Eel antibacterial peptide Anguinin and application thereof

Abstract

The invention discloses an anguilla anguilla antibacterial peptide Anguinin and application thereof. The amino acid sequence of the anguilla anguilla antibacterial peptide Anguinin is shown as SEQ ID NO. 01. The amino acid sequence consists of 18 amino acid residues, and the molecular weight of the amino acid sequence is 2091.41 Daltons. According to the prediction of a HeliQuest tool, the net charge number is + 5, the hydrophobicity value is 0.267, and meanwhile, good water solubility is shown. As a typical cationic polypeptide with positive charges, the cationic polypeptide has the remarkable advantages of being easy to chemically synthesize, wide in antibacterial spectrum, efficient in antibacterial activity, high in safety and the like, a solid foundation is laid for practical application of the cationic polypeptide in the fields of pharmacy and feed addition due to the characteristics, and wide commercialization prospects are shown.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of marine molecular biology, and particularly relates to a eel antibacterial peptide Anguinin and application thereof. BACKGROUND

[0002] The antibacterial mechanism of antibacterial peptides is diverse, and in addition to direct bactericidal effect, it also has immunomodulatory effect and is not prone to drug resistance. Therefore, antibacterial peptides are considered as a potential antibiotic substitute. So far, various antibacterial peptides have been found, whether they are of natural origin or synthetic, and they all have strong broad-spectrum bactericidal activity in vitro. A few of them have been subjected to clinical trials and are mainly used for the treatment of chronic wound infections. Fish is considered an important source of new antibacterial drugs, and existing research has isolated antibacterial peptides from various fish species, such as peptides extracted from carp, salmon, etc. These antibacterial peptides have a certain antibacterial spectrum, but they often have problems such as large molecular weight, poor thermal stability, or high cytotoxicity, which limit their application. For example, existing fish-derived antibacterial peptides have unsatisfactory inhibitory effect on some gram-negative bacteria such as Vibrio, and their anti-infection effect in aquaculture has not been fully verified. In addition, the existing antibacterial peptides have narrow activity spectrum in inhibiting waterborne pathogenic bacteria such as Pseudomonas fluorescens, Pseudomonas putida, Vibrio fluvialis and Vibrio alginolyticus, and are difficult to significantly improve the survival rate of farmed animals. At the same time, the existing antifungal peptides have limited inhibitory effect on molds such as Aspergillus niger and Aspergillus fumigatus, and the safety assessment is insufficient, such as incomplete toxicity test on mammalian cells. These problems lead to the fact that the existing technology cannot effectively deal with the mixed infection of bacteria and fungi in aquaculture, as well as the drug resistance challenge in human health. Eel is an important economic fish in aquaculture, and its immune system is well developed, but there is no report on the screening of high-efficiency broad-spectrum antibacterial peptides from eel. Therefore, it is necessary to develop a fish-derived antibacterial peptide with small molecular weight, high thermal stability, no cytotoxicity and broad-spectrum antibacterial property, in order to solve the limitations of existing antibiotic substitutes and provide a new anti-infection solution. SUMMARY

[0003] The present application aims to overcome the defects of the prior art and provide a eel antibacterial peptide Anguinin.

[0004] Another object of the present application is to provide the application of the above-mentioned eel antibacterial peptide Anguinin.

[0005] The technical solution of the present application is as follows:

[0006] A eel antibacterial peptide Anguinin, the amino acid sequence of which is shown in SEQ ID NO. 01.

[0007] The use of the above-mentioned eel antibacterial peptide Anguinin in the preparation of an antibacterial composition is characterized in that the eel antibacterial peptide Anguinin has an inhibitory effect on Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Vibrio fluviatilis and Vibrio alginolyticus.

[0008] An antibacterial composition, the active ingredient of which includes the above-mentioned eel antibacterial peptide Anguinin.

[0009] In a preferred embodiment of the present invention, the active ingredient is the above-mentioned eel antimicrobial peptide Anguinin.

[0010] The eel antimicrobial peptide Anguinin is used in the preparation of an antifungal composition. The eel antimicrobial peptide Anguinin has an inhibitory effect on Aspergillus niger, Aspergillus fumigatus, Fusarium solani and Penicillium expansum.

[0011] An antifungal composition, the active ingredient of which includes the above-mentioned anguilla antibacterial peptide Anguinin.

[0012] In a preferred embodiment of the present invention, the active ingredient is the above-mentioned eel antimicrobial peptide Anguinin.

[0013] The application of the above-mentioned eel antimicrobial peptide Anguinin in the preparation of aquatic feed additives.

[0014] An aquatic feed additive, whose active ingredient is the eel antibacterial peptide Anguinin.

[0015] The beneficial effects of the present invention are:

[0016] 1. The present invention is composed of 18 amino acid residues with a molecular weight of precisely 2091.41 Daltons. According to the HeliQuest tool, its net charge is +5, its hydrophobicity is 0.267, and it exhibits good water solubility. As a typical positively charged cationic polypeptide, it possesses significant advantages such as ease of chemical synthesis, a broad antimicrobial spectrum, high antimicrobial activity, and high safety. These properties lay a solid foundation for its practical application in the pharmaceutical and feed additive fields, and show broad commercial prospects.

[0017] 2. The present invention exhibits potent broad-spectrum antimicrobial activity, significantly inhibiting a variety of pathogenic microorganisms, including Gram-positive bacteria such as Staphylococcus epidermidis, Enterococcus faecium, and Listeria monocytogenes; Gram-negative bacteria such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Vibrio fluvii, and Vibrio alginolyticus; and fungi such as Aspergillus niger, Aspergillus fumigatus, Fusarium solani, and Penicillium expansum. This multi-target inhibitory effect was experimentally validated, as demonstrated by minimal inhibitory concentration (MIC) determinations and bactericidal kinetic curves, rapid bacterial killing in vitro, and bacterial cell membrane disruption and intracellular substance release observed under scanning electron microscopy, further confirming its highly effective antimicrobial mechanism.

[0018] 3. The present invention exhibits extremely high biocompatibility at the cellular level. MTS-PMS assays revealed no significant cytotoxicity against zebrafish embryonic ZF4 cells and human kidney epithelial HEK-293T cells. Even at high concentrations, cell survival rates remained high, demonstrating its harmlessness to host cells and providing reliable support for its use as a safe anti-infective agent.

[0019] 4. The present invention possesses excellent in vivo anti-infective efficacy. Experimental results show that in zebrafish models infected with Vibrio fluvii and Vibrio algae, it can significantly increase the survival rate of infected fish from a lower level in the control group to a higher proportion. This protective effect was confirmed by statistical analysis of the survival curve (with a significant p-value), highlighting its potential value in aquaculture disease prevention and control. It can be used as a feed additive to effectively inhibit aquatic pathogens, reduce economic losses, and promote sustainable aquaculture development. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the bactericidal kinetic curve of the eel antimicrobial peptide Anguinin against Staphylococcus epidermidis and Acinetobacter baumannii in Example 1 of the present invention.

[0021] Figure 2 This is a thermal stability diagram of the antibacterial activity of the eel antimicrobial peptide Anguinin against Staphylococcus epidermidis and Acinetobacter baumannii in Example 1 of the present invention.

[0022] Figure 3 This is a diagram showing the morphological and structural changes caused by the eel antimicrobial peptide Anguinin treatment on Staphylococcus epidermidis and Acinetobacter baumannii in Example 1 of the present invention.

[0023] Figure 4 This is a graph showing the results of an experiment using the MTS-PMS method to detect the cytotoxicity of the eel antimicrobial peptide Anguinin to ZF4 and HEK-293T cells in Example 1 of the present invention, wherein the abscissa represents the Anguinin concentration (μM) and the ordinate represents the cell survival rate (%).

[0024] Figure 5 This is a graph showing the effect of the eel antimicrobial peptide Anguinin on the survival rate of zebrafish infected with Vibrio fluvialis and Vibrio algae-lyticus in Example 1 of the present invention. The horizontal axis is the time after infection (h) and the vertical axis is the survival rate of zebrafish (%). 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

[0027] The amino acid sequence of the eel antimicrobial peptide Anguinin in this example is SEQ ID NO: 01: KFTGSWTRGAWGRAVVRR.

[0028] In this example, Nanjing GenScript Co., Ltd. was commissioned to synthesize an eel antimicrobial peptide Anguinin with a purity of over 95%. The company also provided information on peptide molecular weight, HPLC, and other testing. HeliQuest was used to predict its charge and hydrophobicity, and ProtParam was used to predict other physicochemical parameters. Table 1 lists the physicochemical parameters of the eel antimicrobial peptide Anguinin.

[0029] Table 1 Physicochemical parameters of eel antimicrobial peptide Anguinin

[0030]

[0031] 1. Determination of Anguinin Minimum Inhibitory Concentration (MIC)

[0032] The strains involved in this section include: Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Vibrio fluvialis, Vibrio alginolyticus, Aspergillus niger, Aspergillus fumigatus, Fusarium solani, and Penicillium expansum. All strains were purchased from the Culture Collection of the Institute of Microbiology, Chinese Academy of Sciences.

[0033] The specific method is as follows:

[0034] (1) Activate the bacterial strains. Streak the preserved bacteria onto appropriate nutrient broth plates, spread Vibrio onto 2216 plates, and spread fungal spores onto potato glucose plates. Incubate the plates at 28°C for 1-7 days.

[0035] (2) Colonies were picked from each plate and cultured to the logarithmic phase. The cells were resuspended in sterile 10 mM sodium phosphate buffer and then diluted with MH liquid medium and Vibrio in TSB + 2% NaCl liquid medium to a final concentration of 5 × 10 5CFU / mL; dilute the fungus with RPMI-MOPS liquid medium to make the final concentration of the fungus 5×10 4 CFU / mL.

[0036] (3) The synthesized Anguinin powder was dissolved in sterile MilliQ water to 3 μM, 6 μM, 12 μM, 24 μM, 48 μM, 96 μM, and 192 μM, respectively, and placed on ice for later use.

[0037] (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:

[0038] a Blank control group: 50 μL of the protein sample to be tested (Anguinin) and 50 μL of culture medium;

[0039] b Negative control group: 50 μL sterile MilliQ water and 50 μL bacterial suspension;

[0040] c Test experimental group: 50 μL of test protein sample (Anguinin) and 50 μL of bacterial suspension.

[0041] Place the 96-well cell culture plate in an incubator at a suitable temperature and culture for 1-2 days, and then observe the MIC results in the experimental group to be tested.

[0042] The antibacterial activity results of the eel antimicrobial peptide Anguinin are shown in Table 2. Anguinin has a broad-spectrum antibacterial activity.

[0043] Table 2 Antimicrobial activity of Anguinin, an antimicrobial peptide from eel

[0044]

[0045] 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.

[0046] 2. Anguinin Bactericidal Kinetic Curve

[0047] The strains involved in this section are: Staphylococcus epidermidis and Acinetobacter baumannii.

[0048] The specific method is as follows:

[0049] The specific method is similar to the method for determining antibacterial activity. After the antimicrobial peptide and bacteria are co-incubated for a certain period of time, the co-incubated mixture is gradiently diluted at different time points and then spread on the plate. After static incubation, the colonies are counted.

[0050] The bactericidal kinetic curves of Anguinin, an eel antimicrobial peptide, against Staphylococcus epidermidis and Acinetobacter baumannii are shown in Figure 2.Figure 1 The antibacterial peptide Anguinin has a rapid bactericidal effect, and 99% of S. epidermidis can be killed by Anguinin at a final concentration of 24 μM in 120 min; 99% of A. baumannii can be killed by Anguinin at a final concentration of 6 μM in 240 min.

[0051] The strains involved in this part are S. epidermidis and A. baumannii.

[0052] The specific method is as follows:

[0053] The specific method is similar to the determination method of antibacterial activity. The antibacterial peptide Anguinin is incubated with the bacteria to be tested for 24 h, and the value of OD 595 is continuously monitored by using an enzyme marker.

[0054] The thermal stability of the eel antibacterial peptide Anguinin on S. epidermidis and A. baumannii is shown in Figure 2 . The antibacterial peptide Anguinin still has high antibacterial activity after being continuously heated at 100°C for 30 min.

[0055] Four, scanning electron microscope observation of morphological structure changes of bacteria treated by Anguinin

[0056] The strains involved in this part are S. epidermidis and A. baumannii.

[0057] The specific experimental method is as follows:

[0058] (1) Scanning electron microscope sample preparation: activate the bacterial strain, and when the clone grows to an appropriate size, randomly pick a single clone into the corresponding culture medium, shake to the logarithmic growth phase, centrifuge to remove the supernatant, and adjust to 5×10 7 cfu / mL with the culture medium. Add an equal volume of antibacterial peptide, and the antibacterial peptide concentration is 1×MIC of the corresponding bacteria. Incubate for 30 min, centrifuge to remove the supernatant, and wash the bacteria once with PBS, then collect the bacteria.

[0059] (2) Fixing, washing, and dropping: 2.5% glutaraldehyde 4°C fixation of the bacteria in step (1) for more than 2 h, then wash the bacteria three times with PBS, prepare a high-concentration suspension, drop onto a glass slide, and after the bacteria adhere, perform ethanol gradient dehydration.

[0060] (3) After critical point drying, 10 mA current, and gold spraying for 60 s; observe under a scanning electron microscope and take pictures for record.

[0061] The morphological structure changes of S. epidermidis and A. baumannii treated by the eel antibacterial peptide Anguinin are shown in Figure 3As shown. Anguinin treatment destroyed the integrity of the bacterial cell membrane, leading to obvious cell membrane structure damage and release of intracellular substances. In contrast, the surface of untreated bacterial cells remained intact and smooth.

[0062] Five, MTS-PMS method for detecting and evaluating the cytotoxicity of Anguinin

[0063] This part selects zebrafish embryo cells (ZF4) and human kidney epithelial cells (HEK-293T) to determine the cytotoxicity of Anguinin, as shown in Figure 3 , the abscissa is the concentration of Anguinin (μM), and the ordinate is the cell survival rate (%).

[0064] The specific method is as follows:

[0065] (1) Collect cells in good growth condition, adjust the concentration of ZF4 and HEK-293T cells to 1×10 5 / mL, add 100 μL of the above cell suspension to each well of a 96-well cell culture plate, and place it in a suitable temperature incubator for culture until more than 80% of the cells adhere.

[0066] (2) Aspirate the culture medium, add culture medium containing different concentrations of Anguinin, and place it in a suitable temperature incubator for culture for 24 h.

[0067] (3) Add 20 μL of MTS-PMS mixed solution to each well, avoid light for 4 h, and read the plate with an enzyme marker OD 492 , and calculate the cell survival rate.

[0068] The results are shown in Figure 4 , Anguinin has no cytotoxicity to ZF4 and HEK-293T cells.

[0069] Six, the anti-infection protective effect of Anguinin on Vibrio infected zebrafish

[0070] This part selects healthy 4-month-old AB wild-type zebrafish and randomly divides them into two groups, 20 tails in each group.

[0071] Collect the logarithmic growth period of Vibrio fluvialis and Vibrio alginolyticus, adjust the concentration of the bacterial solution with phosphate buffer solution (PBS), and inject 8.8 μL of Vibrio fluvialis (concentration of 5×10 6 CFU / tail) or Vibrio alginolyticus (concentration of 2.5×10 6 CFU / tail) per tail by intraperitoneal injection.

[0072] One hour after the treatment group was infected with bacteria, 8.8 μL of Anguinin (concentration of 1 mg / mL) was injected intraperitoneally, and 8.8 μL of PBS solution was injected into the control group.

[0073] The survival of the zebrafish after infection was continuously monitored until 48h, and the survival curve was drawn.

[0074] The results are shown in Table 1. Figure 5 As shown in Table 1, in the Vibrio fluvialis infection experiment, the survival rate of the treatment group was significantly higher than that of the control group (p=0.0196), and the eel antibacterial peptide Anguinin could increase the survival rate of zebrafish from 50% to 85%; in the Vibrio alginolyticus infection experiment, the survival rate of the treatment group was significantly higher than that of the control group (p=0.0285), and the eel antibacterial peptide Anguinin could increase the survival rate of zebrafish from 50% to 80%.

[0075] The above merely describes preferred embodiments of the present application, and therefore cannot limit the scope of the present application, i.e. equivalent changes and modifications made according to the scope of the present application and the content of the specification should still fall within the scope of the present application.

Claims

1. An eel antimicrobial peptide Anguinin, characterized by: Its amino acid sequence is shown in SEQ ID NO.

01.

2. Use of the eel antimicrobial peptide Anguinin according to claim 1 in the preparation of an antibacterial composition, characterized in that: The eel antimicrobial peptide Anguinin has inhibitory effects on Staphylococcus epidermidis, Enterococcus faecium, Listeria monocytogenes, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Vibrio fluviatilis and Vibrio alginolyticus.

3. An antibacterial composition, characterized in that: The effective ingredient comprises the eel antibacterial peptide Anguinin as claimed in claim 1.

4. An antibacterial composition according to claim 3, characterized in that: The active ingredient is the eel antibacterial peptide Anguinin as claimed in claim 1.

5. Use of the eel antimicrobial peptide Anguinin according to claim 1 in the preparation of an antifungal composition, characterized in that: The eel antimicrobial peptide Anguinin has inhibitory effects on Aspergillus niger, Aspergillus fumigatus, Fusarium solani and Penicillium expansum.

6. An antifungal composition, characterized in that: The effective ingredient comprises the eel antibacterial peptide Anguinin as claimed in claim 1.

7. The antifungal composition according to claim 6, wherein: The active ingredient is the eel antibacterial peptide Anguinin as claimed in claim 1.

8. Use of the eel antimicrobial peptide Anguinin according to claim 1 in the preparation of an aquatic feed additive.

9. An aquatic feed additive, characterized in that: The active ingredient is the eel antibacterial peptide Anguinin as claimed in claim 1.

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