Recombinant oyster polypeptide, preparation method and application thereof

Abstract

The application discloses a kind of improved oyster polypeptide, and its amino acid sequence is Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp-Phe.The improved oyster polypeptide obtained by the application can be synthesized by chemistry, especially by adopting liquid phase synthesis method, raw material source is wide, cost is low, yield is high, purity is high, mass production;And the improved oyster polypeptide of the application has high antibacterial efficiency and high stability, can achieve excellent antibacterial effect with significantly less amount, antibacterial effect is obviously stronger, can efficiently inhibit, kill bacteria such as propionic acid bacillus and staphylococcus aureus, can also promote wound healing, and has no cytotoxicity, is a kind of safe improved oyster polypeptide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a modified oyster polypeptide, its preparation method, and its application. Background Technology

[0002] Facial acne is mainly caused by the pathogen Propionibacterium acnes, a Gram-positive bacterium. Staphylococcus aureus is the most common pathogen causing purulent infections in humans and can cause localized purulent infections.

[0003] Antimicrobial peptides (hereinafter referred to as antimicrobial peptides) mainly exert their antimicrobial effects through physical membrane perforation, dissipation of electrochemical potential, induction of lipid asymmetry, and loss of important metabolites and cellular components, ultimately leading to cell atrophy and cell death. Alternatively, after penetrating the plasma membrane, antimicrobial peptides accumulate intracellularly and interfere with normal cell metabolism by specifically binding to intracellular targets, thereby inhibiting and killing bacteria. Therefore, antimicrobial peptides do not induce drug resistance.

[0004] Currently, there are clinical cases of antimicrobial peptides being used for the treatment of pathogenic bacterial infections, wound healing, and cancer. For example, through sequencing of multiple oyster genomes and peptide sequences, combined with large-scale screening technology, the antimicrobial peptide P-AMP108 (whose amino acid sequence is...) has been obtained.

[0005] Met-Asn-Tyr-Val-Ser-Lys-Arg-Trp-Arg-Val-Trp-Phe), but it has low stability and low antibacterial efficiency. Moreover, the source of naturally extracted antimicrobial peptides is limited, the process is complex, and the cost is high. Summary of the Invention

[0006] Based on the needs of the prior art, the purpose of this invention is to modify the structure of natural antimicrobial peptides and provide a method for chemical synthesis. The raw materials are widely available, low in cost, and can be mass-produced. Moreover, the modified antimicrobial peptides have high stability and high antimicrobial efficiency, achieving excellent antimicrobial effects with significantly less quantity. The antimicrobial effect is significantly stronger, effectively inhibiting and killing bacteria such as Propionibacterium acnes and Staphylococcus aureus, and promoting wound healing. At the same time, it is non-cytotoxic and is a safe modified oyster polypeptide.

[0007] This invention provides the following technical solution:

[0008] A first aspect of the present invention provides a modified oyster polypeptide having the amino acid sequence Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp-Phe.

[0009] A second aspect of the present invention provides a gene encoding the modified oyster polypeptide described above.

[0010] A third aspect of the present invention provides a vector comprising the above-described encoding gene.

[0011] A fourth aspect of the present invention provides a host cell comprising the above-described encoding gene or vector.

[0012] A fifth aspect of the present invention provides a method for preparing a modified oyster polypeptide, wherein the modified oyster polypeptide is synthesized by a liquid-phase synthesis method, comprising the following steps:

[0013] (1) Synthesize compound 1: Fmoc-Asn-Trp(Boc)-Val-Osu;

[0014] (2) Synthesize compound 2: tBu-Ser-Lys(Boc)-Arg(Pbf)-OMe;

[0015] (3) Synthesize compound 3: Pbf-Arg-Trp(Boc)-Phe-Osu;

[0016] (4) Synthesize compound 4: Fmoc-Asn-Trp-Val-Ser-Lys-Arg-OMe;

[0017] (5) Synthesized compound 5: Fmoc-Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp-Phe-Osu;

[0018] (6) Synthesized modified oyster polypeptides.

[0019] Preferably, the method further includes the following steps:

[0020] (7) The modified oyster polypeptide was purified by HPLC and its purity was identified;

[0021] (8) The molecular weight of the modified oyster polypeptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF).

[0022] (9) The isoelectric point of the purified modified oyster polypeptide was determined by isoelectric focusing electrophoresis, and the amino acid sequence of the purified oyster polypeptide was determined by an automated amino acid sequencer to determine its structure.

[0023] More preferably, in step (8), the step of purifying and identifying purity by HPLC includes: dissolving 0.1-0.3 mg of the modified oyster polypeptide sample to be tested in 1-5 mL of water containing 0.2%-0.5% trifluoroacetic acid, filtering with a 0.22-0.45 μm filter membrane, using mobile phase A of 0.1%-0.5% trifluoroacetic acid-water and mobile phase B of 0.1%-0.5% trifluoroacetic acid-acetonitrile, and loading the sample after the baseline stabilizes, with a loading volume of 50-100 μL; using a silica alkyl bonded phase C18 column, employing a binary mobile phase gradient elution system for gradient elution, i.e., within 20-80 min, the content of mobile phase B in the eluent increases linearly from 0% to 100%, with a flow rate of 1-3 mL / min, a detection wavelength of 260 nm, and determination at 25 °C. More preferably, the C18 column has a size of (3-5) mm × (100-300) mm, with a particle size of 2-5 μm and a pore size of 100-150 Å.

[0024] The modified oyster polypeptide of the present invention can be chemically synthesized by the above solid-phase synthesis method. Optionally, primers can be designed according to the modified oyster polypeptide sequence of the present invention, and template-free PCR amplification can be performed using the primers to obtain PCR products. Then, an expression vector can be constructed, and the vector can be transformed into host cells for expression to obtain the modified oyster polypeptide product.

[0025] The sixth aspect of the present invention provides the use of the above-described modified oyster polypeptide, encoding gene, vector, or host cell in the preparation of polypeptide antibacterial compositions or in the preparation of acne treatment drugs.

[0026] A seventh aspect of the present invention provides a polypeptide antibacterial composition, wherein the active ingredient comprises the modified oyster polypeptide described above.

[0027] Preferably, the polypeptide antibacterial composition comprises 8-50 μM of modified oyster polypeptide, 1%-10% (w / v, g / ml) chitosan, and 0.2%-1% (w / v, g / ml) glyceryl behenate.

[0028] Optionally, the preparation method of the peptide antibacterial composition is to mix the modified oyster peptide, chitosan, glycerol behenate, and optionally other commonly used adjuvants in the art at room temperature to obtain the peptide antibacterial composition, which contains 8-50 μM of modified oyster peptide, 1%-10% (w / v, g / ml) of chitosan, and 0.2%-1% (w / v, g / ml) of glycerol behenate.

[0029] The eighth aspect of the present invention provides the use of the above-described polypeptide antibacterial composition against Propionibacterium acnes or against Staphylococcus aureus.

[0030] The beneficial effects of this invention are as follows: This invention simplifies and modifies the sequence and structure of the existing oyster antimicrobial peptide P-AMP108. In this invention, the modification is carried out on the basis of retaining activity, including (1) adding Arg and Lys to increase the positive charge of the antimicrobial peptide (antimicrobial peptides are mainly cations, which can interact with anionic substances on the surface of bacteria, thereby increasing the permeability of the bacterial cell membrane, causing the leakage of intracellular macromolecules, and causing the death of pathogenic microorganisms) to increase the interaction strength between the antimicrobial peptide and bacteria. (2) replacing the hydrophobic amino acid in the sequence with tryptophan to reduce its toxicity. (3) amidation of the carboxyl terminus to protect its stability.

[0031] The modified oyster polypeptide obtained through the above modifications can be chemically synthesized, especially using liquid-phase synthesis, which has a wide range of raw material sources, low cost, high yield, high purity, and can be mass-produced. Moreover, the modified oyster polypeptide of this invention has high antibacterial efficiency and high stability, and can achieve excellent antibacterial effects with significantly less quantity. The antibacterial effect is significantly stronger, and it can effectively inhibit and kill bacteria such as Propionibacterium acnes and Staphylococcus aureus. It can also promote wound healing, while having no cytotoxicity. It is a safe modified oyster polypeptide.

[0032] The features and advantages of the present invention will be described in detail in the following detailed description section. Detailed Implementation

[0033] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in the following description are merely illustrative examples of specific implementations of this invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0034] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. It should be noted that, as used in this application, in the numerical values ​​of specific embodiments, the value can vary by ±5% as the numerical protection range of this application.

[0035] In the description of this application, unless otherwise stated, the terms "multiple / areas" and similar terms mean two / a kind or more. Furthermore, the terms "comprising," "including," and any variations thereof are intended to cover non-exclusive inclusion.

[0036] In this invention, unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art.

[0037] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are merely illustrative and should not be construed as limiting the present invention. The present invention will be explained in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0038] Example 1: Preparation of modified oyster polypeptides

[0039] A method for preparing a modified oyster polypeptide Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp-Phe includes the following steps:

[0040] (i) Synthesizing modified oyster polypeptides using liquid-phase synthesis method;

[0041] (1) Synthesize compound 1: Fmoc-Asn-Trp(Boc)-Val-Osu; including the following steps:

[0042] (1-1) Synthesis of Fmoc-Asn-Osu, including the following steps: Weigh 0.8 mol Fmoc-Asn-OH and 0.85 mol HOSu into a container, add 5 L DMF to dissolve, and dissolve 1 mol HBTU in 3 L DMF and add it to the container. React in a -15℃ cold bath. After reacting for 15 min, turn off the cold bath. Monitor the reaction by TLC. The reaction is complete in 2.5 h. Then filter to remove the solid, wash the solid twice with DMF, concentrate the filtrate, add 2 L isopropanol, and place at -20℃ overnight to obtain solid crystals. Filter again and vacuum dry to obtain the target product with a yield of 86.8% and a purity of 92.1%.

[0043] (1-2) Synthesis of Fmoc-Asn-Trp(Boc) includes the following steps: Weigh 0.7 mol Fmoc-Asn-Osu and 0.8 mol Trp(Boc)-OH into a container, add 5 L of DMF to dissolve them, and dissolve 0.6 mol TEA in 1 L of water. Add the solutions to the container and carry out the reaction. Monitor the reaction by TLC. The reaction is complete after 3 h. Filter to remove a small amount of solid, concentrate the filtrate, remove DMF, wash three times with saturated sodium chloride solution, and vacuum dry to obtain the target product with a yield of 82.5% and a purity of 90.2%.

[0044] (1-3) Synthesis of Fmoc-Asn-Trp(Boc)-Val-Osu, including the following steps: Weigh 0.6 mol Fmoc-Asn-Trp(Boc), 0.65 mol H-Val-NH2.HCl and 0.7 mol HOSu into a container, add 5 L DMF to dissolve, and dissolve 0.6 mol HBTU in 1 L DMF. After adding to the container, react in a -15℃ cold bath. After reacting for 30 min, turn off the cold bath. Monitor the reaction by TLC. The reaction is complete in 2.5 h. Filter to remove the solid. Wash the solid three times with DMF. Concentrate the filtrate and wash it three times with saturated sodium chloride solution. Dry under vacuum to obtain the target product with a yield of 87.6% and a purity of 93.5%.

[0045] (2) Synthesizing compound 2: tBu-Ser-Lys(Boc)-Arg(Pbf)-OMe; including the following steps:

[0046] (2-1) Synthesis of tBu-Ser-Osu, including the following steps: Weigh 0.8 mol Fmoc-Ser(tBu)-OH and HOSu into a container, add 5 L DMF to dissolve, and dissolve 1 mol HBTU in 1.5 L DMF and add it to the container. React in a -18℃ cold bath. After reacting for 20 min, turn off the cold bath. Monitor the reaction by TLC. The reaction is complete in 2.5 h. Filter to remove the solid, redissolve with EA, extract (wash twice with acid and three times with saturated sodium chloride), dry with anhydrous sodium sulfate, filter, concentrate to a viscous state, add 3 L each of diethyl ether and isopropyl ether, precipitate the solid, filter to collect the solid, vacuum dry to obtain the solid, wash twice with DMF, concentrate the filtrate, add 5 L isopropanol, place at -25℃ overnight to obtain solid crystals, filter, vacuum dry to obtain the target product, yield 85.7%, purity 94.1%.

[0047] (2-2) Synthesis of tBu-Ser-Lys(Boc) includes the following steps: Weigh 0.75 mol tBu-Ser-Osu and 0.8 mol Lys(Boc) into a container, add 5 L DMF to dissolve, and dissolve 1 mol HBTU in 1.5 L DMF. The reaction is carried out in a -15℃ cold bath. After the reaction is carried out for 30 min, the cold bath is turned off. The reaction is monitored by TLC and the reaction is complete in 4 h. The solid is removed by filtration, the solid is washed 3 times with DMF, the filtrate is concentrated, 2 L NMP is added, and the mixture is placed at -25℃ overnight to obtain solid crystals. The crystals are filtered and dried under vacuum to obtain the target product with a yield of 86.5% and a purity of 90.1%.

[0048] (2-3) Synthesis of tBu-Ser-Lys(Boc)-Arg(Pbf)-OMe, including the following steps: Weigh 0.7 mol tBu-Ser-Lys(Boc) and 0.75 mol Fmoc-Arg(Pbf)-OMe into a container, add 5 L DMF to dissolve, and separately dissolve 0.8 mol DCC in 2 L THF and 1 L EA. The reaction is carried out in a -15℃ cold bath. After the reaction is stopped after 20 min, the reaction is monitored by TLC. The reaction is complete after 3 h. Filter to remove the solid, concentrate the filtrate, add hydrochloric acid solution, stir to precipitate to obtain an off-white solid, filter, wash once with purified water, wash three times with saturated sodium chloride solution, and vacuum dry to obtain the target product with a yield of 84.1% and a purity of 91.7%.

[0049] (3) Synthesize compound 3: Pbf-Arg-Trp(Boc)-Phe-Osu; including the following steps:

[0050] (3-1) Synthesis of Pbf-Arg-Osu, comprising the following steps: Weigh 0.85 mol Fmoc-Arg(Pbf)-OH and 1 mol HOSu into a container, add 5 L DMF to dissolve, and dissolve 0.9 mol HBTU in 2 L DMF. React in a -15℃ cold bath for 12 min, then turn off the cold bath. Monitor the reaction by TLC; the reaction is complete after 2 h. Filter to remove the solid, wash the solid three times with DMF, concentrate the filtrate to a viscous consistency, add 4 L isopropanol, and let stand overnight at -25℃. A crystalline solid is obtained; filter to collect the solid, and vacuum dry to obtain the target product with a yield of 85.3% and a purity of 90.9%.

[0051] (3-2) Synthesis of Pbf-Arg-Trp(Boc)-OMe, comprising the following steps: Weigh 0.7 mol Pbf-Arg-Osu and 0.8 mol Fmoc-Trp(Boc)-OMe into a container, add 5 L DMF, add 0.6 mol TEA to the reaction flask, monitor the reaction by TLC, and the reaction is complete after 2 h. Filter to remove a small amount of solid, concentrate the filtrate, remove DMF, add hydrochloric acid solution to adjust the pH to 5-5.5, add EA, extract, wash twice with water, wash three times with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate to a viscous state, add 3 L isopropyl ether to precipitate the solid, filter, and vacuum dry to obtain the target product, with a yield of 82.4% and a purity of 93.8%.

[0052] (3-3) Synthesis of Pbf-Arg-Trp(Boc)-Phe-Osu, comprising the following steps: Weigh 0.8 mol of Pbf-Arg-Trp(Boc)-OMe and 0.85 mol of Phe-Osu into a container, add 5 mol of DMF, and add 0.7 mol of TEA to the reaction flask. Monitor the reaction by TLC. The reaction is complete after 2 hours. Filter to remove a small amount of solid, concentrate the filtrate to remove most of the DMF, add hydrochloric acid solution to precipitate, filter to collect the solid, dissolve again with EA, extract, wash three times with 8% sodium bicarbonate solution, wash twice with water, wash three times with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, precipitate with isopropyl ether, filter, yield 81.6%, purity 93.5%.

[0053] (4) Synthesis of compound 4: Fmoc-Asn-Trp-Val-Ser-Lys-Arg-OMe, including the following steps: Weighing 0.9 mol Fmoc-Asn-Trp(Boc)-Val-Osu and 0.95 mol

[0054] tBu-Ser-Lys(Boc)-Arg(Pbf)-OMe was placed in a container, and 5 L of DMF was added. 1 mol of DCC was added to the reaction flask, and the reaction was monitored by TLC. The reaction was complete after 3 hours. A small amount of solid was removed by filtration, the filtrate was concentrated, DMF was removed, hydrochloric acid solution was added to precipitate, the solid was filtered, dissolved in EA, extracted, washed three times with 8% sodium bicarbonate solution, washed three times with water, washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, precipitated with isopropyl ether, and filtered. The yield was 74.2%, and the purity was 92.5%.

[0055] (5) Synthesis of compound 5: Fmoc-Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp-Phe-Osu, including the following steps: Weigh 0.85 mol Fmoc-Asn-Trp-Val-Ser-Lys-Arg-OMe and 0.9 mol Pbf-Arg-Trp(Boc)-Phe-Osu into a container, add 5 mol DMF, take 0.7 mol DIPEA, add to the reaction flask and react, monitor the reaction by TLC, and the reaction is complete after 2 h; filter to remove a small amount of solid, concentrate the filtrate, remove DMF, add hydrochloric acid solution to precipitate, filter to take solid, dissolve in EA, extract, wash three times with 8% sodium bicarbonate solution, wash twice with water, wash three times with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, precipitate with isopropyl ether, filter to obtain the target product, with a yield of 87.6% and a purity of 91.9%.

[0056] (6) Synthesizing the modified oyster polypeptide includes the following steps: Weigh 0.75 mol Fmoc-Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp-Phe-Osu, 0.3 mol piperidine and 0.5 mol DCM into a container, add 5 L THF, and after 1.5 h the reaction is complete, filter to remove a small amount of solid, concentrate the filtrate, remove THF, add hydrochloric acid solution to precipitate, filter to take the solid, dissolve in EA, extract, wash three times with 8% sodium bicarbonate solution, wash twice with water, wash three times with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, precipitate with isopropyl ether, filter to obtain the target product, with a yield of 86.3%.

[0057] (II) Identification of the purity and molecular weight of the modified oyster polypeptides

[0058] (7) The modified oyster polypeptide was purified and its purity was identified by HPLC. The purity of the modified oyster polypeptide was 92.6%. The steps of purifying and identifying the purity by HPLC included: dissolving 0.1 mg of the modified oyster polypeptide sample to be tested in 1 mL of water containing 0.2% trifluoroacetic acid, filtering with a 0.35 μm filter membrane, using mobile phase A of 0.1% trifluoroacetic acid-water and mobile phase B of 0.1% trifluoroacetic acid-acetonitrile, and loading the sample after the baseline stabilized, with a loading volume of 50 μL; using a silica alkyl bonded phase C18 column (4.6 mm × 300 mm, particle size 5 μm, pore size 100 Å), and using a binary mobile phase gradient elution system for gradient elution, i.e., within 40 min, the content of mobile phase B in the eluent increased linearly from 0% to 100%, with a flow rate of 1 mL / min, a detection wavelength of 260 nm, and determination at 25 °C.

[0059] (8) The molecular weight of the modified oyster polypeptide was determined to be 1281.6 Da by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF).

[0060] The method includes: dissolving the purified modified oyster polypeptide in deionized water to prepare a 1 μM solution, taking 10 μL and mixing it with an equal volume of saturated matrix solution (preparing a saturated solution by dissolving α-cyano-4-hydroxycinnamic acid in a 50% acetonitrile solution containing 0.1% trifluoroacetic acid, centrifuging, and taking the supernatant) before determination.

[0061] (9) The isoelectric point of the purified modified oyster polypeptide was determined to be 7.98 by isoelectric focusing electrophoresis, and the amino acid sequence of the purified modified oyster polypeptide was determined by an automated amino acid sequencer, and its structure was determined to be Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp-Phe.

[0062] Example 2: Determination of antibacterial effect and minimum inhibitory concentration (MIC)

[0063] The bacterial cultures of Propionibacterium acnes and Staphylococcus aureus in the logarithmic growth phase were both diluted to 1x10⁻¹. 7 CFU / mL. In a 96-well plate, add 10 μL of the bacterial culture to each well, then add 10 μL of the modified oyster peptide solution at different concentrations to each well. For the negative control, add 20 μL of bacterial culture per well; for the blank control, add 20 μL of the corresponding culture medium. Incubate at 37°C for 16 h, and determine the minimum inhibitory concentration (MIC) by measuring the absorbance at 630 nm using a microplate reader. This MIC represents the lowest concentration of modified oyster peptide that inhibits the growth of *Propionibacterium acnes* and *Staphylococcus aureus* by 100%.

[0064] The results showed that the modified oyster polypeptide solution of the present invention had a minimum inhibitory concentration of 5 μM against Propionibacterium acnes and a minimum inhibitory concentration of 8 μM against Staphylococcus aureus, exhibiting excellent antibacterial effects.

[0065] Example 3: Thermal stability determination and pH stability determination

[0066] (I) Thermal stability test: 100 μL of Propionibacterium acnes suspension was mixed with solid culture medium (containing 1.3% agar), and the final bacterial density was 1 x 10⁻⁶. 7 CFU / mL; after incubation at 20, 30, 40, 50, 60, 80, and 100 °C for 15 minutes, 100 μL of 50 μM modified oyster peptide solution was added to the pre-drilled wells in the culture dish (drilling method); after incubation at 37 °C overnight, the diameter of the inhibition zone in the culture dish was measured (mm), and the average value was obtained after four repetitions; the modified oyster peptide without heat incubation (20 °C) served as a control. The results are shown in Table 1.

[0067] Temperature (°C) 20 25℃ 30℃ 35℃ 40℃ 50℃ 60℃ 80℃ 100℃ Diameter of the inhibition zone (mm) 21.3 24.1 27.1 22.5 12.6 8.9 6.8 2.5 1.1

[0068] The results show that the modified oyster polypeptide of the present invention has a good antibacterial effect in the range of 20-35℃, especially at 30℃ it has the best antibacterial effect.

[0069] (II) pH stability test: The pH of the modified oyster peptide solution was adjusted to 3–12 using hydrochloric acid or sodium hydroxide. 100 μL of 50 μM peptide solution was added to each well of a culture dish; after incubation at 37°C overnight, the diameter of the inhibition zone in the culture dish was measured. This was repeated four times to obtain the average value, with buffer solutions of different pH values ​​used as controls. See Table 2.

[0070] pH 3 4 5 6 7 8 9 10 11 12 Diameter of the inhibition zone (mm) 20.8 21.5 22.4 27.5 28.6 26.1 17.2 13.5 11.3 10.7

[0071] The results showed that the modified oyster polypeptide of the present invention had the best antibacterial effect in the pH range of 6 to 8, and also had a good antibacterial effect in a strongly acidic environment.

[0072] Example 4 Cytotoxicity Assay

[0073] The toxicity of the modified oyster peptide of this invention to human skin fibroblast HFF-1 cells was detected using the MTT assay. Fibroblasts were first cultured in DMEM containing 15% fetal bovine serum and double antibiotics (100 U / ml each of penicillin and streptomycin). After confluence, the cells were digested with 0.25% trypsin, washed twice with the aforementioned medium, resuspended, counted, and 100 μl of the cell suspension was added to a 96-well cell culture plate. The sample concentrations of the modified oyster peptide were set at 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, and 150 μM, with each concentration replicated three times. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, 20 μL of 5 mg / ml MTT solution (PBS buffer) was added to each well of the 96-well cell culture plate, and the plates were incubated for another 5 h. The liquid in the wells was then aspirated with a syringe, and 100 μL of the solution was added to each well. Dissolve the purple crystals completely in DMSO using a pipette. Measure the absorbance using a microplate reader at a wavelength of 490 nm.

[0074] Table 3 shows the toxicity of the modified oyster polypeptide of the present invention to HFF-1 cells.

[0075] Sample concentration (μM) 1 10 20 40 50 60 80 100 150 cytotoxicity % 0 0.1±0.03 0.1±0.07 0.2±0.07 0.5±0.02 1.04±0.09 2.14±0.08 2.96±0.11 3.61±0.12

[0076] The results, as shown in Table 3, indicate that the cytotoxicity of the modified oyster polypeptide at a concentration of 50 μM is only about 0.5%. Therefore, the modified oyster polypeptide concentration has extremely low cytotoxicity to human skin fibroblasts and will not harm normal human skin cells.

[0077] Example 5: Analysis of the effects of modified oyster peptides and peptide antibacterial compositions on cellular pro-inflammatory responses.

[0078] (1) 10 4HFF-1 skin fibroblasts were cultured in 12-well plates and added to DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. The culture conditions were 37°C and 5% CO2. (2) After 24 hours of culture, the medium was replaced with serum-free DMEM medium. The experimental groups were treated with antibacterial compositions of modified oyster polypeptides (group A) and polypeptides (group B) with final concentrations of 5, 10, 20, 30, 40 and 50 μM, respectively. The compositions contained 8-50 μM of modified oyster polypeptides and 1% chitosan. -10% (w / v, g / ml), 0.2%-1% (w / v, g / ml) of glycerol behenate, 10% glycerol and deionized water) were treated for 24 hours (the ratio of modified oyster peptide and peptide antibacterial composition to cell culture medium and culture volume was 1:100), and the control group (i.e., the concentration of modified oyster peptide and peptide antibacterial composition were both 0 μM) was treated with an equal volume of PBS for 24 hours, and three replicates were set up; (3) The level of cytokine IL-1α was detected by ELISA kit.

[0079] Table 4 shows the pro-inflammatory cellular responses of the modified oyster polypeptide and polypeptide antibacterial composition of the present invention.

[0080]

[0081]

[0082] As shown in Table 4, the results indicate that the modified oyster peptides and peptide antibacterial compositions do not stimulate cells to release inflammatory factors.

[0083] Example 6: Modified oyster peptides and peptide antibacterial compositions promote wound healing.

[0084] Five-week-old mice (approximately 20g) were randomly selected and anesthetized with 1% sodium pentobarbital solution via intraperitoneal injection. Their dorsal hair was shaved, and after alcohol disinfection, a 2cm long and approximately 0.3cm wide strip incision was made on their backs. PBS solution served as the blank control group; mouse-derived EGF (mEGF, 5μg / mL) and human-derived EGF (hEFG, 5μg / mL) served as positive controls; and modified oyster peptides (group A, 5μg / mL) and a peptide antibacterial composition (group B, containing 5μM modified oyster peptides, 1% chitosan (w / v, g / ml), 0.2% glycerol behenate (w / v, g / ml), 10% glycerol, and deionized water) served as experimental groups, with three mice in each group. Both EGF and modified oyster peptides were prepared using PBS solution. Administered twice daily, 10μL each time.

[0085] Table 5. Modified oyster peptides and peptide antibacterial compositions promote wound healing.

[0086] As shown in Table 5, observation and measurement of wound length after 7 days showed that the modified oyster polypeptide at a concentration of 5 μg / ml had a significantly better wound healing effect than mouse mEGF at a concentration of 5 μg / ml and human hEGF at a concentration of 5 μg / ml. The polypeptide antibacterial composition had an even better wound healing effect than the modified oyster polypeptide. The modified oyster polypeptide and polypeptide antibacterial composition of the present invention can significantly improve wound healing ability.

[0087] Example 7: Evaluation of the human efficacy of the modified oyster peptides and peptide antibacterial compositions

[0088] Human efficacy evaluation includes the following steps:

[0089] (1) 100 volunteers aged 18-30 with obvious acne on their face and facial skin prone to pimples were selected and randomly divided into two groups, half male and half female, to conduct half-face test.

[0090] (2) Method of use: After cleansing the face every morning, Group A applied a solution of 5 μM modified oyster peptides (10% glycerin and deionized water) once (about 0.5 ml), while Group B applied an emulsion containing a peptide antibacterial composition (containing 5 μM modified oyster peptides, 1% chitosan (w / v, g / ml), and 0.2% glyceryl behenate (w / v, g / ml)) once (about 0.5 ml). The treatment was continued for 30 days, and follow-up visits and data collection were conducted on the 15th and 30th days. The control group used an emulsion without modified oyster peptides, and the method of use was the same.

[0091] (3) According to the ISGA scale of the clinical evaluation standard for acne-removing functional skin care products T / CNMIA 0012-2020 (six levels from 0 to 5), the skin lesion reduction percentage was calculated according to the following formula: Skin lesion reduction percentage (%) = (Initial ISGA score - Follow-up ISGA score) / Initial ISGA score × 100%; According to the above clinical standards, clinical volunteers conducted self-evaluation of skin improvement, and the relevant standards were (1 point: clean, completely cleared; 2 points: very good improvement, cleared >75%; 3 points: good improvement, cleared 50-75%; 4 points: moderate improvement, cleared <50%; 5 points: no improvement, not cleared; 6 points: worsened or aggravated), and the improvement rate (%) = (the sum of the number of people with scores of 1-4) / the total number of people × 100%.

[0092] Table 6. Statistics of self-evaluation results of the human efficacy of the modified oyster peptides.

[0093]

[0094] The results of the subjects' self-evaluation are shown in Table 6. The improvement rate of applying the lotion containing the modified oyster peptide and the peptide antibacterial composition was 100% at 15 days and 30 days. Among the parts with improvement of grade 2 or above, the peptide antibacterial composition was more effective than the modified oyster peptide.

[0095] Example 8: Human safety evaluation of modified oyster peptides and peptide antibacterial compositions

[0096] Human efficacy evaluation includes the following steps:

[0097] (1) 100 volunteers aged 18-30 were randomly divided into two groups, with half males and half females, and a repeatable open-label application experiment was conducted; (2) Group A was given 0.05 ml of 5 μM glycerol solution containing modified oyster polypeptide (glycerol content 10%) each time, and Group B was given 0.05 ml of glycerol solution each time. A 5 μM glycerin solution containing a polypeptide antibacterial composition (glycerin content 10%) was applied evenly to the test skin twice a day for 30 consecutive days. The blank group was a 0.05 ml glycerin solution without modified oyster polypeptide (glycerin content 10%). (3) The scoring criteria for open patch test on human skin were described according to the 2015 edition of the "Cosmetic Safety Technical Specifications": (0, negative reaction; 1, weak erythema, dry skin, wrinkles; 2, erythema, edema, papules, wheals, desquamation, fissures; 3, obvious erythema, edema, vesicles; 4, severe erythema, edema, bullae, erosion, pigmentation or hypopigmentation, acne-like changes). After 15 and 30 days of the experiment, the negative reactions of the test group and the blank group were 100%, which proved that the modified oyster polypeptide and polypeptide antibacterial composition of the present invention have very high safety and high gentleness on the skin.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Although the present invention has been described in detail through the above embodiments, those skilled in the art can still make various changes in form and detail based on the technical content described in the Summary of the Invention and the Embodiments, without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A modified oyster polypeptide, characterized in that, Its amino acid sequence is Asn-Trp-Val-Ser-Lys-Arg- Arg-Trp-Phe.

2. A gene encoding the modified oyster polypeptide according to claim 1.

3. A carrier, characterized in that, It includes the coding gene as described in claim 2.

4. A host cell, characterized in that, It includes the coding gene according to claim 2 or the vector according to claim 3.

5. A method for preparing the modified oyster polypeptide as described in claim 1, characterized in that, The modified oyster polypeptide was synthesized by liquid-phase synthesis, including the following steps: (1) Synthesize compound 1: Fmoc-Asn-Trp(Boc)-Val-Osu; (2) Synthesize compound 2: tBu-Ser-Lys(Boc)-Arg(Pbf)-OMe; (3) Synthesize compound 3: Pbf-Arg-Trp(Boc)-Phe-Osu; (4) Synthesized compound 4: Fmoc-Asn-Trp-Val-Ser-Lys-Arg-OMe; (5) Synthesized compound 5: Fmoc-Asn-Trp-Val-Ser-Lys-Arg-Arg-Trp(Boc)-Phe-Osu; (6) Synthetic modified oyster polypeptides.

6. The method for preparing the modified oyster polypeptide according to claim 5, characterized in that, It also includes the following steps: (7) The modified oyster polypeptide was purified by HPLC and its purity was identified; (8) The molecular weight of the modified oyster polypeptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF). (9) The isoelectric point of the purified modified oyster polypeptide was determined by isoelectric focusing electrophoresis, and the amino acid sequence of the purified oyster polypeptide was determined by an automated amino acid sequencer to determine its structure.

7. The use of the modified oyster polypeptide of claim 1, the encoding gene of claim 2, the vector of claim 3, or the host cell of claim 4 in the preparation of antibacterial polypeptide compositions against Propionibacterium acnes or Staphylococcus aureus, or in the preparation of drugs for treating acne.

8. A polypeptide antibacterial composition, characterized in that, Its active ingredient comprises the modified oyster polypeptide as described in claim 1.

9. A polypeptide antibacterial composition, characterized in that, It contains 8-50 μM of the modified oyster polypeptide as described in claim 1, 1%-10% (w / v, g / ml) of chitosan, and 0.2%-1% (w / v, g / ml) of glycerol behenate.

10. The use of the polypeptide antibacterial composition of claim 8 or 9 in the preparation of drugs against Propionibacterium acnes or Staphylococcus aureus.

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