating bacterial infectious diseases, characterized in that, The drug contains the polypeptide of claim 1 or the pharmaceutical salt of claim 2.
4. The drug according to claim 3, characterized in that, The bacteria is Bacillus stearothermiae (Bacillus stearothermiae) Bacillusspizizenii Staphylococcus aureus subsp. aureus Staphylococcus aureus subsp. aureus ), Escherichia coli ( Escherichiacoli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Acinetobacter baumannii ( Acinetobacter baumannii Klebsiella pneumoniae subsp. pneumoniae Klebsiella pneumoniae subsp. pneumoniae ).
5. The drug according to claim 3 or 4, characterized in that, The concentration of the polypeptide used in the drug is 8 to 128 μg / mL.
6. A biomaterial, characterized in that, The biomaterial is any one of the following: C1) A nucleic acid molecule encoding the polypeptide of claim 1; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette described in C2).
7. The use of the polypeptide of claim 1 in the preparation of a medicament for inhibiting bacteria or treating bacterial infectious diseases.
8. The use of the pharmaceutical salt according to claim 2 in the preparation of a medicament for inhibiting bacteria or treating bacterial infectious diseases.
9. The use of the medicament according to any one of claims 3-5 in any of the following: 1) Application in the preparation of products for treating bacterial infectious diseases; 2) Application in the preparation of products for the treatment of bacterial infectious diseases; 3) Applications in the preparation of products that inhibit bacterial activity; 4) Application in the preparation of products for inhibiting bacterial activity.