An antibacterial peptide derived from the skin of bullfrog and application thereof

Abstract

The application discloses an antibacterial peptide derived from the skin of bullfrog and application thereof, and the amino acid sequence of the antibacterial peptide is GSVGPVGPR (as shown in SEQ ID NO:1), and the molecular weight is 824.4504 g / mol. The antibacterial peptide has obvious inhibiting effect on Staphylococcus aureus, and can be used for preparing a medicine for preventing or inhibiting Staphylococcus aureus infection. Meanwhile, the antibacterial peptide and vitamin A have a significant synergistic bactericidal effect on Staphylococcus aureus. The antibacterial peptide and vitamin A can significantly reduce the use concentration of single component, improve the antibacterial efficiency, overcome the bacterial drug resistance problem, and can be used for preparing antibacterial medicines, skin external preparation, medical dressings, feed additives and food preservatives.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antimicrobial peptide derived from bullfrog skin and its applications. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (SAA) is a widespread Gram-positive pathogen that can cause a variety of diseases, ranging from mild skin infections to life-threatening bacteremia and pneumonia. The emergence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) has posed a significant challenge to clinical treatment, necessitating the development of novel antimicrobial strategies that are less likely to induce resistance.

[0003] Antimicrobial peptides (AMPs) are a class of multifunctional small protein molecules renowned for their broad-spectrum antibacterial, antiviral, and antifungal activities. These peptides are ubiquitous in various life forms, including animals, plants, and microorganisms, and have attracted significant scientific interest due to their unique mechanisms of action and potential applications. Unlike traditional antibiotics, AMPs primarily target bacterial cell membranes, disrupting them through multiple pathways and eliminating bacteria at multiple targets, significantly reducing the likelihood of bacterial resistance development. As an alternative to traditional antibiotics, AMPs have been widely used in medicine, animal husbandry, and the food industry.

[0004] Existing research indicates that high concentrations (typically >30 µg / mL) of retinoic acid have a direct antibacterial effect on certain bacteria (such as Propionibacterium acnes and Staphylococcus aureus), but its effective concentration is high and its value for use alone is limited.

[0005] Therefore, finding a novel antimicrobial peptide that can inhibit and kill Staphylococcus aureus without inducing drug resistance and can be used in combination with vitamin A (retinoic acid) to improve antibacterial efficiency is a technical problem that urgently needs to be solved in this industry. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art. This invention provides an antimicrobial peptide derived from bullfrog skin and its application, thus solving the problems mentioned in the background art.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] One of the technical solutions adopted by the present invention to solve its technical problem is: providing an antimicrobial peptide derived from bullfrog skin, wherein the amino acid sequence of the antimicrobial peptide is GSVGPVGPR (as shown in SEQ ID NO: 1), the molecular weight of the antimicrobial peptide is 824.4504 Daltons, the positive charge is +1, the total hydrophobicity ratio is 22%, and the peptide is predicted to have a random structure by APD3.

[0009] The present invention also provides a nucleic acid encoding the above-mentioned antimicrobial peptide, an expression vector containing the nucleic acid, and a recombinant cell containing the expression vector.

[0010] The present invention also provides the use of the above-mentioned antimicrobial peptides or their encoded nucleic acids in the preparation of antimicrobial drugs or feed additives.

[0011] Preferably, the antimicrobial drug or feed additive is used to inhibit and / or kill Staphylococcus aureus.

[0012] The second technical solution adopted by the present invention to solve its technical problem is: to provide an antibacterial drug, wherein the antibacterial drug includes the above-mentioned antibacterial peptide.

[0013] Preferably, the antibacterial drug further includes vitamin A.

[0014] The third technical solution adopted by the present invention to solve its technical problem is: to provide a feed additive, wherein the feed additive includes the above-mentioned antimicrobial peptide.

[0015] Preferably, the feed additive also includes vitamin A.

[0016] The antimicrobial peptides of this invention mainly act on bacterial cell membranes, increasing cell membrane permeability, leading to leakage of intracellular ions, metabolites, and other cellular contents, loss of cell membrane integrity, and damage to membrane structure and function. These changes ultimately lead to the death of bacterial cells.

[0017] The antimicrobial peptides of the present invention can be synthesized using methods known to those skilled in the art, such as solid-phase synthesis, and purified using methods known to those skilled in the art, such as high-performance liquid chromatography.

[0018] Implementing this invention has the following beneficial effects:

[0019] 1. The antimicrobial peptide of the present invention is safe, non-toxic, has good water solubility, stability, and good in vitro inhibitory or killing activity against Staphylococcus aureus, and can be used to prepare antimicrobial drugs that inhibit or kill Staphylococcus aureus.

[0020] 2. Based on the complementary effects of vitamin A's disruption of bacterial cell membrane lipid structure and the membrane-damaging ability of antimicrobial peptides, a cascade synergistic effect of "membrane disruption-bactericidal action" is generated. At the optimal ratio (500 µg / mL antimicrobial peptide + 10 µg / mL vitamin A), this composition can achieve a faster and more thorough bactericidal effect with a lower dose, thereby significantly reducing the cytotoxicity, irritation, and application costs that may be caused by high concentrations of a single component. Furthermore, this composition is stable and easily integrated into various dosage forms such as gels, dressings, feed, or preservatives, showing broad application prospects in the medical, animal husbandry, and food industries. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the antimicrobial peptide of the present invention.

[0022] Figure 2 This is the mass spectrum of the antimicrobial peptide of the present invention.

[0023] Figure 3 This diagram illustrates the effects of the antimicrobial peptides of the present invention, alone and in combination with vitamin A (retinoic acid), on Staphylococcus aureus.

[0024] Figure 4 This is a time-bacterial kinetic curve of the antimicrobial peptide of the present invention and vitamin A (retinoic acid) alone and in combination against Staphylococcus aureus.

[0025] Figure 5 This invention relates to the effect of the antimicrobial peptide on the permeability of the Staphylococcus aureus cell membrane (potassium ion leakage).

[0026] Figure 6 The effect of vitamin A (retinoic acid) on the permeability of Staphylococcus aureus cell membrane (potassium ion leakage).

[0027] Figure 7 The effect of the combination of antimicrobial peptide and vitamin A (retinoic acid) of the present invention on the permeability of Staphylococcus aureus cell membrane (potassium ion leakage).

[0028] Figure 8 The effect of the combination of antimicrobial peptide and vitamin A (retinoic acid) 2 of the present invention on the permeability of Staphylococcus aureus cell membrane (potassium ion leakage).

[0029] Figure 9 The effect of the combination of antimicrobial peptide and vitamin A (retinoic acid) of the present invention on the permeability of Staphylococcus aureus cell membrane (potassium ion leakage).

[0030] Figure 10 The effect of the antimicrobial peptide of this invention on the permeability of Staphylococcus aureus cell membrane (propidium iodide staining analysis).

[0031] Figure 11 The effect of vitamin A (retinoic acid) on the permeability of Staphylococcus aureus cell membrane (propidium iodide staining analysis).

[0032] Figure 12 The effect of the combination of antimicrobial peptide and vitamin A (retinoic acid) of the present invention on the permeability of Staphylococcus aureus cell membrane (propidium iodide staining analysis).

[0033] Figure 13 The effect of the combination of antimicrobial peptide and vitamin A (retinoic acid) 2 of the present invention on the permeability of Staphylococcus aureus cell membrane (propidium iodide staining analysis).

[0034] Figure 14 The effect of the combination of antimicrobial peptide and vitamin A (retinoic acid) 3 of the present invention on the permeability of Staphylococcus aureus cell membrane (propidium iodide staining analysis). Detailed Implementation

[0035] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. However, those skilled in the art will understand that the following embodiments are not intended to limit the scope of protection of the present invention, and any changes and variations made on the basis of the present invention are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1: Identification and Screening of the Antimicrobial Peptides of the Present Invention

[0039] After drying and crushing, bullfrog skin was enzymatically hydrolyzed with alkaline protease at a material-to-liquid ratio of 1:15 (w / v) for 2 hours at a temperature of 55°C. The enzyme dosage was 5000 U / g. After hydrolysis, the enzyme was inactivated at 100°C for 15 minutes, cooled, and centrifuged at 10000 r / min for 10 minutes. The supernatant was collected and ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 3000 Da. The supernatant with a molecular weight <3000 Da was collected, freeze-dried, and then analyzed by mass spectrometry.

[0040] Chromatographic column: 75μm id×25cm, NanoViper C18 1.9μm, 100A

[0041] Mobile phase A: 0.1% formic acid (FA);

[0042] Mobile phase B: 0.1% FA, 80% ACN acetonitrile;

[0043] Flow rate: 2.50 μL / min;

[0044] Analysis time for each component: 7 min;

[0045] The original file was searched using software in the Lithobates catesbeianus protein database. The search results were then used to screen identified peptide sequences using the APD3 online server. The peptide structure was predicted using the Swiss-model server, revealing an antimicrobial peptide with strong antibacterial activity against Staphylococcus aureus. The sequence is GSVGPVGPR (as shown in SEQ ID NO:1), and the molecular weight is 824.4504 Daltons. The structural schematic and mass spectrum of the antimicrobial peptide are shown below. Figure 1 , 2 As shown.

[0046] Example 2: Preparation of a synergistic antibacterial composition

[0047] Weigh out the antimicrobial peptide (solid-phase synthesis, purity >95%) powder, dissolve and dilute it with sterile phosphate buffer (PBS, pH 7.4) to prepare a stock solution with a concentration of 1 mg / mL, and store at -20℃.

[0048] Weigh out all-trans retinoic acid powder, dissolve it in a trace amount of dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 10 mg / mL, and then perform serial dilution with Mueller-Hinton broth (MHB) medium containing 0.5% fetal bovine serum so that the concentration of DMSO in the final test system is less than 0.5% (v / v) and does not affect bacterial growth.

[0049] When using, dilute both stock solutions with MHB medium to the required working concentration. The synergistic group is a mixture of antimicrobial peptide at a final concentration of 500 µg / mL and vitamin A at a final concentration of 10 µg / mL.

[0050] Example 3: Determination of Time-Kill Curve

[0051] Grouping: Four groups were set up: ① PBS negative control; ② Antimicrobial peptide alone (500 µg / mL); ③ Vitamin A alone (10 µg / mL); ④ Synergistic combination group (500 µg / mL + 10 µg / mL); ⑤ Synergistic combination group (400 µg / mL + 5 µg / mL); ⑥ Synergistic combination group (400 µg / mL + 30 µg / mL); ⑦ Synergistic combination group (600 µg / mL + 5 µg / mL); ⑧ Synergistic combination group (600 µg / mL + 30 µg / mL).

[0052] Operation: In a container containing 5×10 5Add equal volumes of each test solution to the CFU / mL Staphylococcus aureus MHB, mix, and incubate at 37°C. Take samples at 0, 1, 2, 3, 4, 5, and 6 hours, dilute appropriately with PBS, spread on MH agar plates, and incubate at 37°C for 24 hours before counting colonies.

[0053] The results are as follows Figure 4 As shown, the vitamin A alone group had only a slight antibacterial effect. The antimicrobial peptide alone group showed bactericidal activity within 4-6 hours. The synergistic combination group showed a rapid bactericidal effect within 2 hours, and the colony count decreased by more than 3 logarithmic orders by 6 hours (i.e., more than 99.9% kill), and no bacterial regrowth was observed within 24 hours. The bactericidal speed and thoroughness were significantly better than any single component.

[0054] Example 4: Effect of antimicrobial peptides on Staphylococcus aureus cell membrane permeability (potassium ion leakage analysis)

[0055] Dilute Staphylococcus aureus cultured to the logarithmic growth phase to 10. 6 -10 7 CFU / mL was incubated with an equal volume of sample. After incubation, 20 µL of the mixture was combined with 180 µL of protein precipitant, and then centrifuged at 3500 r / min for 5 min. 50 µL aliquots of the supernatant were collected and analyzed using a K+ detection kit. Results are as follows: Figure 5-9 As shown, the individual antimicrobial peptide and vitamin A groups exhibited only slight potassium ion leakage. However, the synergistic combination group demonstrated rapid bactericidal effects within 6 hours, with potassium ion leakage rates significantly better than either component alone.

[0056] Example 5: Effect of antimicrobial peptides on bacterial PI uptake (propidium iodide staining analysis)

[0057] Take culture up to 10 6 -10 7 50 μL of CFU / mL bacterial suspension was added to a 1.5 mL sterile centrifuge tube, followed by an equal volume of sample solutions of different concentrations, with sterile water as a blank control. After incubation at 37°C for 3 h, 100 μL LPI (final concentration 10 μM) was added and incubated in the dark for 15 min. The fluorescence intensity of PI was detected at an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

[0058] Propidium iodide (PI) is a DNA-binding dye that cannot penetrate the intact cell membrane of normal cells or early apoptotic cells. However, in late apoptotic cells and dead cells, PI can pass through the cell membrane and stain the cell nucleus red. Figure 10-14As shown, the fluorescence signal of PI in Staphylococcus aureus treated with antimicrobial peptide alone, vitamin A, and synergistic combination was significantly increased compared with the control group, and the fluorescence signal of the synergistic combination was more obvious, indicating that the synergistic use of the two can accelerate the destruction of the cell membrane integrity of Staphylococcus aureus, so that PI can bind to intracellular nucleic acids.

[0059] The synergistic antibacterial composition of antimicrobial peptides and vitamin A provided by this invention has been experimentally demonstrated to have a clear synergistic effect in antibacterial activity against Staphylococcus aureus. This composition can significantly improve the sterilization speed and efficiency, reduce the concentration of each component, and has higher safety potential. Its preparation method is simple, and it is easy to integrate into various product forms, showing broad application prospects in the fields of anti-infection treatment and preservation.

[0060] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. An antimicrobial peptide derived from bullfrog skin, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the antimicrobial peptide as described in claim 1.

3. An expression vector containing the nucleic acid as described in claim 2.

4. Recombinant cells containing the expression vector as described in claim 3.

5. The application of the antimicrobial peptide or its encoded nucleic acid as described in claim 1 in the preparation of antimicrobial drugs, feed additives, or food preservatives, characterized in that, The antimicrobial drugs, feed additives, or food preservatives are used to inhibit and / or kill Staphylococcus aureus.

6. An antibacterial drug, characterized in that, The antimicrobial drug includes the antimicrobial peptide as described in claim 1.

7. The antibacterial drug according to claim 6, characterized in that, The antibacterial drugs also include vitamin A.

8. A feed additive, characterized in that, The feed additive includes the antimicrobial peptide as described in claim 1.

9. The feed additive according to claim 8, characterized in that, The feed additives also include vitamin A.

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