CN117683089A - Boleophtin as antibacterial peptide of Pacific barracuda and application thereof - Google Patents
Boleophtin as antibacterial peptide of Pacific barracuda and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a barracuda antibacterial peptide Boleophtin and application thereof, and the amino acid sequence of the barracuda antibacterial peptide Boleophtin is shown as SEQ ID NO. 01. The antibacterial peptide has broad-spectrum in-vitro antibacterial and antifungal activity, has the characteristic of high sterilization rate, has obvious in-vivo anti-infection activity, and shows a certain immune enhancement activity. The compound is derived from marine teleosts, can be applied to aquatic feed additives with immunity enhancing effect, can also be used for preparing antifungal compositions (mildew-proof preservative), antibacterial compositions and the like, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of marine molecular biology, and particularly relates to a Boleophtin which is an antimicrobial peptide of megalophatherum gracile and application thereof.
Background
Antibacterial peptide (antimicrobial peptides, AMPs) was first discovered in 1939, and researchers extracted clarithromycin (Gramcidin) from Bacillus brevis, and then isolated Cecropin (Cecropin) and bombesin (Magainin) in 1981. Vertebrate antibacterial peptides were first found in amphibians, humans and rabbits in the middle of the 80 s of the 20 th century, and fish AMPs were found in the same period, but were not first detected for antibacterial activity until 1996.
AMPs are generally considered to be an important component of innate immunity, being the first line of defense against a variety of pathogens. The secondary structure of AMPs is a key factor that it acts on pathogenic bacteria, and the antimicrobial peptides recorded in the APD3 database can be divided into four families: α (67.2%), β (12.7%), αβ (17.3%), and non- αβ (2.8%). AMPs in the alpha family contain one alpha helix as the primary structure; the beta family of AMPs comprises at least a pair of beta sheet structures; the alpha beta family of AMPs exist in two different structures at the same time; non- αβ is the absence of both structures. Most AMPs are cationic or amphiphilic, and most AMPs leak the contents by disrupting the bacterial cell membrane, causing cavitation. AMPs rapidly kill invasive pathogens by disrupting cell membranes, making the pathogens less susceptible to drug resistance. AMPs bind to bacterial DNA, RNA to inhibit protein synthesis and cause bacterial death. Some AMPs may exert antibacterial activity by inhibiting cellular respiration and binding to bacterial heat shock proteins.
Marine organisms are living in the marine environment, and an innate immune system which can adapt to the surrounding complex microbial environment is evolved in a long-term evolution process, so that the marine active substances of the microorganisms are essential, and the acquisition of the antibacterial active substances from marine organism resources becomes one of research hotspots. Marine antibacterial actives have been found to include antibacterial peptides, lysozyme, lectins, and a variety of small molecule compounds, which are primarily derived from marine invertebrates, fish and marine microorganisms.
Disclosure of Invention
The invention aims at providing the Boleophtin which is an antimicrobial peptide of Pacific barracuda.
Another object of the invention is to provide the use of the above-mentioned megalophatherum gracile antibacterial peptide Boleophtin.
The technical scheme of the invention is as follows:
the amino acid sequence of the antibacterial peptide Boleophtin of the Pacific barracuda is shown as SEQ ID NO. 01.
The application of the megalophatherum gracile antibacterial peptide Boleophtin in preparing antibacterial composition.
An antibacterial composition comprises the above antibacterial peptide Boleophtin of Lepidoptera.
In a preferred embodiment of the present invention, it has inhibitory and killing effects against Staphylococcus aureus, enterococcus faecium, enterococcus faecalis, acinetobacter baumannii, escherichia coli and Pseudomonas aeruginosa.
The application of the megalophatherum gracile antibacterial peptide Boleophtin in preparing antifungal compositions.
An antifungal composition comprises the above antibacterial peptide Boleophtin of Lepidoptera.
In a preferred embodiment of the invention, it has inhibitory and killing effects against Cryptococcus neoformans, fusarium oxysporum, aspergillus flavus and Fusarium putrescens.
The application of the megalophatherum gracile antibacterial peptide Boleophtin in preparing aquatic feed additives.
An aquatic feed additive comprises the above antibacterial peptide Boleophtin of Pacific barracuda as effective component.
The beneficial effects of the invention are as follows:
1. the invention consists of 20 amino acids, and the molecular formula is C 118 H 188 N 36 O 26 The molecular weight is 2527.02 daltons, which contains 6 positively charged amino acid residues, the isoelectric point of the amino acid residues is predicted to be 12.01 according to the charges of the amino acid residues, the average hydrophilic coefficient is-0.525, and the amino acid residues have very good hydrophilicityStrong water solubility, is a cationic polypeptide with positive charges.
2. The invention has remarkable antibacterial effect on gram-positive bacteria, gram-negative bacteria and mould, and has no cytotoxicity on normal zebra fish embryo cells and normal mammalian cells such as human normal liver cells.
3. The invention has stronger antibacterial activity and antifungal activity, good antibacterial effect, wide antibacterial spectrum, high sterilization rate and certain immunopotentiation activity, is derived from marine teleostea, can be applied to aquatic feed additives with immunopotentiation, can also be used for preparing antifungal compositions (mildew-proof preservative), antibacterial compositions and the like, and has wide application prospect.
Drawings
FIG. 1 is a graph showing the sterilization kinetics of Boleophtin on Pseudomonas aeruginosa and Acinetobacter baumannii in example 3; in FIG. 1, A is Pseudomonas aeruginosa+24 μM Boleophtin; b is Acinetobacter baumannii+6 mu M Boleophtin. The abscissa is time (min) and the ordinate is sterilization index (%)
FIG. 2 is a graph showing the thermostability of the antibacterial activity of the Boleophtin, an antimicrobial peptide from Paenias aeruginosa, in example 4 of the present invention; in FIG. 2A, temperature (. Degree. C.) is plotted on the abscissa and colony count (CFU/mL) is plotted on the ordinate; in FIG. 2B, time (min) is plotted on the abscissa and colony count (CFU/mL) is plotted on the ordinate.
Fig. 3 to 5 are experimental diagrams of the inhibition of mold spore germination by the megalopseed antimicrobial peptide Boleophtin in example 5 of the present invention: wherein, FIG. 3 is Fusarium oxysporum, FIG. 4 is Aspergillus flavus, and FIG. 5 is Fusarium solani. The final concentration of the Boleophtin is A:0 μM; b: 1.5. Mu.M; c: 3. Mu.M; d: 6. Mu.M; e: 12. Mu.M; f: 24. Mu.M; g: 48. Mu.M; h: 96. Mu.M.
FIG. 6 is a photograph of a scanning electron microscope of the antimicrobial peptide Boleophtin of example 6 of the present invention after interaction with Acinetobacter baumannii, staphylococcus aureus and Pseudomonas aeruginosa; wherein A: acinetobacter baumannii; b: acinetobacter baumannii+6 μM; c: staphylococcus aureus; d: staphylococcus aureus +24 μm; e: pseudomonas aeruginosa; pseudomonas aeruginosa +24μm.
FIG. 7 is a scanning electron microscope photograph of the antimicrobial peptides Boleophtin of example 6 of the present invention after having been reacted with spores of Aspergillus flavus, fusarium putrescens and Fusarium oxysporum; wherein A: aspergillus flavus; b: aspergillus flavus +192. Mu.M Boleophtin; c: fusarium solani (L.) Kummer; d: fusarium solani+12. Mu.M Boleophtin; e: fusarium oxysporum; f: fusarium oxysporum +24 μm Boleophtin.
FIG. 8 is a diagram showing the cytotoxicity test of the MTS-PMS method for detecting the antimicrobial peptide Boleophtin of Pacific barrage in example 7 of the present invention; wherein, panel a is 293T cells; panel B shows ZF4 cells. The axis of abscissa shows the concentration of Boleophtin protein (. Mu.M), and the axis of ordinate shows the cell proliferation rate (%).
FIG. 9 is a graph showing experimental results of an anti-infective study of an acute infection model of Aeromonas hydrophila in zebra fish in example 8 of the present invention; wherein, the ordinate is the mortality rate within 72 hours after zebra fish infection; the abscissa is the time after infection, and the sample size n=20 for each group.
Detailed Description
The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.
Example 1 preparation of the antibacterial peptide Boleophtin from Pacific Carnis Pseudosciaenae
The amino acid sequence of the antibacterial peptide Boleophtin of the Pacific barracuda in this example is:
Leu-Lys-Arg-Ser-Asn-Arg-Leu-Pro-Ser-Ile-Phe-Tyr-Arg-Arg-Asn-Lys-Ala-Phe-Ile-Phe(SEQ ID NO.01，LKRSNRLPSIFYRRNKAFIF)
the Boleophtin with the purity of more than 95% can be obtained by adopting the existing solid-phase chemical synthesis method. The antimicrobial peptide Boleophtin of Pacific Tokida in this example was synthesized by solid phase synthesis method, and provided detection information such as polypeptide molecular weight, HPLC, etc.
The physicochemical parameters of the antibacterial peptide Boleophtin are shown in Table 1.
TABLE 1 physical and chemical parameters of antibacterial peptide Boleophtin
From Table 1, it is clear that Boleophtin has small molecular weight, good stability, and strong water solubility, and is a cationic polypeptide with positive charges.
Example 2 determination of the minimum inhibitory concentration (MIC: minimum Inhibition Concentration) and the minimum bactericidal concentration (MBC: minimum Bactericidal Concentration) of the antimicrobial peptide Boleophtin from Pacific Carnis
The strains involved in this example are: staphylococcus aureus (Staphylococcus aureus), acinetobacter baumannii (Acinetobacter baumannii), escherichia coli (Escherichia coli), enterococcus faecium (Enterococcus faecium), enterococcus faecalis (Enterococcus faecalis), pseudomonas aeruginosa (Pseudomonas aeruginosa), cryptococcus neoformans (Cryptococcus neoformans), fusarium oxysporum (Fusarium oxysporum), aspergillus flavus (Aspergillus flavus) and Fusarium solani (Fusarium solani).
The strains are purchased from China general microbiological culture Collection center and are preserved and stored in the laboratory.
The specific method comprises the following steps:
(1) Coating staphylococcus aureus, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, enterococcus faecalis and enterococcus faecium on a nutrient broth plate, and culturing for 18-24 hours in an inversion way at each proper temperature; coating Cryptococcus neoformans on YPD plates, and culturing for 1-3d at 28deg.C in an inverted manner; fusarium oxysporum, aspergillus flavus and Fusarium putrescens spores are coated on a potato dextrose plate, and are inversely cultured for 1-7d at 28 ℃.
(2) Bacterial colonies are picked from each flat plate and inoculated on the corresponding culture medium inclined planes, bacteria and vibrio are continuously cultured for 18-24 hours, yeast fungi are continuously cultured for 1-3 days, and mould is continuously cultured for 1-7 days. Bacteria, vibrio and yeast fungi were washed down the incline with 10mM sodium phosphate buffer (ph=7.4) and the bacterial suspension concentration was adjusted. Diluting bacteria with MH liquid culture medium; dilution of Yeast with YPD liquid MediumA fungus; TSB medium supplemented with 1.5% NaCl dilutes Vibrio so that the final concentration of the cells is 5X 10 5 CFU/mL. Washing mold spores from the slant with 10mM sodium phosphate buffer solution (pH=7.4), diluting the spores with a mixture of potato dextrose liquid medium and sodium phosphate buffer solution, counting the spores under an optical microscope with a blood cell counting plate, and adjusting the spore concentration to give a final mold spore concentration of 5×10 4 And each mL.
(3) The synthesized Boleophtin powder is sterilized with ddH, respectively 2 O water is dissolved, and after filtration through a 0.22 μm filter membrane, the protein concentration is diluted to 3. Mu.M, 6. Mu.M, 12. Mu.M, 24. Mu.M, 48. Mu.M, 96. Mu.M, 192. Mu.M by a multiple ratio, and the mixture is placed on ice for standby.
(4) On a 96-well cell culture plate, each test bacterium is provided with a blank control group, a negative control group and a test experiment group, and each group is provided with three parallels:
a blank control group: 50. Mu.L of protein sample to be tested and 50. Mu.L of culture medium
b negative control group: 50 mu L sterile ddH 2 O-water and 50. Mu.L of bacterial suspension
c test group: 50. Mu.L of protein sample to be tested and 50. Mu.L of bacterial suspension
(5) Placing the 96-well cell culture plate in a 28 ℃ incubator, culturing for 1-2d, and observing MIC results in the experimental group to be tested;
blowing and uniformly mixing the experimental group to be tested, sucking a proper amount of fungus liquid drops on a corresponding solid culture medium plate, inversely culturing for 1-2d at a proper temperature, and observing an MBC result.
The observations of the Boleophtin MIC and MBC are shown in Table 2:
TABLE 2 antibacterial Activity of the antibacterial peptide Boleophtin from Pacific barracuda
Note that: MIC: minimum inhibitory concentration (μM), indicated as a-b. a: the highest protein concentration at which the cells grow is visible to the naked eye; b: minimal protein concentration at which no cell growth was observed by naked eyes
MBC: minimum bactericidal concentration (μm), indicated as a-b. a: the highest protein concentration for visible colony growth on the plate; b: the plates did not see the lowest protein concentration for colony growth.
Example 3 antimicrobial peptide Boleophtin Sterilization kinetics Curve for Pacific
In the embodiment, pseudomonas aeruginosa and acinetobacter baumannii are selected as bacteria to be detected, and the sterilization kinetics of the antibacterial peptide Boleophtin of the megalophatherum gracile is determined.
The specific procedure is similar to the antibacterial activity assay described in example 2. Boleophtin was adjusted to 1-fold MBC (Pseudomonas aeruginosa: 12. Mu.M final concentration; acinetobacter baumannii: 3. Mu.M final concentration). After being hatched with the bacteria to be detected, 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, 150min and 180min are carried out, a blank control group, a negative control group and an experimental group to be detected are taken from a 96-hole cell culture plate, uniformly mixed, diluted by a doubling ratio, coated on a nutrient broth plate, inversely cultured for 1-2d at 37 ℃, the monoclonal number of the bacteria to be detected is recorded, and the sterilization index is calculated.
The bactericidal index is the ratio of the number of clones in the test group to the number of clones in the negative control group after a certain period of co-incubation, expressed as a percentage (see fig. 1).
As shown in FIG. 1, boleophtin can kill more than 95% of Pseudomonas aeruginosa at a final concentration of 12 μM for 90 min; boleophtin has a rapid killing effect on Acinetobacter baumannii at a final concentration of 3 mu M, and can kill more than 90% of live bacteria within 5min.
Example 4 antibacterial Activity and thermal stability of the Marasmius macrocarpa antibacterial peptide Boleophtin under different temperature conditions
In the embodiment, pseudomonas aeruginosa is selected as a bacterium to be detected, and the antibacterial activity and the thermal stability of the antibacterial peptide Boleophtin of the megalopharyngodon idella are measured.
The specific procedure is similar to the antibacterial activity assay described in example 2. The final concentration of Boleophtin was adjusted to 1-fold MBC (Pseudomonas aeruginosa: 12. Mu.M). (1) Treating Boleophtin solution at 0deg.C, 4deg.C, 20deg.C, 30deg.C, 40deg.C, 50deg.C, 60deg.C, 70deg.C, 80deg.C, 90deg.C, 100deg.C, 121deg.C for 30min, cooling on ice for 10min, and co-incubating with bacteria to be tested (control)Equal volume sterile ddH 2 O co-incubation) for 4h, plating, culturing in a constant temperature incubator at 37 ℃ for 10h, and counting (see FIG. 2A); (2) Water-bathing in boiling water at 100deg.C for 5min, 10min, 20min, 30min, 60min, 120min, 240min, and standing on ice. Either Boleophtin or sterile ddH 2 O was incubated with the test bacteria for 4 hours and then incubated in a plate-coated incubator at 37℃for 10 hours and counted (see FIG. 2B). As shown in fig. 2, boleophtin can maintain its antimicrobial activity under different temperature conditions or after continuous heat treatment at 100 ℃.
EXAMPLE 5 optical microscopic observation of mold spore germination following action of the Boleophtin antibacterial peptide
In the embodiment, fusarium oxysporum, aspergillus flavus and Fusarium putrescens are selected as bacteria to be detected, and the influence of the Boleophtin on the germination of all mould spores is observed.
The specific procedure is similar to the antibacterial activity assay described in example 2. Adjusting the concentration of Boleophtin protein to 6 mu M, 12 mu M, 24 mu M, 48 mu M, 96 mu M and 192 mu M, and placing on ice for later use; adjusting the final concentration of each mould spore to 5×10 4 And each mL. Mixing equal volume of Boleophtin with mould spores in 96-well cell culture plates, placing in a 28 ℃ incubator, standing for 24 hours, observing the germination condition of mould spores under an optical microscope, and observing the germination condition of mould spores under the optical microscope, wherein Boleophtin has remarkable inhibition effect on spore germination of Fusarium oxysporum, fusarium flavum and Fusarium putrescens under the final concentrations of 12 mu M, 96 mu M and 6 mu M respectively, as shown in figures 3 to 5.
Example 6 scanning electron microscope observation of the Boleophtin, an antimicrobial peptide of Pacific Carnis, after action with spores of bacteria and fungi
In the embodiment, acinetobacter baumannii, pseudomonas aeruginosa, staphylococcus aureus, aspergillus flavus and fusarium oxysporum are selected as strains to be detected, and the preparation of a scanning electron microscope sample is carried out according to the following steps:
(1) Preparation of Acinetobacter baumannii, pseudomonas aeruginosa, staphylococcus aureus suspension (OD) as described in example 2 600 Preparation of aspergillus flavus and fusarium oxysporum spore suspensions (5×10) =0.4) 6 and/mL), and placed on ice for use.
(2) The synthetic peptide Boleophtin was dissolved in sterile pure water and adjusted to protein concentrations of 6. Mu.M, 24. Mu.M, 96. Mu.M, 192. Mu.M, and placed on ice for further use.
(3) Equal volumes of suspension and protein were mixed and incubated at the appropriate temperature for the appropriate time (according to MIC and sterilization kinetics criteria).
(4) Adding an equal volume of glutaraldehyde fixing solution, fixing for 2 hours at 4 ℃, and centrifuging for 5 minutes at 6000 g.
(5) The supernatant was removed, 1mL of PBS was used to resuspend the pellet, 6000g was centrifuged for 10min, and the procedure was repeated.
(6) A higher concentration bacterial suspension was prepared by adding 10. Mu.L of PBS.
(7) The high-concentration bacterial suspension is dripped on a glass slide, placed on ice, and after standing for 30min, the redundant liquid is sucked by filter paper.
(8) After PBS is soaked for 15min, ethanol with the concentration of 30% -50% -70% -80% -90% -95% -100% (v/v) is dehydrated step by step, and each stage is dehydrated for 15min.
(9) The critical point drying method dries the sample.
(10) After the metal spraying, the metal is observed and photographed by a scanning electron microscope.
As shown in fig. 6 and 7, the bacteria of the control group are normal in morphology, complete in structure and smooth in surface; the bacteria treated by the antibacterial peptide has obvious change of morphology, and folds, holes and even cracks appear on the surface of the membrane, so that the content leaks.
EXAMPLE 7 determination of Boleophtin cytotoxicity of antibacterial peptide of Pacific Carnis Pseudosciaenae
Human kidney epithelial cells (293T) and zebra fish embryonic cells (ZF 4) were selected and tested for the cytotoxicity of the megalobrama ambrosioides antibacterial peptide Boleophtin.
(1) Collecting human kidney epithelial cells and zebra fish embryo cells in good growth state, and regulating cell concentration to 1×10 5 The cells are evenly blown off per mL, 100 mu L of cell suspension is added into each hole of a 96-hole cell culture plate, and the mixture is placed in a proper temperature incubator for culture until more than 80% of cells are attached.
(2) The medium was carefully aspirated, medium containing varying concentrations (0. Mu.M, 6. Mu.M, 12. Mu.M, 24. Mu.M, 48. Mu.M, 96. Mu.M) of Boleophtin was added and incubated in an incubator at the appropriate temperature for 24h.
(3) After incubation for 2h in the dark after addition of 20. Mu.L MTS-PMS solution, OD was measured using an ELISA reader 492 Value, the cytotoxicity of Boleophtin was evaluated.
As shown in FIG. 8, boleophtin concentrations as high as 96. Mu.M had no toxic effect on human kidney epithelial cells and zebra fish embryonic cells.
Example 8 evaluation of anti-infective Activity of the antibacterial peptide Boleophtin from Pacific Carnis
The present example uses the zebra fish infection model to evaluate the in vivo activity of Boleophtin, according to the previous method steps as follows: (1) Aeromonas hydrophila (Aeromonas hydrophila) deposited at-80℃was activated on NB plates and incubated overnight in an incubator with inversion at 28 ℃. The following day, monoclonal colonies were picked up in fresh NB medium and expanded to log phase in a shaker at 28 ℃. (2) The bacterial liquid harvested in the logarithmic phase was centrifuged at 3000rpm for 10min to collect bacteria and resuspended to 1X 10 with PBS 8 CFU/mL. (3) Zebra fish were anesthetized by soaking with 0.02% (w/v) MS-222, and 10.mu.0 Aeromonas hydrophila (about 1X 10) was intraperitoneally injected with a microinjector 6 CFU/tail). (4) At 0.5h post infection, 15 μg of peptide was intraperitoneally administered to each fish in the experimental group, and an equal volume of PBS was injected into the control group, 20 fish in each of the experimental and control groups. (5) And observing the death condition of the fish within 72 hours after infection, fishing out the dead fish in time, and counting the death rate at a specific time point. And survival curves were drawn using GraphPad software.
The results are shown in fig. 9, the survival rate of the control group after bacterial infection is about 52% and the survival rate of the control group after Boleophtin treatment is improved to about 82%, which proves that the Boleophtin has remarkable in vivo anti-infection activity and can be used as an immunopotentiator for further research and application.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, i.e., the invention is not to be limited to the details of the invention.
Claims (9)
1. The antibacterial peptide Boleophtin for the megalopharyngodon piceus is characterized in that: the amino acid sequence is shown as SEQ ID NO. 01.
2. Use of the antimicrobial peptide Boleophtin of megalopseed according to claim 1 for the preparation of an antimicrobial composition.
3. An antibacterial composition characterized in that: the effective component comprises the antibacterial peptide Boleophtin of the megalophatherum gracile in claim 1.
4. An antibacterial composition according to claim 3 wherein: it has inhibiting and killing effects on Staphylococcus aureus, enterococcus faecium, enterococcus faecalis, acinetobacter baumannii, escherichia coli and Pseudomonas aeruginosa.
5. Use of the antimicrobial peptide Boleophtin of megalopseed according to claim 1 for the preparation of an antifungal composition.
6. An antifungal composition characterized in that: the effective component comprises the antibacterial peptide Boleophtin of the megalophatherum gracile in claim 1.
7. An antifungal composition as defined in claim 6 wherein: it has inhibiting and killing effects on Cryptococcus neoformans, fusarium oxysporum, aspergillus flavus and Fusarium putrescens.
8. Use of the antimicrobial peptide Boleophtin of megalophatherum gracile according to claim 1 for the preparation of an aquatic feed additive.
9. An aquatic feed additive, characterized in that: the effective component comprises the antibacterial peptide Boleophtin of the megalophatherum gracile in claim 1.
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