A mudskipper antibacterial polypeptide Bolespleenin and its application

Abstract

The present invention discloses an antibacterial polypeptide, Bolespleenin, derived from a mudskipper, and its applications. Its amino acid sequence is shown in SEQ ID NO.01. The polypeptide exhibits significant antibacterial effects against Gram-positive and Gram-negative bacteria, as well as some fungi. Furthermore, Bolespleenin exhibits no cytotoxic effects on normal zebrafish embryonic cells and normal mammalian cells, such as human liver cells, at concentrations below 48 μM. The polypeptide exhibits excellent antibacterial efficacy, a broad antibacterial spectrum, and a rapid sterilization rate. Derived from marine bony fish, the polypeptide can be used as an aquatic feed additive or developed into antibacterial and antifungal compositions, possessing broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine molecular biology, and in particular relates to a mudskipper antibacterial polypeptide Bolespleenin and an application thereof. Background Art

[0002] As aquaculture density continues to increase, aquatic diseases are becoming increasingly serious. To address these issues, the use of antibiotics in aquaculture is increasing. However, the widespread, irregular, and irrational use of antibiotics has led to a series of problems, including increased bacterial resistance, suppression of animal immunity, impacts on human health, and even damage to the ecological environment. Therefore, the development of new, highly effective antibacterial drugs and the search for effective alternatives to antibiotics have become urgent issues.

[0003] Antimicrobial peptides (AMPs) are a class of small molecule antimicrobial peptides that are widely distributed in animals and plants. They serve as the first line of defense against infection by various pathogenic microorganisms and are an important component of the innate immune system. Their primary anti-disease mechanism is to act on the cell membranes of pathogenic microorganisms, making it difficult for pathogenic microorganisms to develop resistance to them, thereby avoiding the emergence of drug resistance problems and possessing broad-spectrum antibacterial, antifungal, antiviral, and antiparasitic activities. In addition, antimicrobial peptides also have multiple roles such as immunomodulators, signaling molecules, and anti-tumor agents, making them extremely attractive alternatives to traditional antibiotics and important candidate materials for the development of new antibacterial drugs. They also have considerable prospects in terms of translational applications. Summary of the Invention

[0004] The present invention aims to provide a mudskipper antibacterial polypeptide Bolespleenin and its application.

[0005] The technical solutions of the present invention are as follows:

[0006] A mudskipper antibacterial polypeptide, Bolespleenin, has an amino acid sequence as shown in SEQ ID NO.01.

[0007] Application of the above-mentioned mudskipper antibacterial polypeptide Bolespleenin in the preparation of an antibacterial composition.

[0008] In a preferred embodiment of the present invention, the antibacterial composition has inhibitory and killing effects on Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa.

[0009] An antibacterial composition, the active ingredient of which includes the above-mentioned mudskipper antibacterial polypeptide Bolespleenin.

[0010] Application of the above-mentioned mudskipper antibacterial polypeptide Bolespleenin in the preparation of an antifungal composition.

[0011] In a preferred embodiment of the present invention, the antifungal composition has inhibitory and killing effects on Cryptococcus neoformans, Fusarium oxysporum, Aspergillus flavus and Fusarium solani.

[0012] An antifungal composition, the active ingredient of which includes the above-mentioned mudskipper antibacterial polypeptide Bolespleenin.

[0013] Application of the above-mentioned mudskipper antibacterial polypeptide Bolespleenin in the preparation of aquatic feed additives.

[0014] An aquatic feed additive, the effective ingredient of which includes the above-mentioned mudskipper antibacterial polypeptide Bolespleenin.

[0015] In a preferred embodiment of the present invention, the active ingredient is the above-mentioned mudskipper antibacterial polypeptide Bolespleenin.

[0016] The beneficial effects of the present invention are:

[0017] 1. The antibacterial polypeptide Bolespleenin of the present invention is composed of 14 amino acids and has the molecular formula C 83 H 143 N 33 O 16 The molecular weight is 1859.26 Daltons, which contains 5 positively charged amino acid residues. According to the charge of amino acid residues, the isoelectric point of the antimicrobial peptide is predicted to be 12.18, and the average hydrophilicity coefficient is -0.779. It has strong water solubility and is a positively charged cationic polypeptide.

[0018] 2. The present invention has significant antibacterial effects on Gram-positive bacteria, Gram-negative bacteria, and some fungi. In addition, it has no cytotoxic effect on normal zebrafish embryonic cells and normal mammalian cells such as normal human liver cells at a concentration below 48 μM.

[0019] 3. The present invention has good antibacterial effect, a broad antibacterial spectrum, and a fast sterilization rate. It is derived from marine bony fish and can be used as an aquatic feed additive. It can also be developed into antibacterial compositions, antifungal compositions, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a graph showing the bactericidal kinetics of Bolespleenin, an antimicrobial peptide from mudskippers, against Pseudomonas aeruginosa and Acinetobacter baumannii in Example 3 of the present invention; A represents Staphylococcus aureus + 6 μM Bolespleenin; B represents Acinetobacter baumannii + 6 μM Bolespleenin. The horizontal axis represents time (min) and the vertical axis represents the bactericidal index (%).

[0021] Figure 2 This is a thermostability graph of the antibacterial activity of the mudskipper antibacterial polypeptide Bolespleenin against Acinetobacter baumannii in Example 4 of the present invention; in A, the abscissa is temperature (°C) and the ordinate is colony count (CFU / mL); in B, the abscissa is time (min) and the ordinate is colony count (CFU / mL).

[0022] Figure 3 Figure 5 shows an experiment in which the antimicrobial peptide Bolespleenin from mudskippers inhibits the germination of Fusarium oxysporum and Aspergillus flavus spores. The final concentrations of Bolespleenin are A: 0 μM, B: 1.5 μM, C: 3 μM, D: 6 μM, E: 12 μM, F: 24 μM, G: 48 μM, and H: 96 μM.

[0023] Figure 4 These are scanning electron microscopic images of the mudskipper antibacterial polypeptide Bolespleenin in Example 6 of the present invention after interaction with bacteria and fungi; wherein, A: Acinetobacter baumannii; B: Acinetobacter baumannii + 6 μM; C: Staphylococcus aureus; D: Staphylococcus aureus + 6 μM; E: Aspergillus flavus; F: Aspergillus flavus + 96 μM Bolespleenin; G: Fusarium oxysporum; H: Fusarium oxysporum + 12 μM Bolespleenin.

[0024] Figure 5 Figure 7 shows the MTS-PMS assay for detecting the cytotoxicity of the mudskipper antibacterial peptide Bolespleenin. Figure A shows 293T cells, and Figure B shows ZF4 cells. The horizontal axis represents Bolespleenin protein concentration (μM), and the vertical axis represents cell proliferation rate (%). DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0026] Example 1 Preparation of the antibacterial polypeptide Bolespleenin from mudskipper

[0027] The amino acid sequence of the mudskipper antibacterial polypeptide Bolespleenin is: Leu-Ile-Gly-Leu-Tyr-Leu-Leu-His-Arg-Arg-Arg-Arg-Arg-His (as shown in SEQ ID NO.01)

[0028] The mudskipper antibacterial peptide Bolespleenin with a purity of over 95% can be obtained using existing solid-phase chemical synthesis methods. The mudskipper antibacterial peptide Bolespleenin in this example was synthesized by Nanjing GenScript Biotechnology Co., Ltd. using solid-phase synthesis, and the peptide molecular weight, HPLC and other test information are provided.

[0029] The physicochemical parameters of the antimicrobial peptide Bolespleenin are shown in Table 1.

[0030] Table 1 Physicochemical parameters of antimicrobial peptide Bolespleenin

[0031]

[0032] As shown in Table 1, Bolespleenin has a small molecular weight, good stability, strong water solubility, and is a cationic polypeptide with a positive charge.

[0033] Example 2 Determination of the Minimum Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Mudskipper Antibacterial Polypeptide Bolespleenin The strains involved in this example are: Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Pseudomonas aeruginosa, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus flavus, and Fusarium solani.

[0034] All strains were purchased from China General Microbiological Culture Collection Center.

[0035] The specific determination method of the present embodiment is as follows:

[0036] (1) Spread the preserved strain onto nutrient broth, YPD, or potato glucose plates and invert the plate for 1-7 days.

[0037] (2) Pick colonies from each plate and inoculate them on the corresponding culture medium slant and continue to culture for 1-7 days. Use 10mM sodium phosphate buffer (pH=7.4) to wash down the slant and adjust the concentration of bacterial suspension. Dilute the bacteria to make the final concentration of 5×10 5 CFU / mL. Count the spores under an optical microscope using a hemocytometer and adjust the spore concentration so that the final concentration of mold spores is 5×10 4 pieces / mL.

[0038] (3) The synthesized Bolespleenin powder was dissolved in sterile ddH2O water, filtered through a 0.22 μm filter membrane, and then diluted to protein concentrations of 3 μM, 6 μM, 12 μM, 24 μM, 48 μM, 96 μM, and 192 μM, and placed on ice for later use.

[0039] (4) On a 96-well cell culture plate, set up a blank control group, a negative control group, and an experimental group for each test bacterium, with three replicates for each group:

[0040] aBlank control group: 50 μL of protein sample to be tested and 50 μL of culture medium

[0041] b Negative control group: 50 μL sterile ddH2O water and 50 μL bacterial suspension

[0042] c Test group: 50 μL of protein sample and 50 μL of bacterial suspension

[0043] (5) Place the 96-well cell culture plate in a 28°C incubator and culture for 1-2 days, and observe the MIC results of the experimental group to be tested; after mixing the experimental group to be tested, draw an appropriate amount of bacterial liquid droplets onto the corresponding solid culture medium plate, culture it upside down at an appropriate temperature for 1-2 days, and observe the MBC results.

[0044] The MIC and MBC observation results of the mudskipper antimicrobial peptide Bolespleenin are shown in Table 2:

[0045] Table 2 Antimicrobial activity of the antimicrobial peptide Bolespleenin from mudskipper

[0046]

[0047]

[0048] Note: MIC: Minimum inhibitory concentration (μM), expressed as ab. a: The highest protein concentration at which bacterial growth is visible to the naked eye; b: The lowest protein concentration at which no bacterial growth is visible to the naked eye.

[0049] MBC: Minimum bactericidal concentration (μM), expressed as ab. a: The highest protein concentration at which bacterial colonies were observed to grow on the plate; b: The lowest protein concentration at which no bacterial colonies were observed to grow on the plate.

[0050] Example 3 Bactericidal kinetic curve of the antibacterial peptide Bolespleenin from mudskipper

[0051] Staphylococcus aureus and Acinetobacter baumannii were selected as test bacteria, and the bactericidal kinetics of the mudskipper antibacterial peptide Bolespleenin were determined.

[0052] The specific method is similar to the antibacterial activity assay described in Example 2. Adjust Bolespleenin to 1x the MBC (3 μM final concentration for Staphylococcus aureus and 3 μM final concentration for Acinetobacter baumannii). After incubation with the test bacteria for a period of time, mix the blank control group, negative control group, and test group in a 96-well cell culture plate. Draw 6 μL of the bacterial suspension and dilute it to 600 μL of DPBS. After mixing, spread 40 μL onto a nutrient broth plate and incubate inverted at 37°C for 1-2 days. Record the number of monoclonal isolates of the test bacteria and calculate the bactericidal index.

[0053] The bactericidal index refers to the ratio of the number of clones in the experimental group to the number of clones in the negative control group after a certain period of incubation, expressed as a percentage (see Figure 1 ).

[0054] like Figure 1 As shown, Bolespleenin can kill more than 95% of Staphylococcus aureus at a final concentration of 3 μM in 30 minutes; Bolespleenin can kill more than 95% of Acinetobacter baumannii at a final concentration of 3 μM in 60 minutes.

[0055] Example 4 Antimicrobial activity and thermostability of the antimicrobial peptide Bolespleenin from mudskippers at different temperatures

[0056] Staphylococcus aureus was selected as the test bacteria, and the antibacterial activity and thermal stability of the mudskipper antimicrobial peptide Bolespleenin were determined.

[0057] The specific method is similar to the antibacterial activity determination described in Example 2. The final concentration of Bolespleenin was adjusted to 1 times the MBC (Acinetobacter baumannii: 3 μM). (1) After the Bolespleenin solution was treated at different temperatures for 30 minutes, it was placed on ice for 10 minutes and then incubated with the test bacteria (the control was incubated with an equal volume of sterile ddH2O) for 4 hours. After that, the plate was coated and incubated in a constant temperature incubator at 37°C for 10 hours and then counted (see Figure 2A); (2) After being immersed in boiling water at 100℃ for different periods of time, place on ice for later use. Incubate Bolespleenin or sterile ddH2O with the bacteria to be tested for 4 hours, apply the solution to the plate and incubate in a 37℃ constant temperature incubator for 10 hours, then count (see Figure 2 B). Figure 2 As shown in the results, Bolespleenin can still maintain its antibacterial activity under different temperature conditions or after continuous heat treatment at 100°C.

[0058] Example 5 Optical microscopic observation of mold spore germination after the action of the antibacterial polypeptide Bolespleenin from mudskipper

[0059] Fusarium oxysporum and Aspergillus flavus were selected as test fungi to observe the effect of antimicrobial peptide Bolespleenin from mudskipper on the spore germination of each fungus.

[0060] The specific method is similar to the antibacterial activity determination described in Example 2. The concentration of Bolespleenin protein was adjusted to 6 μM, 12 μM, 24 μM, 48 μM, 96 μM, and 192 μM, and placed on ice for later use; the final concentration of each mold spore was adjusted to 5×10 4 Equal volumes of Bolespleenin at different concentrations were mixed with various mold spores in a 96-well cell culture plate, placed in a 28°C incubator, and cultured for 24 hours. The germination of mold spores was observed under an optical microscope. Figure 3 As shown, spore germination was observed under an optical microscope. Bolespleenin had a significant inhibitory effect on the spore germination of Fusarium oxysporum and Aspergillus flavus at final concentrations of 6 μM and 12 μM, respectively.

[0061] Example 6 Scanning electron microscopic observation of the interaction of the antibacterial peptide Bolespleenin with bacteria and fungal spores

[0062] Acinetobacter baumannii, Staphylococcus aureus, Aspergillus flavus, and Fusarium oxysporum were selected as the strains to be tested, and the preparation of scanning electron microscopy samples was carried out according to the following steps:

[0063] (1) Prepare suspensions of Acinetobacter baumannii and Staphylococcus aureus (OD 600 =0.4), prepare Aspergillus flavus and Fusarium oxysporum spore suspension (5×10 6 / mL) and placed on ice for later use.

[0064] (2) Dissolve the synthetic peptide Bolespleenin in sterile pure water and adjust the protein concentration to 6 μM, 12 μM, and 96 μM. Place on ice until ready for use.

[0065] (3) After mixing equal volumes of the suspension and protein, incubate at an appropriate temperature for an appropriate time (based on MIC and bactericidal kinetics standards).

[0066] (4) Add an equal volume of glutaraldehyde fixative and fix at 4°C for 2 h. Centrifuge at 6000 g for 5 min. Remove the supernatant, resuspend the pellet in 1 mL of PBS, and centrifuge at 6000 g for 10 min. Repeat this step.

[0067] (5) Add 10 μL of PBS to prepare a high-concentration bacterial suspension. Place the high-concentration bacterial suspension dropwise on a glass slide, place on ice, and let stand for 30 minutes. Remove excess liquid with filter paper.

[0068] (6) After soaking in PBS for 15 minutes, dehydrate the sample in 30%-50%-70%-80%-90%-95%-100%-100% (v / v) ethanol, with each dehydration step lasting 15 minutes.

[0069] (7) Dry the sample using the critical point drying method. After spraying with gold, observe and photograph using a scanning electron microscope. (See Figure 4 )

[0070] like Figure 4 As shown in the figure, the bacteria in the control group had normal morphology, complete structure and smooth surface; the bacteria treated with antimicrobial peptides had obvious morphological changes, with wrinkles, holes and even ruptures on the membrane surface, causing the contents to leak out.

[0071] Example 7 Cytotoxicity Assay of the Antibacterial Mudskipper Peptide Bolespleenin

[0072] Human renal epithelial cells (293T) and zebrafish embryonic cells (ZF4) were selected to determine the cytotoxicity of the mudskipper antimicrobial peptide Bolespleenin.

[0073] (1) Collect well-growing human renal epithelial cells and zebrafish embryonic cells and adjust the cell concentration to 1×10 5 The cells were evenly dispersed by blowing, and 100 μL of cell suspension was added to each well of a 96-well cell culture plate. The plate was placed in an incubator at a suitable temperature and cultured until more than 80% of the cells adhered to the wall.

[0074] (2) Carefully aspirate the culture medium, add culture medium containing different concentrations of Bolespleenin, and place in an incubator at an appropriate temperature for 24 hours.

[0075] (3) After adding 20 μL of MTS-PMS solution and incubating in the dark for 2 h, the OD was measured using a microplate reader. 492 The cytotoxicity of Bolespleenin was evaluated by ELISA.

[0076] The results are as follows Figure 5As shown in the results, Bolespleenin has certain cytotoxicity to human kidney epithelial cells and zebrafish embryonic cells only at a concentration of up to 96 μM, and has no cytotoxicity below 48 μM.

[0077] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A mudskipper antibacterial polypeptide Bolespleenin, characterized by: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The use of the antibacterial polypeptide Bolespleenin from mudskipper according to claim 1 in the preparation of an antibacterial composition, characterized in that: The antibacterial composition has inhibitory and killing effects on Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Acinetobacter baumannii, Escherichia coli and Pseudomonas aeruginosa.

3. An antibacterial composition, characterized in that: The active ingredient comprises the mudskipper antibacterial polypeptide Bolespleenin according to claim 1.

4. The use of the antimicrobial polypeptide Bolespleenin from mudskipper according to claim 1 in the preparation of an antifungal composition, characterized in that: The antifungal composition has inhibitory and killing effects on Cryptococcus neoformans, Fusarium oxysporum, Aspergillus flavus and Fusarium solani.

5. An antifungal composition, characterized in that: The active ingredient comprises the mudskipper antibacterial polypeptide Bolespleenin according to claim 1.

6. Use of the antibacterial polypeptide Bolespleenin from mudskipper according to claim 1 in the preparation of an aquatic feed additive.

7. An aquatic feed additive, characterized in that: The active ingredient comprises the mudskipper antibacterial polypeptide Bolespleenin according to claim 1.

8. The aquatic feed additive according to claim 7, characterized in that: The active ingredient is the mudskipper antibacterial polypeptide Bolespleenin as claimed in claim 1.

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