Polypeptide PN-VW22 and application thereof

Abstract

The invention provides a polypeptide PN-VW22 and application thereof, and belongs to the technical field of polypeptide drugs. The polypeptide PN-VW22 provided by the invention is specifically as follows A1) or A2): A1) an amino acid sequence as shown in SEQ ID NO.1; a2) a fusion protein obtained by connecting a label to the N end or / and C end of A1). The polypeptide has obvious cell proliferation activity and anti-staphylococcus aureus bifunctional activity, and also has a wound repairing effect. The polypeptide is small in molecular weight and stable in structure, can be industrially produced through solid-phase chemical synthesis, can also be industrially applied to preparation of treatment medicines for promoting skin wound repair, and can be used as a candidate polypeptide medicine or a medical beauty product raw material for treating body surface wounds, repairing tissues, burns and skin ulcers, reducing scars and accelerating scar repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptide drugs, in particular to a polypeptide PN-VW22 and application thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, which is essential for maintaining the integrity and health of the body. After injury, normal wound healing mainly undergoes four overlapping and orderly stages. Coagulation stage: the skin blood vessels are ruptured with bleeding, platelets adhere to the damaged blood vessels, release TGFβ, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), etc., activate the coagulation cascade to form fibrin clots, close the wound and prevent infection. Inflammation stage: after the wound is formed, neutrophils are recruited to clear pathogens by the factors released by platelets, secrete inflammatory factors to activate fibroblasts and keratinocytes apoptosis, monocytes migrate and differentiate into macrophages, early strengthen pro-inflammatory signals to remove foreign bodies, and later secrete VEGF, IL-10, etc. to promote repair and inhibit excessive inflammation. Cell proliferation stage: fibroblasts proliferate and differentiate, new blood vessels, granulation tissue and nerves increase, fibroblasts migrate to the wound to form a loose edematous granulation tissue matrix. Tissue remodeling stage: fibroblasts differentiate into myofibroblasts, secrete type I collagen and crosslink to form scars. Wound healing is a complex process involving the coordinated action of many different tissues and cell lineages. Small wounds will heal within a few days, while larger wound injuries or chronic non-healing wounds caused by diseases may take weeks or even months to heal. The long repair time provides an opportunity for pathogens to invade and multiply, leading to infection, secondary diseases, etc., reducing the patient's quality of life and increasing the economic burden. Traditional wound treatment drugs include antibiotics, growth factors, biological dressings, etc., but have limitations such as drug resistance, high cost, poor biocompatibility, etc., and most have a single mechanism of action, which has limited effect on complex wounds such as chronic wounds and diabetic ulcers. Therefore, it is still necessary to develop new repair drugs. SUMMARY

[0003] The present application aims at the above-mentioned deficiencies of the prior art, and provides a novel polypeptide PN-VW22, which can promote keratinocyte proliferation and migration and has certain antibacterial effect, and has the function of promoting wound repair.

[0004] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The first object of the present application is to provide a polypeptide PN-VW22, which is specifically as follows A1) or A2): A1) the amino acid sequence is shown as SEQ ID NO. 1; A2) a fusion protein obtained by connecting a tag to the N-terminal or / and C-terminal of the protein of A1): In order to facilitate the purification of the protein of A1), a tag such as Poly-Arg (RRRRR), Poly-His (HHHHHH), FLAG (DYKDDDK) can be connected to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 of the sequence listing.

[0005] Further, the 11th and 17th amino acids of the polypeptide are cysteines, forming a disulfide bond.

[0006] A second object of the present application is to provide a biological material related to the above-described polypeptide PN-VW22, which is any one of the following B1) to B6): B1) a nucleic acid molecule encoding the polypeptide PN-VW22; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1); B4) a recombinant microorganism containing the nucleic acid molecule of B1); B5) a recombinant microorganism containing the expression cassette of B2); B6) a recombinant microorganism containing the recombinant vector of B3).

[0007] A third object of the present application is to provide the use of the above-described biological material in the production of the polypeptide PN-VW22.

[0008] A fourth object of the present application is to provide the use of the above-described polypeptide PN-VW22 or biological material in the production of a product for promoting wound healing or an antibacterial product.

[0009] Further, the polypeptide PN-VW22 has a bacteriostatic effect on Staphylococcus aureus, Escherichia coli, and / or Candida albicans.

[0010] Further, the product is any one of a pharmaceutical product, a cosmetic product, or a food product.

[0011] A fifth object of the present application is to provide a pharmaceutical composition comprising the above-described polypeptide PN-VW22 or a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

[0012] Further, the polypeptide PN-VW22 is the only effective active ingredient.

[0013] A sixth object of the present application is to provide a daily use product comprising the above-described polypeptide PN-VW22.

[0014] Further, the daily use product includes a shampoo, a shower gel.

[0015] The pharmaceutical excipients can be those commonly employed in the art of pharmaceutical production. The excipients are used to provide a safe, stable, and functional pharmaceutical composition, and can also provide a means to allow the active ingredient to dissolve at a desired rate or to be effectively absorbed by a subject after administration of the composition. The pharmaceutical excipients can be inert fillers or can provide some function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient of the composition. The pharmaceutical excipients can include one or more of the following: binders, suspending agents, emulsifying agents, diluents, fillers, granulating agents, glidants, disintegrants, lubricants, antiadherents, flow aids, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavorants, and sweeteners.

[0016] The pharmaceutical compositions of the present application can be prepared by any of the methods known to those skilled in the art using, for example, conventional mixing, dissolving, granulating, emulsifying, dragee-making, encapsulating, entrapping or lyophilizing processes.

[0017] The pharmaceutical compositions of the present application can be administered in any form including injection (intravenous), mucosally, orally (both solid and liquid forms), inhalation, ocularly, rectally, topically, or parenterally (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular). The pharmaceutical compositions of the present application can also be in controlled or delayed release dosage forms (e.g., liposomes or microspheres). Examples of solid oral dosage forms include, but are not limited to, powders, capsules, caplets, gelcaps, and tablets. Examples of liquid dosage forms for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical dosage forms include, but are not limited to, creams, gels, ointments, pastes, patches, cataplasms, foams, lotions, drops, or serums. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable dosage forms for the pharmaceutical compositions include, but are not limited to, eye drops and other ophthalmic formulations; aerosols: such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0018] The term "nucleic acid molecule encoding" as used herein means a polynucleotide that specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG or TGA. The coding sequence can be a genomic DNA, cDNA, or a combination thereof.

[0019] The term "expression" as used herein includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0020] The term "recombinant vector" as used herein means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and that is operatively linked to control sequences for its expression.

[0021] The term "recombinant microorganism" as used herein means any type of microorganism susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present application.

[0022] The term "pharmaceutically acceptable" as used herein pertains to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0023] The term "pharmaceutically acceptable salt" refers to salts of conventional salt-forming reactions in the art, e.g., the chemical reaction between an acid and a base to form a salt, such as: NH3+ H2SO4→ (NH4)2SO4.

[0024] Salts can be basic salts, acid salts, or neutral salts. Basic salts produce hydroxyl ions in aqueous solution and acid salts produce hydronium ions. Salts of the polypeptide PN-VW22 of the present application can be formed between cationic or anionic groups of the polypeptide PN-VW22 of the present application and cations or anions, respectively. Such groups can be located in the peptide portion of the polypeptide PN-VW22 of the present application. Anionic groups of the polypeptide PN-VW22 of the present application can include free carboxyl groups of the peptide portion. The peptide portion typically includes a free carboxylic acid group at the C-terminus. Cationic groups of the peptide portion are not limited in the present application and include free amino groups at the N-terminus, if present, and any free amino groups of internal basic amino acid residues (e.g., Arg and Lys).

[0025] "Pharmaceutically acceptable acid addition salt" refers to those salts of the polypeptide PN-VW22 of the present application which are acceptable for use in pharmaceuticals, and which are formed from suitable non-toxic acids, such as inorganic acids, for example hydrochloric, hydrobromic, sulfuric, nitric, carbonic, phosphoric, and organic acids such as aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, for example acetic, propionic, glycolic, gluconic, lactic, pyruvic, oxalic, malic, maleic, malonic, succinic, fumaric, tartaric, citric, aspartic, ascorbic, glutamic, benzoic, cinnamic, mandelic, embonic, phenylacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic and salicylic.

[0026] "Pharmaceutically acceptable base addition salts" refers to those salts which are acceptable in pharmaceutical sciences. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion-exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins.

[0027] Compared with the prior art, the present application has the following advantages: The PN-VW22 polypeptide prepared by the present application has obvious cell proliferation activity and anti-Staphylococcus aureus bifunctional activity, and is also a polypeptide molecule with wound repair efficacy. The polypeptide has small molecular weight and stable structure, can be synthesized by solid phase chemistry to realize industrial production, can be applied to the preparation of drugs for promoting skin wound repair, and can be used as a candidate polypeptide drug or medical and cosmetic product raw material for treating body surface wounds, tissue repair, burns, skin ulcers, reducing scar formation, and accelerating scar repair. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A chromatogram for separation and purification of the polypeptide PN-VW22 prepared by solid phase synthesis; Figure 2 A mass spectrum identification diagram of the polypeptide PN-VW22; Figure 3 The polypeptide PN-VW22 has obvious promoting effect on HaCaT cell proliferation, and has concentration-dependent relationship; Figure 4 A result diagram of no proliferation activity of the polypeptide PN-VW22 on NIH 3T3 cells; Figure 5 The polypeptide PN-VW22 promotes the migration ability of HaCaT cells; Figure 6 Statistical results of the polypeptide PN-VW22 promoting the migration ability of HaCaT cells. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present application clearer, the embodiments of the present application are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference signs represent the same or similar elements or elements with the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0030] The experimental methods used in the following examples are conventional methods, and are generally performed according to conventional conditions such as those described in Molecular Cloning Experiments Guide (Fourth Edition) (Chinese version) published by Science Press, or according to the conditions recommended by the manufacturer, which are well known to those skilled in the art, and are not limited by the present application.

[0031] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0032] Active polypeptide screening The reported proliferation-promoting polypeptides cathelicidin-DM, OM-LV20, brevinin-2Ta, brevinin-2PN, tylotoin, Bv8-AJ and RL-QN15 were selected as templates, and 20 structurally similar polypeptides with potential proliferation-promoting effects were selected from the database according to the literature Characterization and mechanism of action of amphibian-derived wound-healing-promoting peptides. Further structure transformation of the 20 polypeptides was performed by Schrödinger software, and molecular docking was performed with the receptor (human epidermal factor receptor 1 NQL). The data volume was reduced by wet experiments, and finally 5 sequences were selected according to the scoring, and the activity was verified at a concentration of 2 μM. The results are shown in Table 1.

[0033] Table 1.

[0034] Example 1 This example provides a solid-phase synthesis method of polypeptide PN-VW22.

[0035] The synthesis was completed by a solid-phase synthesis method. The specific steps are as follows: (1) 0.2 g of resin was weighed into a reaction tube and activated with DMF for 30 minutes; 1 mM of the first amino acid residue, 150 mg of DMAP, and DMF as the solvent were added, and reacted for 3 hours; after the reaction, the mixture was washed with DMF for 3-6 times, and PIP was used to elute the Fmoc protecting group twice. (2) The subsequent amino acids and HBTU were weighed, 0.5 ml of DIEA was added, and reacted for 40 minutes, and then washed with DMF for 3-6 times; the Fmoc protecting group was eluted twice (10 minutes each time), washed with DMF for 4 times, washed with methanol for 2 times, and the above steps were repeated until the last amino acid. (3) The product was cut with a TFA:H2O:EDT=92:6:2 solution for 2 hours, and then filtered; the product was precipitated with ether, centrifuged and washed for 3-5 times to obtain a white solid. (4) The obtained crude product was dissolved in pure water, centrifuged at 10000 rpm for 10 minutes, and then the supernatant was filtered with a 0.22 μm filter. (5) The filtered sample was separated and purified on a semi-preparative reverse-phase high-performance liquid chromatography column using a C18 reverse-phase chromatography column, the elution peaks were collected and identified by mass spectrometry, and the correct elution peak was transferred to a vacuum freeze dryer for freeze-drying. (6) At room temperature, 50 mL of renaturation solution (0.1 M NaCl, 0.1 M Tris-HCl, 5 mM GSH and 0.5 mM GSSG) was prepared, the pH value was adjusted to 7.5, 5 mg of the sample purified by HPLC was weighed and dissolved in 1 mL of ultrapure water. (7) The prepared renaturation solution was transferred to a 50 mL centrifuge tube wrapped with tin foil, the dissolved sample was slowly dropped into the centrifuge tube, the opening was sealed with sealing film, and the tube was placed in a 4°C refrigerator, 0.5 mL of 50% TFA was added after 24 hours to terminate the reaction. (8) After the termination of the reaction, the renatured polypeptide needs to be further purified by HPLC and detected by mass spectrometry to determine whether the renaturation is successful.

[0036] The polypeptide solid-phase synthesis was used to complete the chemical synthesis and preparation of PN-VW22. The product was purified and identified as shown in Figure 1 and Figure 2 PN-VW22 was purified by high-performance liquid chromatography, the acetonitrile elution concentration before renaturation was 28%, and the purity was more than 95%. After redox renaturation, the target peak elution time was 13 min, and the acetonitrile concentration was 25%. The mass spectrometry identification result shows that the molecular weight of the renatured PN-VW22 is 2297.4 Da, which is consistent with the theoretical molecular weight 2297.6 Da, indicating that a pair of disulfide bond is formed and the pairing is successful. After freeze-drying, it can be used for subsequent activity identification research.

[0037] Example 2 The embodiment provides a biological synthesis method of a polypeptide PN-VW22.

[0038] Prokaryotic expression of polypeptide PN-VW22.

[0039] The polypeptide PN-VW22 amino sequence was optimized by online codon optimization software (https: / / www.novopro.cn / tools / codon-optimization.html). An enterokinase cleavage site (GACGACGACGACAAG) was added to the front end of the optimized nucleotide sequence encoding PN-VW22 (as shown in SEQ ID NO. 6), a stop codon (TAA) was added to the end, and EcoRI / HindIII enzyme cleavage sites were added to the front and back ends of the above nucleotide sequence. The above sequence was artificially synthesized (completed by Nanjing Kingsrui Biotechnology Co., Ltd.) and constructed into a pET32a vector through EcoRI / HindIII enzyme cleavage sites. The constructed PN-VW22-pET32a plasmid was transformed into BL21 (DE3) E. coli, a single colony was inoculated in LB medium containing ampicillin resistance (ST007, Biyun Tian), 37°C, 220 rpm shaking culture for 6-8 hours, 300 μL was dispensed and sent to GenScript Biotech Corporation for sequencing, and the correct bacterial liquid was used for subsequent prokaryotic expression. The bacteria liquid after resuscitation was inoculated in 1 L of LB medium containing resistance at a ratio of 1:100, and cultured at 37°C, 220 rpm in a shaking incubator. When the OD 600nm value reached 0.6-0.8, 1 mM of IPTG was added to induce polypeptide expression, and the induction was carried out at 28°C, 100 rpm for 12-16 hours. After induction, the bacterial liquid was centrifuged and washed twice with ultrapure water, and then centrifuged to remove the supernatant. The bacterial body was resuspended with PBS until no obvious bacterial body sedimentation, the bacterial liquid was poured into a homogenizer and broken for 3 times in succession, and the bacterial liquid was collected after centrifugation.

[0040] Enzymatic digestion and separation and purification of polypeptide PN-VW22: The pretreated bacterial supernatant sample was loaded into a balanced nickel column, and 10 mM and 30 mM imidazole solutions were used for impurity elution, and 300 mM imidazole solution was used for target protein elution and collection. Enzymatic digestion was performed at a ratio of 0.2 U enterokinase (P4237-1000U, Biyun Tian) per 0.1 mg of Flag fusion protein in a 25 mM Tris-HCl (pH 8.0) system, and the enzyme digestion conditions were 25°C for 12-14 hours. After breaking the bacterial cells, the supernatant sample, the sample after nickel column purification, and the sample after enzyme digestion were subjected to SDS-PAGE electrophoresis analysis. After the analysis was completed, the polypeptide sample after enzyme digestion was separated and purified by a self-packed C18 reverse column. The specific conditions were as follows: 0.1% TFA in 5% acetonitrile was used to balance the column, the sample after enzyme digestion was loaded into the balanced column, 3 column volumes of 0.1% TFA in 5% acetonitrile were used for desalting and impurity washing, and 2 column volumes of 0.1% TFA in 40% acetonitrile were used to elute the target protein, thereby obtaining the polypeptide PN-VW22.

[0041] Example 3 Cell viability assay of polypeptide PN-VW22.

[0042] Cell culture: The frozen HaCaT cells were taken out from the liquid nitrogen tank and placed in a preheated 37°C constant temperature water bath to quickly melt the cells. After melting, the cells were transferred from the freezing tube to a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After centrifugation, the supernatant was discarded. The cells were resuspended in DMEM culture medium containing 10% FBS and 1% double antibody, and then transferred to a culture bottle and placed in a cell culture incubator at 37°C, 5% CO2 saturated humidity. When the cell growth confluence reached 80%, the original culture medium was discarded, and the cells were washed twice with room temperature PBS. 0.25% trypsin (1 ml / 25 cm 2 ) was used to cover the cells at the bottom of the cell culture bottle, and the cells were placed in a 37°C incubator for 3-5 minutes. An appropriate amount of serum-containing culture medium was added to terminate trypsin digestion, and centrifuged at 1000 rpm for 5 min. The cells were transferred to a new culture bottle at a ratio of 1:3 and cultured at 37°C, 5% CO2 saturated humidity. After three generations, the cells were plated.

[0043] CCK8 colorimetric method for detecting HaCaT cell proliferation rate: HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody, and the culture conditions were 37°C, 5% CO2, pH 7.2-7.4, and relative humidity 95%-98%. HaCaT cells in the logarithmic growth phase were trypsinized, centrifuged at 1000 rpm for 5 min, resuspended with an appropriate amount of culture medium, and a small amount of suspension was counted. According to the counting result, the cells were diluted to 8x104 CFU / mL. Add 100 μL of cell culture to each well of a 96-well plate. After 4 hours of cell adhesion, observe the cell density under a microscope until it reaches 70%. Then add PN-VW22 and EGF. Set up four groups: blank (culture medium only), normal group (cells + culture medium), control group (cells + culture medium + EGF), and experimental group (cells + culture medium + PN-VW22). Discard the culture medium in each well (except the blank group). Add 100 μL of PN-VW22 diluted with serum-free culture medium to final concentrations of 0.125, 0.25, 0.5, 1, and 2 μM, respectively, to the experimental groups. Add 100 μL of EGF to the control group at a final concentration of 2.5 nM. After incubation for 24 hours, discard the culture medium in each well to avoid interference from the old medium. Prepare a new DMEM culture medium: CCK8 indicator solution at a ratio of 10:1, add 100 μL of the mixture to each well, and continue incubation for 2 hours. Remove the 96-well plate, incubate for 10 minutes, and measure the absorbance at 450 nm using a microplate reader. Calculate the cell proliferation rate: Cell proliferation rate = (Experimental group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × ... Figure 4 As shown, 100%.

[0044] The cytotoxic effects of PN-VW22 on HaCaT and NIH 3T3 cells were detected using the CCK8 cytotoxicity assay kit. The results showed that, compared with the control group, PN-VW22 at different concentrations had no proliferative activity against NIH 3T3 cells. Figure 3 As shown, PN-VW22 exhibited significant proliferative activity against HaCaT, with cell viability at concentrations of 0.125, 0.25, 0.5, 1, and 2 μM being (126.5±5.173), (139.6±15.21), (164.2±23.58), (162.2±3.051), and (158.9±3.969), respectively. These results indicate that PN-VW22 cell viability significantly increased with increasing concentration, demonstrating a clear concentration-dependent effect.

[0045] Example 4 Scratch repair assay of peptide PN-VW22.

[0046] Scratch assay to detect HaCaT cell migration: Cells were seeded into 12-well plates (5 × 10⁻⁶ cells / well). 5The scratch wound healing assay was performed as follows: 1 mL of DMEM medium was added to each well of a 6-well plate, and 1 mL of DMEM medium containing 1 x 105cells / well was added to each well of a 6-well plate. The cells were incubated in an incubator until they were completely adherent and the monolayer cells covered the well plate. A vertical line was drawn along the center of the well plate using a 100 μL sterile pipette tip, and the cells were washed with PBS three times to remove the detached cells. The cells were divided into the following groups: a normal group (cells + medium), a control group (cells + medium + EGF), and an experimental group (cells + medium + PN-VW22). Each group was diluted with serum-free DMEM medium, and the experimental group was diluted to a final concentration of 0.5, 1, and 2 μM, and the control group was diluted to a final concentration of 2.5 nM EGF. Each well contained 1 mL. Photographs were taken under a 10x optical microscope at 0, 6, 12, and 24 h. The scratch areas at 0, 6, 12, and 24 h were measured using image J software, and the cell migration rate was calculated as follows: cell migration rate = total area of migrated cells in the fixed scratch area / initial scratch area x 100%.

[0047] In this experiment, serum-free short-term culture (within 24 h) was used to reduce the effects of serum on cell proliferation and drug action, while also ensuring cell survival. Figure 3 As can be seen, under the incubation of 0.5 μM PN-VW22 polypeptide, the HaCaT cells did not exhibit obvious migration activity within 0-12 h, but exhibited obvious migration activity at 24 h, and the wound healing rate was 64.44% (***P < 0.001).

[0048] Example 5 Antibacterial activity determination of polypeptide PN-VW22.

[0049] Staphylococcus aureus glycerol bacteria, Escherichia coli glycerol bacteria, and Candida albicans glycerol bacteria were taken out of a -80°C refrigerator and streaked on LB plates, and incubated at 37°C overnight. Single colonies were picked and cultured in LB liquid medium at 220 rpm and 37°C until the logarithmic phase (OD600=0.6-0.8 CFU / mL). The bacterial solution was diluted to 2 x 10 5 CFU / mL, and the following groups were set up: a normal group (bacteria + medium), a control group (bacteria + medium + ampicillin / fluconazole), and an experimental group (bacteria + medium + PN-VW22). 100 μL of bacterial solution was added to each well, and PN-VW22 was added to a final concentration of 4, 8, 16, 32, 64, and 128 μg / mL. The positive control group was added with 100 μL of ampicillin (final concentration 8 μg / mL), and the Candida albicans control group was added with 100 μL of fluconazole (final concentration 0.25 μg / mL). The absorbance at 630 nm was measured after overnight incubation at 37°C.

[0050] In this experiment, the common wound infection bacteria including Staphylococcus aureus, Escherichia coli, Candida albicans were determined for antibacterial activity. As shown in Table 2, PN-VW22 had significant antibacterial effect on Staphylococcus aureus at 32 μg / mL. But had no significant antibacterial activity on Escherichia coli and Candida albicans. The antibacterial effect of PN-VW22 provides a good microenvironment for wound repair, which is conducive to wound healing.

[0051] Table 2.

[0052] Example 6 The present embodiment provides a shampoo.

[0053] Basic formula: purified water 80 parts, sodium lauryl sulfate 3 parts, sodium laureth sulfate 5 parts, sodium chloride 2 parts, cocamidopropyl betaine 4 parts, citric acid 0.5 parts, cocamide MEA 2 parts, sodium xylene sulfonate 0.2 parts, sodium benzoate 0.2 parts, glycerol 5 parts, disodium EDTA 0.5 parts, methylchloroisothiazolinone 0.3 parts, methylisothiazolinone 0.3 parts, polypeptide PN-VW22 0.2-1 parts.

[0054] Comparative Example 1: purified water 80 parts, sodium lauryl sulfate 3 parts, sodium laureth sulfate 5 parts, sodium chloride 2 parts, cocamidopropyl betaine 4 parts, citric acid 0.5 parts, cocamide MEA 2 parts, sodium xylene sulfonate 0.2 parts, sodium benzoate 0.2 parts, glycerol 5 parts, disodium EDTA 0.5 parts, methylchloroisothiazolinone 0.3 parts, methylisothiazolinone 0.3 parts.

[0055] Manufacturing process: Add water and disodium EDTA to the main pot and stir to dissolve; Slowly add sodium lauryl sulfate, sodium laureth sulfate, cocamidopropyl betaine, and cocamide MEA, emulsify and stir evenly; Add sodium xylene sulfonate, sodium benzoate, and glycerol, and stir evenly; Add fragrance, methylchloroisothiazolinone, and methylisothiazolinone, and stir evenly; Adjust the pH to weakly acidic or neutral with citric acid solution; Slowly add polypeptide PN-VW22 and stir evenly; Slowly add sodium chloride to adjust the viscosity to the appropriate range.

[0056] The shampoo prepared by the formula containing 0.2 parts of polypeptide PN-VW22 and Comparative Example 1 was used as a test sample for antibacterial effect test.

[0057] The antibacterial effect was detected by the method of inhibition zone. The culture medium was agar medium, and the test strains were Staphylococcus aureus and Malassezia furfur standard strains. The detection results are shown in Table 3.

[0058] Table 3. In vitro antibacterial test results.

[0059] As can be seen from Table 3, compared with Comparative Example 1, Example 6 has significant antibacterial efficacy.

[0060] Example 7 The present embodiment provides a shower gel.

[0061] Basic formula: 80 parts of purified water, 5 parts of sodium laureth sulfate, 3 parts of cocamidopropyl betaine, 2 parts of sodium chloride, 3 parts of sodium lauryl sulfate, 0.5 parts of sodium benzoate, 0.5 parts of citric acid, 0.5 parts of pyrrolidone carboxylic acid, and 0.2-1 parts of polypeptide PN-VW22.

[0062] Manufacturing process: Sufficient water is added to sodium laureth sulfate, sodium lauryl sulfate, and cocamidopropyl betaine, and emulsified uniformly; Sodium benzoate, citric acid, and pyrrolidone carboxylic acid are added, and stirred uniformly; PN-VW22 biological polypeptide is added, and stirred uniformly; Sodium chloride is added to adjust the viscosity.

[0063] The above-mentioned aspects not covered are applicable to the prior art.

[0064] Although some specific embodiments of the present application have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration, and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications or supplements to the described specific embodiments or replace them with similar ways, without deviating from the direction of the present application or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modification, equivalent replacement, improvement, etc. made according to the technical essence of the present application to the above embodiments should be included in the protection scope of the present application.

Claims

1. A polypeptide PN-VW22, characterized in that, The polypeptide PN-VW22 is specifically as follows (A1) or (A2): A1) The amino acid sequence is shown in SEQ ID NO.1; A2) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1).

2. The polypeptide PN-VW22 according to claim 1, characterized in that, The polypeptide PN-VW22 has cysteine ​​residues at positions 11 and 17, forming a disulfide bond.

3. The biomaterial related to the polypeptide PN-VW22 of claim 1 is any one of B1) to B6) below: B1) The nucleic acid molecule encoding the polypeptide PN-VW22; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecules described in B1); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1); B5) Recombinant microorganisms containing the expression cassette described in B2); B6) Recombinant microorganisms containing the recombinant vector described in B3).

4. The use of the biomaterial described in claim 3 in the preparation of polypeptide PN-VW22.

5. Use of the polypeptide PN-VW22 as described in claim 1 or the biomaterial as described in claim 3 in the preparation of wound healing products or antibacterial products.

6. The use according to claim 5, characterized in that, The polypeptide PN-VW22 has antibacterial activity against Staphylococcus aureus, Escherichia coli, and / or Candida albicans.

7. The use according to claim 5 or 6, characterized in that, The products include any one of the following: pharmaceuticals, daily chemical products, or food.

8. A pharmaceutical composition, characterized in that, It includes the polypeptide PN-VW22 as described in claim 1 or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The polypeptide PN-VW22 is the only effective active ingredient.

10. A daily chemical product, characterized in that, Includes the polypeptide PN-VW22 as described in claim 1.

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