An anti-vibrio polypeptide Lvvibriocin-GK and its application

Abstract

This invention discloses an anti-Vibrio polypeptide, Lvvibriocin-GK, and its applications, the amino acid sequence of which is shown in SEQ ID NO. 01. This invention is a positively charged cationic, highly effective anti-Vibrio polypeptide that exhibits significant antibacterial effects against common aquatic pathogens such as Vibrio. Furthermore, Lvvibriocin-GK showed no cytotoxic effects on zebrafish embryonic fibroblast cell line (ZF4) and human kidney epithelial cell line (HEK-293T); its antibacterial activity is unaffected by high temperature, salt ion concentration, or pH changes, demonstrating good environmental stability.

Description

Technical Field

[0001] This invention belongs to the field of marine molecular biology technology, specifically relating to a highly efficient anti-Vibrio polypeptide Lvvibriocin-GK and its applications. Background Technology

[0002] The whiteleg shrimp (Penaeus vannamei), also known as the Litopenaeus vannamei, belongs to the class Crustacea of ​​the phylum Arthropoda, holding a significant place in the animal classification system. This shrimp is not only highly nutritious but also has significant economic value, making it one of the most widely farmed shrimp species globally. Vibrio spp. are common aquatic pathogens in crustaceans, with Vibrio parahaemolyticus being one of the main pathogens causing acute hepatopancreatic necrosis disease in crustaceans, leading to extremely high mortality rates in infected shrimp.

[0003] Antibiotics have long been the primary drugs for preventing and treating diseases in aquaculture, with significant efficacy, but their drawbacks are becoming increasingly apparent. Bacterial resistance poses a serious challenge to traditional antibiotics, prompting researchers to accelerate the development of novel antimicrobial drugs. Antimicrobial peptides (AMPs) are an important component of an organism's innate immune system, known to inhibit and kill various pathogens, including bacteria, fungi, and viruses. Compared to traditional antibiotics, antimicrobial peptides are less likely to induce bacterial resistance, and are therefore considered an ideal alternative to antibiotics.

[0004] To date, the APD3 antimicrobial peptide database has included 5,099 antimicrobial peptides, which perform a variety of biological functions and demonstrate great application potential. The development of novel antimicrobial peptides from Litopenaeus vannamei is expected to provide new strategies and solutions for screening novel antimicrobial drugs, preventing and controlling aquaculture diseases, and preventing spoilage of fresh food. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient anti-Vibrio polypeptide, Lvvibriocin-GK.

[0006] Another object of the present invention is to provide the application of the above-mentioned highly effective anti-vibrio polypeptide Lvvibriocin-GK.

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

[0008] A highly effective anti-Vibrio polypeptide, Lvvibriocin-GK, has the amino acid sequence shown in SEQ ID NO.01.

[0009] The use of the aforementioned highly effective anti-Vibrio polypeptide Lvvibriocin-GK in the preparation of anti-Vibrio compositions.

[0010] In a preferred embodiment of the present invention, the anti-vibrio composition is an aquatic anti-vibrio composition.

[0011] In a preferred embodiment of the present invention, the highly effective anti-vibrio polypeptide Lvvibriocin-GK has inhibitory and killing effects on Vibrio parahaemolyticus, Vibrio harzianum, Vibrio fluvialis, Vibrio alginolyticus, and luminescent bacillus mermaidus.

[0012] An anti-vibrio composition comprising the above-mentioned highly effective anti-vibrio polypeptide Lvvibriocin-GK.

[0013] In a preferred embodiment of the present invention, it is an aquatic anti-Vibrio composition.

[0014] In a preferred embodiment of the present invention, it has inhibitory and killing effects on Vibrio parahaemolyticus, Vibrio harzianum, Vibrio fluvialis, Vibrio alginolyticus and luminescent bacillus mermaidus.

[0015] The beneficial effects of this invention are:

[0016] 1. The highly efficient anti-vibrio polypeptide Lvvibriocin-GK of the present invention consists of 17 amino acids with a molecular weight of approximately 2.043 kDa, including 5 positively charged amino acid residues. Based on the predicted amino acid residue charges, the isoelectric point of this highly efficient anti-vibrio polypeptide is 10.30, and its hydrophobicity is 41%, making it a positively charged cationic highly efficient anti-vibrio polypeptide.

[0017] 2. The highly effective anti-Vibrio polypeptide Lvvibriocin-GK of this invention has a significant antibacterial effect against Vibrio, a common aquatic pathogen. Furthermore, Lvvibriocin-GK did not exhibit cytotoxicity against zebrafish embryonic fibroblast cell line (ZF4) and human kidney epithelial cell line (HEK-293T).

[0018] 3. The antibacterial activity of the highly efficient anti-vibrio polypeptide Lvvibriocin-GK of the present invention is not affected by high temperature, salt ion concentration, or pH changes, showing good environmental stability.

[0019] 4. The highly efficient anti-vibrio polypeptide Lvvibriocin-GK of the present invention has the activity of inhibiting biofilm formation, which is of great significance for preventing bacteria from forming difficult-to-remove biofilms on the surface.

[0020] 5. The highly efficient anti-vibrio polypeptide Lvvibriocin-GK of the present invention has a small molecular weight, good water solubility, strong antibacterial activity, and is safe and non-toxic. It can be developed into antibacterial drugs or antibacterial agents, etc., and has high application value. Attached Figure Description

[0021] Figure 1 The image shows the bactericidal kinetic curve of the highly efficient anti-Vibrio polypeptide Lvvibriocin-GK against Vibrio parahaemolyticus in Example 3 of this invention; where the horizontal axis represents time (min) and the vertical axis represents the bactericidal index (%).

[0022] Figure 2 This is a thermostability graph of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK against Vibrio parahaemolyticus in Example 4 of this invention; where the horizontal axis represents time (h) and the vertical axis represents OD. 600 nm value.

[0023] Figure 3 This is a graph showing the tolerance of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK to Vibrio parahaemolyticus in Example 5 of this invention; where the horizontal axis represents time (h) and the vertical axis represents OD. 600 nm value.

[0024] Figure 4 This is a pH stability graph of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK against Vibrio parahaemolyticus in Example 6 of the present invention; where the horizontal axis represents time (h) and the vertical axis represents OD. 600 nm value.

[0025] Figure 5 This is an experimental graph showing the inhibition of Vibrio parahaemolyticus biofilm formation by the highly efficient anti-Vibrio polypeptide Lvvibriocin-GK in Example 7 of the present invention; where the horizontal axis represents the final concentration of Lvvibriocin-GK (μg / mL) and the vertical axis represents the biofilm formation rate (%).

[0026] Figure 6 This is a graph from Example 8 of the present invention, showing the MTS-PMS assay for detecting the cytotoxicity of the highly effective anti-Vibrio peptide Lvvibriocin-GK; where the horizontal axis represents the final concentration of Lvvibriocin-GK (μg / mL) and the vertical axis represents the cell proliferation rate (%). Detailed Implementation

[0027] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0028] Example 1: Preparation of highly effective anti-Vibrio peptide Lvvibriocin-GK

[0029] The amino acid sequence of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK of this invention is as follows:

[0030] Gly-Lys-Lys-Val-Trp-Lys-Lys-Tyr-Tyr-Phe-Val-Leu-Arg-Ala-Ser-Gly-Leu (SEQ ID NO: 01, GKKVWKKYYFVLRASGL)

[0031] The highly efficient anti-Vibrio polypeptide Lvvibriocin-GK with a purity of over 95% can be obtained by chemical solid-phase synthesis. Its physicochemical parameters are shown in Table 1.

[0032] Table 1 Physicochemical parameters of the highly effective anti-Vibrio peptide Lvvibriocin-GK

[0033]

[0034] As shown in Table 1, Lvvibriocin-GK is a small-molecule, highly hydrophobic cationic polypeptide.

[0035] Example 2: Determination of the minimum inhibitory concentration (MIC) of the highly effective anti-Vibrio peptide Lvvibriocin-GK against Vibrio, a pathogenic aquatic bacterium.

[0036] The strains involved in this embodiment are: *Vibrio parahaemolyticus*, *Vibrio alginolyticus*, *Vibrio fluvialis*, *Vibrio harveyi*, and *Photobacterium damselae*. *Photobacterium damselae* was donated by the Fujian Provincial Fisheries Research Institute, while the other strains were purchased from the Culture Collection Center of Microbiology, Chinese Academy of Sciences (CGMCC) and preserved in our laboratory.

[0037] Specifically, the steps include the following:

[0038] (1) Activation of strains: Vibrio strains frozen at -80℃ (i.e. the above test strains) were streaked on solid plates, cultured for 24 hours, and colonies were picked and cultured overnight with shaking.

[0039] (2) Preparation of bacterial suspension: Collect bacteria by centrifugation, and dilute the liquid culture medium to a final concentration of 1×10⁻⁶. 6 CFU / mL;

[0040] (3) Preparation of high-efficiency anti-vibrio peptide working solution: Adjust the concentration of high-efficiency anti-vibrio peptide Lvvibriocin-GK to 6, 12, 25, 50, and 100 μg / mL;

[0041] (4) Determination of minimum inhibitory concentration: Experimental group, negative control group and blank control group were set up for the experiment, and three parallel groups were set up for each group; the MIC results were observed after culturing in sterile 96-well cell culture plates for 24 hours.

[0042] The reagents added to each group are as follows:

[0043] Experimental group: 50 μL antimicrobial peptide working solution + 50 μL bacterial suspension

[0044] Negative control group: 50 μL sterile MilliQ water + 50 μL bacterial suspension

[0045] Blank control group: 50 μL antimicrobial peptide working solution + 50 μL liquid culture medium

[0046] The antibacterial activity results of Lvvibriocin-GK are shown in Table 2. It can be seen that Lvvibriocin-GK has significant antibacterial activity against all tested Vibrio species.

[0047] Table 2 Antibacterial activity of Lvvibriocin-GK

[0048]

[0049] Note: MIC stands for Minimum Inhibitory Concentration (μg / mL), denoted by ab. a represents the highest protein concentration at which bacterial growth is visible to the naked eye; b represents the lowest protein concentration at which no bacterial growth is visible to the naked eye.

[0050] Example 3: Kinetic curve of bactericidal activity of the highly effective anti-Vibrio peptide Lvvibriocin-GK

[0051] In this embodiment, Vibrio parahaemolyticus was selected as a representative of aquatic pathogens to determine the bactericidal kinetics of the highly effective anti-vibrio polypeptide Lvvibriocin-GK.

[0052] After co-incubating the test bacteria with 25 μg / mL Lvvibriocin-GK for a certain period of time, the co-incubated mixture was serially diluted at different time points and plated onto tryptone soybean broth plates. The plates were incubated upside down at a suitable temperature for 18-24 hours for colony counting. Samples incubated with sterile MilliQ water and bacterial suspension for 0 min served as positive controls. Colony counts were performed using the same dilution method and plating technique, and the colony count was defined as 100%. The bactericidal index was the percentage of the number of colonies in the experimental group relative to the number of colonies in the positive control after a certain incubation time. Results are as follows: Figure 1 As shown, Lvvibriocin-GK has rapid bactericidal activity. When co-incubated with Vibrio parahaemolyticus for 1 min, the bactericidal index exceeds 90%, and when co-incubated for 40 min, the bactericidal index reaches 100%.

[0053] Example 4: Thermal stability of the antibacterial activity of the highly effective anti-vibrio peptide Lvvibriocin-GK

[0054] In this embodiment, Vibrio parahaemolyticus was selected as the test bacterium to determine the thermostability of the antibacterial activity of Lvvibriocin-GK. The specific method was similar to the antibacterial activity determination described in Example 2. 25 μg / mL of Lvvibriocin-GK was incubated in boiling water at 100℃ for 10, 20, 30, and 60 min, and then placed on ice for later use. Lvvibriocin-GK or sterile MilliQ water was co-incubated with the test bacteria for 24 h, and the OD was measured using a microplate reader at 0, 12, and 24 h. 600 nm The value. The result is as follows: Figure 2 As shown, Lvvibriocin-GK still exhibits good antibacterial activity against Vibrio parahaemolyticus after being bathed in boiling water at 100℃ for 10, 20, 30, and 60 minutes.

[0055] Example 5: Ion tolerance of the highly effective antibacterial peptide Lvvibriocin-GK

[0056] In this example, the tolerance of Vibrio parahaemolyticus to the antibacterial activity of Lvvibriocin-GK was determined. 25 μg / mL of Lvvibriocin-GK was kept on ice for later use. The specific method was similar to the antibacterial activity determination described in Example 2, with 150 mM NaCl and 4.5 mM KCl added to the culture medium, respectively. Lvvibriocin-GK or sterile MilliQ water was co-incubated with the test bacteria for 24 h, and the OD was measured using a microplate reader at 0, 12, and 24 h. 600 nm The value. The result is as follows: Figure 3 As shown, Lvvibriocin-GK can inhibit the growth of Vibrio parahaemolyticus under the conditions of additional addition of 150mM NaCl or 4.5mM KCl, and has good ion tolerance.

[0057] Example 6: Highly effective anti-vibrio peptide Lvvibriocin-GK antibacterial activity and pH stability

[0058] In this embodiment, the pH stability of the antibacterial activity of Vibrio parahaemolyticus against Lvvibriocin-GK was determined. The pH of the stock solution of the highly effective anti-Vibrio peptide Lvvibriocin-GK was adjusted to 2, 4, 6, 8, 10, and 12 using 1M HCl or NaOH, and treated at 37℃ for 4 h. After treatment, the pH of each sample was adjusted back to neutral, and antibacterial activity was tested. The specific method was similar to the antibacterial activity determination described in Example 2. The concentration of the highly effective anti-Vibrio peptide Lvvibriocin-GK was adjusted to 25 μg / mL, and Lvvibriocin-GK or sterile MilliQ water was co-incubated with the test bacteria for 24 h. The OD was measured using a microplate reader at 0, 12, and 24 h. 600 nm The value. The result is as follows: Figure 4 As shown, the antibacterial activity of the highly effective anti-Vibrio peptide Lvvibriocin-GK is not affected by pH changes, and the antibacterial activity of the highly effective anti-Vibrio peptide Lvvibriocin-GK is pH stable.

[0059] Example 7: Highly effective anti-Vibrio peptide Lvvibriocin-GK inhibits biofilm formation

[0060] In this embodiment, Vibrio parahaemolyticus was selected as the test strain. The crystal violet staining method was used to detect the inhibition of bacterial biofilm formation by Lvvibriocin-GK, which specifically included the following steps:

[0061] (1) Adjust the concentration of the test bacteria suspension to 1×10⁻⁶ 6 CFU / mL;

[0062] (2) The final concentrations of the highly effective anti-Vibrio peptide Lvvibriocin-GK were adjusted to 0.75 μg / mL, 1.5 μg / mL, 3 μg / mL, and 6 μg / mL;

[0063] (3) Take 50 μL of bacterial suspension and 50 μL of high-efficiency anti-Vibrio peptide Lvvibriocin-GK or sterile water into a 96-well sterile cell culture plate and incubate at 28°C for 24 h.

[0064] (4) Remove the supernatant, add 100 μL of methanol, and fix at room temperature for 15 min;

[0065] (5) Add 100 μL of 0.1% crystal violet staining solution and stain for 15 min;

[0066] (6) Remove the supernatant, wash away the floating color with PBS buffer, add 95% ethanol, and shake at room temperature for 30 min;

[0067] (7) OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader 570nm The value of .

[0068] The results are as follows Figure 5 As shown, 6 μg / mL Lvvibriocin-GK can significantly reduce the adhesion of Vibrio parahaemolyticus, meaning that the highly effective anti-vibrio peptide Lvvibriocin-GK can inhibit the biofilm formation of Vibrio parahaemolyticus.

[0069] Example 8: Cytotoxicity assay of the highly effective anti-Vibrio peptide Lvvibriocin-GK

[0070] In this embodiment, zebrafish embryonic fibroblast cell line (ZF4) and human kidney epithelial cell line (HEK-293T) were selected to determine the cytotoxicity of the highly effective anti-Vibrio peptide Lvvibriocin-GK. The specific steps included the following:

[0071] (1) Collect zebrafish embryonic fibroblasts (ZF4) and human kidney epithelial cells (HEK-293T) in good growth condition, and adjust the cell concentration to 1×10⁻⁶. 5 Add 100 μL of cell suspension to each well of a 96-well sterile cell culture plate and incubate overnight in an incubator at a suitable temperature.

[0072] (2) Remove the culture medium and add a culture medium containing different concentrations of highly effective anti-vibrio peptide Lvvibriocin-GK. Incubate in a suitable temperature incubator for 24 hours.

[0073] (3) After adding 20 μL MTS-PMS solution, incubate in the dark for 2 h, and then measure the OD using a microplate reader. 492 nm The value was used to evaluate the cytotoxicity of Lvvibriocin-GK.

[0074] The results are as follows Figure 6 The results showed that there was no significant difference in cell viability after co-incubation of different concentrations of Lvvibriocin-GK with normal cells for 24 hours. Therefore, the experiment demonstrated that Lvvibriocin-GK had no cytotoxicity against zebrafish embryonic fibroblasts (ZF4) and human kidney epithelial cells (HEK-293T).

[0075] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An anti-Vibrio polypeptide Lvvibriocin-GK, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The use of the anti-vibrio polypeptide Lvvibriocin-GK according to claim 1 in the preparation of anti-vibrio compositions, characterized in that: The anti-vibrio composition is an anti-aquatic Vibrio composition.

3. The use as described in claim 2, characterized in that: The aquatic Vibrio species were selected from Vibrio parahaemolyticus, Vibrio harzianum, Vibrio fluvibrio, Vibrio alginolyticus, and luminescent bacillus.

4. An anti-Vibrio composition, characterized in that: Its active ingredient includes the anti-vibrio polypeptide Lvvibriocin-GK as described in claim 1.

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