CN116874564B - Human-derived broad-spectrum antibacterial peptide and application thereof - Google Patents
Human-derived broad-spectrum antibacterial peptide and application thereof Download PDFInfo
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- CN116874564B CN116874564B CN202311077083.XA CN202311077083A CN116874564B CN 116874564 B CN116874564 B CN 116874564B CN 202311077083 A CN202311077083 A CN 202311077083A CN 116874564 B CN116874564 B CN 116874564B
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention discloses a human-derived broad-spectrum antibacterial peptide and application thereof, belonging to the technical field of biology. According to the invention, the short peptide KR-12 of the humanized antibacterial peptide LL-37 is selected as a template peptide, and the design and the modification are carried out according to the characteristics of narrow bacteriostasis spectrum and the like of the short peptide KR-12, so that 12 target peptides are obtained, and the amino acid sequence of the 12 target peptides is shown as SEQ ID NO:2-13, the experiment shows that 12 target peptides have broad-spectrum antibacterial activity and basically have no cytotoxicity, wherein the peptide fragment (KK-I) with the best in-vitro antibacterial effect shows the effects of inhibiting the growth of escherichia coli and reducing inflammation in vivo. The invention provides a new thought for the design of the human antibacterial peptide, the short chain selection also saves the synthetic cost of the antibacterial peptide, and provides an effective candidate peptide for developing novel antibacterial drugs.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a human-derived broad-spectrum antibacterial peptide and application thereof.
Background
Antibacterial peptides are a class of basic small molecule substances that have defensive inhibitory effects on a variety of pathogens (bacteria, fungi, viruses, parasites, etc.). Compared with the traditional antibiotics, the antibacterial peptide has more stable effect and lower tendency to generate drug resistance. Although the antibacterial peptide has high application value, the length and complexity of the amino acid chain of the antibacterial peptide lead to high synthesis cost and complicated extraction process, which limit the application and scientific research of the antibacterial peptide in food industry. In order to solve these problems, development of novel antibacterial peptides as alternatives to antibiotics to combat superbacteria is urgently required to promote human health.
The humanized antimicrobial peptide LL-37 of the Cathelicidins family of antimicrobial peptides is an essential host defense molecule in human innate immunity. In the study of the antibacterial peptide LL-37, fragment KR-12 (residues 18-29 of LL-37) is the shortest amino acid chain, smallest molecular weight antibacterial peptide of LL-37. However, KR-12 has a narrow antibacterial spectrum and has no inhibitory effect on Staphylococcus aureus. Therefore, screening short-chain antimicrobial peptides with broad-spectrum antibacterial activity is important to solve the existing technical problems.
Disclosure of Invention
The invention aims to provide a human-derived broad-spectrum antibacterial peptide and application thereof, which are used for solving the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides an antibacterial peptide, the amino acid sequence of which is SEQ ID NO:1, wherein glutamine at position 5 of the short peptide sequence is replaced with any one of alanine, leucine, phenylalanine, tryptophan, valine or isoleucine, and aspartic acid at position 9 is replaced with lysine or arginine.
Preferably, the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO: 2-13. The specific sequence is as follows: KRIVARIKKFLR (KK-A, SEQ ID NO: 2), KRIVLRIKKFLR (KK-L, SEQ ID NO: 3), KRIVFRIKKFLR (KK-F, SEQ ID NO: 4), KRIVWRIKKFLR (KK-W, SEQ ID NO: 5), KRIVVRIKKFLR (KK-V, SEQ ID NO: 6), KRIVIRIKKFLR (KK-I, SEQ ID NO: 7), KRIVARIKRFLR (KR-A, SEQ ID NO: 8), KRIVLRIKRFLR (KR-L, SEQ ID NO: 9), KRIVFRIKRFLR (KR-F, SEQ ID NO: 10), KRIVWRIKRFLR (KR-W, SEQ ID NO: 11), KRIVVRIKRFLR (KR-V, SEQ ID NO: 12), KRIVIRIKRFLR (KR-I, SEQ ID NO: 13).
The invention also provides application of the antibacterial peptide in preparation of antibacterial agents, wherein the antibacterial agents comprise bacterial agents for inhibiting gram-negative bacteria and gram-positive bacteria, the gram-negative bacteria comprise escherichia coli, salmonella typhimurium, salmonella pullorum, salmonella and enterobacter sakazakii, and the gram-positive bacteria comprise staphylococcus aureus, staphylococcus epidermidis, listeria monocytogenes and bacillus cereus.
The invention also provides application of the antibacterial peptide in preparing a medicament for treating peritonitis infection.
The invention also provides a bacteriostatic agent comprising the antibacterial peptide.
The invention also provides a medicine for treating peritonitis infection, which comprises the antibacterial peptide.
The invention discloses the following technical effects:
the antibacterial peptide designed by the invention is 12 new antibacterial peptides obtained by carrying out site-directed mutagenesis on the basis of parent peptide KR-12 (KRIVQRIKDFLR), and experiments show that the 12 new antibacterial peptides have broad-spectrum antibacterial activity and are all obviously higher than the parent peptide and basically have no cytotoxicity. Wherein the Geometric Mean (GM) of the minimum inhibitory concentration was 6.25 and the Therapeutic Index (TI) of the resulting peptide was 40.99. In an in vitro experiment, the target peptide with the optimal biological activity is KK-I, and the KK-I still has good antibacterial activity in physiological salt concentration and protease environment, has certain serum stability and has higher application value; in vivo experiments, KK-I showed inhibitory effect on E.coli growth and reduced inflammation. The invention provides a new thought for the design of the human antibacterial peptide, the short chain selection also saves the synthetic cost of the antibacterial peptide, and provides an effective candidate peptide for developing novel antibacterial drugs.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of a template peptide and a target peptide alpha-helical turn structure; KR-12 represents a parent peptide, and 1-12 represent KK-A, KK-L, KK-F, KK-W, KK-V, KK-I, KR-A, KR-L, KR-F, KR-W, KR-V and KR-I, respectively;
FIGS. 2A-2M are chromatograms of parent peptide KR-12 and target peptides KK-A, KK-L, KK-F, KK-W, KK-V, KK-I, KR-A, KR-L, KR-F, KR-W, KR-V and KR-I, respectively;
FIGS. 3A-3M are mass spectra of template peptide KR-12 and target peptides KK-A, KK-L, KK-F, KK-W, KK-V, KK-I, KR-A, KR-L, KR-F, KR-W, KR-V and KR-I, respectively;
FIG. 4 is a circular dichroism spectrum of a template peptide and a target peptide in three different environments; KR-12 represents a parent peptide, and 1-12 represent KK-A, KK-L, KK-F, KK-W, KK-V, KK-I, KR-A, KR-L, KR-F, KR-W, KR-V and KR-I, respectively;
FIG. 5 is a laser confocal and ultra-high resolution fluorescence microscopy image of E.coli ATCC25922 and S.aureus ATCC29213 after treatment with FITC-labeled peptide KK-I; a (1) -A (4), B (1) -B (3) are respectively E.coli ATCC25922 laser confocal and ultra-high resolution fluorescence microscopy images, A (5) -A (8), B (4) -B (6) are respectively S.aureus ATCC29213 laser confocal and ultra-high resolution fluorescence microscopy images;
FIG. 6 is an SEM and TEM image of peptides KK-I treated E.coli ATCC25922 and S.aureus ATCC 29213; A-A3 is an SEM image of E.collATCC 25922; B-B3 is a TEM image of E.collATCC 25922; C-C3 is an SEM image of S.aureus ATCC 29213; D-D3 is a TEM image of S.aureus ATCC 29213;
FIG. 7 shows the concentrations of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (ALP) and Blood Urea Nitrogen (BUN) in mouse serum;
FIG. 8 shows colony counts of various tissues of mice;
FIG. 9 is a graph showing the effect of antimicrobial peptides on the levels of mouse serum tumor necrosis factors TNF- α, IL-6 and IL-1β.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 sequence design of broad-spectrum antibacterial peptides
The humanized short peptide KR-12 is used as a template peptide, and according to the characteristics of an amino acid sequence (KRIVQRIKDFLR) of the antibacterial peptide KR-12, lysine (Lys) with positive charges is used for replacing aspartic acid (Asp) with negative charges, and meanwhile, the hydrophilic amino acid glutamine (Gln) is replaced by hydrophobic amino acid isoleucine (Ile), so that 12 antibacterial peptides are designed. The amino acid sequences and physicochemical parameters of the template peptide and the target peptide KK-I are shown in Table 1. The amphiphilicity of the peptide sequence is represented by the alpha-helix pattern, as shown in figure 1.
TABLE 1 amino acid sequence and physicochemical parameters of target peptides
Note that: a: measured by mass spectrometer; b: hydrophobicity; c: average hydrophobic moment
From the alpha-helix pattern of the template peptide KR-12 molecule, KR-12 forms an amphipathic alpha-helix structure, hydrophilic residues are on one side of the helix, and hydrophobic residues are polymerized to the other side of the helix; the alpha-helical structure of the designed peptide after amino acid substitution is that one side of hydrophilic amino acid contains hydrophobic amino acid, and the other side is all hydrophobic amino acid, so that a non-perfect amphipathic structure is formed. To ensure high antimicrobial activity and stability of the peptide fragments, the C-terminus of all short peptides is amidated.
EXAMPLE 2 preparation of antibacterial peptide by solid phase Synthesis
1. Synthesis of antibacterial peptides
Weighing a proper amount of resin, adding dichloromethane to soak for 5 minutes, cleaning with N, N-dimethylformamide, pumping out the dichloromethane, and removing Fmoc protecting groups (uncapping) on the resin by using a prepared deprotection agent, wherein the uncapping time is 20 minutes; weighing the second amino acid at the C end, adding a condensing agent and N, N-dimethylformamide, and putting into a reaction column for reaction (air blowing); the ninhydrin method is used for detection, the detection result is that the solution is bright yellow, the resin is transparent, and no variegation exists; removing Fmoc protecting group on the 2 nd amino acid by using a deprotection solution, cleaning for 6 times by using N, N-dimethylformamide, and repeating the rest amino acids until the last amino acid; removing the last Fmoc protecting group by using a deprotection solution, and then cleaning by using N, N-dimethylformamide; after the reaction is finished, respectively washing and shrinking with dichloromethane and methanol; cutting polypeptide with lysate, and precipitating with diethyl ether; purification was performed by Mass Spectrometry (MS) and High Performance Liquid Chromatography (HPLC).
2. Purification of antibacterial peptides
(1) Pre-analysis: an appropriate amount of the sample was taken and placed in a 0.5mL tube, and dissolved with ultrapure water. Filtering the sample by using a 0.45 mu m membrane, and then analyzing the sample by using a rapid gradient high performance liquid chromatography (10-100%) for sample injection analysis;
(2) Sample treatment: 200mg of sample was added to a 20mL beaker, followed by 15mL of H 2 O and 5mL of Methanol (Methanol). Sonicating the sample until the sample is completely dissolved, and then filtering the solution with a 0.45 μm membrane;
(3) High Performance Liquid Chromatography (HPLC): samples were collected from 0 to 25min, pump A was 100% Acetonitrile (Acetonitrile) plus 0.1% trifluoroacetic acid (Trifluoroacetic acid), pump B was 100% water plus 0.1% trifluoroacetic acid (Trifluoroacetic acid). The collected fractions were analyzed by High Performance Liquid Chromatography (HPLC) and checked for purity.
The chromatograms of the purified template peptide and target peptide are shown in fig. 2A-2M. FIGS. 2A-2M show that each designed peptide has a distinct absorption peak. The purity of the purified peptide reaches more than 95 percent.
3. Identification of antimicrobial peptides
And (3) collecting the purified polypeptide and identifying a target peak by an LC 6000 type reversed phase preparation chromatograph and a Waters 2000 mass spectrometer, and finally analyzing the actual molecular weight and purity of the peptide. Mass spectrometry of the purified peptides is shown in fig. 3A-3M.
Example 3 determination of secondary Structure and biological Activity of antibacterial peptide KK-I
1. Determination of secondary structure of antibacterial peptide
The secondary structure of the antibacterial peptide was determined by circular dichroism spectroscopy (CD), respectively.
Structures in three different solution environments simulated (aqueous environment, cell membrane environment of prokaryotic cells and hydrophobic environment of biological membranes). The polypeptides were dissolved in 10mM PBS buffer, 30mM Sodium Dodecyl Sulfate (SDS) and 50% Trifluoroethanol (TFE), respectively, at ph=7.4, to prepare a peptide solution at a final concentration of 150 μm for use. The prepared polypeptide solution is added into a cuvette (optical path: 1 mm), a circular dichroism spectrometer is used for scanning in a wavelength range of 190 nm-250 nm at room temperature, the ellipticity (resolution is 0.5nm, bandwidth is 1.0nm, scanning speed is 10nm per minute, optical path of a quartz sample cell is 0.1 cm) of the polypeptide is measured, and the method is repeated for three times and finally average value is obtained. The average ellipticity is converted and calculated as follows:
wherein θ is M : average residue ellipticity (deg.m2.dmol-1), θ obs : actually measured ellipticity (mdeg), c: peptide sample concentration (mM), l: optical path length (mm), n: number of amino acids of the peptide.
The calculation formula of the alpha-helix ratio is as follows:
wherein θ is 222 Is the average residue ellipticity of the peptide sample at 222 nm; θ 100 Is the average residue ellipticity at 222nm for the peptide sample at 100% helicity; θ 0 Is the average ellipticity of the peptide sample at 222nm without the helix. The results are shown in FIG. 4 and Table 2.
TABLE 2 helicity of peptides in three different solution environments
Both the template peptide and 12 target peptides exhibited random coil structures under PBS conditions. Under SDS conditions, peptides KK-F, KK-W, KR-L, KR-F, KR-V and KR-I showed no significant negative absorbance peaks at 222nm, so these 6 peptides were randomly coiled. The other 6 short peptides showed positive absorption peaks at 195nm and 198nm and negative absorption peaks at 205nm and 222nm, so that the 6 peptides all have alpha-helical structures in the simulated negatively charged prokaryotic cell environment. Under TFE conditions, the absorption peaks of the alpha-helix structure characteristics appear in the 12 designed peptides, which indicates that the 12 peptides are of the alpha-helix structure in the simulated hydrophobic environment and meet the design requirements of the helical wheel structure.
2. Determination of antibacterial Activity
Determination of Minimum Inhibitory Concentration (MIC): the antibacterial activity is measured by micro double dilution method, firstly, the frozen bacteria are streaked, single colony is selected, purified and inoculated in MHB culture medium, placed in a shaking table (168 rpm,37 ℃) and incubated to logarithmic phase, and the concentration of bacterial liquid is regulated to about 1 multiplied by 10 5 CFU/mL. Then, 90 μLBSA solution was added to column 1 of the 96-well plate, 50 μLBSA solution was added to each of the remaining 7 columns, and 10 μL of peptide stock solution (2.56 mM) was added to column 1 of the wells, and the mixture was stirred uniformly by a pipette tip. Adding 50 mu L of the mixed solution in the 1 st row into the 2 nd row of holes, uniformly mixing the mixed solution in the 2 nd row of holes, absorbing 50 mu L, adding the mixed solution into the 3 rd row of holes, and the like until the mixed solution is added into the 10 th row of holes, uniformly mixing, absorbing 50 mu L, and discarding the mixed solution into a waste liquid bottle. Then, 50. Mu.L of the logarithmic growth phase bacterial liquid was added to the wells 1 to 11, 50. Mu.LMHB was added to the well 12, and the 96-well plate 1-12 columns of the liquid was stirred and mixed well. At this time, the concentrations of the antimicrobial peptides were sequentially diluted to 128, 64, 32, 16, 8, 4, 2, and from column 1 to column 10,1. 0.5, 0.25. Mu.M. Column 11 with bacterial liquid and no antibacterial peptide as positive control group; column 12 was negative control with no added bacteria and antimicrobial peptide. Each peptide sample test was repeated at least 3-4 times. Note that the positive control group, if kept clear, was not contaminated with bacteria throughout the test run, column 12 wells; if turbid, the bacteria are polluted, and the operation steps should be carried out again. The minimum inhibitory concentration value of the antibacterial peptide for the test bacteria is the concentration value corresponding to the critical hole without turbidity or precipitation visible to the naked eye in the hole (column 1-column 10).
Determination of Minimum Bactericidal Concentration (MBC): after completion of MIC determination, 10 μl of the sample was taken from the non-macroscopic culture medium turbid well, and after incubation at 37 ℃ for 18h, colony counting was performed. The minimum peptide concentration to kill 99.9% of the bacteria is the minimum bactericidal concentration of the peptide. The above experiment was independently repeated three times. The results are shown in Table 3 and Table 4.
TABLE 3 MIC (MBC) value (μM) of short peptides for gram-negative bacteria
TABLE 4 MIC (MBC) value (μM) of short peptides for gram-positive bacteria
The antibacterial activity of the short peptide KR-12 on gram-negative bacteria is stronger than that on gram-positive bacteria. The 12 target Tai designed by replacing the cationic amino acid and the hydrophobic amino acid show stronger antibacterial activity to gram-negative bacteria and positive bacteria than the template peptide, and the MBC value measured by the 12 designed peptides is 1-4 times of the MIC value. The proper increase of the hydrophobicity and charge number of the short peptide is illustrated to improve the bactericidal performance and the antibacterial broad spectrum of the short peptide.
3. Determination of hemolytic Activity
1-2 mL of healthy human blood is taken and stored in a heparin sodium tube, 1000g of the blood is centrifuged for 10min at the temperature of 4 ℃, and the supernatant is discarded to collect red blood cells.After continuing to wash 3 times with PBS, the suspension was resuspended for further use. Mixing antibacterial peptide solution with equal volume of red blood cells after dilution in 96-well cell plate, culturing at 37deg.C for 4 hr, centrifuging in 96-well plate centrifuge (1000 g,15 min), transferring supernatant into new 96-well plate, and measuring OD with enzyme-labeled instrument 570nm The absorbance was measured. The negative control was untreated blood cells, the positive control was 0.1% triton X-100 treated blood cells, and the Minimum Hemolysis Concentration (MHC) was defined as the minimum polypeptide concentration corresponding to 10% hemolytic activity.
TABLE 5 MHC, GM (μM) and TI values of short peptides
Note that: a: the concentration of the peptide causing 10% hemolysis was the minimum hemolysis concentration, calculated as 256 μm when the hemolysis concentration was > 128 μm; b: geometric mean of MIC values of peptides, calculated with 128 μm when > 64 μm; c: ti=mhc/GM, a larger Therapeutic Index (TI) indicates a stronger cell selectivity.
The GM values of the 12 designed peptides are smaller than those of the short peptide KR-12, which shows that the antibacterial activity of the short peptide containing 6 positive charges is obviously better than that of the short peptide containing 4 positive charges, and the antibacterial capability of the short peptide is improved to different degrees due to the imperfect amphipathic structure. The peptide KK-I has the smallest GM (gm=6.25) and the largest therapeutic index TI (ti=40.99), the larger the therapeutic index the more cell selective. Therefore, the peptide KK-I with strong antibacterial activity is screened out and used as the short peptide with clinical application potential to conduct deep antibacterial mechanism research.
4. Stability study of antibacterial peptides
(1) Salt ion stability
Preparing salt ion solution with BSA diluent according to different concentrations, wherein the salt ion concentration is 150mM NaCl,4.5mM KCl,1mM MgCl respectively 2 ，6μM NH 4 Cl，8mM ZnCl 2 ，4mM FeCl 3 And 2.5mM CaCl 2 The blank is antimicrobial peptide which is not treated by salt ions, so that the stability of the antimicrobial peptide under different salt ion concentrations is detected. The results are shown in Table 6 and Table 7.
TABLE 6 MIC values (μM) of short peptide pairs E.coilATCC25922 under salt ion treatment
TABLE 7 MIC value of short peptide pair S.aureus ATCC29213 (μM) under salt ion treatment
(2) Serum stability
The stability of the antimicrobial peptides in serum was determined in the same manner as described above for the salt ion stability, and different concentrations of fetal bovine serum were prepared with BSA dilutions, with final serum concentrations of 50%, 25% and 12.5%. The blank is a peptide that has not been subjected to serum treatment, whereby the stability of the antimicrobial peptide was tested at different serum concentrations. The results are shown in tables 8 and 9.
TABLE 8 MIC values (μM) of peptides for E.coilATCC25922 in serum Environment
TABLE 9 MIC values (μM) of peptides for S.aureus ATCC29213 in serum Environment
(3) Enzyme stability
To determine the stability of the antimicrobial peptides under enzyme treatment, enzyme-antimicrobial peptide reaction solutions of trypsin, pepsin, papain and proteinase K were prepared at a final concentration of 1mg/mL, and after 1h of action, the test was performed according to the method for determining the stability of salt ions and serum, and the blank control was the antimicrobial peptides without protease treatment. The stability of the antibacterial peptide under different enzyme treatments is detected. The results are shown in Table 10.
TABLE 10 minimum inhibitory concentration of peptides under different enzyme treatments
The results show that 12 target peptides still have good antibacterial activity in physiological concentration salt ions and high concentration serum environments, and the results show that the peptides have higher salt ion and serum stability. In addition, the antibacterial activity can be basically maintained under different enzyme treatment conditions. The antibacterial property of the peptide KK-I to E.coli ATCC25922 and S.aureus ATCC29213 under the treatment of the salt ion solution is more stable than other designed peptides, and the peptide KK-I has better stability of salt ion resistance and has a certain clinical application potential.
Example 4 antibacterial mechanism of antibacterial peptide
1. FITC labeled polypeptide localization assay
FITC-labeled peptide with concentration of 1 XMBC is mixed with bacterial liquid, incubated for 15min at 37 ℃, centrifuged for 5min at 6000-8000 g, and the supernatant is discarded. After three washes with PBS, the suspension was resuspended, and after incubation at 4℃for 15min with Propidium Iodide (PI) at a final concentration of 10. Mu.g/mL, the free PI was washed off by centrifugation. And after the operation is finished, tabletting. The sample was kept overnight at 4℃and was observed under laser confocal microscopy and ultra-high resolution fluorescence microscopy at excitation wavelengths 488nm and 535 nm. The control was untreated cells as the cells. The results are shown in FIG. 5.
As shown in FIG. 5, FITC-labeled antimicrobial peptide KK-I overlaps the fluorescent green signal covering the E.coli and Staphylococcus aureus surfaces with the red fluorescent signal generated by Propidium Iodide (PI), indicating that peptide KK-I kills bacteria by disrupting the integrity of the bacterial membrane.
2. Observation of cell membrane integrity
The extent of disruption of the bacterial cell outer membrane by the antibacterial peptide was studied by observing the cell outer membrane morphology of the test strains e.coli ATCC25922 and s.aureus ATCC29213 using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The experimental procedure was as follows:
(1) Preparation of a bacterial strain: transferring the frozen bacteria to second generation, culturing to logarithmic phase, centrifuging to collect bacterial mud, washing with sterile PBS for three times, and re-suspending to OD 600nm =0.15; (2) short peptide-bacteria reaction liquid treatment: short peptides were added to the bacterial suspensions to give final concentrations of 1 XMIC, and the bacterial suspensions were reacted at 37℃for 1 hour on a shaker, with untreated bacterial solutions as a blank. After the reaction is finished, the bacterial liquid is centrifuged, the supernatant is discarded, bacterial sediment is collected and washed 3 to 4 times by PBS (pH 7.2); (3) fixing and dehydrating: 2.5% glutaraldehyde (1 mL) was added to the bacterial pellet and the pellet was frozen in a refrigerator at 4℃for 12h. After 12h, the bacterial sludge was collected and eluted with a gradient of 50%, 70%, 90% and 100% ethanol solution for 10 min/time. Mixing 100% ethanol with tert-butanol solution in equal volume, treating for 30min, centrifuging (8000-10000 g,5 min), treating with pure tert-butanol for 1 hr, and storing at-20deg.C; (4) drying and coating: and (3) after the sample is freeze-dried by a freeze dryer, plating a layer of metal film on the surface of the sample. (5) observation: and observing the change condition of the surface of the bacteria by using a field scanning electron microscope (transmission electron microscope). The results are shown in FIG. 6.
The results are shown in FIG. 6. Blank (SEM: A, C; TEM: B, D): the escherichia coli and staphylococcus aureus are complete and full in state and smooth in surface; the cell is full and complete in structure. When treated with 1×mic target peptides, holes appeared on both negative and positive bacterial surfaces; as the treatment time is increased, holes on the surfaces of the two bacteria deepen, the folds are uneven, the escherichia coli has signs of fracture, and staphylococcus aureus is spherically broken; eventually, neither bacteria are intact, and cell disruption and disruption occurs.
Example 5 in vivo biological Activity assay of antibacterial peptides
1. Mouse in vivo compatibility test
The test animals were 40 female ICR mice (24.09.+ -. 2.648g in weight) of 6 weeks old purchased from Liaoning long Biotechnology Co., ltd. And (5) carrying out adaptive feeding for one week, and feeding with standard feed. Mice were randomly divided into 4 groups of 10, each, and the test groups are shown in table 11. The mice were given intraperitoneal injections of 10mg/kg, 20mg/kg and 40mg/kg of polypeptide solution, each for 12h at intervals of 3d. The control group was treated with physiological saline alone. The viability and status of the animals were observed continuously during this period. After 3d, all mice were subjected to eyeball blood collection, followed by centrifugation for 10min at 1000g, and serum was analyzed for glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), alkaline phosphatase (ALP), and Blood Urea Nitrogen (BUN) content using the kit. The results are shown in FIG. 7.
TABLE 11 in vivo mouse compatibility experiments
As shown in FIG. 7, the enzyme levels of the treated group were not significantly different from the control group (P > 0.05) at peptide doses of 10mg/kg, and alanine Aminotransferase (ALT) and Blood Urea Nitrogen (BUN) were significantly increased (P < 0.01) at peptide doses increased to 20mg/kg and 40mg/kg as compared to the control group. With the increase of the dosage of the short peptide, the four enzyme levels are reduced and finally tend to be stable, which indicates that the peptide KK-I has stress response in mice in a short period, but does not cause hepatorenal toxicity, and has higher biocompatibility in vivo. The results show that the peptide KK-I has better stability and safety in mice and has wide clinical application prospect.
2. Mouse peritonitis experiment
The test 32 female ICR mice (body weight: 24.88.+ -. 1.43 g) were purchased from Liaoning long biotechnology Co., ltd. 8 mice per treatment group. After one week of the adaptive breeding, the seeds are fed,
the mice were intraperitoneally injected with physiological saline alone in the healthy group and 100. Mu.L of 1.5X10 s in the control group 8 CFU/mL e.collatcc 25922 was subjected to peritonitis infection. After infection for 0.5h, 2h and 6h, intraperitoneal injection of the mice with physiological saline, 10mg/kg of the target peptide and 5mg/kg of gentamicin was continued, respectively. Mice were observed for their active state after intraperitoneal injection and continued to survive after 24 h. All surviving mice were then treated and the peritoneal fluid was collected from the mice by intraperitoneal injection of 5mL of sterile saline solution, asSpleen and liver were also collected at this time for colony count analysis. The experimental groups are shown in table 12.
Table 12 mouse peritonitis experiment
FIG. 8 shows that the colony count of each tissue of the peptide KK-I treated group was extremely significantly reduced (P < 0.0001) compared to the control group, and that the colony count of the gentamicin group was significantly reduced compared to the peptide treated group. The colony count of the abdominal cavity is extremely remarkably reduced by about 4-Log (P < 0.0001). The colony numbers of the liver, spleen, lung and kidney are respectively reduced by 2-Log (P is less than 0.0001), which proves that the activity of the peptide KK-I in the mouse is stable and a certain killing capacity is reserved, thereby improving the survival rate of the mouse.
The results of the expression levels of each inflammatory factor in the serum of mice in the different treatment groups are shown in FIG. 9. The levels of proinflammatory factors IL-1 beta, IL-6 and TNF-alpha expressed in serum of mice from the pathogenic infected group were higher compared to the healthy group (normal saline). By establishing a mouse peritonitis model, after the peptide KK-I is injected into the abdominal cavity of a mouse, the IL-1 beta, IL-6 and TNF-alpha levels in the mice in the group of injection peptide KK-I are found to be obviously lower than those in the mice in the pathogenic group (P is less than 0.01), which indicates that the target peptide KK-I has a certain relieving effect on the peritonitis of the mouse.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (5)
1. The antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is SEQ ID NO:1, wherein glutamine at position 5 of the short peptide sequence is replaced with any one of alanine, leucine, phenylalanine, tryptophan, valine or isoleucine, and aspartic acid at position 9 is replaced with lysine or arginine;
the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 3-13.
2. The use of an antimicrobial peptide according to claim 1 for the preparation of a bacteriostatic agent, wherein the bacteriostatic agent is a bacterial agent that inhibits gram-negative bacteria, such as escherichia coli, salmonella typhimurium, salmonella pullorum, salmonella and enterobacter sakazakii, and gram-positive bacteria, such as staphylococcus aureus, staphylococcus epidermidis, listeria monocytogenes and bacillus cereus.
3. Use of an antibacterial peptide according to claim 1 for the preparation of a medicament for the treatment of a peritonitis infection.
4. A bacteriostatic agent comprising the antimicrobial peptide of claim 1.
5. A medicament for treating a peritonitis infection comprising the antimicrobial peptide of claim 1.
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