CN116874614A - Antibacterial polypeptide APH171 with high activity and low cracking effect, and preparation method and application thereof - Google Patents
Antibacterial polypeptide APH171 with high activity and low cracking effect, and preparation method and application thereof Download PDFInfo
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- CN116874614A CN116874614A CN202311142929.3A CN202311142929A CN116874614A CN 116874614 A CN116874614 A CN 116874614A CN 202311142929 A CN202311142929 A CN 202311142929A CN 116874614 A CN116874614 A CN 116874614A
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- aph171
- polypeptide
- lys
- antibacterial
- high activity
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- 230000000694 effects Effects 0.000 title claims abstract description 35
- 238000005336 cracking Methods 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
-
- 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
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Insects & Arthropods (AREA)
- Tropical Medicine & Parasitology (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an antibacterial polypeptide APH171 with high activity and low cracking effect, a preparation method and application thereof, wherein the antibacterial polypeptide APH171 has the sequence as follows: lys-Ile-Lys-Leu-Phe-Lys-Lys-Ile-Lys-Phe-Leu-His-Lys-Ala-Leu-Lys-Phe-NH 2 . The antibacterial polypeptide APH171 prepared by the invention not only maintains the original antibacterial activity of the polypeptide P18, but also obviously enhances the antibacterial activity on staphylococcus aureus; in addition, in the case of the optical fiber,has extremely low cracking effect on erythrocytes, remarkably enhances the therapeutic index of APH171, and is expected to become a novel medicament for treating infectious diseases.
Description
Technical Field
The invention belongs to the field of biological polypeptide medicines, and particularly relates to an antibacterial polypeptide APH171 with high activity and low cracking effect, and a preparation method and application thereof.
Background
In recent years, bacterial drug resistance caused by abuse of antibiotics is highly concerned, and novel antibiotic development difficulty is great, and antibacterial peptides which are not easy to cause bacterial drug resistance are becoming research hotspots of vast researchers.
Antibacterial peptides (AMPs), also known as host defenses, are a class of polypeptides that are produced by the body when it is exposed to pathogen invasion. Antibacterial peptides tend to be rich in lysine and arginine and are cationic in nature, with secondary structures typically being α -helical, β -sheet and random coil. Most of the antibacterial peptides have good antibacterial activity against gram-positive bacteria, gram-negative bacteria and fungi; in addition, part of the antibacterial peptide also has the functions of resisting tumor, regulating immunity and the like. The antibacterial peptide has extremely complex antibacterial mechanism, but is mainly combined with bacterial cell membranes to destroy the cell membranes to achieve the aim of sterilization, so that the antibacterial peptide is not easy to generate drug resistance, has great advantages compared with the existing antibiotics easy to generate drug resistance, and is expected to become a novel antibacterial drug for replacing antibiotics.
P18（KWKLFKKIPKFLHLAKKF-NH 2 ) The derivative peptide is a natural antibacterial peptide Cecropin A and Magainin 2 hybrid peptide, has strong antibacterial activity, but also has certain toxicity and has strong cracking effect on erythrocytes. Therefore, a novel antibacterial peptide with good antibacterial effect and low toxicity is required to be obtained through structural transformation and optimization.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention aims to obtain the novel antibacterial peptide with good antibacterial effect and low toxicity through structural transformation and optimization. Specifically, the invention provides an antibacterial polypeptide APH171 with high activity and low cracking effect, and the antibacterial polypeptide APH171 not only maintains the original antibacterial activity of P18, but also obviously enhances the antibacterial activity to staphylococcus aureus; in addition, the therapeutic index of APH171 is significantly enhanced by having very low lysis on erythrocytes. APH171 is expected to be a novel drug for treating bacterial infection.
The invention also provides a preparation method and application of the antibacterial polypeptide APH171.
The technical scheme is as follows: in order to achieve the above objective, the antibacterial polypeptide APH171 with high activity and low cleavage effect of the present invention has the sequence: lys-Ile-Lys-Leu-Phe-Lys-Lys-Ile-Lys-Phe-Leu-His-Lys-Ala-Leu-Lys-Phe-NH 2 。
Wherein, the polypeptide APH171 takes the polypeptide P18 as a template, and the amino acid W, P, L, K of the 2 nd, 9 th, 14 th and 16 th of the P18 are mutated into the amino acid I, K, K, L to obtain the APH171.
The invention relates to a preparation method of an antibacterial polypeptide APH171 with high activity and low cracking effect, which uses polypeptide P18 (KWKLFKKIPKFLHLAKKF-NH) 2 ) As templates, amino acids W, P, L, K of P18 at positions 2, 9, 14 and 16 were mutated to amino acid I, K, K, L, respectively, and APH171 was synthesized by using a solid phase peptide synthesis method.
The invention relates to an application of an antibacterial polypeptide APH171 with high activity and low cracking effect in preparing an anti-pathogenic bacteria infection medicament.
Wherein the anti-pathogenic bacterial infection drug is an anti-pathogenic bacterial infection drug.
Wherein the pathogenic bacteria are one or more of staphylococcus aureus, escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa and micrococcus luteus.
The application of the antibacterial polypeptide APH171 with high activity and low cracking effect in preparing medicaments for resisting pathogenic bacterial infection with low cracking effect on erythrocytes is provided.
The invention relates to a medicinal composition for resisting pathogenic bacteria infection, which comprises an antibacterial polypeptide APH171 and a pharmaceutically acceptable carrier thereof.
Wherein the pharmaceutical composition is in the form of capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.
The pathogenic bacteria are one or more of staphylococcus aureus, escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa and micrococcus luteus.
The invention combines the relation between the hydrophobicity, amphipathy and secondary structure of the antibacterial peptide and the activity and toxicity of the antibacterial peptide to design the antibacterial peptide. APH171 not only maintains the original antibacterial activity of P18, but also significantly enhances the antibacterial activity against Staphylococcus aureus; in addition, the composition has extremely low cracking effect on erythrocytes, remarkably enhances the therapeutic index of APH171, and is expected to be developed into clinically used antibacterial drugs.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. compared with the polypeptide P18, the antibacterial polypeptide APH171 prepared by the invention has better broad-spectrum antibacterial activity, not only has obvious activity on escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa and micrococcus luteus, but also obviously enhances the antibacterial activity on staphylococcus aureus.
2. The antibacterial polypeptide APH171 has broad-spectrum antibacterial activity, small toxic and side effects on normal cells, extremely low cracking effect on erythrocytes, good stability and the like, and is expected to become a novel medicament for treating bacterial infection.
3. The design and preparation method of the antibacterial polypeptide APH171 is simple and convenient, novel in design, easy in raw material source and applicable to industrial production.
Drawings
FIG. 1 shows helix projection of the antimicrobial polypeptide P18 and its derivative peptides:
FIG. 2 secondary structure of antibacterial polypeptide P18 and its derivative peptides predicted by alphafold 2;
FIG. 3 is a reverse liquid chromatogram of antimicrobial polypeptide APH 171;
FIG. 4 shows a mass spectrum of the antimicrobial polypeptide APH 171;
FIG. 5 cleavage of murine erythrocytes by antibacterial polypeptide P18 and its derivative peptides;
FIG. 6 haemolysis at an APH171 concentration of 256. Mu.g/mL for antibacterial polypeptide P18.
Detailed Description
The invention is further described below with reference to examples and figures.
The experimental methods described in the examples, unless otherwise specified, are all conventional; the reagents and materials, unless otherwise specified, are commercially available.
The polypeptide designed in the invention is synthesized directly by biological company, or can be synthesized according to the existing polypeptide synthesis method.
Example 1
Derived peptide P18 (KWKLFKKIPKFLHLAKKF-NH) obtained by hybridizing natural antibacterial peptide Cecropin A with Magainin 2 2 ) As templates, APH171 was synthesized using a solid phase peptide synthesis method, substituting the nonpolar amino acid alanine (a) in the sequence with the basic amino acid arginine (R), substituting W, P, L, K of P18 with I, K, K, L, and then substituting 2, 9, 14, 16.
An antimicrobial polypeptide APH171, having the sequence: lys-Ile-Lys-Leu-Phe-Lys-Lys-Ile-Lys-Phe-Leu-His-Lys-Ala-Leu-Lys-Phe-NH 2 。
Solid phase synthesis method for synthesizing polypeptide APH171
Synthesis of the polypeptide: the synthesis of polypeptide APH171 proceeds from C-terminal to N-terminal one by one. Soaking Fmoc-Phe-Rink Resin in dichloromethane for 15 min, and pumping out dichloromethane after the Resin swells; a1:4 volume ratio of piperidine/DMF solution (10 per gram of resin mL) was added, nitrogen was introduced, the reaction was carried out 2 times for 5 min and 15 min, and after the reaction was completed, the resin was washed 6 times with DMF. Adding 2-3 drops of color-testing agent ABC (solution A: ninhydrin/absolute ethanol solution; solution B: pyridine; solution C: phenol/absolute ethanol solution) into a small amount of washed resin, and heating at 100deg.C for 3 min to obtain blue solution and resin, which indicates that amino protection is completely removed. Fmoc-Lys-Phe-OH and HOBT were added in an excess of twice the number of moles, dissolved in DMF 10 per gram of resin ml, DIC and Collidine were added in twice the number of moles, nitrogen sparged, and reaction 1 h. After the reaction, the resin is washed by DMF for 6 times, the condensation reaction is repeatedly carried out, all Fmoc protected amino acids are sequentially connected, the synthesis of the linear sequence is completed, and the resin is soaked by dichloromethane and diethyl ether and then is pumped out. TFA was added and reacted in a constant temperature shaker at a speed of 110 r/min and a temperature of 25℃for 2 h. Filtering to remove resin, adding anhydrous diethyl ether into the filtrate, centrifuging to obtain solid, adding anhydrous diethyl ether for washing, centrifuging again, repeating for several times, and oven drying to obtain APH171 crude polypeptide.
Purification of the polypeptide: weighing a certain amount of crude product, adding a proper amount of acetonitrile, performing ultrasonic treatment until the crude product is clarified, removing large-particle impurities by using a filter, and then performing preparative liquid chromatograph to collect samples in sections. The sample with the required purity is reserved by using an analytical chromatograph for gradient analysis. Then, freeze-drying treatment was performed.
Purity determination of polypeptide (HPLC method) and mass spectrometry analysis results: and (3) synthesizing the polypeptide, purifying to obtain a finished product, and identifying the finished product by high performance liquid chromatography and mass spectrometry.
Liquid chromatography conditions: c18 chromatographic column (4.6x250 mm, 5 μm); mobile phase a was acetonitrile containing 0.1% trifluoroacetic acid and mobile phase B was purified water containing 0.1% tfa. The detection wavelength is 220 nm; the flow rate is 1.0 ml/min; and (5) carrying out gradient elution with a sample injection amount of 20 mu l.
The amino acid sequence of the polypeptide is shown in SEQ ID NO:1, the purity is more than 95%. The molecular weight of APH171 is: 2257.49; the spiral projection diagram and the alpha fold2 predicted secondary structure of the antibacterial polypeptide APH171 are shown in figures 1 and 2 respectively: HPLC and MS are shown in FIGS. 3 and 4, respectively, and are consistent with theory.
Example 2
Determination of in vitro antibacterial Activity of polypeptide APH171 of the invention
The strains involved in this experiment were Pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii and Staphylococcus aureus.
The experimental method comprises the following steps:
1. configuration of culture Medium
Taking 24g of MHB culture medium, adding into 1000ml of distilled water, heating, boiling for dissolving, and packaging.
2. Preparation and sterilization of laboratory instruments
Placing MHB culture medium, matched gun head, gun discharge groove and test tube together into autoclave, sterilizing at 121deg.C for 20min. The ultra-clean workbench and the operation room are sterilized by ultraviolet lamp for more than 30min before use.
3. Preparation of antibacterial peptide mother liquor
Weighing appropriate amount of polypeptide, dissolving with physiological saline, preparing 1024 μg/ml mother liquor, filtering with 0.22 μm water phase filter head, sterilizing, packaging, and storing at 4deg.C (after use within one week). For a sample insoluble or poorly soluble in physiological saline, DMSO may be used at an appropriate concentration for dissolution, depending on its characteristics.
4. Preparation of bacterial suspension
Extracting strain preserved with glycerol from-80deg.C refrigerator, adding 200 μL of strain solution into 4mL MHB culture medium, culturing in shaking table at 37deg.C for 16 hr for activation, collecting activated strain suspension, transferring 500 μL of strain suspension into 2.5mL MHB, culturing for 4-6 hr (the objective of this step is to obtain logarithmic growth phase of bacteria, which is selected according to bacterial growth curve), and diluting the strain suspension with MHB to OD 600 =0.3 (colony count about 10) 8 CFU/mL).
5. Sample dilution and addition of bacteria
Adding 100 mu L of MHB broth culture medium into each well of a 96-well plate, then carrying out double dilution on the sample, namely adding 100 mu L of the sample into the first well of three rows A/B/C, fully blowing (at least three times) the sample and broth by a row gun to fully mix the sample and broth, then sucking 100 mu L of the sample into the second well, fully blowing again and mixing the sample and broth, repeating until the last well, and then adding 100 mu L of diluted bacterial liquid into each well to ensure that the colony number of a final system is 5 multiplied by 10 5 CFU was repeated three times (three A/B/C rows).
Meanwhile, the 10 th column of the same plate was blank (medium only), 11 th column was negative (bacteria only), and 12 th column was positive (antibacterial peptide P18).
6. Observation result
The 96-well plate was placed in a constant temperature incubator at 37℃for 16-20 hours, and the results were observed, and the lowest sample concentration at which no growing bacteria were visible to the naked eye was designated as MIC, and the results are shown in Table 1.
As can be seen from Table 1, APH171 has good antibacterial activity, and has remarkable activity on Escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa and Micrococcus luteus, MIC values are below 10 mug/ml, and meanwhile, compared with antibacterial polypeptide P18, the antibacterial activity on Staphylococcus aureus can be remarkably enhanced, and the antibacterial activity on the Micrococcus luteus is better.
Example 3
Polypeptide APH171 hemolytic Activity assay of the invention
The experimental method comprises the following steps:
fresh Red Blood Cells (RBCs) were collected from ICR mice. RBCs were washed at least 3 times (after three centrifugation, approximately 3ml in volume) with 0.01mM PBS buffer until no color was visible in the suspension. RBCs were then diluted with PBS to obtain a volume fraction of 2.0% RBCs solution. 100 μl RBCs were mixed with an equal volume of the antimicrobial peptide APH171 to give final polypeptide concentrations of: 256. 128, 64, 32, 16. Mu.g/ml, incubated at 37℃for 1 hour. RBCs after 1 hour incubation with the samples were centrifuged (1500×g, 5 min) to collect supernatant, and 0.1ml of supernatant was transferred to a 96 well flat bottom plate, and a mixture of 2% triton X-100 (Sigma-Aldrich) solution and an equal volume of 2.0% red blood cell solution was used as positive control, and a mixture of PBS and an equal volume of 2.0% red blood cell solution was used as negative control. Hemoglobin absorbance (OD) was measured at 570 nm using a microplate reader 570 ). The formula is as follows:
hemolysis ratio (%) = [ (A-A) 0 ）/（A 100 -A 0 ）]×100。
A represents the absorbance of group APH171. A is that 0 Represents the absorbance of the PBS group, A 100 The absorbance of Triton X-100 group is shown.
Three independent replicates were performed. Hemolytic activity of APH171 and P18 on mouse Red Blood Cells (RBCs) was evaluated, and experimental results are shown in fig. 5 and 6. At a concentration of 256 μg/mL, APH171 hemolytic toxicity was significantly reduced (P < 0.05) compared to P18, and there was no hemolytic toxicity within the pharmaceutically effective dose range.
Example 4
1. Experiment of pH stability
The experimental method comprises the following steps:
the polypeptide APH171 solutions prepared in example 2 were placed under different pH conditions (ph=3, 5, 7 and 9) for 2 hours, respectively, and then an experiment (pH adjustment of sample solution with HCl and NaOH) was performed according to the MIC determination method of example 2 described above, and the experimental results are shown in table 2.
The results in Table 2 show that APH17 has no effect on the activity of Klebsiella pneumoniae at all four pH conditions tested, and still has an activity of 2 μg/ml, indicating that the polypeptide APH17 of the invention has excellent pH stability.
Example 5
Comparison analysis of antimicrobial polypeptide APH171 with other antimicrobial polypeptides
The invention uses the alpha-helical antibacterial peptide P18 (KWKLFKKIPKFLHLAKKF-NH) with 18 amino acids 2 ) Designing new antibacterial peptide for the template. The secondary structure was predicted by AlphaFold2 (fig. 2), and it was found that it did not have a complete alpha helix structure, and that amino acids following proline at position 9 were not alpha helices. According to the invention, leucine and arginine are respectively used for replacing proline at the 9 th position on the basis of P18 to obtain APH11-1 and APH11-2, and the antibacterial activity of the APH11-1 and APH11-2 on the staphylococcus aureus is improved by 4 times compared with that of parent peptide by measuring that the APH11-1 and APH11-2 have higher spiral degrees; however, where APH11-1 is significantly more toxic than P18, APH11-2 is comparable to P18, probably due to the too high hydrophobicity of APH11-1 (Table 3). However, further researches find that the antibacterial activity and toxicity of the antibacterial peptide are influenced by the secondary structure and amphipathy of the antibacterial peptide, and the amphipathy of the P18 antibacterial peptide is further designed to obtain APH79-APH83 (shown in figure 1) based on spiral projectionThe figure adjusts the positions of hydrophilic and hydrophobic amino acids of the hydrophilic surface and the hydrophobic surface of the antibacterial peptide, and balances the activity and toxicity of the antibacterial peptide. Wherein, APH80 is obtained by inserting hydrophilic amino acid on the hydrophobic surface, the hydrophobicity of the APH80 is equal to that of P18, and the amphipathy of the APH80 is higher than that of parent peptide (table 3), so that the antibacterial activity of the APH80 on staphylococcus aureus is obviously enhanced, but the APH has higher hemolysis rate at the same time; although APH79 has the strongest amphiphilicity in the series of peptides, the antibacterial activity is not improved compared with P18, but the antibacterial activity is lower than APH80, which means that the amphiphilicity of the antibacterial peptide and the antibacterial activity are not in a simple linear relationship and have an optimal range. The specific parameters of the antibacterial peptide are not independent through multiple rounds of design, if the peptide with low toxicity and high activity is obtained, the relation among all factors is required to be comprehensively considered, and specific mutation is carried out, wherein APH119 and APH171 are peptides which are designed by combining all factors, wherein the antibacterial activity of APH119 in the peptides is strongest, but the erythrocyte lysis capacity of the peptides is also very strong; the invention then replaces tryptophan (W) at position 2 with specific isoleucine (i.e., polypeptide APH171 of the invention) to significantly reduce the erythrocyte lysis capacity, but maintains its original antibacterial property (its related parameters are shown in table 4), while other positions (tryptophan other than position 2) are replaced with other similar amino acids, which cannot significantly reduce the erythrocyte lysis capacity, nor maintain its original antibacterial property.
Table 3 below shows a comparison of the properties of the antimicrobial polypeptide APH171 with other antimicrobial polypeptides.
Table 4 below shows the MIC values of the antimicrobial polypeptide APH171 and of the different antimicrobial polypeptides for the four bacteria Acinetobacter baumannii ATCC19606, klebsiella pneumoniae ATCC10031, staphylococcus aureus ATCC25923 and Pseudomonas aeruginosa ATCC27853, as determined in example 2. The results show that: APH171 was significantly more active against staphylococcus aureus than P18.
Further, the hemolytic activity of the antibacterial polypeptide APH171 and other antibacterial polypeptides was measured as in example 3, and the results are shown in fig. 5. The results show that: even if the hemolysis rate of APH171 is lower than 10% at high concentration of 256 mug/mL, the effect is significantly better than other antibacterial peptides.
Claims (10)
1. An antibacterial polypeptide APH171 with high activity and low cracking effect, which is characterized in that the sequence is as follows: lys-Ile-Lys-Leu-Phe-Lys-Lys-Ile-Lys-Phe-Leu-His-Lys-Ala-Leu-Lys-Phe-NH 2 。
2. The antibacterial polypeptide APH171 with high activity and low cleavage efficiency according to claim 1, wherein the polypeptide APH171 uses the polypeptide P18 as a template, and the amino acids W, P, L, K of the 2 nd, 9 th, 14 th and 16 th of the P18 are mutated into the amino acids I, K, K, L to obtain the APH171.
3. A method for preparing an antibacterial polypeptide APH171 with high activity and low cleavage effect according to claim 1, wherein the derivative peptide P18 (KWKLFKKIPKFLHLAKKF-NH) of the natural antibacterial peptide Cecropin A and Magainin 2 hybrid peptide is used 2 ) As templates, amino acids W, P, L, K of P18 at positions 2, 9, 14 and 16 were mutated to amino acid I, K, K, L, respectively, and APH171 was synthesized by using a solid phase peptide synthesis method.
4. Use of an antimicrobial polypeptide APH171 with high activity and low cleavage efficiency according to claim 1 for the preparation of a medicament for combating pathogenic bacterial infections.
5. The use according to claim 4, wherein the antipathogenic bacterial infection drug is an antipathogenic bacterial infection drug.
6. The use according to claim 4, wherein the pathogenic bacteria are one or more of staphylococcus aureus, escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa, micrococcus luteus.
7. Use of an antimicrobial polypeptide APH171 with high activity and low lytic effect according to claim 1 for the preparation of a medicament against pathogenic bacterial infection with low lytic effect on erythrocytes.
8. A pharmaceutical composition for combating pathogenic bacterial infection comprising the antimicrobial polypeptide APH171 of claim 1 and a pharmaceutically acceptable carrier therefor.
9. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository, or patch.
10. Use of the pharmaceutical composition for resisting pathogenic bacteria infection according to claim 8 in the preparation of medicines for resisting pathogenic bacteria infection with low lysis effect on erythrocytes, wherein the pathogenic bacteria is one or more of staphylococcus aureus, escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa and micrococcus luteus.
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| CN101903011A (en) * | 2007-12-21 | 2010-12-01 | 巴斯夫欧洲公司 | The anti-dandruff compositions that contains peptide |
| US20120065126A1 (en) * | 2009-05-15 | 2012-03-15 | Basf Se | Pharmaceutical Compositions Containing Antifungal Peptides |
| WO2013039857A1 (en) * | 2011-09-12 | 2013-03-21 | modeRNA Therapeutics | Engineered nucleic acids and methods of use thereof |
| CN105017384A (en) * | 2015-07-13 | 2015-11-04 | 长春普莱医药生物技术有限公司 | Novel antibacterial peptide and application thereof |
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| CN101903011A (en) * | 2007-12-21 | 2010-12-01 | 巴斯夫欧洲公司 | The anti-dandruff compositions that contains peptide |
| US20120065126A1 (en) * | 2009-05-15 | 2012-03-15 | Basf Se | Pharmaceutical Compositions Containing Antifungal Peptides |
| WO2013039857A1 (en) * | 2011-09-12 | 2013-03-21 | modeRNA Therapeutics | Engineered nucleic acids and methods of use thereof |
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