Anoprolin modified peptide with strong antibacterial activity and synthetic method and application thereof
阅读说明：本技术 具有强抗菌活性的阿诺普林改造肽及其合成方法和应用 (Anoprolin modified peptide with strong antibacterial activity and synthetic method and application thereof ) 是由 常明明 郭婷婷 邵祺 刘双鹤 李泊瑶 华艺 于 2021-07-23 设计创作，主要内容包括：本发明提供系列具有强抗菌活性的阿诺普林改造肽,其氨基酸序列依次为序列表中SEQ ID No.2-8。本发明还提供其制备方法和其在抑菌中的应用。本发明的系列阿诺普林改造肽对金黄色葡萄球菌和大肠杆菌具有显著的抗菌活性,其氨基酸序列短、结构简单、合成方便、抗菌活性高,可用于金黄色葡萄球菌和大肠杆菌引起的疾病的预防与治疗。在生理盐条件下以及血清条件下均对金黄色葡萄球菌和大肠杆菌具有一定的抗菌活性,当与抗生素联合使用时,部分组合存在协同功效。本发明多肽ANP-7的环状结构有利于提高多肽稳定性,且其抗菌活性优于相同氨基酸序列的直链结构阿诺普林。(The invention provides a series of anoprost modified peptides with strong antibacterial activity, and the amino acid sequences of the peptides are SEQ ID No.2-8 in a sequence table. The invention also provides a preparation method and application thereof in bacteriostasis. The series of the anoprost modified peptides have remarkable antibacterial activity on staphylococcus aureus and escherichia coli, are short in amino acid sequence, simple in structure, convenient to synthesize and high in antibacterial activity, and can be used for preventing and treating diseases caused by staphylococcus aureus and escherichia coli. Has certain antibacterial activity on staphylococcus aureus and escherichia coli under physiological salt condition and serum condition, and when the antibacterial activity is combined with antibiotics, the synergistic effect exists in partial combination. The cyclic structure of the polypeptide ANP-7 is beneficial to improving the stability of the polypeptide, and the antibacterial activity of the polypeptide ANP-7 is superior to that of the linear structure of the same amino acid sequence of the annoprine.)
1. A series of anoprost modified peptides with strong antibacterial activity are characterized in that: the anoprost modified peptide is ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6 and ANP-7, and the amino acid sequences of the anoprost modified peptide are SEQ ID No.2-8 in a sequence table in sequence.
2. A method for synthesizing a series of anoprost modified peptides with strong antibacterial activity is characterized by comprising the following steps: the synthesis method comprises the following steps:
(1) synthesis of ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6
Synthesizing polypeptide by adopting an Fmoc classical solid-phase synthesis method, taking MBHA resin as a carrier, eluting an Fmoc protective group by using 20% piperidine/DMF (dimethyl formamide) solution, taking HBTU and HOBt (HoBt) as peptide chain coupling agents, extending a peptide chain from a C end to an N end until the required length is reached, then cutting the peptide chain by using 0.95% TFA/water solution, performing rotary evaporation on the concentrated peptide solution by using DCM (DCM), performing RP-HPLC (high Performance liquid chromatography) purification and separation, and performing freeze-drying to obtain Anoplin modified peptides ANP-1, ANP-2, ANP-3, ANP-4, ANP-5 and ANP-6;
(2) synthesis of ANP-7
The method comprises the steps of synthesizing polypeptide by adopting an Fmoc classical solid-phase synthesis method, using MBHA resin as a carrier, eluting an Fmoc protective group by using 20% piperidine/DMF (dimethyl formamide/dimethyl formamide) solution, using HBTU and HOBt as peptide chain coupling agents, extending a peptide chain from a C end to an N end until the required length is reached, cutting the peptide chain by using 0.95% TFA/water solution, performing rotary evaporation and concentration on the peptide chain by using DCM (DCM) to obtain an ANP-7 single-chain crude product, performing disulfide bond connection on the single peptide chain by using an iodine oxidation method, concentrating to obtain an ANP-7 crude product, performing RP-HPLC (high performance liquid chromatography) purification and separation, and performing freeze-drying to obtain the Anoplin modified peptide ANP-7.
3. A pharmaceutical composition comprising one or more of the series of apocrin-modified peptides of claim 1.
4. The pharmaceutical composition of claim 3, wherein: the pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.
5. The pharmaceutical composition according to claim 3 or 4, characterized in that: the amino acid sequence of the anoprost modified peptide is SEQ ID No.2, 6 or 8 in a sequence table.
6. The pharmaceutical composition of claim 5, wherein: the medicament further comprises an antibiotic selected from gentamicin, vancomycin or polymyxin B.
7. The pharmaceutical composition of claim 6, wherein: the active ingredients of the pharmaceutical composition are as follows: the amino acid sequence of the apracloprene modified peptide is SEQ ID No.2 in the sequence table and gentamicin; or
The active ingredients of the pharmaceutical composition are as follows: the amino acid sequence of the anoprost modified peptide is SEQ ID No.2 or SEQ ID No.8 in the sequence table and vancomycin; or
The active ingredients of the pharmaceutical composition are as follows: the amino acid sequence of the anoprost modified peptide is SEQ ID No.2 in the sequence table and polymyxin B.
8. Use of a series of anoprost modified peptides of claim 1 having potent antibacterial activity, or of pharmaceutical compositions of claims 3-7, for the preparation of inhibitors of staphylococcus aureus and/or escherichia coli.
9. Use of the series of anoprost modified peptides of claim 1 having potent antibacterial activity, or the pharmaceutical compositions of claims 3-7, for the preparation of a medicament for the prevention and/or treatment of staphylococcus aureus infections and escherichia coli infections.
Technical Field
The invention belongs to the technical field of antibacterial biological agents, and particularly relates to an amopholin modified peptide with strong antibacterial activity, a synthetic method and application thereof.
Background
The rapid development of antimicrobial peptides is facilitated by the increasing resistance to traditional antibiotics. Antimicrobial Peptides (AMPs) are a class of polypeptides with Antimicrobial activity, are widely available, and can be found in various organisms as the first line of defense against invading pathogens.
With the continuous and deep research on antibacterial peptides, the cationic antibacterial peptide in the natural antibacterial peptide has generally strong antibacterial activity because the sequence of the cationic antibacterial peptide is rich in arginine or lysine, and the action mechanism of the cationic antibacterial peptide is that after amino or guanidino and other basic groups of a cationic amino acid side chain in the antibacterial peptide are combined with hydrogen ions in the in-vivo environment, through the electrostatic action of a phospholipid bilayer of a negatively charged bacterial cell membrane, a hydrophobic end of a peptide chain is inserted into the hydrophobic interior of the phospholipid bilayer and loaded on the bacterial cell membrane to interfere the osmotic pressure inside and outside the bacterial cell and crack the cell membrane structure of the bacteria, and finally, the death of pathogens is caused. The electropositivity of the antimicrobial peptide and the membrane disruption caused by the hydrophobic interaction of the amino acid residues are the main antimicrobial means different from conventional antibiotics, and thus bacterial resistance is not easily developed. In addition, antimicrobial peptides also rely on their secondary structure to exert specific antimicrobial effects. Based on the advantages of broad antibacterial spectrum, rapid and strong effect, difficult occurrence of bacterial drug resistance and the like of the antibacterial peptide, the antibacterial peptide is considered as an ideal substitute of the traditional antibiotics. Therefore, a safe and effective antibacterial peptide is developed to replace antibiotics in the antibacterial field, and has important application value.
The natural antimicrobial peptide, Anoplin (Anoplin, GLLKRIKTLL-NH)2) As the shortest alpha-helical amphiphilic antimicrobial peptide found in natural sources, has attracted extensive research interest to researchers due to its simple and unique structure and diverse biological activities. The research of the last two decades shows that the anoplolin mainly plays the function through the direct interaction of an ion channel and the hydrophobic end of an anionic phospholipid bilayer membrane, and has the capabilities of selectively binding bacterial DNA and inhibiting ATP synthase. As a result of its typical non-receptor mediated membrane mechanism, the arnoprim has particular advantages over conventional antibiotics and chemotherapies, with a wide range of biological activities, including antibacterial, mast cell degranulation, anti-tumor, antimalarial, antifungal and anti-inflammatory activities. In addition, the anoprost has the advantages of simple structure, easy synthesis, no hemolytic activity on mammalian erythrocytes and the like, shows superiority in aspects of chemical operation, structure-activity relation research, action mechanism and medical application, and has huge potential as a novel antibiotic and anticancer drug.
Disclosure of Invention
The invention provides a series of anoprost modified peptides, and the modified peptides have antibacterial activity on escherichia coli and staphylococcus aureus.
The invention provides a series of anoploline modified peptides with strong antibacterial activity, wherein the anoploline modified peptides comprise ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6 and ANP-7, and amino acid sequences of the anoploline modified peptides are SEQ ID No.2-8 in a sequence table in sequence.
The series of modified peptides are modified on the basis of anoprine (Anoplin, ANP for short).
The amino acid sequence of ANP is GLLKRIKTLL-NH2。
The sequence characteristics of the ANP-1 modified peptide are as follows: length of 10 amino acids, and Arg in the sequence5Substitution with a cationic amino acid Lys5The specific sequence is as follows: GLLKKIKTLL-NH2。
The sequence characteristics of the ANP-2 modified peptide are as follows: 10 amino acids in length, and Lys in the sequence4And Lys7Respectively replaced by cationic amino acids Arg4And Arg7The specific sequence is as follows: GLLRRIRTLL-NH2。
The sequence characteristics of the ANP-3 modified peptide are as follows: the length is 11 amino acids, 1 Lys is introduced into the N-terminal of Anoplin, and the specific sequence is as follows: KGLLKRIKTLL-NH2。
The sequence characteristics of the ANP-4 modified peptide are as follows: the length is 12 amino acids, 2 Lys are introduced into the N-terminal of Anoplin, and the specific sequence is as follows: KKGLLKRIKTLL-NH2。
The sequence characteristics of the ANP-5 modified peptide are as follows: the length is 13 amino acids, 3 Lys are introduced into the N-terminal of Anoplin, and the specific sequence is as follows: KKKGLLKRIKTLL-NH2。
The sequence characteristics of the ANP-6 modified peptide are as follows: the length of the protein is 14 amino acids, 4 Lys are introduced into the N terminal of the Anoplin, and the specific sequence is as follows: KKKKGLLKRIKTLL-NH2。
The sequence characteristics of the ANP-7 modified peptide are as follows: the length of the protein is 22 amino acids, and a disulfide bond is formed between two cysteine residues in the amino acid sequence of the protein, wherein the specific sequence is as follows: LLTKIRKLLGCCGLLKRIKTLL-NH2。
The invention also provides a synthetic method of the series of the anoprost modified peptides with strong antibacterial activity, which comprises the following steps:
(1) synthesis of ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6
Synthesizing polypeptide by adopting an Fmoc classical solid-phase synthesis method, taking MBHA resin as a carrier, eluting an Fmoc protective group by using 20% piperidine/DMF (dimethyl formamide) solution, taking HBTU and HOBt (HoBt) as peptide chain coupling agents, extending a peptide chain from a C end to an N end until the required length is reached, then cutting the peptide chain by using 0.95% TFA/water solution, performing rotary evaporation on the concentrated peptide solution by using DCM (DCM), performing RP-HPLC (high Performance liquid chromatography) purification and separation, and performing freeze-drying to obtain Anoplin modified peptides ANP-1, ANP-2, ANP-3, ANP-4, ANP-5 and ANP-6;
(2) synthesis of ANP-7
The method comprises the steps of synthesizing polypeptide by adopting an Fmoc classical solid-phase synthesis method, using MBHA resin as a carrier, eluting an Fmoc protective group by using 20% piperidine/DMF (dimethyl formamide/dimethyl formamide) solution, using HBTU and HOBt as peptide chain coupling agents, extending a peptide chain from a C end to an N end until the required length is reached, cutting the peptide chain by using 0.95% TFA/water solution, performing rotary evaporation and concentration on the peptide chain by using DCM (DCM) to obtain an ANP-7 single-chain crude product, performing disulfide bond connection on the single peptide chain by using an iodine oxidation method, concentrating to obtain an ANP-7 crude product, purifying and separating by RP-HPLC (RP-high performance liquid chromatography), and performing freeze-drying to obtain the Anoplin modified peptide ANP-7.
The invention also provides a pharmaceutical composition which comprises one or more of the series of the anoprost modified peptides.
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the amino acid sequence of the anoprost modified peptide is SEQ ID No.2, 6 or 8 in the sequence table.
Preferably, the medicament further comprises an antibiotic selected from gentamicin, vancomycin or polymyxin B.
Preferably, the active ingredients of the pharmaceutical composition are: the amino acid sequence of the apracloprene modified peptide is SEQ ID No.2 in the sequence table and gentamicin; or
The active ingredients of the pharmaceutical composition are as follows: the amino acid sequence of the anoprost modified peptide is SEQ ID No.2 or SEQ ID No.8 in the sequence table and vancomycin; or
The active ingredients of the pharmaceutical composition are as follows: the amino acid sequence of the anoprost modified peptide is SEQ ID No.2 in the sequence table and polymyxin B.
The invention also provides the series of the anoprost modified peptides with strong antibacterial activity, or the application of the pharmaceutical composition in preparing staphylococcus aureus and/or escherichia coli inhibitors.
The invention also provides the application of the series of the anoprost modified peptides with strong antibacterial activity or the pharmaceutical composition in preparing medicines for preventing and/or treating staphylococcus aureus infection and escherichia coli infection.
The series of the anoprost modified peptides have remarkable antibacterial activity on staphylococcus aureus and escherichia coli, are short in amino acid sequence, simple in structure, convenient to synthesize and high in antibacterial activity, and can be used for preventing and treating diseases caused by staphylococcus aureus and escherichia coli. Has certain antibacterial activity on staphylococcus aureus and escherichia coli under physiological salt condition and serum condition, and when the antibacterial activity is combined with antibiotics, the synergistic effect exists in partial combination. The polypeptide ANP-7 is beneficial to improving the stability of the polypeptide, and the antibacterial activity of the polypeptide ANP-7 is superior to that of the anoplolin which is not connected by a disulfide bond.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIGS. 1 to 8 are MS identification spectra of ANP, ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6 and ANP-7 in this order.
FIGS. 9-16 are RP-HPLC analysis spectrograms of ANP, ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6, and ANP-7 in that order.
FIG. 17 shows the trend of the inhibition rate of E.coli by the arnoprim series of modified peptides.
FIG. 18 shows the trend of the inhibition rate of the ANOPOLIN series modified peptides against Staphylococcus aureus.
FIG. 19 shows the bacteriostatic effect of different concentrations of the series of the aprepilin modified peptides after 18h of culture. In the figure, 1 is ANP; 2 is ANP-1; 3 is ANP-5; 4 is ANP-7.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
Example 1 Synthesis of antibacterial peptides having the amino acid sequences ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6, ANP-7
(1) Pretreatment: the amount of 0.2mmoL product required was calculated from the MBHA substitution value, and 0.55g of MBHA resin (substitution value 0.365mmoL/g) was added to a 25mL solid phase synthesizer along with 15mL of LDCM and allowed to swell at room temperature for 30 min. After the resin is fully swelled, the resin is pumped and filtered by a vacuum pump diaphragm pump, and then a proper amount of DMF is added for washing once.
(2) Deprotection of peptide chain: an appropriate amount of 20% piperidine/DMF solution was added to the solid phase synthesis tube and shaken thoroughly for 30min (39 ℃, 180 rpm). After the reaction, DCM and DMF were repeatedly washed several times to remove the residual piperidine solution. And detecting exposed amino acid by using a Kaiser method, weighing a small amount of resin, heating in ninhydrin solution for 1-2min, developing according to different colors of amino acid resin, namely completing deprotection, and repeating the detection for three times.
(3) Peptide chain condensation reaction: after ninhydrin detection and color development, amino acids can be connected step by step according to the peptide chain sequence. The preparation method of the amino acid solution comprises the following steps: 3eq Fmoc-AA-OH and peptide coupling reagents 3eq HBTU, 3eq HOBt were weighed on an analytical balance and dissolved completely in 15mL sp.DMF by sonication for 3 min. Then 5eq DIEA as reaction starter was taken and added into the solid phase synthesis tube together with the prepared amino acid solution, and the shaking table was shaken well for reaction for 3h (39 ℃, 180 rpm).
After the reaction is finished, the Kaiser method detects the connection effect, the ninhydrin detection does not develop color to prove that the reaction is successful, and the amino acid is connected on the resin, so that the next step can be carried out.
a. ANP-1 peptide chain synthesis
Repeating the steps (2) and (3), and sequentially connecting amino acids Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ile-OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH and Fmoc-Gly-OH from the C end to the N end according to the sequence of ANP-1 to obtain the ANP-1.
b. ANP-2 peptide chain synthesis
And (3) repeating the steps (2) and (3), and connecting amino acids Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH and Fmoc-Gly-OH sequentially from the C end to the N end according to the sequence of ANP-2 to obtain the ANP-2.
c. ANP-3 peptide chain synthesis
And (3) repeating the steps (2) and (3), and connecting amino acids Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH and Fmoc-Lys (Boc) -OH sequentially from the C end to the N end according to the ANP-3 sequence to obtain the ANP-3.
d. ANP-4 peptide chain synthesis
Repeating the steps (2) and (3), and connecting amino acids Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH and Fmoc-Lys (Boc) -OH sequentially from the C terminal to the N terminal according to the sequence of ANP-4 to obtain the ANP-4.
e. ANP-5 peptide chain synthesis
Repeating the steps (2) and (3), and sequentially connecting amino acids Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Lys (OH), (Fmoc) -OH and Fmoc-Lys (Boc) -OH from the C end to the N end according to the ANP-5 sequence to obtain the ANP-5.
f. ANP-6 peptide chain synthesis
Repeating the steps (2) and (3), sequentially connecting amino acids Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-Lys (OH), (Fmoc) -OH, Fmoc-Lys (Boc) -OH and Fmoc-Lys (Boc) -OH from the C-terminal to the N-terminal according to the ANP-6 sequence to obtain the ANP-6.
g. ANP-7 single-chain peptide chain synthesis
Repeating the steps (2) and (3), and connecting amino acids Fmoc-Leu-OH, Fmoc-Thr-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Gly-OH and Fmoc-Cys (Acm) -OH sequentially from the C terminal to the N terminal according to the ANP-7 sequence to obtain the ANP-7 single chain.
(4) Peptide chain cleavage
And after all amino acids are connected, adding 20% piperidine/DMF solution, heating and shaking for 30min, detecting ninhydrin to ensure that Fmoc protective groups are removed, and repeatedly washing the resin until no piperidine solution exists, so that the peptide chain can be cut from the MBHA resin. The cleavage step was performed in a fume hood and the cleavage solution was formulated as TFA: h2O is 0.95: 0.05. Pouring the cutting fluid into a solid-phase synthesis tube for a plurality of times in a small amount to transfer the resin into a beaker, stirring for 3 hours on a magnetic stirrer at room temperature, pouring the cutting fluid into the solid-phase synthesis tube again, repeatedly washing the resin by using DCM solution, carrying out suction filtration on the polypeptide solution after the resin is cut off to a round-bottom flask, and washing until the solution is colorless. Concentrating and collecting the polypeptide solution by adopting a rotary evaporation mode (the water bath temperature is not more than 39 ℃, polypeptide degradation is prevented), concentrating and drying all the polypeptide solution in the round-bottom flask, pouring anhydrous ether into the round-bottom flask to form white powder crude peptide, precipitating at-4 ℃ overnight, centrifuging the next day and removing supernatant to obtain the crude peptide product.
(5) Disulfide bond formation in ANP-7
Dissolving the ANP-7 single-chain crude peptide product obtained in the step (4) with a small amount of water, and weighing 101mg of I2To a 500mL Erlenmeyer flask was added 300mL of 75% methanol-water solution, and the polypeptide solution was slowly added dropwise and stirred for 30 min. Transferring the solution in the conical flask into a 500mL eggplant-shaped flask, and carrying out reduced pressure distillation on a rotary evaporator to obtain an ANP-7 product.
(6) Purification and analysis of antimicrobial peptides
The purification of the antibacterial peptide preparation adopts a preparation liquid phase for purification, the model of a chromatographic column is SHIM-PACK, and the PRC-ODS size: 20mm × 25cm, mobile phase A as 0.1% TFA/acetonitrile solution, mobile phase B as 0.1% TFA/water solution, single sample size of 2mL, collection of 220nm peak sample.
HPLC is adopted for liquid phase analysis in the preparation of the antibacterial peptide, the type of a chromatographic column is Diamonsil C18, a mobile phase A is 0.1% TFA/acetonitrile solution, a mobile phase B is 0.1% TFA/water solution, elution treatment is carried out before the mobile phase is used, the flow rate is 1mL/min, the single sample introduction amount is 10 mu L, the peak condition at 220nm is detected, a target sample is collected after the target peak to be detected is finished, the peptide solution is concentrated in a rotary evaporation mode until the peptide solution is dried in a rotary manner, then the peptide sample is dissolved in deionized water, and freeze drying is carried out to obtain the product. And detecting the absorption peak at 220nm again by using a high liquid chromatograph, and calculating the purity of the crude peptide product by using an area normalization method.
(7) Molecular weight identification of antibacterial peptide
The identification of the antibacterial peptide is carried out by adopting a triple quadrupole liquid chromatography-mass spectrometer, a chromatographic column is Agilent eclipse plus C18 RRHD 1.8 mu M, a mobile phase is 50% formic acid/water solution, the sample injection amount of a single sample is 10 mu L, the sample is subjected to full-sweep identification of a product after being separated by the chromatographic column, the molecular mass of the product is verified by utilizing mass-to-charge ratio calculation, and the result is shown in Table 1.
TABLE 1 design sequence, product purity, charge number, mass-to-charge ratio and molecular mass table of the Aminopril series antibacterial peptides
The purity of the synthetic anoprost series antibacterial peptides in the experiment reaches more than 90%. And mass-to-charge ratio is detected by mass spectrometry to obtain molecular mass, the theoretical mass-to-charge ratio is consistent with the actual mass-to-charge ratio, the error is in a reasonable range, and the identification result shows that the synthesis is correct.
FIGS. 1 to 8 are MS identification spectra of ANP, ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6 and ANP-7 in this order.
FIGS. 9-16 are RP-HPLC analysis spectrograms of ANP, ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6, and ANP-7 in that order.
Tables 2-9 are sequentially peak analyses of RP-HPLC.
TABLE 2
Peak meter
PDA Ch1 220nm 4nm
TABLE 3
Peak meter
PDA Ch1 220nm 4nm
TABLE 4
Peak meter
PDA Ch1 220nm 4nm
TABLE 5
Peak meter
PDA Ch1 220nm 4nm
TABLE 6
Peak meter
PDA Ch1 220nm 4nm
TABLE 7
Peak meter
PDA Ch1 220nm 4nm
TABLE 8
Peak meter
PDA Ch1 220nm 4nm
TABLE 9
Peak meter
PDA Ch1 220nm 4nm
Example 2 determination of minimum inhibitory concentration of the aprepilin series of antimicrobial peptides prepared in example 1
In the experiment, the Minimum Inhibitory Concentration (MIC) of the anoplolin series antibacterial peptide is determined by adopting a classical trace double dilution method, and standard strains are staphylococcus aureus ATCC 25923 and escherichia coli ATCC 25922.
Culturing standard strain to logarithmic growth phase, and diluting the strain solution with MH broth to 1 × 106CFU/mL. Antibacterial peptide solutions were prepared according to the respective concentrations, and were dissolved in deionized water and serially diluted with the sterile filter membrane double dilution method to prepare antibacterial peptide solutions of 256. mu.M, 128. mu.M, 64. mu.M, 32. mu.M, 16. mu.M, 8. mu.M, 4. mu.M, and 2. mu.M. Subsequently, 50 μ L of bacterial liquid and 50 μ L of antibacterial peptide solutions with different concentrations were added into a 96-well plate, three groups were paralleled, gentamicin was used as a positive control, PBS was used as a negative control, the growth of the strain was observed after culturing at 37 ℃ for 18h, the lowest antibacterial peptide concentration in the wells that inhibited the growth of escherichia coli and staphylococcus aureus was recorded as the lowest antibacterial concentration, and the results are shown in table 10.
TABLE 10 minimum inhibitory concentrations of the Aminopril series of antimicrobial peptides
The result shows that except that ANP-2 has no antibacterial activity to staphylococcus aureus (the MIC value is more than 128 mu M, which shows that the medicine has no obvious antibacterial effect), the rest of the anoprost series antibacterial peptides have antibacterial effects to staphylococcus aureus and escherichia coli:
(1) cationic amino acid substitutions:
ANP-1 and ANP-2 adopt substitution transformation of a mother peptide, namely the anoprost, by cationic amino acids of arginine and lysine, and find that MIC values of the mother peptide ANP to escherichia coli and staphylococcus aureus are 64 mu M, the MIC value of the lysine substitution peptide ANP-1 to the escherichia coli is 32 mu M, the MIC value is better than that of the mother peptide, but the MIC value to the staphylococcus aureus is 64 mu M, and the minimum inhibitory concentration is the same as that of the mother peptide. The MIC values of the arginine replacement peptide ANP-2 to escherichia coli and staphylococcus aureus are 128 mu M and 256 mu M respectively, the antibacterial effect is lower than that of the mother peptide and ANP-1, and the fact that the antibacterial activity of the anoprost antibacterial peptide to gram-negative bacteria is improved to a certain extent by introducing arginine compared with lysine is shown, but the influence on the antibacterial activity of the gram-positive bacteria is not obvious.
(2) Peptide chain length:
the serial polypeptides ANP-3 to ANP-6 in the anoplolin series antibacterial peptides are respectively added with 1 to 4 lysines at the N end of the anoplolin. It was found that except that the antibacterial activity of ANP-3 was equivalent to that of the parent peptide, ANP-4 to ANP-6 were all enhanced in antibacterial activity against escherichia coli and staphylococcus aureus as compared to the parent peptide, and the antibacterial activity was gradually enhanced with the increase in the amount of lysine, wherein the MIC values of ANP-5 against escherichia coli and staphylococcus aureus were 8 μ M and 4 μ M, respectively, the antibacterial activity was improved 8 times and 16 times as compared to the parent peptide, and the MIC values of ANP-6 against escherichia coli and staphylococcus aureus were 8 μ M, respectively, and 8 times as compared to the parent peptide. It can be seen that the antibacterial activity is significantly increased with the increase in the number of N-terminal lysines of amoloprine. However, when the number of N-terminal lysines is more than 3, the antibacterial activity is not changed. Because the length of the ANP-5 amino acid sequence is shorter than that of ANP-6 and the antibacterial activity is slightly better than that of ANP-6, the ANP-5 is screened to carry out subsequent antibacterial mechanism research on the sequence.
(3) Disulfide bond:
compared with the anoprin parent peptide, the antibacterial activity of the ANP-7 to escherichia coli and staphylococcus aureus is 4 times of that of the parent peptide; the method is characterized in that on the basis of not changing the sequence of the mother peptide of the anoprost, the number of the amino acid sequences of the anoprost in unit molar concentration is increased by introducing a disulfide bond, so that the antibacterial activity of the anoprost is improved.
Example 3 determination of bacteriostatic ratio of the apraline series antibacterial peptides prepared in example 1
In order to further examine the antibacterial activity of the anoplolin series antibacterial peptide, staphylococcus aureus ATCC 25923 and escherichia coli ATCC 25922 are selected as experimental standard strains, a turbidimetry method is adopted to measure the bacterial concentration in the same time, and the bacteriostasis rate of the antibacterial peptide is calculated, wherein the experimental steps are as follows:
the standard bacterial liquid grown to logarithmic growth phase was diluted to 1X 10 bacterial concentration with MH broth6CFU/mL. The sterile water dissolves the crude peptide product, and the crude peptide product is continuously diluted by a two-fold dilution method to prepare polypeptide solutions with the concentration of 256 MuM, 128 MuM, 64 MuM, 32 MuM, 16 MuM and 8 MuM in sequence. Respectively adding 50 μ L of bacterial liquid and 50 μ L of polypeptide solution with different concentrations into 96-well plate, culturing at 37 deg.C for 18h, and measuring OD in microplate reader600Absorbance of each group was replicated three times, with gentamicin as a positive control and PBS as a negative control, and the experiment was repeated three times. The formula for calculating the bacteriostasis rate is as follows:
the bacteriostasis rate is (A negative control-A experimental group)/(A negative control-A positive control group) x 100%
The experimental results of the escherichia coli bacteriostatic rate are shown in a table 11 and a table 17, the experimental results of the staphylococcus aureus bacteriostatic rate are shown in a table 12 and a table 18, all data are subjected to one-factor variance analysis, comparative significance difference between groups is carried out, when P is less than 0.05, the significance difference between two groups of data is shown, and the statistical analysis significance is achieved; when P is less than 0.01, the difference between two groups of data is very significant and has statistical analysis significance. When P is more than or equal to 0.05, the data shows no obvious difference between the two groups of data and has no statistical analysis significance.
The results in table 11 show that the differences in the antibacterial effects of the series of antibacterial peptides on the antibacterial rates of gram-negative bacteria escherichia coli and gram-positive bacteria staphylococcus aureus under different concentration conditions are elaborated by comparing the antibacterial activities of different series of antibacterial peptides under different concentrations.
(1) Comparison of the bacteriostatic Effect on Escherichia coli
Results of the measurement of the antibacterial rate of the anoprost series antibacterial peptides on escherichia coli and the antibacterial trend are shown in table 11 and fig. 17, all the antibacterial peptides show good antibacterial activity at a concentration of about 128 μ M, but the antibacterial activity gradually decreases with the decrease of the antibacterial peptide concentration.
a. Antibacterial rates of the antibacterial peptides ANP-1 and ANP-2 are not obviously different from those of the mother peptide ANP (both are 100%) at the concentration of about 64 mu M, and the antibacterial rates of the antibacterial peptides ANP-1 and ANP-2 are reduced along with the reduction of the concentration of the antibacterial peptides when the concentration is 32 mu M to 8 mu M, but have no significant difference compared with the mother peptide, which indicates that the biological activities of arginine and lysine on the antibacterial peptides are not greatly changed;
b. the bacteriostatic effect is gradually enhanced with the increase of the number of N-terminal lysine, and when the concentration of the antibacterial peptide is lower than 64 mu M, the bacteriostatic effect is remarkably reduced with the reduction of the concentration of the antibacterial peptide and shows concentration dependence. When the number of N-terminal lysine is increased to 3 or more (ANP-5 and ANP-6), the antibacterial peptide keeps the same antibacterial effect as the high concentration under the experimental concentration, which shows that the N-terminal lysine is added, thereby being beneficial to increasing the antibacterial effect of the antibacterial peptide;
c. two identical parent peptides are linked by introducing a disulfide bond, and a modified peptide ANP-7 has a higher bacteriostatic rate than the parent peptide at each concentration compared with the parent peptide ANP, and the introduction of the disulfide bond is supposed to have a certain stabilizing effect on the structure of the antibacterial peptide and increase the number of the parent peptides in unit molar mass.
TABLE 11 determination of antibacterial rate of Escherichia coli by Aminopril series antibacterial peptides
Remarking: p <0.01, i.e. the difference between the two groups of data is very significant and has statistical analytical significance; p <0.05, i.e. significant difference between the two sets of data; no mark indicates that P is more than or equal to 0.05, namely, no obvious difference exists between two groups of data, and the statistical analysis significance is not achieved.
As shown in FIG. 17, it can be seen by comparing the bacteriostatic ratio of the anoprost series antibacterial peptides to Escherichia coli; the antibacterial rates of ANP-5 and ANP-6 are close to 100%, and the antibacterial rate is improved by 1 time compared with that of the mother peptide ANP, and the antibacterial peptide has good antibacterial activity on escherichia coli; with the increase of the concentration of the antibacterial peptide, the ANP-5, the ANP-6 and the ANP-7 all show obvious antibacterial activity, and the bacteriostasis rate is close to 100 percent; when the concentration of the antibacterial peptide is higher than 64 mu M, the antibacterial activity of ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6 and ANP-7 on escherichia coli reaches more than 95%, the result is consistent with the result of the lowest antibacterial concentration in the table 11, and the antibacterial activity of the anoprost modified peptide is proved, and the antibacterial activity of the anoprost series antibacterial peptide can be obviously improved by modifying lysine at the N-terminal and increasing the number of the anoprost molecules in unit molar mass by introducing a disulfide bond.
FIG. 17 shows the variation trend of the bacteriostasis rate of E.coli by the arnoprim series antibacterial peptides.
(2) Comparison of bacteriostatic effects on Staphylococcus aureus
The results and the bacteriostatic trend of the arnoprim series antibacterial peptides on staphylococcus aureus are shown in table 12 and fig. 18, all the antibacterial peptides show good antibacterial activity at high concentrations of 256 μ M and 128 μ M, but when the concentration is lower than 128 μ M, the antibacterial rate is gradually reduced along with the reduction of the antibacterial peptide concentration.
a. With the decrease of the concentration of the antibacterial peptide, the antibacterial peptide ANP-1 is decreased from 98% to 80% of staphylococcus aureus, the bacteriostasis rate of ANP-2 to staphylococcus aureus is decreased from 91% to 66%, the bacteriostasis rate of ANP-1 at each concentration is about 80%, and the bacteriostasis effect of the modified peptide at each concentration is decreased compared with that of the mother peptide. The anoprost antibacterial peptide has sequence specificity;
b. the sequence that the number of lysine is added at the N end of the anoploline increases gradually, the bacteriostasis rate of the series of modified peptides to staphylococcus aureus is increased gradually along with the gradual increase of the number of lysine, wherein the bacteriostasis effect of ANP-5 is optimal, the bacteriostasis rate of each concentration of an experimental group is close to 100%, the bacteriostasis rate of ANP-6 to staphylococcus aureus is lower than that of ANP-5, the result further indicates that 3 lysine is added at the N end to be the optimal quantity for the structural modification of the N end of the anoploline, and the antibacterial activity cannot be improved continuously by continuously increasing the quantity of N-terminal lysine;
c. compared with the arnoprim mother peptide, the antibacterial effect of ANP-7 on staphylococcus aureus is not obviously improved, which indicates that the antibacterial effect of the formation of disulfide bond on staphylococcus aureus is not obvious.
TABLE 12 determination of antimicrobial rates of Aminopril series antimicrobial peptides against Staphylococcus aureus
Remarking: p <0.01, i.e. the difference between the two groups of data is very significant and has statistical analytical significance; p <0.05, i.e. significant difference between the two sets of data; no mark indicates that P is more than or equal to 0.05, namely, no obvious difference exists between two groups of data, and the statistical analysis significance is not achieved.
The determination result and the bacteriostatic trend of the anoprost series antibacterial peptides on the staphylococcus aureus are shown in fig. 18, and the higher the bacteriostatic effect of the anoprost series antibacterial peptides with the higher antibacterial peptide concentration can be obviously observed in fig. 18, wherein the modified peptide with the good bacteriostatic rate is ANP-5, and the minimum concentration of 8 mu M still has the complete bacteriostatic rate close to 100%; the ANP-1 and the ANP-2 also show more obvious difference, and the bacteriostatic effect of the ANP-1 is obviously better than that of the ANP-2 in the range of 8-256 mu M, which indicates that the bacteriostatic ability of the ANP-2 to staphylococcus aureus is lower than that of escherichia coli; with reference to fig. 18, ANP-1 demonstrated good antibacterial activity against both gram-negative and gram-positive bacteria, so we selected ANP-1 as one of the best peptides for subsequent antibacterial studies; the bacteriostasis rate of ANP-7 is similar to that of the parent peptide ANP, and the introduction of disulfide bonds is not supposed to enhance the antibacterial ability of the ANP to staphylococcus aureus; therefore, the optimal peptides ANP-1, ANP-5 and ANP-7 are selected for further antibacterial study in subsequent antibacterial experiments.
FIG. 18 shows the variation trend of the bacteriostasis rate of the anoprost series antibacterial peptides on Staphylococcus aureus.
EXAMPLE 4 antibacterial drug susceptibility testing of the optimized peptides ANP-1, ANP-5, ANP-7 of example 1
In order to more intuitively investigate the bacteriostatic effect of the anoprost series antibacterial peptide, staphylococcus aureus ATCC 25923 and escherichia coli ATCC 25922 are used as experimental standard strains, an oxford cup method is adopted to observe the size of bacteriostatic circles of different series antibacterial peptide at the same concentration in the same time, and the bigger the bacteriostatic circle is, the better the bacteriostatic effect of the antibacterial peptide is proved, and the specific experimental steps are as follows:
preparing MH (A) solid culture medium, preparing according to SN standard formula, weighing 36.5g of culture medium, adding into 1000mL of deionized water, heating to boil and dissolve, subpackaging, and sterilizing at 121 ℃ for 15min for later use. And taking out the prepared solid culture medium, pouring the solid culture medium into a flat plate, wherein the dosage of each flat plate is about 30mL, and the surface of each flat plate needs to be as flat and smooth as possible so as to ensure that the antibacterial peptide solution in the oxford cup is uniformly diffused. After the culture medium is solidified, 100. mu.L of bacterial liquid (10) growing to logarithmic phase is taken6CFU/mL), air-drying, vertically placing an oxford cup, adding 200 mu of LANP, ANP-1, ANP-5 and ANP7 antimicrobial peptide solutions (256 mu M-64 mu M) into the oxford cup, standing the culture medium face upwards after the oxford cup is filled with the solutions, culturing at 37 ℃ for 18 hours, observing and comparing the sizes of inhibition zones, and recording the obtained result as shown in figure 19.
ANP-1, ANP-5 and ANP-7 in the ANP series antibacterial peptides are respectively co-cultured with Escherichia coli and Staphylococcus aureus for 18h according to the same concentration, and the inhibition zones are observed, and the specific results are shown in FIG. 19. In FIG. 19 (top), the antibacterial peptides ANP (No. 1), ANP-1 (No. 2), ANP-5 (No. 3) and ANP-7 (No. 4) all have the generation of inhibition zones at the concentration of 64 μ M to 256 μ M, which proves that the antibacterial peptides have the antibacterial ability, the inhibition zones of the experiment group with the comparative administration concentration of 128 μ M can be obtained, the inhibition ability of ANP and ANP-5 to Escherichia coli is higher than that of ANP-1 and ANP-7, and the result further verifies that the antibacterial effect of ANP-5 to Escherichia coli is the best in the synthesized series of antibacterial peptides.
FIG. 19 (bottom) shows the inhibition of different ANP series ANP (No. 1), ANP-1 (No. 2), ANP-5 (No. 3), and ANP-7 (No. 4) after co-culturing with Staphylococcus aureus at a concentration of 64 μ M to 256 μ M for 18h, as shown in the figure, the ANP series peptides all show good inhibition zones, and at a concentration of 256 μ M, the modified peptides ANP-1, ANP-5, and ANP-7 have larger diameter of inhibition zones for Staphylococcus aureus than the ANP parent peptide, which proves that Staphylococcus aureus is more sensitive to the modified peptides; the antibacterial peptide after structural modification has different degrees of antibacterial zone generation, and the result proves that the antibacterial effect of the anoprin peptide on gram-positive bacteria can be improved after structural modification.
FIG. 19 shows the inhibition of bacteria after 18h of incubation of different concentrations of different series of antimicrobial peptides. In the figure, 1 is ANP; 2 is ANP-1; 3 is ANP-5; 4 is ANP-7.
EXAMPLE 5 determination of the antimicrobial Activity of the optimally selected peptides ANP-1, ANP-5, ANP-7 of example 1 under different culture conditions
In order to investigate the antibacterial effect of the anoprine series antibacterial peptides under different conditions, the antibacterial peptides ANP-1, ANP-5 and ANP-7 are cultured in different physiological environments and the antibacterial activity of the antibacterial peptides is researched, and the specific experimental steps are as follows:
(1) determination of antibacterial Activity under physiological salt conditions
Selecting staphylococcus aureus ATCC 25923 and escherichia coli ATCC 25922 standard strains as experimental strains, and culturing until the number of diluted colonies is 10 after logarithmic growth phase6CFU/mL. Antimicrobial peptides ANP, ANP-1, ANP-5 and ANP-7 were diluted twice with 154mM NaCl solution to prepare 128. mu.M, 64. mu.M, 32. mu.M, 16. mu.M, 8. mu.M, 4. mu.M and 2. mu.M antimicrobial peptide solutions in this order. Subsequently, 50 μ L of the bacterial solution and 50 μ L of the antimicrobial peptide solution were put into a 96-well plate, three groups were performed in parallel, the growth of the colonies was observed after culturing at 37 ℃ for 18 hours, the minimum antimicrobial peptide concentration in the wells that could inhibit the growth of the microorganisms was recorded as MIC value, and the experiment was repeated three times, and the results are shown in tables 13 and 14.
(2) Determination of antibacterial Activity under serum conditions
Selecting staphylococcus aureus ATCC 25923 and escherichia coli ATCC 25922 standard strains as experimental strains, and culturing until the number of diluted colonies is 10 after logarithmic growth phase6CFU/mL. Preparing solution of antibacterial peptides ANP, ANP-1, ANP-5 and ANP-7, diluting twice, and sequentially preparing into 128 μ M, 64 μ M, 32 μ M, 16 μ M and 8 μ Mmu.M, 4 mu.M, 2 mu.M, etc., adding 10% FBS, culturing at 37 deg.C for 1h, immediately placing in 60 deg.C water bath for inactivating for 15 min. Subsequently, 50 μ L of the bacterial solution and 50 μ L of the antimicrobial peptide solution were put into a 96-well plate, three groups were performed in parallel, the growth of the colonies was observed after culturing at 37 ℃ for 18 hours, the minimum antimicrobial peptide concentration in the wells that could inhibit the growth of the microorganisms was recorded as MIC value, and the experiment was repeated three times, and the results are shown in tables 13 and 14.
TABLE 13 MIC values of the Aminopril series of antibacterial peptides against E.coli under different physiological conditions
In Table 13, the change of MIC values of ANP-1, ANP-5, and ANP-7 in different physiological environments was shown, and the change of the minimum inhibitory concentrations of ANP-1, ANP-5, and ANP-7 in 154mM NaCl in physiological environments was shown. The result shows that the minimum inhibitory concentration of ANP-1, ANP-5 and ANP-7 to escherichia coli is improved by 0.5-1 time and the antibacterial effect of the parent peptide is improved by 4 times under a 154mM NaCl physiological environment, which indicates that sodium ions in 154mM NaCl have small influence on the antibacterial activity of the three antibacterial peptides, wherein the minimum inhibitory concentration of ANP-1 and ANP-7 to escherichia coli ATCC 25922 has no obvious change, and the result indicates that the modified peptide basically maintains the original antibacterial activity in a cationic environment.
The MIC value multiple change range of ANP-1, ANP-5 and ANP-7 to escherichia coli in 10% FBS physiological environment is also 0.5-1, which indicates that 10% FBS physiological environment has small influence on the three antibacterial peptides, the minimum antibacterial concentration of ANP-5 in 10% FBS physiological environment does not change, which indicates that the environment has no influence on the antibacterial action of ANP-5, the antibacterial ability of ANP-1 in 10% FBS physiological environment is improved by 1 time, ANP-7 is reduced by 1 time, and the antibacterial effect of the parent peptide is changed to 4 times.
Table 14 shows the change of MIC values of the minimum inhibitory concentrations of ANP-1, ANP-5 and ANP-7 to Staphylococcus aureus in different physiological environments, as shown in Table 14, the minimum inhibitory concentrations of the parent peptide and ANP-1 were not significantly changed under the 154mM NaCl culture condition, and the inhibitory effects of ANP-5 and ANP-7 were respectively 0.125 times and 0.5 times the initial values. At 10% FBS, the inhibitory effect of the parent peptide becomes 4 times of the initial value, ANP-1 becomes 2 times of the original value, and ANP-5 and ANP-7 decrease to 0.25 times and 0.5 times of the initial value, respectively. The cationic amino acid substitution on the parent peptide sequence improves the antibacterial activity and the stability of the antibacterial peptide, and the modification of the N end of the parent peptide is beneficial to improving the antibacterial activity of the parent peptide, but has certain influence on the stability under different physiological conditions.
TABLE 14 MIC values of the Aminopril series of antimicrobial peptides against Staphylococcus aureus under different physiological conditions
EXAMPLE 6 evaluation of antibacterial Effect of combination of the best suited peptides ANP-1, ANP-5 and ANP-7 of example 1
Through earlier stage of antibacterial activity investigation of the anoprine series antibacterial peptides, ANP-1, ANP-5, ANP-7 and mother peptide ANP with good antibacterial activity are screened out to carry out antibiotic combined antibacterial activity investigation, and three traditional antibiotics with different action mechanisms are selected to carry out combined antibacterial with the anoprine series antibacterial peptides: (1) gentamicin, the mechanism of action of which is acting on ribosomes in bacteria, inhibits bacterial protein synthesis and destroys the integrity of bacterial cell membranes. (2) Polymyxin B, the mechanism of action of which is the interaction with lipopolysaccharides of the outer membrane of gram-negative bacteria, leading to the lytic death of bacterial cells. (3) Vancomycin, a glycopeptide antibiotic, causes lytic death of cells by inhibiting synthesis of bacterial cell walls. In the experiment, a chessboard microdilution method is adopted to determine the antibacterial activity of the antibiotic combined with the standard strains of staphylococcus aureus ATCC 25923 and escherichia coli ATCC 25922. The specific experimental steps are as follows:
the concentration is prepared by adopting a two-fold dilution method in sequence: polypeptide solution 4 × MIC, 2 × MIC, 1 × MIC antimicrobial peptide solution and antibiotic solution. Diluting and culturing standard strain bacterial liquid to logarithmic phase to make its colony number be 106CFU/mL. 100 mu L of bacterial liquid, 50 mu L of polypeptide solution and 50 mu L of antibiotic solution are sequentially put into a 96-well plate, three groups are paralleled, gentamicin is used as positive control, PBS is used as negative control, the growth condition of the strain is observed after culturing for 18h at 37 ℃, the lowest antibacterial peptide concentration which can inhibit the growth of the microorganism in the hole is recorded as MIC value by naked eyes, the effect of the drug combination is evaluated according to the antibacterial concentration index FICI, the experiment is repeated for three times, and the results are shown in tables 15 and 16.
FICI ═ value (MIC combined with drug a)/(MIC independent drug a) + (MIC combined with drug B)/(independent drug B).
When the FICI is less than or equal to 0.5, the synergistic effect exists between the antibacterial peptide and the traditional antibiotic, when the FICI is more than 0.5 and less than or equal to 1, the synergistic effect exists between the antibacterial peptide and the traditional antibiotic, when the FICI is more than 1 and less than or equal to 4, the no obvious effect exists between the antibacterial peptide and the traditional antibiotic, and when the FICI is more than or equal to 4, the obvious antagonistic effect exists between the antibacterial peptide and the traditional antibiotic.
TABLE 15 study of the efficacy of the combination of Anoprolin series antibacterial peptides and antibiotics in inhibiting Escherichia coli
Remarking: FICI less than or equal to 0.5 shows that the composition has a synergistic effect; b, FICI of more than 0.5 and less than or equal to 1 represents synergistic effect; c.1< FICI <4 indicates no significant effect; FICI is more than or equal to 4, which shows that the composition has antagonistic action.
(1) The results in Table 15 show that when the anoprost series antibacterial peptides are respectively used in combination with gentamicin, vancomycin and polymyxin B, the differences of the inhibition effects on Escherichia coli can be compared, and the following results can be obtained:
a. the combined application of gentamicin and ANP mother peptide shows a more obvious synergistic effect. The combined application of the modified ANP-1 and gentamicin shows stronger synergistic effect. ANP-5 and ANP-7 had no significant interaction. The antibacterial action mechanism of ANP and ANP-1 is suspected to be consistent with the action mode that gentamicin obstructs the synthesis of bacterial protein, and the combined use of the ANP and the ANP-1 in clinic can be considered;
b. the joint application of vancomycin and ANP parent peptide has obvious synergistic effect, the combined application of the modified ANP-1 and ANP-7 and vancomycin respectively shows good synergistic effect, and the antibacterial mechanism of the modified ANP-1 and ANP-7 and vancomycin are suspected to be possibly cooperated with vancomycin to inhibit the synthesis of bacterial cell walls, so that the cracking and death of escherichia coli cells are caused.
c. The FICI value of the combined application of the polymyxin B and 4 antibacterial peptides is between 1 and 4, which shows that the polymyxin B has no obvious influence on the antibacterial action of the antibacterial peptides.
(2) The results in table 16, which show the differences in the inhibitory effects of the aprepilin series antibacterial peptides on staphylococcus aureus when used in combination with gentamicin, vancomycin and polymyxin B, respectively, are as follows:
a. the combined application of the gentamicin, the ANP mother peptide, the ANP-1 and the ANP-5 has no obvious influence. The combined application of the modified ANP-7 and gentamicin shows antagonism, and the introduction of the disulfide bond of the ANP-7 is supposed to cause the difference between the antibacterial effect of the ANP and the antibacterial effect of the parent peptide ANP.
b. In the vancomycin group, the combined effect of all the antibacterial peptides is not affected.
c. In polymyxin B group, the combination of ANP, ANP-1 and polymyxin B resulted in synergistic effect, the combination of ANP-5 and ANP-7 showed antagonistic effect, and the substitution of lysine in ANP-1 was not supposed to affect the antibacterial effect of ANP parent peptide, which will be further investigated later.
TABLE 16 study of the efficacy of a combination of anoprine series antibacterial peptides and antibiotics in inhibiting Staphylococcus aureus
Remarking: FICI less than or equal to 0.5 shows that the composition has a synergistic effect; b, FICI of more than 0.5 and less than or equal to 1 represents synergistic effect; c.1< FICI <4 indicates no significant effect; FICI is more than or equal to 4, which shows that the composition has antagonistic action.
In conclusion, the invention carries out modification design on the natural antibacterial peptide anoplolin and synthesizes anoplolin modified peptides with strong antibacterial activity, such as ANP-1, ANP-2, ANP-3, ANP-4, ANP-5, ANP-6, ANP-7 and the like. The research result of antibacterial experiments shows that the modified peptide has obvious antibacterial effect on staphylococcus aureus and escherichia coli, has short amino acid sequence, simple structure, convenient synthesis and high antibacterial activity, and has good application prospect in clinical antibacterial drug development.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.