Efficient anti-vibrio polypeptide Lvvibrio-GK and application thereof

Abstract

The invention discloses an efficient anti-vibrio polypeptide Lvvibrio-GK and application thereof. The amino acid sequence of the efficient anti-vibrio polypeptide Lvvibrio-GK is as shown in SEQ ID NO. 01. The Lvvibriocin-GK is a cationic efficient anti-vibrio polypeptide with positive charges and has a remarkable antibacterial effect on common aquatic pathogenic bacteria vibrio, and in addition, the Lvvibriocin-GK has no cytotoxic effect on a zebrafish embryo fibroblast cell line (ZF4) and a human kidney epithelial cell line (HEK-293T), and the Lvvibriocin-GK has no cytotoxic effect on the zebrafish embryo fibroblast cell line (ZF4) and the human kidney epithelial cell line (HEK-293T). The antibacterial activity is not influenced by high temperature, salt ion concentration and pH change, and good environmental stability is shown.

Description

Technical Field

[0001] The invention belongs to the technical field of marine molecular biology, and specifically relates to a highly effective anti-Vibrio polypeptide Lvvibriocin-GK and an application thereof. Background Art

[0002] Penaeus vannamei, also known as Penaeus vannamei, belongs to the class Crustacea of ​​the phylum Arthropoda and occupies a place in the animal classification system. This shrimp not only has high nutritional value, but also has significant economic value, and has become one of the most farmed shrimp species in the world. Vibrio spp. is a common aquatic pathogen of crustaceans, among which Vibrio parahaemolyticus is one of the main pathogens that cause acute hepatopancreatic necrosis in crustaceans. After infection, the mortality rate of diseased shrimp is extremely high.

[0003] Antibiotics have always been the main drugs for preventing and treating aquaculture diseases. They are effective, but their drawbacks are also becoming increasingly prominent. The problem of bacterial resistance poses a severe challenge to traditional antibiotics, prompting researchers to accelerate the pace of research and development of new antimicrobial drugs. Antimicrobial peptides (AMPs) are an important part of the innate immune system of organisms. They are known to have inhibitory and killing effects on a variety of pathogens such as bacteria, fungi, and viruses. Compared with traditional antibiotics, antimicrobial peptides are less likely to induce bacterial resistance and are therefore regarded as an ideal alternative to antibiotics.

[0004] To date, the antimicrobial peptide database APD3 has included 5,099 antimicrobial peptides, which play a variety of biological functions and show great application potential. The development of new antimicrobial peptides from white shrimp is expected to provide new strategies and solutions for screening new antimicrobial drugs, preventing aquaculture diseases, and preventing the corruption of fresh food. Summary of the invention

[0005] The purpose of the present invention is to provide a highly effective 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, whose amino acid sequence is shown in SEQ ID NO.01.

[0009] Use of the above-mentioned highly effective anti-Vibrio polypeptide Lvvibriocin-GK in preparing an anti-Vibrio composition.

[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 harveyi, Vibrio fluvialis, Vibrio alginolyticus and Photobacterium mermanii.

[0012] An anti-Vibrio composition, the effective component of which includes the above-mentioned high-efficiency 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 harveyi, Vibrio fluvialis, Vibrio alginolyticus and Photobacterium mermanii.

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

[0016] 1. The highly effective anti-Vibrio polypeptide Lvvibriocin-GK of the present invention is composed of 17 amino acids, with a molecular weight of about 2.043 kDa, and contains 5 positively charged amino acid residues. According to the prediction of the charge of the amino acid residues, the isoelectric point of the highly effective anti-Vibrio polypeptide is 10.30, and the hydrophobicity is 41%, and it is a positively charged cationic highly effective anti-Vibrio polypeptide.

[0017] 2. The highly effective anti-Vibrio polypeptide Lvvibriocin-GK of the present invention has a significant antibacterial effect on Vibrio, a common aquatic pathogen. In addition, no cytotoxic effect of Lvvibriocin-GK was detected on zebrafish embryonic fibroblast cell line (ZF4) and human kidney epithelial cell line (HEK-293T).

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

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

[0020] 5. The highly effective anti-Vibrio polypeptide Lvvibriocin-GK of the present invention has small molecular weight, good water solubility, strong antibacterial activity, safety and non-toxicity, can be developed into antibacterial drugs or antibacterial agents, etc., and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the bactericidal kinetic curve of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK against Vibrio parahaemolyticus in Example 3 of the present invention; wherein the abscissa is time (min) and the ordinate is the bactericidal index (%).

[0022] Figure 2 This is a thermal stability diagram of the antibacterial activity of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK against Vibrio parahaemolyticus in Example 4 of the present invention; wherein the abscissa is time (h) and the ordinate is OD 600 nm value.

[0023] Figure 3 This is a graph showing the tolerance of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK to the antibacterial activity ions of Vibrio parahaemolyticus in Example 5 of the present invention; wherein the abscissa is time (h) and the ordinate is OD 600 nm value.

[0024] Figure 4 This is a pH stability diagram of the antibacterial activity of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK against Vibrio parahaemolyticus in Example 6 of the present invention; wherein the abscissa is time (h) and the ordinate is OD 600 nm value.

[0025] Figure 5 This is a graph showing the inhibition of biofilm formation of Vibrio parahaemolyticus by the highly effective anti-Vibrio polypeptide Lvvibriocin-GK in Example 7 of the present invention; wherein the abscissa is the final concentration of Lvvibriocin-GK (μg / mL), and the ordinate is the biofilm formation rate (%).

[0026] Figure 6 This is a graph showing the cytotoxicity test of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK using the MTS-PMS method in Example 8 of the present invention; wherein the abscissa is the final concentration of Lvvibriocin-GK (μg / mL), and the ordinate is the cell proliferation rate (%). DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the 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 the present invention is:

[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 chemical solid phase synthesis method can be used to obtain a highly effective anti-Vibrio peptide Lvvibriocin-GK with a purity of more than 95%. Its physical and chemical parameters are shown in Table 1.

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

[0033]

[0034] As shown in Table 1, Lvvibriocin-GK is a cationic polypeptide with small molecular weight and strong hydrophobicity.

[0035] Example 2: Determination of the Minimum Inhibition Concentration (MIC) of the Highly Effective Anti-Vibrio Peptide Lvvibriocin-GK against Aquatic Pathogenic Bacteria Vibrio

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

[0037] The specific steps include:

[0038] (1) Strain activation: streak the Vibrio strain (i.e., the test strain) frozen at -80°C on a solid plate, incubate for 24 hours, pick a colony and culture overnight with shaking;

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

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

[0041] (4) Determination of minimum inhibitory concentration: An experimental group, a negative control group, and a blank control group were set up for the experiment, with three parallels in each group; the cells were cultured in a sterile 96-well cell culture plate for 24 h and the MIC results were observed.

[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, which shows that Lvvibriocin-GK has significant antibacterial activity against all tested Vibrio.

[0047] Table 2 Antibacterial activity of Lvvibriocin-GK

[0048]

[0049] Note: MIC is the minimum inhibitory concentration (μg / mL), expressed as ab. a is the highest protein concentration at which bacterial growth can be seen with the naked eye; b is the lowest protein concentration at which bacterial growth cannot be seen with the naked eye.

[0050] Example 3: Bactericidal kinetic curve of highly effective anti-Vibrio peptide Lvvibriocin-GK

[0051] In this example, Vibrio parahaemolyticus was selected as a representative of aquatic pathogens, and the bactericidal kinetics of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK was measured.

[0052] After 25 μg / mL of Lvvibriocin-GK was incubated with the test bacteria for a certain period of time, the incubated mixture was diluted gradiently at different time points and spread on a tryptone soy broth plate, and inverted at an appropriate temperature for 18-24 hours for colony counting. The sample incubated with sterile MilliQ water and bacterial suspension for 0 min was used as a positive control. The clone number was counted using the same dilution method and coating method, and its clone number was defined as 100%. The bactericidal index is the percentage of the number of clones in the experimental group after a certain incubation time relative to the number of clones in the positive control. The results are shown in Figure 1 As shown, Lvvibriocin-GK has rapid bactericidal activity. When it is co-incubated with Vibrio parahaemolyticus for 1 min, the bactericidal index exceeds 90%, and when it is co-incubated for 40 min, the bactericidal index reaches 100%.

[0053] Example 4: Thermostability of the antibacterial activity of the highly effective anti-Vibrio peptide Lvvibriocin-GK

[0054] In this example, Vibrio parahaemolyticus was selected as the test bacteria to measure the thermal stability of the antibacterial activity of Lvvibriocin-GK. The specific method is similar to the antibacterial activity determination described in Example 2. 25 μg / mL of Lvvibriocin-GK was bathed in 100°C boiling water for 10, 20, 30, and 60 minutes, and then placed on ice for later use. Lvvibriocin-GK or sterile MilliQ water was incubated with the test bacteria for 24 hours, and the OD was measured with an enzyme marker at 0, 12, and 24 hours. 600 nm The result is Figure 2 As shown, Lvvibriocin-GK still had good antibacterial activity against Vibrio parahaemolyticus after being bathed in 100°C boiling water for 10, 20, 30, and 60 min.

[0055] Example 5: Antimicrobial activity and ion tolerance of highly effective anti-Vibrio peptide Lvvibriocin-GK

[0056] In this example, Vibrio parahaemolyticus was selected to measure the tolerance of Lvvibriocin-GK to the antibacterial activity ions. 25 μg / mL of Lvvibriocin-GK was placed on ice for later use. The specific method was similar to the antibacterial activity determination described in Example 2, and 150 mM NaCl and 4.5 mM KCl were added to the culture medium, respectively. Lvvibriocin-GK or sterile MilliQ water was incubated with the test bacteria for 24 hours, and the OD was measured with an enzyme marker at 0, 12, and 24 hours. 600 nm The result is Figure 3 As shown, Lvvibriocin-GK can inhibit the growth of Vibrio parahaemolyticus under the condition of additional addition of 150mM NaCl or 4.5mM KCl, and has good ion tolerance.

[0057] Example 6: pH stability of antibacterial activity of highly effective anti-Vibrio peptide Lvvibriocin-GK

[0058] In this example, Vibrio parahaemolyticus was selected to determine the pH stability of the antibacterial activity of Lvvibriocin-GK. 1M HCl or NaOH was used to adjust the pH of the mother solution of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK to 2, 4, 6, 8, 10, or 12, and treated at 37°C for 4 hours. After treatment, the pH of each sample was adjusted back to neutral, and the antibacterial activity was tested. The specific method is similar to the antibacterial activity determination described in Example 2. The concentration of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK was adjusted to 25 μg / mL, and Lvvibriocin-GK or sterile MilliQ water was incubated with the test bacteria for 24 hours, and the OD was measured with an enzyme marker at 0, 12, and 24 hours. 600 nm The result is 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 example, Vibrio parahaemolyticus was selected as the test strain, and 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 bacterial suspension to 1×10 6 CFU / mL;

[0062] (2) The final concentration of the highly effective anti-Vibrio peptide Lvvibriocin-GK was 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 highly effective anti-Vibrio peptide Lvvibriocin-GK or sterile water to a 96-well sterile cell culture plate and culture 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 by aspiration, wash with PBS buffer to remove the floating color, add 95% ethanol, and shake at room temperature for 30 min;

[0067] (7) OD determination by microplate 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 amount of Vibrio parahaemolyticus, that is, the highly efficient 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 example, 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, which specifically includes the following steps:

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

[0072] (2) The culture medium was removed by aspiration, and culture medium containing different concentrations of the highly effective anti-Vibrio peptide Lvvibriocin-GK was added, and the cells were placed in an incubator at an appropriate temperature for 24 h.

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

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

[0075] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

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

01.

2. Use of the highly effective anti-Vibrio polypeptide Lvvibriocin-GK according to claim 1 in preparing an anti-Vibrio composition.

3. The use according to claim 2, characterized in that: The anti-Vibrio composition is an aquatic anti-Vibrio composition.

4. The use according to claim 2, characterized in that: The high-efficiency anti-Vibrio polypeptide Lvvibriocin-GK has inhibitory and killing effects on Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvii, Vibrio alginolyticus and Photobacterium mermanii.

5. An anti-Vibrio composition, characterized in that: The effective component thereof comprises the highly effective anti-Vibrio polypeptide Lvvibriocin-GK as claimed in claim 1.

6. An anti-Vibrio composition according to claim 5, characterized in that: The invention is an aquatic anti-Vibrio composition.

7. An anti-Vibrio composition according to claim 5, characterized in that: It has inhibitory and killing effects on Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, Vibrio alginolyticus and Photobacterium mermanii.

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