Eel antibacterial polypeptide Ajapkidin and application thereof in preparation of anti-vibrio composition

Abstract

The invention discloses an eel antibacterial polypeptide Ajaspkidin and application thereof in preparation of an anti-vibrio composition. The amino acid sequence of the eel antibacterial polypeptide Ajaspkidin is shown as SEQ ID NO.01. The invention further discloses an application of the eel antibacterial polypeptide The zebra fish antibacterial peptide has the characteristics of excellent molecular characteristics, broad-spectrum antibacterial activity, efficient bactericidal ability, high thermal stability, high biological safety, anti-infection effect on zebra fish infected by vibrio vulnificus, capability of remarkably improving the survival rate of bred animals and the like, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to an eel antibacterial polypeptide Ajaspkidin and its application in preparing an anti-vibrio composition. BACKGROUND

[0002] With the rapid development of global aquaculture industry, Japanese eel (Anguilla japonica) as a unique and important economic fish in East Asia, its farming production has increased significantly in recent years, with important economic and cultural value. The progress of farming technology and the growth of market demand have driven the expansion of this industry. However, as the scale of aquaculture continues to expand, disease problems have gradually emerged, becoming the main bottleneck restricting the sustainable development of Japanese eel aquaculture industry. In particular, bacterial diseases such as ulcer disease and red spot disease not only seriously threaten the health of farmed fish, but also have a significant negative impact on the economic benefits and biological safety of aquaculture industry. Traditionally, antibiotics have been widely used to control such diseases, although some results have been achieved in the short term, but its long-term use has led to the widespread emergence of bacterial drug resistance. This drug resistance problem has made many traditional antibiotics gradually ineffective, bringing new challenges to aquaculture industry, and also prompting researchers to seek safer and more efficient alternatives.

[0003] Antibacterial peptides as a new type of antibacterial substances, due to their unique antibacterial mechanism and excellent biological characteristics, have received widespread attention from academia and industry in recent years. Antibacterial peptides are a class of small molecule polypeptides widely existing in nature, usually composed of 10 to 50 amino acids, and are an important part of the innate immune system of organisms. Compared with traditional antibiotics, antibacterial peptides have significant advantages such as broad-spectrum antibacterial activity, low toxicity, and not easy to induce drug resistance. Its antibacterial mechanism is diverse, mainly including forming pores in the phospholipid bilayer of microbial cell membranes through interaction with the phospholipid bilayer, leading to leakage of cell contents and killing microorganisms; in addition, antibacterial peptides can also interfere with cell wall synthesis, disrupt membrane potential, or penetrate the cell membrane and bind to the DNA or RNA of microorganisms, inhibit the replication and transcription of nucleic acids, and further block the proliferation of microorganisms. Some antibacterial peptides can also interfere with protein synthesis or enzyme activity, further weakening the survival ability of microorganisms. Due to these multiple mechanisms of action, antibacterial peptides significantly reduce the possibility of bacterial drug resistance, especially in dealing with multi-drug resistant pathogens, they show stronger antibacterial effect. Therefore, antibacterial peptides are considered as one of the important candidates to solve the crisis of antibiotic resistance, with broad application prospects.

[0004] Japanese eel experiences multiple environmental transitions from freshwater to seawater during its life cycle, which endows it with strong ecological adaptability and immune defense capacity. This unique life history suggests that Japanese eel may contain rich resources of antimicrobial peptides that have not been fully explored and utilized. Research on Japanese eel antimicrobial peptides not only helps to solve the disease problems faced in the process of aquaculture, but also may provide new options for antibacterial drugs in the field of human medicine. However, current research on Japanese eel antimicrobial peptides is still limited, especially in-depth exploration of their antibacterial activity, mechanism of action, and practical application is still blank. Although some antimicrobial peptides derived from other species have been reported in the prior art, research on antimicrobial peptides specific to Japanese eel is still relatively rare, especially the lack of systematic development and application verification of specific antimicrobial peptides. SUMMARY

[0005] The present application aims to overcome the defects of the prior art and provide an eel antimicrobial peptide Ajaspkidin.

[0006] Another object of the present application is to provide the use of the above-mentioned eel antimicrobial peptide Ajaspkidin in the preparation of an anti-Vibrio composition.

[0007] The technical solution of the present application is as follows:

[0008] An eel antimicrobial peptide Ajaspkidin, the amino acid sequence of which is shown in SEQ ID NO. 01.

[0009] The use of the above-mentioned eel antimicrobial peptide Ajaspkidin in the preparation of an anti-Vibrio composition is characterized in that the eel antimicrobial peptide Ajaspkidin has inhibitory and killing effects on Vibrio vulnificus, Vibrio fluvialis, Vibrio harveyi and Vibrio alginolyticus.

[0010] An anti-Vibrio composition, the effective component of which comprises the eel antimicrobial peptide Ajaspkidin of claim 1.

[0011] The use of the above-mentioned eel antimicrobial peptide Ajaspkidin in the preparation of an antibacterial composition, the eel antimicrobial peptide Ajaspkidin has inhibitory and killing effects on Enterococcus faecium, Staphylococcus epidermidis, Listeria, Staphylococcus aureus, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas aeruginosa, Shigella flexneri, Acinetobacter baumannii, Vibrio fluvialis, Escherichia coli, Vibrio vulnificus, Vibrio harveyi and Vibrio alginolyticus.

[0012] An antibacterial composition, the effective component of which comprises the above-mentioned eel antimicrobial peptide Ajaspkidin.

[0013] The use of the above-mentioned eel antimicrobial peptide Ajaspkidin in the preparation of an antifungal composition.

[0014] In a preferred embodiment of the present application, the eel antibacterial polypeptide Ajaspkidin has inhibitory and killing effects on Cryptococcus neoformans.

[0015] An antifungal composition, the effective component of which comprises the above-mentioned eel antibacterial polypeptide Ajaspkidin.

[0016] The application of the above-mentioned eel antibacterial polypeptide Ajaspkidin in the preparation of aquatic feed additives.

[0017] An aquatic feed additive, the effective component of which comprises the above-mentioned eel antibacterial polypeptide Ajaspkidin.

[0018] The beneficial effects of the present application are:

[0019] 1. The eel antibacterial polypeptide Ajaspkidin of the present application is composed of 16 amino acids, and has a molecular formula of C 93 H 157 N 27 O 18 , a molecular weight of 1941.42 Dalton, 6 positively charged amino acid residues, an isoelectric point of 12.03, a net charge of +6, and a hydrophobicity of 35.5%. The small molecular weight, good stability and good water solubility provide a basis for its application in various environments.

[0020] 2. The present application has significant inhibitory and killing effects on various gram-positive bacteria (such as Enterococcus faecium, Staphylococcus epidermidis, Listeria monocytogenes, Staphylococcus aureus), gram-negative bacteria (such as Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas aeruginosa, Shigella flexneri, Acinetobacter baumannii, Vibrio fluvialis, Escherichia coli, Vibrio harveyi, Vibrio alginolyticus) and fungi (such as Cryptococcus neoformans), and exhibits excellent broad-spectrum antibacterial ability.

[0021] 3. The Ajaspkidin of the present application can kill 99.99% of Acinetobacter baumannii at a concentration of 6 μM in 120 min, and can kill 99.99% of Pseudomonas aeruginosa at a concentration of 12 μM in 180 min, showing a rapid and efficient bactericidal effect.

[0022] 4. The present application still retains good antibacterial activity after being heated in boiling water at 100℃ for 60 min, showing excellent thermal stability and being suitable for high-temperature processing environments.

[0023] 5. The present application has no cytotoxicity to human kidney epithelial cells (HEK-293T) and zebrafish embryo cells (ZF4), indicating that it has good biological safety and can be safely applied in biological related fields.

[0024] 6、The application can be used as an antibacterial agent and an aquatic feed additive to prevent and treat bacterial and fungal diseases in aquaculture, effectively improving the economic benefits and biological safety of aquaculture.

[0025] 7、The eel antibacterial polypeptide Ajaspkidin has good anti-infection effect, experiments show that it can significantly improve the survival rate of Vibrio vulnificus infected zebrafish, and has potential application value in the prevention and control of aquatic diseases. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 6 is a bactericidal kinetic curve of the eel antibacterial polypeptide Ajaspkidin on Acinetobacter baumannii and Pseudomonas aeruginosa in Example 3 of the application, wherein the abscissa is time (min), and the ordinate is the number of colonies (log 10 CFU / mL).

[0027] Figure 2 Figure 7 is a thermal stability diagram of the antibacterial activity of the eel antibacterial polypeptide Ajaspkidin on Acinetobacter baumannii and Pseudomonas aeruginosa in Example 4 of the application, wherein the abscissa is time (h), and the ordinate is the OD 600 value.

[0028] Figure 3 Figure 8 is a scanning electron microscope observation diagram of the eel antibacterial polypeptide Ajaspkidin on Acinetobacter baumannii and Pseudomonas aeruginosa in Example 5 of the application, wherein A: Acinetobacter baumannii, B: Acinetobacter baumannii+12μM Ajaspkidin, C: Pseudomonas aeruginosa, D: Pseudomonas aeruginosa+24μM Ajaspkidin.

[0029] Figure 4 Figure 9 is a cell toxicity experiment diagram of the MTS-PMS method for detecting the eel antibacterial polypeptide Ajaspkidin in Example 6 of the application, wherein the abscissa is the concentration of the antibacterial polypeptide Ajaspkidin (μM), and the ordinate is the cell proliferation rate (%).

[0030] Figure 5 Figure 10 is a diagram of the effect of Ajaspkidin on the survival rate of Vibrio vulnificus infected zebrafish in Example 7 of the application, wherein the abscissa is the time after infection (h), and the ordinate is the survival rate of zebrafish (%). DETAILED DESCRIPTION

[0031] The technical solutions of the application will be further described and explained through specific embodiments in combination with the accompanying drawings.

[0032] Example 1 Preparation of the eel antibacterial polypeptide Ajaspkidin

[0033] The amino acid sequence of the eel antibacterial polypeptide Ajaspkidin in this embodiment is as follows:

[0034] Pro-Ala-Trp-Phe-Lys-Arg-Lys-Leu-Lys-Arg-Leu-Val-Ser-Ala-Leu-Lys (SEQ ID NO. 01, PAWFKRKLKRLVSALK).

[0035] The eel antibacterial polypeptide Ajaspkidin can be obtained by using the existing solid-phase chemical synthesis method, and the purity of the eel antibacterial polypeptide Ajaspkidin can reach more than 95%. In this embodiment, the eel antibacterial polypeptide Ajaspkidin is synthesized by the Jinersi Biotechnology Co., Ltd. by using the solid-phase synthesis method, and the polypeptide molecular weight and HPLC detection information are provided. The physicochemical parameters of the eel antibacterial polypeptide Ajaspkidin are shown in Table 1.

[0036] Table 1 Physicochemical parameters of Ajaspkidin

[0037]

[0038] As shown in Table 1, the Ajaspkidin has a small molecular weight and good stability, and is a cationic polypeptide with positive charge.

[0039] Example 2 Determination of minimum inhibition concentration (MIC) of eel antibacterial polypeptide

[0040] The bacterial strains involved in this embodiment include: Enterococcus faecium, Staphylococcus epidermidis, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas aeruginosa, Shigella fiexneri, Acinetobacter baumannii, Vibrio fluvialis, Escherichia coli, Vibrio harveyi, Vibrio alginolyticus, Vibrio vulnificus and Cryptococcus neoformans. Among them, Vibrio vulnificus originated from Guangdong Haid Group Co., Ltd. and was isolated from diseased materials in the farm. The remaining strains were purchased from the China Center for the Collection of General Microorganisms.

[0041] The specific method is as follows:

[0042] (1) The preserved Enterococcus faecium, Staphylococcus epidermidis, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas aeruginosa, Shigella flexneri, Acinetobacter baumannii and Escherichia coli were spread on nutrient broth plates and cultured inverted; Vibrio fluvii, Vibrio harveyi and Vibrio alginolyticus were spread on 2216 plates and cultured inverted; Cryptococcus neoformans was spread on YPD plates and cultured inverted.

[0043] (2) Pick colonies from each plate and inoculate them into the corresponding liquid culture medium, then place them in a constant temperature shaker for overnight culture.

[0044] (3) The cells were collected by centrifugation and resuspended in sterile 10 mM sodium phosphate buffer (pH = 7.4). The bacteria were then diluted with MH liquid medium and Vibrio was diluted with TSB + 2% NaCl liquid medium to make the final concentration of the cells 5 × 10 5 CFU / mL; dilute the fungus with RPMI-MOPS liquid medium to make the final concentration of the fungus 5×10 4 CFU / mL.

[0045] (4) Ajaspkidin powder synthesized in Example 1 was dissolved with sterilized MilliQ water, filtered through a 0.22 μM filter, and diluted by a factor of 2 to obtain a protein concentration of 3 μM, 6 μM, 12 μM, 24 μM, 48 μM, and 96 μM, and placed on ice for standby.

[0046] (5) In the 96-well cell culture plate, a blank control group, a negative control group, and a test group were set up for each bacterium to be tested, and three parallel groups were set up for each group.

[0047] a Blank control group: 50 μL of the protein sample to be tested and 50 μL of the culture medium;

[0048] b Negative control group: 50 μL of sterile MilliQ water and 50 μL of the bacterial suspension;

[0049] c Test group: 50 μL of the protein sample to be tested and 50 μL of the bacterial suspension.

[0050] (6) The 96-well cell culture plate was placed in a corresponding incubator at a suitable temperature for bacteria and fungi, and incubated for 1-2 days. The MIC results in the test group were observed.

[0051] The antibacterial activity results of Ajaspkidin are shown in Table 2, which has broad-spectrum antibacterial activity

[0052] Table 2 Antibacterial activity of eel antibacterial peptide Ajaspkidin

[0053]

[0054]

[0055] Note: MIC: minimum inhibitory concentration (μM), represented by a-b. a: the highest protein concentration at which bacterial growth is visible to the naked eye; b: the lowest protein concentration at which bacterial growth is not visible to the naked eye.

[0056] Example 3 Bactericidal kinetics curve of eel antibacterial peptide Ajaspkidin

[0057] In this example, Acinetobacter baumannii and Pseudomonas aeruginosa were selected as the bacteria to be tested, and the bactericidal kinetics of the eel antibacterial peptide Ajaspkidin obtained in Example 1 was determined.

[0058] The specific method is similar to the antibacterial activity determination described in Example 2. After the antibacterial peptide Ajaspkidin and the bacteria were co-incubated for a certain time, an appropriate amount of the co-incubated bacterial suspension was diluted and spread on nutrient broth plates, which were incubated at 37°C for 1-2 days for colony counting. The bactericidal kinetics curves of the eel antibacterial peptide Ajaspkidin on Acinetobacter baumannii and Pseudomonas aeruginosa are shown in Figure 1As shown in the table, Ajaspkidin can kill 99.99% of Acinetobacter baumannii at a final concentration of 6 μΜ for 120 min; Ajaspkidin can kill 99.99% of Pseudomonas aeruginosa at a final concentration of 12 μΜ for 180 min.

[0059] Example 4 Thermal stability of antibacterial activity of eel antibacterial peptide Ajaspkidin

[0060] In this example, Acinetobacter baumannii and Pseudomonas aeruginosa were selected as the test bacteria, and the thermal stability of the antibacterial activity of the eel antibacterial peptide Ajaspkidin obtained in Example 1 was determined.

[0061] The specific method was similar to the antibacterial activity determination described in Example 2. The final concentration of Ajaspkidin was adjusted to 1 MIC, and the antibacterial peptide Ajaspkidin was heated in boiling water at 100°C for 10 min, 20 min, 30 min and 60 min, and placed on ice for standby. Subsequently, the antibacterial peptide Ajaspkidin was incubated with the test bacteria for 24 h, and the value of OD 600 was continuously monitored using a microplate reader. As shown in the table, Ajaspkidin still retains good antibacterial activity after being heated at 100°C for 60 min. Figure 2

[0062] Example 5 Scanning electron microscopy observation of morphological changes of bacteria after Ajaspkidin treatment

[0063] In this example, Acinetobacter baumannii and Pseudomonas aeruginosa were selected as the test strains, and the morphological changes of bacteria after Ajaspkidin treatment were observed by scanning electron microscopy. The preparation of scanning electron microscopy samples was carried out according to the following steps:

[0064] (1) Activate the bacterial strain and culture to the logarithmic growth phase, measure OD 600 , centrifuge to remove the supernatant, resuspend the bacterial body with MH liquid medium, adjust the OD 600 to 0.2, and place on ice for standby.

[0065] (2) Dissolve the Ajaspkidin powder obtained in Example 1 with sterilized MilliQ water, mix the bacterial suspension and the antibacterial peptide in equal volumes, and the final concentration of the antibacterial peptide Ajaspkidin is 2 MIC. After incubation at 37°C for 1 h, centrifuge to remove the supernatant, wash once with PBS, and collect the bacterial body.

[0066] (3) Resuspend the bacterial body with 2.5% glutaraldehyde, fix at 4°C for 2 h. After fixation, wash the bacterial body with PBS three times, prepare a high-concentration bacterial suspension, drop onto a glass slide, and adhere for 30 min. After the bacterial body adheres, perform ethanol gradient dehydration.

[0067] ​(4) Critical point drying, 10 mA current, gold spraying for 60 s; scanning electron microscope observation and photographing.

[0068] Results as shown in Figure 3 Fig. 1, the control group of bacteria had normal morphology, complete structure and smooth surface without shrinkage. The bacteria treated with antibacterial peptide Ajaspkidin had significant changes in morphology, surface shrinkage and cytoplasmic content outflow.

[0069] Example 6 Cell toxicity assay of eel antibacterial peptide Ajaspkidin

[0070] In this example, human kidney epithelial cells (HEK-293T) and zebrafish embryo cells (ZF4) were selected to determine the cell toxicity of the eel antibacterial peptide Ajaspkidin of Example 1.

[0071] (1) Collect human kidney epithelial cells and zebrafish embryo cells in good growth state, count with cell counting plate and adjust the cell concentration to 1 × 10 5 6 / mL with culture medium, mix the cell suspension, add 100 μL cell suspension to each well of 96-well cell culture plate, and place in a culture incubator with suitable cell conditions for culture until more than 80% of cells adhere.

[0072] (2) Carefully aspirate the culture medium, add culture medium containing different concentrations of Ajaspkidin, and place in a culture incubator with suitable cell conditions for culture for 24 h.

[0073] (3) After adding 20 μL MTS-PMS solution to each well, avoid light incubation for 4 h, measure the OD 492 value with enzyme marker instrument to evaluate the cell toxicity of Ajaspkidin.

[0074] Results as shown in Figure 4 Fig. 2, 96 μM of Ajaspkidin had no cell toxicity on human kidney epithelial cells and zebrafish embryo cells.

[0075] Example 7 Anti-infection protective effect of eel antibacterial peptide Ajaspkidin on Vibrio vulnificus infected zebrafish

[0076] This example aims to verify whether the eel antibacterial peptide Ajaspkidin obtained in Example 1 has antibacterial protective ability in farmed animals. A Vibrio vulnificus infected zebrafish model was constructed and the survival rate of farmed animals was detected.

[0077] (1) Healthy 4-month-old zebrafish were selected and adaptively fed for 7 days, then randomly divided into two groups, 20 fish in each group.

[0078] (2) Collect the Vibrio vulnificus in logarithmic growth phase, adjust the concentration of the bacteria solution with phosphate buffer solution (PBS), and inject 8.8 μL of the Vibrio vulnificus (1×10 5 CFU / mL) into each zebrafish by intraperitoneal injection.

[0079] (3) One hour after the zebrafish are infected with the Vibrio vulnificus, 8.8 μL of the eel antibacterial peptide Ajaspkidin (1 mg / mL) mother liquor is injected into each zebrafish in the experimental group, and 8.8 μL of PBS solution is injected into each zebrafish in the control group.

[0080] (4) The survival of the zebrafish is continuously monitored after the infection, and the survival is recorded every 12 hours until the end of 48 hours, and a survival curve is drawn.

[0081] The results are shown in Table 1. Figure 5 The survival rate of the experimental group is significantly higher than that of the control group (p=0.0195), and the eel antibacterial peptide Ajaspkidin can increase the survival rate of the infected zebrafish from 20% to 55%.

[0082] The above description is only a preferred embodiment of the present application, and therefore cannot limit the scope of the present application. Any equivalent changes and modifications made according to the scope and content of the present patent should still be within the scope of the present application.

Claims

1. An eel antibacterial polypeptide Ajaspkidin, characterized by: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The use of the eel antibacterial polypeptide Ajaspkidin according to claim 1 in the preparation of an anti-Vibrio composition, characterized in that: The eel antibacterial polypeptide Ajaspkidin has inhibitory and killing effects on Vibrio vulnificus, Vibrio fluvialis, Vibrio harveyi and Vibrio alginolyticus.

3. An anti-Vibrio composition, characterized in that: The effective ingredient includes the eel antibacterial polypeptide Ajaspkidin according to claim 1.

4. Use of the eel antibacterial polypeptide Ajaspkidin according to claim 1 in the preparation of an antibacterial composition, characterized in that: The eel antibacterial polypeptide Ajaspkidin has inhibitory and killing effects on Enterococcus faecium, Staphylococcus epidermidis, Listeria, Staphylococcus aureus, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas aeruginosa, Shigella flexneri, Acinetobacter baumannii, Vibrio fluvii, Escherichia coli, Vibrio vulnificus, Vibrio harveyi and Vibrio alginolyticus.

5. An antibacterial composition, characterized in that: The effective ingredient includes the eel antibacterial polypeptide Ajaspkidin according to claim 1.

6. Use of the eel antibacterial polypeptide Ajaspkidin according to claim 1 in the preparation of an antifungal composition.

7. The use according to claim 6, characterized in that: The eel antibacterial polypeptide Ajaspkidin has inhibitory and killing effects on Cryptococcus neoformans.

8. An antifungal composition, characterized in that: The effective ingredient includes the eel antibacterial polypeptide Ajaspkidin according to claim 1.

9. Use of the eel antibacterial polypeptide Ajaspkidin according to claim 1 in the preparation of an aquatic feed additive.

10. An aquatic feed additive, characterized in that: The effective ingredient includes the eel antibacterial polypeptide Ajaspkidin according to claim 1.

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