A sea dragon source antimicrobial peptide SsPle for antibacterial use in complex tropical island reef environments

Abstract

The present invention discloses a sea dragon-derived antimicrobial peptide, SsPle, for antimicrobial applications in the complex environments of tropical islands and reefs. The amino acid sequence of this antimicrobial peptide is shown in SEQ ID NO. 3. This antimicrobial peptide, SsPle, exhibits good water solubility, broad antimicrobial activity, and highly stable antimicrobial properties under high temperature, high salt, and ultraviolet irradiation conditions. It can inhibit the release of free bacterial endotoxins and has significant application prospects as an eco-friendly antimicrobial agent in the unique environments of South China Sea islands and reefs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a sea dragon-derived antimicrobial peptide SsPle for use in complex tropical island and reef environments for antibacterial purposes. Background Art

[0002] The islands and reefs in the South China Sea are typical tropical islands and reefs, marine "oases" formed through coral reef biogeomorphic processes. Their ecosystems are fragile and their environmental carrying capacity is low. Therefore, to meet the sustainable development needs of these islands and reefs, a range of products with minimal impact on the ecological environment is needed. Antimicrobial drugs are indispensable for maintaining human health, but traditional drugs such as antibiotics generally carry risks such as residual contamination, bioaccumulation, the development of microbial resistance, and disruption of ecological community structure. Excessive use of antibiotics could cause significant disturbances in the local environment of these islands and reefs, threatening ecological and environmental safety.

[0003] Antimicrobial peptides (AMPs), a class of small molecule polypeptides commonly found in nature, have shown broad-spectrum inhibitory effects on bacteria, fungi, viruses, tumor cells, etc. It is worth noting that unlike antibiotics that exert their antibacterial effects by acting on specific molecular receptors or targets of pathogens, antimicrobial peptides mainly exert their antibacterial effects through non-receptor-mediated physical destruction of bacterial cell membranes. This rapid and unique mechanism of action makes it difficult for pathogenic microorganisms to develop resistance to antimicrobial peptides. Therefore, antimicrobial peptides can exhibit good antibacterial activity against both antibiotic-sensitive and resistant strains. However, the South China Sea islands and reefs are located in a tropical marine environment with special environmental conditions such as high temperature, high salt, high humidity, and high radiation. Some common antimicrobial peptides have defects such as salt sensitivity, pH sensitivity, or UV sensitivity, which hinder their further application.

[0004] Xue's sea dragon ( Syngnathus schlegeli ) is an important marine bony fish of the family Syngnathidae and the genus Syngnathus, which inhabits the shallow sea environment of nearshore and island reefs. According to the "Chinese Pharmacopoeia" (2020 edition), sea dragon is mainly a traditional Chinese medicine that warms the kidneys and strengthens yang, disperses knots and reduces swelling. Modern pharmacology has further confirmed that sea dragons have significant effects in heat, anti-inflammatory and analgesic effects. However, due to the large demand in the Chinese medicine market and the destruction of habitats and ecological environments, wild sea dragon resources are becoming increasingly scarce. At present, many species of fish in the family Syngnathidae are listed in the "Red List of Endangered Species of the World Conservation Union". Therefore, it is feasible to extract antimicrobial peptides with antibacterial activity from sea dragons, and it is expected to reduce the over-exploitation of resources. Summary of the Invention

[0005] The purpose of the present invention is to provide a new antimicrobial peptide SsPle derived from Scheherazade that can be used for antibacterial purposes in the complex environments of tropical islands and reefs. The antimicrobial peptide has good water solubility, broad antimicrobial activity, and very stable antimicrobial properties under high temperature, high salt, and ultraviolet irradiation. It can inhibit the release of free bacterial endotoxins and can be used as an eco-friendly antimicrobial agent in the special environments of South China Sea islands and reefs. Its application will help reduce the negative impact of traditional chemical preparations on the ecological environment of South China Sea islands and reefs, and provide a new solution for the protection of fragile ecosystems.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The invention relates to an antimicrobial peptide Pleurocidin from Pleurocidin schoeni, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence encoding the amino acid thereof is shown in SEQ ID NO.1.

[0008] A SsPle polypeptide fragment with antibacterial activity was designed and synthesized based on the amino acid sequence of the antibacterial peptide Pleurocidin from Pseudomonas schrenkiana, and its amino acid sequence is shown in SEQ ID NO.3.

[0009] The present invention also provides a nucleic acid encoding the antimicrobial peptide SsPle.

[0010] The present invention also provides a biological material containing the above nucleic acid.

[0011] Preferably, the biological material comprises an expression cassette, a vector or a host cell.

[0012] The present invention also provides the use of the antimicrobial peptide SsPle, nucleic acid or biomaterial in the preparation of antimicrobial products.

[0013] Preferably, the antibacterial agent is one or more of Gram-negative bacteria, Gram-positive bacteria and fungi.

[0014] Preferably, the antibacterial agent is one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Salmonella typhimurium, and Candida albicans.

[0015] Preferably, the antibacterial agent is one or more of multidrug-resistant Escherichia coli, multidrug-resistant Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus.

[0016] Preferably, the product is a medicine, feed, feed additive, disinfectant, detergent or preservative.

[0017] The present invention has the following beneficial effects:

[0018] This invention provides, for the first time, a novel antimicrobial peptide, SsPle, derived from Pieris schoeni, for antimicrobial applications in the complex environments of tropical islands and reefs. This peptide can be prepared in large quantities through solid-phase chemical synthesis and exhibits strong antibacterial activity against common pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. It also exhibits strong inhibitory activity against common clinically resistant bacteria, including multidrug-resistant Escherichia coli, multidrug-resistant Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus. Its inhibitory concentration against multidrug-resistant Acinetobacter baumannii is approximately 3-6 μM, and its bactericidal concentration is approximately 6 μM. It maintains strong antibacterial activity in high-temperature environments (boiling water bath for 30 minutes), high-salt environments (0.9% NaCl), and strong ultraviolet (UV) irradiation for 30 minutes. It also effectively inhibits the release of free bacterial endotoxins, preventing pathogen infection. This peptide has promising application prospects in the complex environments of tropical islands and reefs, and could serve as an eco-friendly antimicrobial agent in the unique environments of South China Sea islands and reefs, reducing the impact of traditional chemical agents on the ecological environment. In addition, its good antibacterial ability and excellent stability give it broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The thermal stability of SsPle antibacterial activity.

[0020] Figure 2 Ion tolerance for the antibacterial activity of SsPle.

[0021] Figure 3 UV resistance for the antimicrobial activity of SsPle.

[0022] Figure 4 The pH tolerance of SsPle antimicrobial activity is shown in Figure 1. A: SsPle peptide and microorganisms were incubated at pH 4; B: SsPle peptide and microorganisms were incubated at pH 5; C: SsPle peptide and microorganisms were incubated at pH 6; D: SsPle peptide and microorganisms were incubated at pH 7; E: SsPle peptide and microorganisms were incubated at pH 8; and F: SsPle peptide and microorganisms were incubated at pH 9.

[0023] Figure 5 The SsPle polypeptide from H. schoenle can inhibit the release of bacterial free endotoxins. DETAILED DESCRIPTION

[0024] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0025] Example 1. Pleurocidin sequence of Pisces xuei

[0026] Based on the genome database of the sea dragon Xue's genotype, the present invention screened and obtained the gene sequence of Pleurocidin, and translated the full-length cDNA of the Pleurocidin antimicrobial peptide gene into an amino acid sequence. The sequence information is as follows:

[0027] The nucleotide sequence of the gene encoding the antimicrobial peptide Pleurocidin from Pseudomonas schrenkiana:>SEQ ID NO.1

[0028] ATGAAGTTTGTCACGCTCTTCCTGGTGTTGTCGCTGGTGATGCTCATGGCTGAGCCCGGCGATTGCTTCTTTAGAAGTCTCTGGAAAGGCGTCAAGGAGGGATTCAGAGCAGGACGCTCGGCATATAAAGCACCGACAACTAAAGAGGATGGGCTGGGAGAACCAACAACCCAACCACTACCCGCCACCACAGCAGCAATACATGCCGATCATCGTTGAGGATTGA

[0029] Amino acid sequence of the antimicrobial peptide Pleurocidin from Pseudomonas schoenostoma:>SEQ ID NO.2

[0030] MKFVTLFLVLSLVMLMAEPGDCFFRSLWKGVKEGFRAGRSAYKAHRQLKRMGWENQQPNHYPPPQQQYMPIIVED

[0031] Example 2. Obtaining polypeptide fragments with antibacterial activity

[0032] Pleurocidin is an antimicrobial peptide composed of 75 amino acid residues. By improving and designing a polypeptide fragment with antimicrobial activity, a 22-amino acid polypeptide fragment was obtained and named SsPle, with the amino acid sequence: FFRSLWKGVKEGFRAGRSAYKA (SEQ ID NO. 3).

[0033] Example 3. Preparation of SsPle polypeptide from Heterosaur xuei

[0034] The amino acid sequence of the antimicrobial peptide SsPle from Schaefera schreiberensis was submitted to Nanjing GenScript Biotechnology Co., Ltd., and the company was commissioned to synthesize the crude product of the antimicrobial peptide SsPle from Schaefera schreiberensis using solid-phase chemical synthesis. The product was purified by high-performance liquid chromatography, concentrated using a freeze dryer, and finally identified by mass spectrometry using a mass spectrometer. After qualified identification, high-purity Schaefera schreiberensis antimicrobial peptide SsPle was prepared.

[0035] Example 4. Determination of antibacterial activity of the SsPle polypeptide from the genus Heterodon schoeni

[0036] The antibacterial activity of the synthetic peptides was determined by incubating bacteria with different concentrations of SsPle peptides. For this assay, Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus), Gram-negative bacteria (Escherichia coli, multidrug-resistant Escherichia coli, Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhimurium) and fungi (Candida albicans) were used. In a 96-well microtiter plate, 50 μL of each bacterial suspension was mixed with 50 μL of different concentrations of SsPle peptide (final peptide concentrations of 1.5, 3, 6, 12, 24, 48, 96 and 192 μM; final bacterial suspension concentration: 10 μM). 4 CFU / mL, fungus 10 3 CFU / mL) and incubated at room temperature for 12-24 hours. A control group was incubated with the peptide solvent and bacterial solution. Bacterial growth was monitored by reading the optical density of the culture at 600 nm. All experiments were performed in triplicate.

[0037] The results are shown in Table 1. The SsPle polypeptide has a good inhibitory effect on the tested Gram-negative bacteria, Gram-positive bacteria and fungi, especially on drug-resistant bacteria, indicating that the SsPle polypeptide has a broad-spectrum antibacterial activity.

[0038] Table 1. Inhibitory concentration of SsPle peptide against microorganisms (unit: μM)

[0039]

[0040] Example 5. Determination of the thermal stability of the SsPle polypeptide from the sea dragon

[0041] The thermal stability of the SsPle polypeptide antibacterial activity was tested using Pseudomonas aeruginosa, multidrug-resistant Escherichia coli, and MRSA. The polypeptide at a concentration twice the MBC of the corresponding strain was placed in a boiling water bath for 10 minutes, 20 minutes, and 30 minutes, and then an antibacterial experiment was performed (see Example 4). The absorbance of the bacteria at a wavelength of 600 nm was measured using a full-wavelength microplate reader at 0 hours, 12 hours, 24 hours, and 36 hours. The results showed that the antibacterial activity against Pseudomonas aeruginosa, MRSA, and multidrug-resistant Escherichia coli was still maintained after 10, 20, and 30 minutes of boiling water bath, and the boiling water bath had no effect on the antibacterial activity ( Figure 1 The results showed that sustained high temperatures of 100°C did not affect the antibacterial activity of SsPle. The SsPle polypeptide exhibited good thermal stability and could stably exert its antibacterial activity in high-temperature environments, suggesting that it could be further applied in the unique natural environments of tropical islands and reefs.

[0042] Example 6. Determination of high salt tolerance of the SsPle polypeptide of the sea dragon

[0043] The high salt tolerance test of antibacterial activity was conducted using Pseudomonas aeruginosa, multidrug-resistant Escherichia coli, and MRSA. After the microorganisms were cultured in fresh nutrient broth to the logarithmic phase, the bacterial solution was adjusted to the required concentration (see Example 4). The bacterial solution of the control group was not added with antimicrobial peptides and no additional sodium ions were added. It was placed under suitable conditions for normal growth. At this time, the sodium ion concentration in the antibacterial experimental solution system was about 122.2 mM. + In the experimental groups with different concentrations, the bacterial solution was first incubated with a peptide with a concentration of 2 times the MBC of the corresponding strain (volume ratio 1:1, final peptide concentration 1 MBC), and Na was additionally added at concentrations of 0 mM, 10 mM, 20 mM, 40 mM, 80 mM, and 160 mM. + (sodium chloride). The results showed that as the concentration of added sodium ions increased, the SsPle peptide still had antibacterial activity against Pseudomonas aeruginosa, MRSA, and multidrug-resistant Escherichia coli. When 80 and 160 mM were added, the SsPle peptide completely lost its antibacterial activity ( Figure 2 When an additional 40 mM sodium ion was added, the total sodium ion concentration was approximately 162.2 mM, compared to 154 mM in normal saline. The results demonstrated that the SsPle peptide maintained good activity even at normal saline concentrations, exhibiting stable antimicrobial activity in high-salt environments (normal saline concentrations), making it suitable for further application in the unique natural environments of tropical islands and reefs.

[0044] Example 7. Determination of UV tolerance of the SsPle polypeptide of the Sea Dragon

[0045] The UV tolerance test of antibacterial activity was carried out using multidrug-resistant Escherichia coli and MRSA. After the solid peptide and liquid peptide (0.5 mg / mL) were irradiated under UV light for 30 minutes, the antibacterial experiment was carried out using the treated peptide at a concentration of 2 times the MBC of the corresponding strain (refer to Example 4). The absorbance value of the bacteria at a wavelength of 600 nm was measured using a full-wavelength microplate reader at 0 h, 12 h, 24 h, 36 h and 48 h. The results showed that the antibacterial activity against MRSA and multidrug-resistant Escherichia coli was still maintained after 30 minutes of UV irradiation, and the antibacterial activity effect was not affected by UV irradiation ( Figure 3 The results showed that UV irradiation treatment did not affect the antibacterial activity of SsPle polypeptide, and it had good UV tolerance. It could stably exert its antibacterial activity in a strong UV environment and could be used in the unique natural environment of tropical islands and reefs.

[0046] Example 8. Determination of pH tolerance of the SsPle polypeptide of the Sea Dragon

[0047] The pH tolerance of antibacterial activity was determined using multidrug-resistant Escherichia coli. The peptides were dissolved in MES buffer at different pH values ​​and subjected to antibacterial experiments, with the final pH values ​​being 4, 5, 6, 7, 8, and 9. The final peptide concentration was the MBC concentration of the corresponding microorganism. The cells were incubated at the optimal temperature and the growth of the microorganisms was monitored using a full-wavelength microplate reader. The results showed that under conditions of pH 4, 5, 6, 7, 8, and 9, the control group grew normally, while the growth of the microorganisms in the experimental group was inhibited ( Figure 4 The results showed that SsPle peptide can stably exert antibacterial activity in an environment with a pH of 4-9 and can be further applied in some extreme environments such as tropical islands and reefs.

[0048] Example 9. Determination of the endotoxin neutralization activity of the SsPle polypeptide of the Sea Dragon

[0049] The SsPle peptide from Heterodon schoenostoma was incubated with Pseudomonas aeruginosa at concentrations of 0, 6, and 12 μM for 15 min. After 30 min, SsPle could significantly inhibit the release of free endotoxin from bacteria with increasing concentrations ( Figure 5 That is, at 15 minutes, the levels of free bacterial endotoxins at 0, 6, and 12 μM concentrations were all between 2 and 4 EU / mL. At 30 minutes, the free bacterial endotoxin levels in the 0 μM control group were approximately 20.37 EU / mL, 3 EU / mL at 6 μM, and 2.59 EU / mL at 12 μM. As the concentration increased, the release of free bacterial endotoxins was inhibited. While the free bacterial endotoxin levels in the control group increased with treatment time, the SsPle peptide in the experimental group successfully inhibited the release of free bacterial endotoxins, particularly at concentrations as low as 6 and 12 μM. These results demonstrate that the SsPle peptide effectively inhibits the release of free bacterial endotoxins. Bacterial endotoxins can lead to host infection, and the SsPle peptide can effectively inhibit the release of free bacterial endotoxins, thereby mitigating infection. This suggests that the SsPle peptide has potential for further application in tropical island reef environments.

[0050] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide SsPle, characterized in that Its amino acid sequence is shown in SEQ ID NO.

3.

2. A nucleic acid, characterized in that It encodes the antimicrobial peptide SsPle according to claim 1.

3. A biological material containing the nucleic acid according to claim 2.

4. The biomaterial according to claim 3, characterized in that The biological material includes an expression cassette, a vector or a host cell.

5. Use of the antimicrobial peptide according to claim 1, the nucleic acid according to claim 2, or the biomaterial according to claim 3 in the preparation of an antimicrobial product; the antimicrobial is one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Salmonella typhimurium, and Candida albicans.

6. The use according to claim 5, characterized in that The anti-Escherichia coli includes anti-multi-drug resistant Escherichia coli; the anti-Acinetobacter baumannii includes anti-multi-drug resistant Acinetobacter baumannii; and the anti-Staphylococcus aureus includes anti-methicillin-resistant Staphylococcus aureus.

7. The use according to claim 5, characterized in that The product is a medicine, feed, feed additive, disinfectant, cleaning agent or preservative.

Images