Production and use of propionibacterium acnes-targeting antibacterial protein

Abstract

Provided is a use of an antibacterial protein in a product for preventing and treating acne, or an infection caused by Propionibacterium acnes, or a condition related to Propionibacterium acnes, wherein the antibacterial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment or analog thereof. The active fragment or analog can inhibit the growth of Propionibacterium acnes. The provided antibacterial protein can specifically and effectively kill Propionibacterium acnes without affecting hosts and other common skin microorganisms, can provide a safe and effective acne solution, and is suitable for long-term use without causing the risks of skin microecological imbalance and microbial drug resistance.

Description

Production and application of antibacterial protein targeting propionibacterium acnes TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to production and application of antibacterial protein targeting propionibacterium acnes. BACKGROUND

[0002] Acne is a common skin disease, which is most serious in adolescents and brings great psychological burden to the adolescent population. Different degrees of acne will appear different symptoms, leading to different degrees of facial damage. The common manifestations mainly include comedones, papules, pustules, and severe cysts, nodules and scars, etc., often accompanied by sebum excretion.

[0003] Propionibacterium acnes is a common gram-positive bacterium, which is often colonized on human skin and is an important component of skin microbiome. Skin microbiome and host jointly build human skin barrier to maintain host health. Other important components of skin microbiome include Staphylococcus epidermidis, Corynebacterium xerosis and Staphylococcus capitis, etc. Propionibacterium acnes has been considered as the microorganism most closely related to acne, and a large amount of evidence has shown that it plays an important role in the pathogenesis of acne. At present, the means to resist propionibacterium acnes depends on traditional antibiotic drugs. At present, for mild and moderate acne patients, the first choice is still traditional antibiotic treatment, although it reduces the number of propionibacterium acnes to a certain extent, but at the same time affects other skin symbiotic bacteria and destroys the skin barrier. In addition, antibiotics inevitably have cytotoxicity. The problem of microbial drug resistance caused by antibiotic abuse is also serious. For severe acne patients, isotretinoin is the most commonly used retinoid drug, but its potential risk to male reproduction is controversial, and there are also effects on human liver and kidney function and potential risk of causing depression in patients, which greatly limits its use scenarios. Oral hormone drugs are another means, which can alleviate the occurrence of acne to a certain extent, but also have risks of allergic reactions and hormone secretion level disorders.

[0004] Prior art solutions:

[0005] For the treatment of acne, Eichenfield DZ et al. proposed topical treatments such as retinoids (e.g. tretinoin, adapalene), benzoyl peroxide, azelaic acid and combination therapy with topical drugs. In a randomized trial involving 207 patients, the use of 0.025% tretinoin gel treatment can reduce the acne lesion count by 63% at 12 weeks when prescribed as a monotherapy compared to the control group. This fully demonstrates the effectiveness of topical drugs, but the problem is that topical drug treatment often leads to recurrence and to some extent skin irritation and damage. For more severe acne symptoms, oral antibiotics (e.g. doxycycline or minocycline), hormone therapy (e.g. combined oral contraceptive or spironolactone) or isotretinoin are the most effective, but also mean more potential risks.

[0006] CN102482655A and CN114908077A both disclose bacteriophage lytic enzymes that can inhibit P. acnes, but neither of them has cases and data to show that the bacteriophage lytic enzymes target the elimination of P. acnes. Since the lytic enzymes disclosed in CN114908077A are derived from multiple different bacterial species bacteriophages, the lytic enzymes disclosed will not only have targeting specificity for P. acnes. According to the publicly available literature (PMID:37239874), some bacteriophage lytic enzymes derived from P. acnes bacteriophage genomes have not been shown to have targeting specificity for P. acnes. In addition, the lytic enzymes that have been disclosed are all soluble proteins in the supernatant after E. coli heterologous expression, and there is no disclosure of inclusion body renaturation to obtain bacteriophage lytic enzymes with activity and targeting specificity for P. acnes.

[0007] In summary, there is a great demand in the current medical and skin care industry for the development of new antibacterial agents that inhibit P. acnes, especially those that can specifically inhibit P. acnes without affecting normal skin flora. Currently, there is still a lack of sufficient data and cases to support bacteriophage lytic enzymes that can target the elimination of P. acnes. SUMMARY

[0008] The purpose of the present application is to provide an antibacterial agent that targets P. acnes, which can target the elimination of P. acnes and inhibit its growth without damaging the human skin microecology, thereby alleviating skin discomfort symptoms such as acne-related inflammatory reactions caused by P. acnes, regulating skin microecology and improving skin condition.

[0009] The present application finds potential antibacterial proteins from the genome of bacteriophages, which can target and inhibit various P. acnes. The bioactive antibacterial proteins can be obtained by heterologous expression, which can target and eliminate P. acnes without destroying the skin microecology on the surface of human skin, inhibit the growth of P. acnes, relieve the skin symptoms such as inflammation caused by P. acnes, regulate the skin microecology, and improve the skin condition.

[0010] The present application provides, in a first aspect, the use of an antibacterial protein in the preparation of a product for preventing, treating, or alleviating the symptoms of acne or an infection or a condition associated with P. acnes, wherein the antibacterial protein comprises an amino acid sequence as set forth in SEQ ID NO. 6, or an active fragment or an analog thereof, which can inhibit the growth of P. acnes.

[0011] In the present application, the active fragment or the analog has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 6.

[0012] As an embodiment of the present application, the active fragment or the analog has at least 85% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 6 or has a substitution amino acid sequence with the same functional group.

[0013] In some embodiments, the substitution amino acid sequence is obtained by conservative substitution of amino acids in the same amino acid group, which includes:

[0014] Aliphatic: glycine, alanine, valine, leucine, or isoleucine;

[0015] Hydroxyl or sulfur / selenium containing: serine, cysteine, threonine, or methionine;

[0016] Cyclic: proline;

[0017] Aromatic: phenylalanine, tyrosine, or tryptophan;

[0018] Basic: histidine, lysine, or arginine;

[0019] Acidic and their amides: aspartic acid, glutamic acid, asparagine, glutamine.

[0020] As an embodiment of the present application, the nucleic acid encoding the antibacterial protein comprises a sequence as set forth in SEQ ID NO. 1, or a synonymous codon sequence thereof.

[0021] As an embodiment of the present application, the product is a medicine, a food additive, a disinfectant, or a cosmetic.

[0022] As an embodiment of the present application, the product is a cosmetic.

[0023] As an embodiment of the present application, the cosmetic further comprises an ionic additive, a preservative.

[0024] In the present application, the ionic additive includes, but is not limited to, dipotassium glycyrrhizinate, disodium EDTA, sodium hyaluronate.

[0025] In the present application, the preservative includes, but is not limited to, benzoic acid or its salt.

[0026] As an embodiment of the present application, the condition associated with P. acnes includes prostatitis leading to cancer, SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome, sarcoidosis, or sciatica.

[0027] As an embodiment of the present application, the infection caused by P. acnes includes invasive infection, postoperative infection, and / or instrument-related infection.

[0028] The second aspect of the present application provides an application of an antibacterial protein or a binding domain thereof in the preparation of a tool for diagnosing or detecting P. acnes, wherein the antibacterial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment, an analog thereof.

[0029] As an embodiment of the present application, the active fragment, the analog has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 6 or has a substitution amino acid sequence with the same functional group.

[0030] As an embodiment of the present application, the active fragment, the analog can inhibit the growth of P. acnes.

[0031] In some embodiments of the present application, the substitution amino acid sequence is obtained by conservative substitution of amino acids in the same amino acid group, which includes:

[0032] Aliphatic: glycine, alanine, valine, leucine, or isoleucine;

[0033] Hydroxyl or sulfur / selenium containing: serine, cysteine, threonine, or methionine;

[0034] Cyclic: proline;

[0035] Aromatic: phenylalanine, tyrosine, or tryptophan;

[0036] Basic: Histidine, Lysine, or Arginine;

[0037] Acidic and their amides: Aspartic acid, Glutamic acid, Asparagine, Glutamine.

[0038] As an embodiment of the present application, the antibacterial protein or its binding domain is combined with a signaling molecule.

[0039] As an embodiment of the present application, the antibacterial protein or its binding domain is combined with a signaling molecule through genetic fusion or chemical coupling to form a fusion.

[0040] As an embodiment of the present application, the fusion is used to directly detect P. acnes on microscope slides by fluorescence or other means, to label P. acnes by immunohistochemistry, as a detection reagent in ELISA assays, as a detection reagent on Western blots, for attachment to magnetic beads in MACS or other pull-down assays, or as a detection reagent in assays where antibodies are the detection reagent.

[0041] In the present application, the signaling molecule includes but is not limited to a protein or chemical fluorescent dye, a protein tag, an enzyme, avidin, streptavidin, ovalbumin, biotin, a tag sensitive to click chemistry labeling, an intein, or other molecules capable of recruiting secondary proteins or molecules that generate a signal.

[0042] In the present application, the fluorescent dye includes but is not limited to GFP, RFP, mCherry, FITC, TRITC, Alexafluor 488, Cy3, or Cy5.

[0043] In the present application, the protein tag includes but is not limited to a Flag-tag, a myc-tag, a halo-tag, a his-tag, or any tag capable of binding to an antibody or other high-affinity molecule to generate a signal.

[0044] In the present application, the enzyme includes but is not limited to firefly luciferase, beta-lactamase, alkaline phosphatase, horseradish peroxidase, or an enzyme that causes a reaction such as light, color change, substrate deposition, or any other reaction that can be detected in an experiment.

[0045] The third aspect of the present application provides a method for inhibiting the growth of P. acnes, the method comprising adding an effective amount of an antibacterial protein to a system in need thereof, wherein the antibacterial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment, an analog thereof, the active fragment, the analog being capable of inhibiting the growth of P. acnes.

[0046] As an embodiment of the present application, the active fragment, analog has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO. 6 or has a substitution amino acid sequence with the same functional group.

[0047] In some embodiments of the present application, the substitution amino acid sequence is obtained by conservative substitution of amino acids in the same amino acid group, which includes:

[0048] Aliphatic: glycine, alanine, valine, leucine or isoleucine;

[0049] Hydroxyl or sulfur / selenium containing: serine, cysteine, threonine or methionine;

[0050] Cyclic: proline;

[0051] Aromatic: phenylalanine, tyrosine or tryptophan;

[0052] Basic: histidine, lysine or arginine;

[0053] Acidic and their amides: aspartic acid, glutamic acid, asparagine, glutamine.

[0054] As an embodiment of the present application, the nucleic acid encoding the antibacterial protein comprises a sequence as set forth in SEQ ID NO. 1, or a synonymous codon sequence thereof.

[0055] The fourth aspect of the present application provides a method for preventing or treating acne or an infection caused by P. acnes or a condition associated with P. acnes, the method comprising administering an antibacterial protein to a subject in need thereof, wherein the antibacterial protein comprises an amino acid sequence as set forth in SEQ ID NO. 6, or an active fragment, analog thereof. The active fragment, analog can inhibit the growth of P. acnes.

[0056] As an embodiment of the present application, the active fragment, analog has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO. 6 or has a substitution amino acid sequence with the same functional group.

[0057] In some embodiments of the present application, the substitution amino acid sequence is obtained by conservative substitution of amino acids in the same amino acid group, which includes:

[0058] Aliphatic: glycine, alanine, valine, leucine or isoleucine;

[0059] Hydroxyl or sulfur / selenium containing: serine, cysteine, threonine or methionine;

[0060] Cyclic: Proline;

[0061] Aromatic: Phenylalanine, Tyrosine or Tryptophan;

[0062] Basic: Histidine, Lysine or Arginine;

[0063] Acidic and their amides: Aspartic acid, Glutamic acid, Asparagine, Glutamine.

[0064] As an embodiment of the present application, the nucleic acid encoding the antibacterial protein comprises the sequence as shown in SEQ ID NO. 1, or a synonymous codon sequence thereof.

[0065] In the present application, the drug further comprises a pharmaceutically acceptable carrier and / or adjuvant, which refers to a substance that does not cause significant stimulation to organisms and does not affect the biological activity and properties of the administered antibacterial protein.

[0066] In the present application, the pharmaceutically acceptable carrier and / or adjuvant includes but is not limited to diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, disintegrants. The diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, water, etc. The binders include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid, alginic acid salt, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc. The surfactants include but are not limited to polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, stearic acid monoglyceride, cetyl alcohol, etc. The wetting agents include but are not limited to glycerol, starch, etc. The adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, soap clay, etc. The lubricants include but are not limited to zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauryl sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc. The fillers include but are not limited to mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupled sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc. The disintegrants include but are not limited to cross-linked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0067] In the present application, the subject includes a human or other mammal, including but not limited to a mouse, rat, guinea pig, rabbit, cat, dog, sheep, pig, cow, monkey, baboon, chimpanzee.

[0068] In the present application, the cosmetic preparation further comprises any active ingredient having a cosmetic activity. Examples of these active ingredients are emollients, humectants, free radical inhibitors, anti-inflammatory agents, vitamins, depigmenting agents, anti-acne agents, keratolytic agents, slimming agents, skin coloring agents, and sunscreens, etc., such as linoleic acid, retinol, retinoic acid, ascorbyl alkyl ester, polyunsaturated fatty acid, nicotinic acid ester, tocopheryl nicotinate, un-saponified rice, soybean or beef tallow, ceramide, hydroxy acid such as glycolic acid, selenium derivative, antioxidant, beta-carotene, gamma-orizanol, and stearoyl glycerol ester.

[0069] In the present application, the antibacterial protein can be directly administered or can be formulated into various dosage forms using known pharmaceutical preparation methods. For example, the drug can be orally administered as a sugar-coated tablet, capsule, elixir, and microcapsule, as needed, or can be non-orally administered in the form of an injection by being formulated into a sterile solution or suspension with water or any other pharmaceutically acceptable liquid. For example, the compound can be mixed with a pharmaceutically acceptable carrier or medium, including but not limited to sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., in a unit dosage form required for a generally accepted pharmaceutical administration method. Depending on the content of the active ingredient in these formulations, a suitable administration amount within a specified range can be obtained.

[0070] The administration dose of the antibacterial protein according to the present application is not limited as long as a desired therapeutic or prophylactic effect can be obtained, and can be determined depending on the symptoms, gender, age, etc. of the subject. The administration dose of the antibacterial protein according to the present application can be determined in detail using, for example, the therapeutic or prophylactic effect on a disease as an index.

[0071] In the present application, the treatment and / or prevention means to slow down, interrupt, retard, alleviate, stop, reduce, or reverse the progression or severity of an existing symptom, disorder, condition, or disease (e.g., acne). The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state.

[0072] In a fifth aspect, the present application provides a method for producing the antibacterial protein as described above, wherein the antibacterial protein comprises an amino acid sequence as set forth in SEQ ID NO. 6, or an active fragment or an analog thereof, the method comprising heterologous expression in E. coli, breaking and separating the obtained inclusion bodies, denaturing and renaturing with urea, and purifying by affinity chromatography. In some embodiments, the breaking method comprises ultrasonic breaking, high-pressure homogenization, and chemical breaking. In some embodiments, the affinity chromatography is performed using a metal ion column.

[0073] As an embodiment of the present application, the antibacterial protein comprises an amino acid sequence as set forth in SEQ ID NO. 6, or an active fragment or an analog thereof.

[0074] The present application has been simulated, experimented and verified, proving that the antibacterial protein of the present application can effectively kill and inhibit the growth of P. acnes, has good targeting property and does not affect other common microorganisms on the skin surface, and the influence of different environments on the antibacterial protein of the present application and the minimum inhibitory concentration (MIC) of the antibacterial protein of the present application on P. acnes have been tested. Compared with other lytic enzymes and antibiotics, the antibacterial protein of the present application has the characteristics of high efficiency, high specificity, no drug resistance risk, etc.

[0075] (1) High efficiency: Compared with other lytic enzymes, the antibacterial protein involved in the present application has a higher antibacterial rate and a lower minimum inhibitory concentration (MIC).

[0076] (2) Instantaneity: Compared with antibiotics and organic acids and other antibacterial substances, the antibacterial protein involved in the present application can act in a short time (usually within a few minutes), which is confirmed in the SEM electron microscope results.

[0077] (3) Universality: The antibacterial protein involved in the present application has universality to P. acnes and is effective on P. acnes strains of different sources (including drug-resistant strains).

[0078] (4) No drug resistance risk: The abuse of antibiotics leads to a very serious problem of antibiotic resistance of P. acnes. Based on the unique mechanism of action of phage lytic enzyme, it almost does not cause similar problems such as drug resistance.

[0079] (5) Targeting property: Compared with other lytic enzymes and antibiotics, the antibacterial protein involved in the present application has high specificity to specific bacterial species or strains, which means that it can accurately kill P. acnes without affecting other symbiotic bacteria on the skin.

[0080] (6) Safety: Due to the specificity of the antibacterial proteins involved in the present application, they are generally harmless to mammalian cells, have higher safety and fewer side effects.

[0081] (7) pH compatibility: The antibacterial proteins involved in the present application can tolerate different acid-base levels and can function in an environment with pH = 5-7. Since the human skin and sweat are neutral / weakly acidic, they exactly meet the interval for the stable function of the antibacterial proteins.

[0082] (8) Thermal stability: The antibacterial proteins involved in the present application can be treated at different temperatures, and after being treated at 40-60℃ for a certain period of time, they can still effectively function, showing good thermal stability. This provides great convenience for the application of antibacterial proteins in skin care product processing, greatly increasing the possibility of production and application of antibacterial proteins.

[0083] (9) No need for long-term use: Compared with antibiotic treatment which needs long-term use, the antibacterial proteins involved in the present application can only need short-term application to achieve therapeutic effect.

[0084] Since the application of antibacterial proteins on the skin surface is more targeted than the traditional antibiotic treatment method, it does not destroy the skin microecology on the skin surface and has no effect on other common skin microorganisms, which is the biggest advantage and characteristic of the application of antibacterial proteins in the field of skin care products. The present application shows the effect of antibacterial proteins on other common skin microorganisms and proves the targeting of the antibacterial proteins of the present application, which provides strong evidence for the use of antibacterial proteins to target the removal of P. acnes on the skin surface without destroying the skin microecology. BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1 is the result of sequence alignment of LysCA001 with other phage lytic enzymes.

[0086] Figure 2 is the plasmid map of the expression vector of lytic enzyme LysCA001.

[0087] Figure 3 is the result of SDS-PAGE analysis of the heterologous expression of lytic enzyme LysCA001 protein in BL21 (DE3) in Example 2.

[0088] Figure 4 is the result of SDS-PAGE analysis of inclusion body purification of lytic enzyme LysCA001 protein by affinity chromatography in Example 3.

[0089] Figure 5 is the result of antibacterial activity of phage lytic enzyme LysCA001 on P. acnes in Example 4.

[0090] Figure 6 is the result of lytic activity of lytic enzyme LysCA001 on P. acnes in a buffer system in Example 5.

[0091] Figure 7 is a scanning electron microscope (SEM) image of the lysis of P. acnes by the lytic enzyme LysCA001.

[0092] Figure 8 is a graph of the targeting assay of P. acnes by the lytic enzyme LysCA001.

[0093] Figure 9 is a bar graph of the tolerance of the lytic enzyme LysCA001 to different pH.

[0094] Figure 10 is a bar graph of the tolerance of the lytic enzyme LysCA001 to different temperatures.

[0095] Figure 11 is a bar graph of the tolerance of the lytic enzyme LysCA001 to different ion additives and preservatives.

[0096] Figure 12 is a graph of the results of the MTT cytotoxicity assay of the lytic enzyme LysCA001 on human immortalized keratinocytes.

[0097] Figure 13 is a graph of the results of the assay of the lytic enzyme LysCA001 on the inflammatory cytokines IL-6 and IL-1 β of acne mice. DETAILED DESCRIPTION

[0098] Before further describing specific embodiments of the present application, it is to be understood that the scope of the protection is not limited to the particular specific embodiments described below; it is also to be understood that the terminology used in the embodiments of the present application is for the purpose of describing the particular specific embodiments only and is not intended to limit the scope of the present application.

[0099] When the embodiments give numerical ranges, it is to be understood that, unless otherwise specified by the present application, both endpoints of each numerical range and any number falling within the range can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material similar or equivalent to those described in the embodiments of the present application can be used to implement the present application according to the knowledge of the prior art possessed by those skilled in the art and the description of the present application.

[0100] Unless otherwise specified, the experimental methods, detection methods, preparation methods not disclosed in the present application all use conventional techniques in the art.

[0101] Example 1 Identification of lytic enzyme gene targeting P. acnes and construction of expression vector

[0102] The genomic sequences of the bacteriophages targeting P. acnes were collected, and the lytic enzyme genes were identified by sequence alignment and domain alignment using BLAST and Pfam. A total of 124 potential bacteriophage lytic enzyme gene sequences were found. The identified lytic enzymes were clustered according to sequence similarity, and classified according to the set threshold (e.g., sequence similarity and sequence coverage are greater than or equal to 90%). According to the threshold of sequence similarity and sequence coverage being greater than or equal to 90%, the identified bacteriophage-derived lytic enzyme genes were classified into 5 categories, and a representative sequence was selected from each category for expression vector construction. One of the preferred representative sequences is named LysCA001, and the amino acid full-length sequence similarity of LysCA001 with the published bacteriophage lytic enzyme is less than 80%. The remaining representative sequences of the other categories are named LysCA002, LysCA003, LysCA004, and LysCA005, respectively. The selected bacteriophage lytic enzyme map is shown in FIG. 1, in which LysCA001 has an extra structure from the 150th amino acid to the 185th amino acid sequence, which is significantly different from other bacteriophage lytic enzymes. Among the 124 mined bacteriophage lytic enzyme genes, only LysCA001 has a unique sequence and structure.

[0103] The identified lytic enzyme genes were codon-optimized for E. coli, and then the corresponding genes were cloned into the pET28a(+) vector with a 6x-His tag at the C-terminus, and the genes were controlled by the lactose operon. The pET28a(+) plasmid was used as the expression vector for the identified lytic enzyme genes, and the BamHI / XhoI enzyme digestion site was used for construction. FIG. 2 is a plasmid map of the constructed lytic enzyme expression vector.

[0104] The nucleic acid sequences of the 5 categories of lytic enzymes codon-optimized for E. coli are as follows:

[0105] The nucleic acid sequence of LysCA001 is as follows:

[0106] The nucleic acid sequence of LysCA002 is as follows:

[0107] The nucleic acid sequence of LysCA003 is as follows:

[0108] The nucleic acid sequence of LysCA004 is as follows:

[0109] The nucleic acid sequence of LysCA005 is as follows:

[0110] LysCA001 amino acid sequence:

[0111] LysCA002 amino acid sequence:

[0112] LysCA003 amino acid sequence:

[0113] LysCA004 amino acid sequence:

[0114] LysCA005 amino acid sequence:

[0115] Example 2 Heterologous expression of lytic enzyme protein LysCA001

[0116] The constructed plasmid containing the lytic enzyme LysCA001 gene was transformed into BL21(DE3) competent cells, then uniformly coated on LB plates (containing 50 μg / mL kanamycin sulfate), then inverted in a 37°C incubator overnight. Single colonies were selected from the transformed plate and inoculated into 1 L of TB medium (containing 50 μg / mL kanamycin sulfate). When the culture reached OD 600 600.8, IPTG was added to the test tube culture to a final concentration of 0.1-1 mM, then placed at 16-37°C, 100 rpm, 18 h to induce expression of LysCA001 protein.

[0117] The induced culture was centrifuged at 12000 rpm for 5 min, the medium was removed, and the strain was resuspended and washed with PBS solution. Finally, SDS-PAGE loading buffer was added to heat the sample at 100°C for 30 min, and the supernatant was electrophoresed after centrifugation. 10 min before electrophoresis, 100-150 V constant voltage electrophoresis was performed, and after the bromophenol blue indicator entered the separation gel, 200 V constant voltage electrophoresis was performed until the bromophenol blue band migrated to the bottom of the gel by 1 cm. The gel was stained with Coomassie brilliant blue staining solution, then transferred to the decolorizing solution, and decolorized until the background was clear.

[0118] The whole bacteria was lysed by ultrasonic or high pressure homogenization in PBS buffer, and the Ni-IDA affinity chromatography column was equilibrated with PBS buffer for at least three times, then the target protein was eluted with equilibration buffer containing different concentrations of imidazole, and each elution fraction was collected for SDS-PAGE analysis. The SDS results are shown in Figure 3 (in the figure, M is SDS-PAGE Protein marker, 0 is the control, 1 is the low temperature induction for 16-24h, 2 is the supernatant of induced expression, and 3 is the inclusion body of induced expression). Figure 3 shows that LysCA001 can be heterologously expressed in E. coli. After breaking and separating, it is shown that the lytic enzyme LysCA001 protein is expressed in the form of inclusion body in E. coli.

[0119] Example 3 Denaturation, renaturation and purification of lytic enzyme protein LysCA001

[0120] After the inclusion body was washed with 200ml of washing solution, it was centrifuged at 12000rpm for at least 15min at 4℃, and the supernatant was discarded. The inclusion body was dissolved in about 2L of denaturation buffer, and the dissolution was complete (about 30min) under magnetic stirring at 4℃. At the same time, the Ni-IDA column was equilibrated with denaturation buffer. The denaturation buffer containing metal ions was combined with the column by using a four-dimensional mixer. After sufficient combination for at least 1h, the target protein was eluted with equilibration buffer containing different concentrations of imidazole, and each elution fraction was collected for SDS-PAGE analysis. After Ni-IDA affinity chromatography purification analysis, Lane 3-9 with higher purity was collected and added to the treated dialysis bag. The LysCA001 protein was dialyzed into buffer at 4℃ using a 5-10KD dialysis bag, and was renatured overnight at low temperature. After renaturation, the dialysate was changed, and the LysCA001 protein was dialyzed again at low temperature for 6-8h. The supernatant was collected, and 1ml was taken for SDS electrophoresis for identification. The SDS results are shown in Figure 4 (in the figure, M is SDS-PAGE Protein marker, 1 is the supernatant of whole bacteria breaking and centrifugation, 2 is the supernatant of Ni-IDA incubation effluent, and 3-9 is the elution fraction of imidazole). The protein concentration was determined by Nanodrop and Bradford method, showing that a high concentration of LysCA001 protein was finally obtained. Figure 4 shows that LysCA001 can be renatured by inclusion body and purified by affinity chromatography. A large amount of lytic enzyme LysCA001 protein was obtained by separation and purification from the precipitate.

[0121] After the dialysis renaturation was completed, the supernatant was filtered by 0.22um filter, then was divided and stored at -80℃. One of the divided LysCA001 protein stored at -80℃ was placed in an ice water mixture and allowed to thaw slowly. After thawing, there was no abnormal phenomenon, indicating that the LysCA001 protein was normal after freezing and thawing.

[0122] Example 4 Determination of antibacterial activity of lytic enzyme protein on P. acnes

[0123] P. acnes was inoculated into anaerobic BHI liquid medium, and after the bacteria were cultured to the stationary phase, the bacterial solution was diluted to 105CFU / mL in BHI medium. LysCA001 and other four lytic enzymes, i.e., LysCA002, LysCA003, LysCA004, and LysCA005, were added to a final concentration of 100 ug / mL, and the 96-well plate was placed in an enzyme marker for continuous determination of the change in absorbance. After 24 h, the change in absorbance was calculated.

[0124] Under the conditions of an enzyme concentration of 100 ug / mL, an initial inoculum of 105CFU / mL, a gas of 5% H2, 5% CO2, 90% N2, and a temperature of 37°C, LysCA001 exhibited complete inhibitory activity on P. acnes, indicating that LysCA001 has strong antibacterial performance on P. acnes. The antibacterial activity results are shown in FIG. 5. Compared with other lytic enzymes, LysCA001 exhibited stronger antibacterial activity on P. acnes.

[0125] Example 5 Determination of lytic activity of LysCA001 on P. acnes in a buffer system

[0126] P. acnes was inoculated into anaerobic BHI liquid medium, and P. acnes was cultured to OD 600 = 0.45. The bacterial solution was centrifuged at 4000 rpm for 5 min, washed twice with a buffer (50 mM Tris-HCl [pH 7.0], 100 mM NaCl), and resuspended in the buffer after washing. The bacterial solution containing the buffer was added to a 96-well plate, and LysCA001 lytic enzyme protein was added to a final concentration of 100 ug / mL, and the final volume of each well was 200 ul. The experimental data were arranged, and the lytic activity was calculated.

[0127] It was observed that the number of bacteria in the buffer added with LysCA001 lytic enzyme changed significantly, and it was observed that the curvature was maximum near 30 min, and after 60 min, the curve gradually slowed down, and OD 600 almost no longer changed. It was proved that LysCA001 could effectively lyse P. acnes in the buffer, and the lytic performance of LysCA001 on P. acnes was proved.

[0128] FIG. 6 shows that LysCA001 can efficiently kill P. acnes in a Tris-HCl buffer system.

[0129] FIG. 6 shows that LysCA001 can significantly reduce the number of P. acnes bacteria in the buffer within two hours.

[0130] The lysis rate formula: [Tested ΔOD600 (with lysis enzyme) - ΔOD600 control (buffer only)] / initial OD600.

[0131] Example 6 LysCA001 Minimum Inhibitory Concentration (MIC) Assay

[0132] The expression and purification of LysCA001 was performed according to the above method, briefly: E. coli BL21 (DE3) containing each recombinant expression plasmid was incubated in LB medium for 2-3 h, and then the OD 600 = 0.6, followed by the addition of IPTG to a final concentration of 0.1 Mm, and incubation at 16°C, 100 rpm for 18 h. The cells were collected by centrifugation, lysed by ultrasonication, and purified by affinity chromatography. The antibacterial protein obtained according to the above method was used to determine the minimum inhibitory concentration (MIC).

[0133] The minimum inhibitory concentration (MIC) was determined as follows. P. acnes was streaked on brain heart infusion plates (1.5% agar in brain heart infusion medium BHI). The plates were incubated anaerobically at 37°C for 72 h, and single colonies were picked and subcultured in liquid BHI medium for 48 h to an OD 600 = 0.6, diluted 10-fold with BHI liquid medium, at which time there were 10 7 colony forming units (CFU / ml) per ml of liquid, and further diluted 20-fold into 5 x 10 5 cfu / ml in liquid BHI medium. Each lysis enzyme was prepared in sterile PBS buffer as a 2-fold serial dilution, and added to no more than one-tenth of the total culture volume. The cultures were incubated anaerobically at 37°C for 72-96 h. The MIC was the lowest concentration of lysis enzyme at which no growth of P. acnes was observed.

[0134] Table 1 records the minimum inhibitory concentration (MIC) of LysCA001 against different strains of P. acnes. C. acnes strain 1 is the type strain numbered ATCC 6919. C. acnes strain 2 and C. acnes strain 3 were derived from a sample collection. C. acnes strain 4 is the standard strain numbered ATCC 11827. Both LysCA001 and LysCA004 have strong antibacterial effects on different strains of C. acnes. The minimum inhibitory concentration (MIC) test found that the minimum inhibitory concentration of LysCA001 was lower than that of LysCA004, indicating that LysCA001 has a stronger antibacterial effect on C. acnes strains than other phage lysis enzymes.

[0135] The MIC results of phage lysis enzymes against P. acnes are shown in Table 1:

[0136] Table 1

[0137] Example 7 LysCA001 lysing P. acnes in buffer Scanning electron microscope SEM images

[0138] P. acnes was inoculated into BHI liquid medium, and after reaching the stationary phase, sequencing verification was performed. After centrifugation at 4000 rpm for 10 min, the medium was discarded, and the P. acnes was washed three times with 50 mM Tris-HCl buffer, and then resuspended with an equal amount of buffer as the medium. Phage lysing enzyme was added to a final concentration of 100 μg / ml, and the control group was added with an equal amount of lysing enzyme preservation solution without phage lysing enzyme. The bacterial bodies of the experimental and control groups at 15 min, 30 min and 1 h were each placed in a 2 ml centrifuge tube, and after centrifugation to remove the buffer, 2% glutaraldehyde solution was used for fixation to maintain its morphology. After 6-8 h of fixation, the samples were thoroughly dried to avoid artifacts caused by water vapor when observed in SEM. A conductive coating was used to cover the surface of the sample to enhance the conductivity of the sample to obtain clear images in SEM. The sample was placed on the SEM sample holder and its position and focal length were adjusted. The high-energy electron beam of the SEM was used to scan the surface of the sample to obtain the image of the sample surface topography.

[0139] As shown in Figure 7, LysCA001 can effectively lyse the cell wall of P. acnes in the Tris-HCl buffer system, causing the cell to rupture and release the contents, losing the intact morphology of the cell. Under the same environmental conditions, the lysing efficiency of LysCA001 for P. acnes is significantly better than that of lysozyme (derived from egg white) for P. acnes in unit time. These results demonstrate the high efficiency of LysCA001 for in vitro lysis of P. acnes. It is of great significance for the application of LysCA001 in skin care products, biomedicine and medical devices, etc.

[0140] Example 8 LysCA001 Targeting Assay

[0141] The bacteriostatic targeting of LysCA001 was determined in solid medium. The tested strains were uniformly coated on BHI plates, LysCA001 was added dropwise and its position was recorded, antibiotics were used as controls, and the plates were incubated at 37°C for 3 days under anaerobic conditions, and the growth of the colonies was observed; no bacteriostatic zone was observed near it, and as a control group, an obvious bacteriostatic zone appeared near the antibiotic drop, proving that LysCA001 had no inhibitory effect on other common skin strains and protein expression chassis E. coli.

[0142] Determination of bacteriostatic targeting of LysCA001 and other lytic enzymes in liquid medium. Common skin microorganism strains were inoculated into BHI liquid medium, cultured for 24 h to the logarithmic phase, and then 20 ul of bacterial solution containing 10^6 CFU / ml was transferred into BHI medium containing 25 ug / ml LysCA001, with a final volume of 200 ul, and placed in a 96-well plate in an enzyme marker for continuous determination of the change in absorbance. A control group without LysCA001 was also set up. After 24 h, the change in light intensity was calculated. As shown in Figure 8, LysCA001 can significantly inhibit the growth of P. acnes in vitro. However, for other microorganisms, LysCA001 does not exhibit significant inhibitory activity. This indicates that LysCA001 has significant targeting ability and can target and eliminate P. acnes on the skin surface, thereby maximizing the protection of the skin microecology from being destroyed, which is of great significance for the application of LysCA001 in skin care products and biological medicine.

[0143] Example 9 Determination of pH tolerance of LysCA001

[0144] LysCA001 was placed in the same Tris-HCl buffer and the pH was adjusted with concentrated hydrochloric acid and sodium hydroxide to pH = 5, 6, 7, and 8. P. acnes was inoculated into BHI medium and cultured to the logarithmic phase and diluted to 10 5 CFU / ml, 100 ug / ml of LysCA001 with different pH values was added, and the absorbance was continuously determined in an enzyme marker to observe the growth state.

[0145] Figure 9 records the effect of different pH on the effect of LysCA001. At pH = 5-7, LysCA001 showed good bacteriostatic performance. Considering that human skin is an acidic environment, LysCA001 can inhibit the growth of P. acnes under acidic conditions, which is of great significance for subsequent application and production.

[0146] Example 10 Determination of temperature tolerance of LysCA001

[0147] LysCA001 was heated in a water bath at temperatures of 40℃, 50℃, and 60℃ for 120 min. After heating, LysCA001 was removed. P. acnes was inoculated into BHI medium and diluted to 10^5 CFU / ml, and 100 ug / ml of LysCA001 treated at different temperatures was added, and the absorbance was continuously determined in an enzyme marker to observe the growth state.

[0148] After different temperature treatments, the highest temperature is 60℃ for 120 min, LysCA001 still exhibits antibacterial activity in vitro, which proves that LysCA001 can tolerate higher temperature treatment in vitro and has good in vitro thermal stability, which is of great reference value for the application of LysCA001 in production and processing.

[0149] Figure 10 records the influence of different temperature conditions on the function of LysCA001. LysCA001 was treated at different temperatures for 2 hours, respectively, which proves that LysCA001 preserved at low temperature has better bactericidal activity.

[0150] Example 11 Determination of the tolerance of LysCA001 to different ionic additives and preservatives

[0151] After P. acnes was inoculated into BHI liquid medium and cultured to the stationary phase, sequencing verification was performed, and then the bacterial solution was diluted to 10 to the power of 5 CFU / ml in BHI medium. Different LysCA001 was added to a final concentration of 100 ug / ml and different concentrations of ionic additives or preservatives, and another group without LysCA001 was added only as a control. The change in absorbance was continuously measured in a microplate reader. After 48 h, the change in absorbance was calculated.

[0152] As shown in Figure 11, the ionic additives dipotassium glycyrrhizinate, disodium EDTA, sodium hyaluronate and the preservative sodium benzoate did not affect the inhibitory effect of LysCA001 on P. acnes, indicating that LysCA001 can coexist with common ionic agents and preservatives and maintain activity. Dipotassium glycyrrhizinate, disodium EDTA, sodium hyaluronate and other ionic additives are commonly used to adjust the pH and stability of skin care products, and have the functions of water locking and moisturizing, and are the most important additives in skin care products. Benzoic acid and its salts are commonly used broad-spectrum antimicrobial agents in skin care products, which have good effects on yeast, mold and some bacteria. These results have important reference value for the application of LysCA001 in skin care products.

[0153] Example 12 MTT cytotoxicity assay of LysCA001 on human immortalized keratinocytes

[0154] HaCat cells were cultured in T75 culture bottles to 80% confluence, and 2 mL of 0.25% TE enzyme was used for incubation and digestion at 37°C for 4 min to make the cells suspended. The culture medium was removed at 900 rpm for 5 min, and the cells were collected. Resuspend with 10% FBS DMEM medium to make the cell concentration to 3*10 5 / mL. 96-well plates were inoculated with 100 ul of resuspended cells per well, and the cells were allowed to grow for 18-24 hours. The lysin stock solution was diluted using DMEM base medium to prepare a test medium containing 20% lysin stock solution, and 8 concentrations were prepared by 2-fold gradient dilution. The original medium in each well of the 96-well plate was removed to remove suspended cells, and the residual FBS was removed by rinsing with 1X PBS. 100 ul of test medium at different concentrations was added to each well, and 3 wells were prepared in parallel for each concentration. Incubation was performed for 24 hours. The medium was removed to remove floating dead cells, and PBS was used for rinsing once. 100 uL of cell lysis solution was added to each well, and incubation was performed at 37 degrees for 30 minutes. The lysate supernatant was collected by centrifugation at 9000 rpm for 5 minutes, and transferred to a new well plate. MTT reagent was prepared and added to the wells, and incubation was performed for 30 minutes. OD 570 The results are shown in FIG. 12. It was shown that LysCA001 did not show cytotoxicity to human immortalized keratinocytes at 10 times the effective dose, indicating that LysCA001 has good safety for human cells.

[0155] Example 13 LysCA001 binding domain combined with fluorescent protein for detection of P. acnes

[0156] According to the binding domain of LysCA001 and the gene sequence of GFP (green fluorescent protein), primers were designed to amplify the two genes. Using the designed primers, the genes of the binding domain of LysCA001 and GFP were amplified by PCR. The two fragments obtained by PCR amplification were recombined to generate a gene fusion fragment containing the binding domain of LysCA001 and the fluorescent protein. The gene fragment obtained by recombination was ligated by restriction enzyme, and the complete fusion gene was constructed. The constructed fusion gene was inserted into the expression vector, and the IPTG-induced low-temperature expression of the fusion protein was performed in the host cell E. coli containing the fusion gene. Then the purified fusion protein was obtained by affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was used to test the specific binding ability of the fusion protein of the phage lysin binding domain and the fluorescent protein to P. acnes. The fusion protein labeled with fluorescent protein was treated with P. acnes, and then the sample was observed using a microscope to observe the intensity and position of the fluorescent signal to confirm that the fusion protein specifically binds to P. acnes.

[0157] Example 14 Determination of LysCA001 gel in reducing inflammatory cytokines in a mouse model of acne

[0158] SPF grade C57BL / 6J male mice, 12. Normal feed, conventional water, no limit to the amount of food and water, 12h / 12h light rhythm, adaptive feeding for a week. Animal modeling, skin preparation (with tape to peel off 10 times) subcutaneous injection of Propionibacterium acnes, skin preparation with oil smearing, for three consecutive days; intervention treatment, C57BL / 6J with LysCA001 gel to C (treatment group) smearing skin preparation, twice a day, for a week. Record sampling, C57BL / 6J after intervention treatment, after blood sampling (ophthalmic blood sampling) decapitated animals. Skin preparation tissue (aluminum foil flat fixed) placed in 4% paraformaldehyde for fixation, serum placed in -80°C refrigerator for storage. Whole blood samples were placed at room temperature for 1 hour or overnight at 2-8°C, then centrifuged at 2-8°C, 1000xg for 20 minutes, and the supernatant was detected. Centrifugation for 15 minutes, the supernatant can be detected. The tissue was rinsed with pre-cooled PBS (0.01M, pH = 7.4) to remove residual blood, weighed, and then cut into small pieces. The cut tissue was added to a glass homogenizer with the corresponding volume of PBS and ground thoroughly on ice. To further lyse the tissue cells. Finally, the homogenate was centrifuged at 2-8°C, 5000xg for 5-10 minutes, and the supernatant was detected. Adherent cells were gently washed with cold PBS, then trypsin-digested, centrifuged at 1000xg for 5 minutes, and the cells were collected. The collected cells were washed with cold PBS 3 times. Add 150-200μL PBS per 10 6 cells to resuspend the extract, centrifuge at 2-8°C, 1500xg for 10 minutes, and the supernatant was detected. After collecting the liquid, centrifuge at 2-8°C, 1000xg for 20 minutes, remove impurities and cell debris. The supernatant was detected. The results are shown in Figure 13. It is shown that LysCA001 can significantly reduce the inflammatory cytokines IL-6 and IL-1β in acne mice at an effective dose concentration.

[0159] The above description of the embodiments is to facilitate those of ordinary skill in the art to understand and use the application. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. Use of an antibacterial protein in a product for the prevention, treatment of acne or an infection caused by Propionibacterium acnes or a condition associated with Propionibacterium acnes, wherein, The antibacterial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment or an analogue thereof, the active fragment or the analogue inhibiting growth of P. acnes, preferably, the active fragment or the analogue has at least 85% sequence identity with the amino acid sequence as shown in SEQ ID NO. 6 or has a substitution amino acid sequence with the same functional group.

2. The use according to claim 1, wherein, The substitution amino acid sequence is obtained by conservative substitution of amino acids in the same amino acid group, the amino acid group comprising: aliphatic: glycine, alanine, valine, leucine or isoleucine; hydroxyl or sulfur / selenium containing: serine, cysteine, threonine or methionine; cyclic: proline; aromatic: phenylalanine, tyrosine or tryptophan; basic: histidine, lysine or arginine; acidic and their amides: aspartic acid, glutamic acid, asparagine, glutamine.

3. The use according to claim 1, wherein, The nucleic acid encoding the antibacterial protein comprises a sequence as shown in SEQ ID NO. 1, or a synonymous codon sequence thereof.

4. The use according to claim 1, wherein, The product comprises a medicine, a food additive, a disinfectant, a cosmetic or a medical device, Preferably, the product is a cosmetic, Preferably, the cosmetic further comprises an ionic additive, a preservative, Preferably, the ionic additive comprises one or more of dipotassium glycyrrhizinate, disodium EDTA, sodium hyaluronate, Preferably, the preservative comprises benzoic acid or a salt thereof.

5. The use according to any one of claims 1 to 4, wherein, The condition related to P. acnes comprises prostatitis leading to cancer, SAPHO (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome, sarcoidosis, or sciatica, the infection caused by P. acnes comprising invasive infection, postoperative infection and / or instrument-related infection.

6. Use of an antibacterial protein or a binding domain thereof in the preparation of a tool for the diagnosis or detection of P. acnes, wherein, The antibacterial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment or an analogue thereof, preferably, the active fragment or the analogue has at least 85% sequence identity with the amino acid sequence as shown in SEQ ID NO. 6 or has a substitution amino acid sequence with the same functional group, Preferably, the antibacterial protein or the binding domain thereof is used in combination with a signal molecule, preferably, the antibacterial protein or the binding domain thereof is combined with the signal molecule to form a fusion by gene fusion or chemical coupling; Preferably, the substitution amino acid sequence is obtained by conservative substitution of amino acids in the same amino acid group, the amino acid group comprising: aliphatic: glycine, alanine, valine, leucine or isoleucine; hydroxyl or sulfur / selenium containing: serine, cysteine, threonine or methionine; cyclic: proline; aromatic: phenylalanine, tyrosine or tryptophan; basic: histidine, lysine or arginine; acidic and their amides: aspartic acid, glutamic acid, asparagine, glutamine. The nucleic acid encoding the antibacterial protein comprises a sequence as shown in SEQ ID NO. 1, or a synonymous codon sequence thereof.

7. Use according to claim 6, wherein, The fusion is used to directly detect P. acnes on microscope slides by fluorescence or other means, to label P. acnes by immunohistochemistry, as a detection reagent in ELISA assays, as a detection reagent on Western blots, for attachment to magnetic beads in MACS or other pull-down assays, or as a detection reagent in assays where the antibody is the detection reagent.

8. Use according to claim 6 or 7, wherein, The signal molecule comprises a protein or chemical fluorescent dye, a protein tag, an enzyme, avidin, streptavidin, ovalbumin, biotin, a tag susceptible to labeling by click chemistry, an intein, or other molecule capable of recruiting secondary proteins or molecules that generate a signal, Preferably, the fluorescent dye comprises GFP, RFP, mCherry, FITC, TRITC, Alexafluor 488, Cy3, or Cy5. Preferably, the protein tag comprises a Flag-tag, a myc-tag, a halo-tag, a his-tag, or any tag capable of binding to an antibody or other high affinity molecule to generate a signal. Preferably, the enzyme comprises firefly luciferase, beta-lactamase, alkaline phosphatase, horseradish peroxidase, or an enzyme that causes a reaction such as light, color change, substrate deposition, or any other reaction that can be detected in an experiment.

9. A method of inhibiting the growth of P. acnes comprising adding to a system in need thereof an effective amount of an antibacterial protein, wherein, The antibacterial protein comprises an amino acid sequence as shown in SEQ ID NO. 6, or an active fragment, analog thereof; preferably, the active fragment, analog can inhibit the growth of P. acnes, preferably, the active fragment, analog has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO. 6 or has a substitution amino acid sequence with the same functional group.

10. A method for producing the antibacterial protein as claimed in any one of claims 1-9, the method comprising denaturation and renaturation of the inclusion bodies obtained after heterologous expression in E. coli by using urea, and purification by affinity chromatography; the method of breaking comprises ultrasonic breaking, high pressure homogenization and chemical breaking; the method of affinity chromatography is affinity chromatography by using a metal ion column of His-tag.

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