Syngnathus source antibacterial peptide SsPle applied to antibiosis in complex environment of tropical islands and reefs

Abstract

The invention discloses a syngnathus source antibacterial peptide SsPle applied to antibiosis in a complex environment of tropical islands and reefs. The amino acid sequence of the antibacterial peptide SsPle is as shown in SEQ ID NO. 3. The antibacterial peptide SsPle is relatively good in water solubility and wide in antibacterial activity, has very stable antibacterial performance under high temperature, high salt and ultraviolet irradiation, can inhibit release of free endotoxin of bacteria, can be used as an eco-friendly antibacterial agent in a special environment of islands and reefs in the South China Sea, and has an important application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a syngnathid-derived antimicrobial peptide SsPle for antibacterial use in the complex environment of tropical islands and reefs. Background Art

[0002] The South China Sea islands and reefs are typical tropical islands and reefs, which are "oases" on the sea formed by coral reef biogeomorphic processes. The ecosystem is fragile and the environmental carrying capacity is low. Therefore, in response to the needs of the sustainable development of the South China Sea islands and reefs, a batch of products with little impact on the ecological environment are required. Antibacterial drugs are indispensable for maintaining human life and health. However, traditional drugs such as antibiotics generally have risks such as residual pollution, bioaccumulation, easy formation of microbial drug resistance, and interference with the ecological community structure. The large-scale use of antibiotics may cause severe disturbances in the local environment of the South China Sea islands and reefs, threatening the ecological environment security.

[0003] Antimicrobial peptides (AMPs), as a class of small molecular polypeptides widely present in nature, have shown broad-spectrum inhibitory effects on bacteria, fungi, viruses, tumor cells, etc. It is worth noting that different from antibiotics that play antibacterial roles by acting on specific molecular receptors or targets of pathogens, antimicrobial peptides mainly play antibacterial roles through the physical destruction of bacterial cell membranes mediated by non-receptors. This rapid and unique mechanism of action makes it difficult for pathogenic microorganisms to develop drug resistance to antimicrobial peptides. Therefore, whether it is antibiotic-sensitive strains or drug-resistant strains, antimicrobial peptides can show good antibacterial activities. 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 ultraviolet sensitivity, which hinder their further application.

[0004] Syngnathus schlegelii Syngnathus schlegeli is an important marine teleost fish of the family Syngnathidae and the genus Syngnathus, inhabiting the shallow sea environment of the inshore and islands and reefs. According to the "Chinese Pharmacopoeia" (2020 Edition), syngnathus is mainly a traditional Chinese medicine for warming the kidney and strengthening yang, dispelling stasis and relieving swelling. Modern pharmacology has further confirmed that syngnathus has significant effects in aspects such as... and anti-inflammatory and analgesic effects. However, due to the large demand in the Chinese medicine market and the destruction of habitats and ecological environments, the wild resources of syngnathus are becoming increasingly scarce. At present, many species of the family Syngnathidae have been listed in the "Red List of Endangered Species of the International Union for Conservation of Nature". Therefore, it is feasible to extract antimicrobial peptides with antibacterial activities from syngnathus, and it is expected to reduce the overexploitation of resources. Summary of the Invention

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

[0006] To solve the above technical problems, the present invention provides the following technical solutions: A pleurocidin antibacterial peptide from Syngnathus schlegeli, its amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence encoding its amino acid is shown in SEQ ID NO.1.

[0007] A polypeptide fragment SsPle with antibacterial activity designed and synthesized based on the amino acid sequence of the pleurocidin antibacterial peptide from Syngnathus schlegeli, its amino acid sequence is shown in SEQ ID NO.3.

[0008] The present invention also provides a nucleic acid encoding the above antibacterial peptide SsPle.

[0009] The present invention also provides a biological material containing the above nucleic acid.

[0010] Preferably, the biological material includes an expression cassette, a vector or a host cell.

[0011] The present invention also provides the application of the above antibacterial peptide SsPle, nucleic acid or biological material in the preparation of antibacterial products.

[0012] Preferably, the antibacterial is one or more of anti-Gram-negative bacteria, Gram-positive bacteria and fungi.

[0013] Preferably, the antibacterial is one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Salmonella typhimurium, Candida albicans.

[0014] Preferably, the antibacterial is one or more of multi-drug resistant Escherichia coli, multi-drug resistant Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus.

[0015] Preferably, the product is a drug, feed, feed additive, disinfectant, cleaner or preservative.

[0016] The present invention has the following beneficial effects: The present invention for the first time provides a novel antibacterial peptide SsPle derived from Syngnathus schlegeli that can be applied to the complex environment of tropical islands and reefs. This antibacterial peptide can be prepared in large quantities through solid-phase chemical synthesis. Moreover, this antibacterial peptide has a strong antibacterial effect on common pathogenic bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus, and also has strong inhibitory activity against clinically common drug-resistant bacteria such as multidrug-resistant Escherichia coli, multidrug-resistant Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus. Its antibacterial concentration against multidrug-resistant Acinetobacter baumannii is about 3 - 6 μM and the bactericidal concentration is about 6 μM. It can still maintain good antibacterial activity under high-temperature environments (boiling water bath for 30 min), high-salt environments (0.9% NaCl), and strong ultraviolet environments (ultraviolet irradiation for 30 min). It can also effectively inhibit the release of bacterial free endotoxin and prevent pathogen infection. It is very suitable for the application prospects in the complex environment of tropical islands and reefs and can be used as an eco-friendly antibacterial agent in the special environment of the South China Sea islands and reefs to reduce the impact of traditional chemical agents on the ecological environment. In addition, its good antibacterial ability and excellent stability endow it with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the thermal stability of the antibacterial activity of SsPle.

[0018] Figure 2 shows the ion tolerance of the antibacterial activity of SsPle.

[0019] Figure 3 shows the ultraviolet tolerance of the antibacterial activity of SsPle.

[0020] Figure 4 shows the pH tolerance of the antibacterial activity of SsPle. Among them, A: SsPle polypeptide is co-incubated with microorganisms at pH = 4; B: SsPle polypeptide is co-incubated with microorganisms at pH = 5; C: SsPle polypeptide is co-incubated with microorganisms at pH = 6; D: SsPle polypeptide is co-incubated with microorganisms at pH = 7; E: SsPle polypeptide is co-incubated with microorganisms at pH = 8; F: SsPle polypeptide is co-incubated with microorganisms at pH = 9.

[0021] Figure 5 shows that the SsPle polypeptide of Syngnathus schlegeli can inhibit the release of bacterial free endotoxin. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following examples are further illustrations of the present invention rather than limitations thereof.

[0023] Example 1. The sequence of Pleurocidin from Syngnathus schlegeli Based on the genome database of Syngnathus schlegeli, the gene sequence of Pleurocidin was screened, and the full-length cDNA of the Pleurocidin antimicrobial peptide gene was translated into an amino acid sequence. The sequence information is as follows: Nucleotide sequence of the coding gene of Syngnathus schlegeli antimicrobial peptide Pleurocidin: >SEQ ID NO.1 ATGAAGTTTGTCACGCTCTTCCTGGTGTTGTCGCTGGTGATGCTCATGGCTGAGCCCGGCGATTGCTTCTTTAGAAGTCTCTGGAAAGGCGTCAAGGAGGGATTCAGAGCAGGACGCTCGGCATATAAAGCACACCGACAACTAAAGAGGATGGGCTGGGAGAACCAACAACCCAACCACTACCCGCCACCACAGCAGCAATACATGCCGATCATCGTTGAGGATTGA Amino acid sequence of Syngnathus schlegeli antimicrobial peptide Pleurocidin: >SEQ ID NO.2 MKFVTLFLVLSLVMLMAEPGDCFFRSLWKGVKEGFRAGRSAYKAHRQLKRMGWENQQPNHYPPPQQQYMPIIVED Example 2. Obtaining a polypeptide fragment with antibacterial activity

[0024] Syngnathus schlegeli antimicrobial peptide Pleurocidin is an antimicrobial peptide composed of 75 amino acid residues. By modifying and designing a polypeptide fragment with antibacterial activity of Syngnathus schlegeli Pleurocidin, a polypeptide fragment with a length of 22 amino acids was obtained and named SsPle. Its amino acid sequence is: FFRSLWKGVKEGFRAGRSAYKA (SEQ ID NO.3).

[0025] Example 3. Preparation of Syngnathus schlegeli SsPle polypeptide The amino acid sequence of Syngnathus schlegeli antimicrobial peptide SsPle was submitted to Nanjing Genscript Biotech Co., Ltd., and the company was commissioned to synthesize the crude product of Syngnathus schlegeli antimicrobial peptide SsPle using solid-phase chemical synthesis. It was purified by high-performance liquid chromatography, then concentrated using a freeze dryer, and finally mass spectrometry identification was carried out using a mass spectrometer. After passing the identification, high-purity Syngnathus schlegeli antimicrobial peptide SsPle was prepared.

[0026] Example 4. Determination of the antibacterial activity of Syngnathus schlegeli SsPle polypeptide The antibacterial activity of the synthetic peptide was determined by incubating bacteria with different concentrations of SsPle polypeptide. 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 SsPle polypeptide at different concentrations (final polypeptide concentrations of 1.5, 3, 6, 12, 24, 48, 96 and 192 μM; final bacterial suspension concentration: bacteria 10 4 CFU / mL, fungi 10 3 CFU / mL), and incubated at room temperature for 12 - 24 hours. The control group was incubated with the polypeptide solvent and the bacterial suspension together. The optical density of the culture was read at 600 nm to monitor bacterial growth. All experiments were performed in triplicate.

[0027] As shown in Table 1, the SsPle polypeptide had good inhibitory effects on the tested Gram-negative bacteria, Gram-positive bacteria and fungi, especially on drug-resistant bacteria. This indicates that the SsPle polypeptide has broad-spectrum antibacterial activity.

[0028] Table 1. Antibacterial concentrations of SsPle polypeptide against microorganisms (in μM)

[0029] Example 5. Determination of the thermal stability of SsPle polypeptide from Syngnathus schlegelii The thermal stability of the antibacterial activity of SsPle polypeptide was determined using Pseudomonas aeruginosa, multidrug-resistant Escherichia coli, and MRSA. The polypeptides at 2-fold MBC concentration of the corresponding strains were boiled in water bath for 10 min, 20 min, and 30 min respectively, and then antibacterial experiments were carried out (refer to Example 4). The absorbance values of the bacteria at 600 nm wavelength were measured using a full-wavelength microplate reader at 0 h, 12 h, 24 h, and 36 h respectively. It was found that after boiling in water bath for 10, 20, and 30 min, the antibacterial activity against Pseudomonas aeruginosa, MRSA, and multidrug-resistant Escherichia coli still remained, and the boiling water bath had no effect on the antibacterial activity effect ( Figure 1 ). The results showed that continuous high temperature at 100 °C did not affect the antibacterial activity of SsPle. The SsPle polypeptide had good thermal stability of antibacterial activity and could stably exert its antibacterial activity in a high-temperature environment, and could be further applied in the unique natural environment of tropical islands and reefs.

[0030] Example 6. Determination of the high salt tolerance of SsPle polypeptide from Syngnathus schlegelii The high-salt tolerance of antibacterial activity was determined using Pseudomonas aeruginosa, multidrug-resistant Escherichia coli, and MRSA. When the microorganisms were cultured to the logarithmic phase in fresh nutrient broth medium, the bacterial solution was adjusted to the required concentration (see Example 4). For the bacterial solution in the Control group, no antibacterial peptide was added, and no additional sodium ions were added, and it was allowed to grow normally under appropriate conditions. At this time, the sodium ion concentration in the antibacterial experiment solution system was approximately 122.2 mM. In the experimental groups with different Na + concentrations, the bacterial solution was first co-incubated with the polypeptide at 2 times the MBC of the corresponding strain (volume ratio 1:1, final polypeptide concentration 1 MBC), and at the same time, sodium ions with concentrations of 0 mM, 10 mM, 20 mM, 40 mM, 80 mM, and 160 mM were respectively added additionally. + (sodium chloride). The results showed that as the concentration of added sodium ions increased, the SsPle polypeptide still had antibacterial activity against Pseudomonas aeruginosa, MRSA, and multidrug-resistant Escherichia coli. When 80 and 160 mM were added additionally, the SsPle polypeptide completely lost its antibacterial activity ( Figure 2 ). When 40 mM was added additionally, the total sodium ion concentration was approximately 162.2 mM, and the sodium ion concentration of physiological saline was 154 mM. The results indicated that the SsPle polypeptide still had good activity at the concentration of physiological saline, could stably exert its antibacterial activity in a high-salt environment (physiological saline concentration), and was suitable for further application in the unique natural environment of tropical islands and reefs.

[0031] Example 7. Determination of the ultraviolet tolerance of the SsPle polypeptide of Syngnathus schlegelii The ultraviolet tolerance of antibacterial activity was determined using multidrug-resistant Escherichia coli and MRSA. After irradiating the solid polypeptide and liquid polypeptide (0.5 mg / mL) under ultraviolet light for 30 min respectively, antibacterial experiments were carried out using the treated polypeptide at 2 times the MBC concentration of the corresponding strain (refer to Example 4). The absorbance values of the bacteria at a wavelength of 600 nm were measured using a full-wavelength microplate reader at 0 h, 12 h, 24 h, 36 h, and 48 h respectively. It was found that after ultraviolet irradiation treatment for 30 min, it still had antibacterial activity against MRSA and multidrug-resistant Escherichia coli, and the antibacterial activity effect was not affected after ultraviolet irradiation treatment ( Figure 3 ). The results showed that ultraviolet irradiation treatment did not affect the antibacterial activity of the SsPle polypeptide, had good ultraviolet tolerance, could stably exert its antibacterial activity in a strong ultraviolet environment, and could be applied in the unique natural environment of tropical islands and reefs.

[0032] Example 8. Determination of the pH tolerance of the SsPle polypeptide of Syngnathus schlegelii The pH tolerance of antibacterial activity was determined using multidrug-resistant Escherichia coli. The polypeptide was dissolved in MES buffer at different pH values and the antibacterial experiment was carried out, with the final pH = 4, 5, 6, 7, 8, 9. The final concentration of the polypeptide was the MBC concentration of the corresponding microorganism. Incubated at the optimal temperature, the growth of the microorganism was monitored with a full-wavelength microplate reader. The results showed that in the environment with pH = 4, 5, 6, 7, 8, 9, the control group grew normally while the microorganism in the experimental group was inhibited from growing ( Figure 4 ). The results indicate that the SsPle polypeptide can stably exert its antibacterial activity in the environment with pH = 4 - 9 and can be further applied in some extreme environments in tropical island reefs.

[0033] Example 9. Determination of the endotoxin-neutralizing activity of the SsPle polypeptide of Syngnathus schlegeli The SsPle polypeptide of Syngnathus schlegeli was co-incubated with Pseudomonas aeruginosa at concentrations of 0, 6, and 12 μM for 15 min and 30 min respectively. With the increase in concentration, SsPle could significantly inhibit the release of free bacterial endotoxin ( Figure 5 ). That is, the content of free bacterial endotoxin at 0, 6, and 12 μM was between 2 - 4 EU / mL after 15 minutes. The content of free bacterial endotoxin in the 0 μM control group was about 20.37 EU / mL after 30 minutes, about 3 EU / mL for 6 μM, and about 2.59 EU / mL for 12 μM. The release of free bacterial endotoxin was inhibited with the increase in concentration. With the increase in treatment time, the free bacterial endotoxin in the control group also increased, while the SsPle polypeptide in the experimental group successfully inhibited the release of bacterial endotoxin, and could well inhibit the release of bacterial endotoxin at low concentrations of 6 and 12 μM. The results indicate that the SsPle polypeptide can effectively inhibit the release of free bacterial endotoxin. Bacterial endotoxin can cause infection in the host, and the SsPle polypeptide can effectively inhibit the release of bacterial endotoxin to slow down the infection. This shows that the SsPle polypeptide can be further applied in the tropical island reef environment.

[0034] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as a limitation of the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art of this technology, without departing from the spirit and scope of the present invention, several improvements and refinements can also be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An antibacterial 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 described in claim 1.

3. A biological material containing the nucleic acid described in claim 2.

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

5. Use of the antimicrobial peptide described in claim 1, the nucleic acid described in claim 2 or the biological material described in claim 3 in the preparation of an antimicrobial product.

6. The application according to claim 5, characterized in that, The antimicrobial is one or more of anti-Gram-negative bacteria, Gram-positive bacteria and fungi.

7. The application according to claim 5, characterized in that, The antimicrobial is one or more of anti-Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Salmonella typhimurium, Candida albicans.

8. The application according to claim 5, wherein The antimicrobial is one or more of anti-multidrug-resistant Escherichia coli, multidrug-resistant Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus.

9. The application according to claim 5, wherein The product is a drug, feed, feed additive, disinfectant, cleaner or preservative.

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