An antibacterial peptide LP-P1 from large yellow croaker collagen and its application

Abstract

The present invention discloses a large yellow croaker collagen antibacterial peptide LP-P1, whose amino acid sequence is KRVRAGMMMRKVAV and molecular weight is 1633 daltons. Experiments have proved that the antibacterial peptide LP-P1 of the present invention can effectively inhibit the growth of Vibrio parahaemolyticus and Vibrio alginolyticus, and is not prone to drug resistance, and can be used as a good substitute for antibiotics for the prevention and treatment of Vibrio parahaemolyticus and Vibrio alginolyticus. The present invention lays a foundation for further research on using large yellow croaker collagen antibacterial peptides as feed additives and biomedical development in the future.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a large yellow croaker collagen antibacterial peptide LP-P1 and its application. Background Art

[0002] The large yellow croaker (Pseudosciaena crocea) is one of the four major economic fish species in the East China Sea of China, and is widely cultured across the country. Fujian Province has the highest output, accounting for about 90% of the total national output. In recent years, with the continuous expansion of the large yellow croaker breeding scale, the breeding density has been continuously increased, the breeding environment has been continuously deteriorated, and diseases have occurred frequently and become increasingly serious. Among them, the diseases caused by Vibrio bacteria have caused great economic losses to aquaculture practitioners every year due to their high incidence, wide prevalence and the most serious harm. After the large yellow croaker is infected with Vibrio and gets sick, antibiotics are often used in aquaculture, which not only causes secondary pollution to the environment but also brings food safety problems. And overuse will lead to many pathogenic microorganisms developing varying degrees of drug resistance to almost all drugs, which has become a shackle to the sustainable development of China's animal husbandry, aquaculture and food industry. Therefore, the research and development of safe and effective "antibiotic substitutes" has important practical significance and application value for the sustainable development of the large yellow croaker aquaculture industry.

[0003] Antimicrobial peptides are known as "natural antibiotics" and are a crucial part of the innate immune system. They are short peptides formed by organisms to resist the invasion of pathogenic microorganisms and have antibacterial effects. Antimicrobial peptides have the characteristics of a broad antibacterial spectrum and strong bactericidal activity, and are considered to be the preferred drugs that can replace antibiotics, with good application prospects. Summary of the Invention

[0004] The purpose of the present invention is to provide a large yellow croaker collagen antibacterial peptide LP-P1 and its application. Through the research on the antibacterial activities of the large yellow croaker collagen antibacterial peptide LP-P1 against Vibrio parahaemolyticus and Vibrio alginolyticus, it provides experimental basis for finding new food preservatives, biological medicines and aquaculture feed additives, and promotes the healthy and sustainable development of China's food, medicine and aquaculture industries.

[0005] One of the technical solutions adopted by the present invention to solve its technical problems is: to provide a large yellow croaker collagen antibacterial peptide LP-P1. Its amino acid sequence is KRVRAGMMMRKVAV, as shown in SEQ ID NO: 1.

[0006] The molecular weight of the antibacterial peptide LP-P1 is 1633 daltons, the positive charge carried is +5, and the total hydrophobicity ratio is 57%.

[0007] The antibacterial peptide LP-P1 can damage bacteria in the following ways: First, it adsorbs on the surface of bacteria, penetrates the lipid bilayer of the cell membrane, and then binds to the genomic DNA of bacteria, inhibiting the synthesis of bacterial DNA, thereby causing bacterial death. The present invention provides an experimental basis for riboflavin combined with the antibacterial peptide LP-P1 of large yellow croaker collagen as a food preservative and an aquatic feed additive.

[0008] The second technical solution adopted by the present invention to solve its technical problems is: to provide the application of the antibacterial peptide LP-P1 of large yellow croaker collagen in the preparation of antibacterial drugs, and the antibacterial drugs are used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0009] The third technical solution adopted by the present invention to solve its technical problems is: to provide an antibacterial drug, the active ingredient of which is the antibacterial peptide LP-P1 of large yellow croaker collagen, and the amino acid sequence of the antibacterial peptide LP-P1 is SEQ ID NO: 1.

[0010] In a preferred embodiment of the present invention, the antibacterial drug is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0011] The fourth technical solution adopted by the present invention to solve its technical problems is: to provide a feed additive, the active ingredient of which is the antibacterial peptide LP-P1 of large yellow croaker collagen, and the amino acid sequence of the antibacterial peptide LP-P1 is SEQ ID NO: 1.

[0012] In a preferred embodiment of the present invention, the feed additive is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0013] The fifth technical solution adopted by the present invention to solve its technical problems is: to provide a food preservative, the active ingredient of which is the antibacterial peptide LP-P1 of large yellow croaker collagen, and the amino acid sequence of the antibacterial peptide LP-P1 of large yellow croaker collagen is SEQ ID NO: 1.

[0014] In a preferred embodiment of the present invention, the food preservative is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0015] The sixth technical solution adopted by the present invention to solve its technical problems is: to provide an antibacterial drug, the active ingredient of which is the antibacterial peptide LP-P1 of large yellow croaker collagen and vitamin B2, and the amino acid sequence of the antibacterial peptide LP-P1 is SEQ ID NO: 1.

[0016] In a preferred embodiment of the present invention, the antibacterial drug is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0017] The antimicrobial peptide of the present invention can be synthesized by methods known to those skilled in the art, such as solid-phase synthesis, and purified by methods known to those skilled in the art, such as high-performance liquid chromatography.

[0018] Implementing the present invention has the following beneficial effects:

[0019] The present invention takes large yellow croaker collagen as the research object. Through bioinformatics technology screening, a polypeptide LP-P1 with a brand-new amino acid sequence is discovered. The antimicrobial peptide LP-P1 is combined with vitamins B1, B2, B6, and B12, and the combination of vitamin B2 (riboflavin) and antimicrobial peptide LP-P1 with the best antibacterial effect is screened out. The antibacterial activity of riboflavin combined with large yellow croaker collagen antimicrobial peptide LP-P1 against Vibrio parahaemolyticus and Vibrio alginolyticus is studied; and its antibacterial mechanism is studied. The experimental results show that the combined peptide has a strong inhibitory effect on Vibrio parahaemolyticus and Vibrio alginolyticus, and is significantly superior to the inhibitory effect when using antimicrobial peptide LP-P1 alone, thereby reducing the usage amount of antimicrobial peptide LP-P1 and thus reducing the usage cost. Its antibacterial mechanism is to first adsorb on the surface of bacteria, penetrate the lipid bilayer of the cell membrane, and then combine with bacterial genomic DNA to inhibit the synthesis of bacterial DNA, thereby causing bacteria to die. The present invention provides an experimental basis for riboflavin combined with large yellow croaker collagen antimicrobial peptide LP-P1 as a food preservative and aquaculture feed additive. Description of the Drawings

[0020] Figure 1 It is the antibacterial result diagram of the large yellow croaker collagen-derived antimicrobial peptide of the present invention against Vibrio alginolyticus and Vibrio parahaemolyticus.

[0021] Figure 2 It is the control diagram for measuring the minimum inhibitory concentration (MIC) of the large yellow croaker collagen antimicrobial peptide LP-P1 of the present invention against Vibrio alginolyticus (Figure A) and Vibrio parahaemolyticus (Figure B). Among them,

[0022] a: Antimicrobial peptide concentration 0 μg / mL;

[0023] b: Antimicrobial peptide concentration 62.5 μg / mL;

[0024] c: Antimicrobial peptide concentration 31.3 μg / mL;

[0025] d: Antimicrobial peptide concentration 15.6 μg / mL;

[0026] e: Antimicrobial peptide concentration 7.8 μg / mL;

[0027] f: Antimicrobial peptide concentration 3.9 μg / mL;

[0028] Figure 3 It is the 3D structure prediction diagram of the large yellow croaker collagen antimicrobial peptide LP-P1 of the present invention.

[0029] Figure 4 This is the antibacterial result diagram of the combined action of yellow croaker collagen antibacterial peptide LP-P1 (1 / 2MIC) of the present invention against Vibrio alginolyticus and Vibrio parahaemolyticus.

[0030] Figure 5 This is the time-kill curve diagram of the combined action of yellow croaker collagen antibacterial peptide LP-P1 (1 / 2MIC) of the present invention against Vibrio parahaemolyticus. Diluted to 10 4-5 CFU / mL in 0.01M pH 7.2 phosphate buffer. The concentration of the antibacterial peptide is 1.9 μg / mL.

[0031] Figure 6 This is the intracellular K + leakage diagram of yellow croaker collagen antibacterial peptide LP-P1 of the present invention against Vibrio parahaemolyticus.

[0032] Figure 7 This is the electrophoresis diagram of the binding of yellow croaker collagen antibacterial peptide LP-P1 of the present invention to Vibrio parahaemolyticus DNA. Among them,

[0033] Lane 7: Blank control;

[0034] Lanes 1-6: The mass ratios of the binding peptide to DNA are 100 / 1, 50 / 1, 25 / 1, 25 / 2, 25 / 4, and 25 / 8, respectively.

[0035] Figure 8 This is the bar chart of the effect of yellow croaker collagen antibacterial peptide LP-P1 of the present invention on the viability of L929 cells. Detailed implementation manners

[0036] To better understand the present invention, the present invention will be further described in detail below in conjunction with examples and drawings. However, those skilled in the art understand that the following examples do not limit the protection scope of the present invention, and any changes and variations made on the basis of the present invention are within the protection scope of the present invention.

[0037] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0038] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0039] Example 1: Screening of yellow croaker collagen antibacterial peptide

[0040] The possible antibacterial sequences in the collagen antibacterial peptide sequence of large yellow croaker were predicted using three online software: the AntiBP server, the CAMPR3 server, and the apd2 (Antibacterial Peptide Calculator and Predictor). The charge and hydrophobicity of the possible antibacterial sequences were analyzed, and finally, the chemical syntheses of the sequences numbered LP-P1, LP-P2, LP-P6, LP-P7, and LP-P8 were selected and verified for antibacterial activity. The screening results are shown in the following table.

[0041] Table 1 Bioinformatics analysis results of large yellow croaker collagen peptide segments

[0042] Serial number Peptide sequence Molecular weight (Da) Total hydrophobic ratio (%) Charge LP-P1 KRVRAGMMMRKVAV 1633.125 57％ +5 LP-P2 EVSPNRVRVGIV 1324.55 42％ +1 LP-P3 ESGSIGTPNF 1008.054 20％ -1 LP-P4 KASTGSRISSKISQN 1563.724 20％ +3 LP-P5 EGIKQIGGGT 959.065 20％ 0 LP-P6 CCIKPPGPINPKIGF 1583.978 40％ +2 LP-P7 KKQLIESVKNIA 1370.646 42％ +2 LP-P8 EKIKQKVVDTICKSKP 1844.232 31％ +3 LP-P9 EMQQIGGGTHTG 1215.311 17％ -0.75 LP-P10 DKGVQQVAVVIT 1256.464 50％ 0

[0043] Example 2: Verification of antibacterial activity of antibacterial peptides

[0044] Vibrio alginolyticus and Vibrio parahaemolyticus were cultured at 37 °C for 12 h until the logarithmic growth phase and diluted to 10 4-5 CFU / mL in 0.01 M phosphate buffer at pH = 7.2. Equal volumes of the above five 0.5 mg / mL peptide solutions were mixed with the bacterial solutions and incubated at 37 °C. Samples were taken every 30 minutes and spread on plates, and the total number of colonies was recorded after overnight culture at 37 °C. The inhibition rate was calculated according to the formula: inhibition rate = (number of colonies in blank control - number of colonies in experimental group) / number of colonies in blank control × 100%. From the results, it can be seen that the antibacterial peptide LP-P1 had the most obvious inhibitory effect on Vibrio alginolyticus and Vibrio parahaemolyticus, with inhibition rates as high as 99.31 ± 0.22% and 99.71 ± 0.49% ( Figure 1 ).

[0045] Example 3: Determination of the minimum inhibitory concentration (MIC) of antibacterial peptide LP-P1

[0046] The minimum inhibitory concentration (MIC) of the large yellow croaker collagen antibacterial peptide LP-P1 against Vibrio alginolyticus and Vibrio parahaemolyticus was determined by the microplate method on a 96-well plate. Vibrio alginolyticus or Vibrio parahaemolyticus diluted to 10 4-5 CFU / mL in 0.01 M phosphate buffer at pH 7.2 was added to the wells, and then equal volumes of different concentrations of the large yellow croaker collagen antibacterial peptide LP-P1 diluted in gradient were added to the wells. The 96-well plate was sealed and cultured in a 37 °C constant temperature incubator for 18 - 24 h. The concentration corresponding to the dilution with the minimum antibacterial effect was the minimum inhibitory concentration MIC. As Figure 2 shown, the minimum inhibitory concentrations (MIC) of the antibacterial peptide LP-P1 against Vibrio alginolyticus and Vibrio parahaemolyticus were 7.8125 μg / mL and 3.9063 μg / mL.

[0047] Example 4: Prediction of the 3D structure of antibacterial peptide LP-P1

[0048] Using the online structure prediction server Swiss-model, the structure of the antimicrobial peptide LP-P1 was predicted, and it was edited and modified using the Pymol software to obtain the secondary structure of the antimicrobial peptide LP-P1, as Figure 3 shown.

[0049] Example 4: Study on the antibacterial effect of the combined use of the antimicrobial peptide LP-P1 with vitamin B1, B2, B6 and B12

[0050] Weighed a certain amount of the antimicrobial peptide LP-P1 and dissolved it in distilled water with vitamin B1, B2, B6 and B12 respectively. The peptide solution and the riboflavin solution were mixed at a molar ratio of 10:1. The prepared mixed solution was magnetically stirred in the dark at room temperature for 3 h, and the sample was freeze-dried in vacuo into a powder. The above powder was formulated into a mixed peptide solution with a 1 / 2 MIC value of the antimicrobial peptide LP-P1 against Vibrio alginolyticus and Vibrio parahaemolyticus when used alone, and the antibacterial activity verification experiment in Example 2 was repeated. The results are as Figure 4 shown. The antibacterial effect of the antimicrobial peptide LP-P1 was significantly improved after being compounded with vitamin B complex. Among them, the peptide solution obtained after compounding with vitamin B2 had the best antibacterial effect. When the antimicrobial peptide LP-P1 changed from the original 1 MIC to 1 / 2 MIC, it could still inhibit the growth of Vibrio alginolyticus and Vibrio parahaemolyticus, indicating that the inhibitory effect of the compound use of the antimicrobial peptide LP-P1 and vitamin B2 on Vibrio alginolyticus and Vibrio parahaemolyticus was greater than that of the antimicrobial peptide LP-P1 used alone. Example 3 was repeated, and the minimum inhibitory concentration (MIC) of the compound peptide solution of the antimicrobial peptide LP-P1 and vitamin B2 against Vibrio alginolyticus and Vibrio parahaemolyticus was 1.95312 μg / mL.

[0051] Example 5: TIMEKILL determination of the antimicrobial peptide LP-P1

[0052] Using Vibrio alginolyticus, Vibrio alginolyticus was cultured at 37 °C for 12 h until the logarithmic growth phase, and diluted to 10 in 0.01 M phosphate buffer at pH 7.2 4-5CFU / mL. Take the antibacterial peptide LP-P1 at the MIC concentration and the vitamin B2-binding peptide, mix them with the bacteria in equal volumes at 37 °C, and then incubate. Samples are taken every 30 minutes and spread on plates, and then cultured at 37 °C for 24 h. Observe and count the number of viable bacteria. With time as the abscissa and the logarithm (log) of the colony-forming unit number (CFU·mL-1) as the ordinate, plot the time-killing curve, and the antibacterial peptide LP-P1 at the MIC concentration is used as the blank group. From the results, it can be seen that the combined use of vitamin B2 and the antibacterial peptide can reduce the number of Vibrio alginolyticus within 30 min, and the bactericidal effect can be achieved after 1 h. Under the combined action of vitamin B2 and the antibacterial peptide LP-P1, the number of bacteria decreases faster. It shows that the combination of vitamin B2 and the antibacterial peptide LP-P1 has an obvious inhibitory effect on Vibrio alginolyticus with the increase of the action time( Figure 5 ).

[0053] Example 6: Effect of antibacterial peptide LP-P1 on the leakage of K + in bacterial cells

[0054] Taking Vibrio alginolyticus as an example, the Vibrio alginolyticus bacterial suspension (bacterial concentration is 10 6-7 CFU / mL) is respectively mixed with the antibacterial peptide LP-P1 and the vitamin B2-binding peptide at the mass concentration of MIC, incubated at 37 °C for a certain time, centrifuged at 10000 r / min for 10 min. The control group is the bacterial suspension plus deionized water. The atomic absorption spectrometer is used to measure the change of the K + mass concentration in the above supernatant, recorded once every 10 min, and recorded 7 times in total. From Figure 6 the results, it can be seen that after Vibrio alginolyticus is treated with the antibacterial peptide LP-P1 and the vitamin B2-binding peptide, the release amount of intracellular K + shows a rapid growth trend with the extension of time, while the change in the control group is not obvious. It shows that the combination of the antibacterial peptide LP-P1 and vitamin B2 can damage the integrity of the cell membrane of Vibrio alginolyticus.

[0055] Example 7: Interaction between antibacterial peptide LP-P1 and vitamin B2-binding peptide and bacterial DNA

[0056] The interaction between antibacterial peptide LP-P1 and vitamin B2-binding peptide with the genomic DNA of Vibrio parahaemolyticus was studied by DNA gel retardation assay. Vibrio parahaemolyticus was cultured in 50 mL of nutrient broth medium (NB) at 37 °C for 12 h, and the genomic DNA of the bacteria was extracted using a bacterial genomic DNA extraction kit. The purity of the extracted genomic DNA was evaluated by the optical density ratio at 260 and 280 nm (OD260 / OD280 ≥ 1.90). Next, 10 μL of DNA (218 ng / μL) was mixed with vitamin B2 combined with large yellow croaker collagen peptide LP-P1 at 25 °C for 90 min, and the mixture was electrophoresed on a 0.8% agarose gel. Gel retardation was observed under ultraviolet light using a GelDocXR gel imaging system (Bio-Rad, USA), as Figure 7 shown. After the antibacterial peptide LP-P1 and vitamin B2-binding peptide at different mass concentrations acted on Vibrio parahaemolyticus, no obvious migration phenomenon occurred in the bacterial DNA bands. This binding effect could still retard the migration of DNA when the ratio of peptide to DNA was 25 / 8. And some DNA remained in the loading wells, which might be due to the change in charge electronegativity or the change in the DNA double helix structure, thus affecting the DNA migration rate.

[0057] Example 8: Cytotoxicity experiment of antibacterial peptide LP-P1 and vitamin B2-binding peptide

[0058] In this experiment, the L929 cell line of mouse lung fibroblast cells was used as the experimental object, and the MTS method was used to evaluate the cytotoxicity of the effective antibacterial drug combination. First, the cell line was inoculated into high-glucose DMEM medium and cultured in an incubator at 37 °C, 5% CO2, and saturated humidity for 24 h. The cells adhered and grew, and after continuing to incubate for 5 - 7 d, they were digested and passaged with 0.25% trypsin. The cells in the logarithmic phase were selected as the experimental object, and the cell suspension concentration was uniformly diluted to 1.0×10 6 CFU / mL for standby. 100 μL of cell suspension (about 10 5 CFU / mL) was added to each well of a 96-well microplate, and after culturing in an incubator at 5% CO2 and saturated humidity for 24 h, different concentrations of antibacterial peptide LP-P1 and vitamin B2-binding peptide were added to each well, and no drug was added to the control group. After continuing to culture for 24 h, 20 μL of MTS solution was added and cultured for another 4 h, and the absorbance value (OD value) was measured at 490 nm, and the cell survival rate was calculated.

[0059] In summary, the present invention provides a novel antibacterial peptide LP-P1. The minimum inhibitory concentrations (MIC) of LP-P1 against Vibrio parahaemolyticus and Vibrio alginolyticus are 7.8125 μg / mL and 3.9063 μg / mL, respectively, and it can inhibit the growth of Vibrio parahaemolyticus and Vibrio alginolyticus. The antibacterial peptide LP-P1 of the present invention first adsorbs on the surface of bacteria, penetrates the lipid bilayer of the cell membrane, and then binds to the genomic DNA of bacteria, inhibiting the synthesis of bacterial DNA, thereby causing the death of bacteria.

[0060] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative rather than used to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope protected by the claims of the present invention.

Claims

1. A large yellow croaker collagen antibacterial peptide LP-P1, whose amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the large yellow croaker collagen antibacterial peptide LP-P1 as described in claim 1 in the preparation of antibacterial drugs, characterized in that: The antibacterial agent is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

3. An antibacterial drug, characterized in that: Its active ingredient is the large yellow croaker collagen antibacterial peptide LP-P1, and the amino acid sequence of the antibacterial peptide LP-P1 is SEQ ID NO:

1.

4. The antibacterial agent according to claim 3, wherein: The antibacterial agent is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

5. A feed additive, characterized in that: Its active ingredient is the large yellow croaker collagen antibacterial peptide LP-P1, and the amino acid sequence of the antibacterial peptide LP-P1 is SEQ ID NO:

1.

6. The feed additive according to claim 5, characterized in that: The feed additive is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

7. A food preservative, characterized in that: Its active ingredient is the large yellow croaker collagen antibacterial peptide LP-P1, and the amino acid sequence of the antibacterial peptide LP-P1 is SEQ ID NO:

1.

8. The food preservative according to claim 7, characterized in that: The food preservative is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

9. An antibacterial drug, characterized in that: Its active ingredients are the large yellow croaker collagen antibacterial peptide LP-P1 and vitamin B2, and the amino acid sequence of the antibacterial peptide LP-P1 is SEQ ID NO:

1.

10. The antibacterial agent according to claim 9, wherein: The antibacterial agent is used to inhibit and / or kill Vibrio parahaemolyticus and / or Vibrio alginolyticus.

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