Anti-acne recombinant protein vaccine, its preparation method and use

Abstract

An anti-acne recombinant protein vaccine, its preparation method, and use are provided. To solve the current problem of a lack of effective drugs for the prevention and treatment of acne disease, a recombinant protein vaccine targeting the CAMP pathogenic factor of Cutibacterium acnes is provided, and this vaccine is mainly used to induce immune responses such as antibody production in vivo to neutralize the CAMP pathogenic factor and inhibit Cutibacterium acnes-induced skin inflammation, so as to exert the effects of preventing and treating acne.

Description

[Technical Field]

[0001] The present invention belongs to the field of biomedicine, in particular to an anti-acne recombinant protein vaccine, its preparation method and its use. [Background technology]

[0002] Acne is the eighth largest epidemic in the world, affecting more than 640 million people worldwide. According to the Chinese Guideline for Diagnosis and Treatment of Acne (2019 edition), cross-sectional statistics indicate that the incidence of acne in the Chinese population is 81%, but that over 95% of people experience acne to varying degrees. Acne typically develops with hormonal changes during adolescence, affecting approximately 85% of teenagers and young adults between the ages of 12 and 25. Acne can also persist into adulthood or develop during adulthood. Acne lesions and / or scarring can persist in adults. While acne is not a life-threatening disease, the psychosocial burden caused by acne is significant and often underestimated. Recent studies have shown that acne negatively impacts the quality of life of 90% of patients. It is noteworthy that over half of acne patients experience serious emotional disorders, including anxiety, the most common symptom. Depression and obvious emotional distress may be accompanied by suicidal thoughts. Acne patients, especially those with the most severe acne, need effective and safe treatments.

[0003] Our understanding of acne pathogenesis is constantly evolving, and research has shown that acne development can be influenced by familial genetics, diet, stress, the environment, and other factors. As a chronic inflammatory disease affecting the pilosebaceous unit of the skin, key pathogenic factors involved include a complex interplay between multiple factors, such as abnormal keratinization of the pilosebaceous duct, dysregulated sebaceous gland activity, abnormal microbial colonization, imbalances in the hormonal microenvironment, and dysfunction of innate and adaptive immunity. As a globally recognized opportunistic pathogen of acne vulgaris, Cutibacterium acnes (C. acnes) has attracted significant attention from scientists. The close balance between Cutibacterium acnes and the skin microbiota plays a crucial role in the pathogenesis of acne. However, antibiotics currently used clinically for acne treatment are unable to specifically target the target microorganism. If we develop a more specific and targeted therapeutic agent or treatment regimen against Cutibacterium acnes (the causative bacterium of acne), this will provide a promising intervention candidate for the clinical treatment of acne.

[0004] Cutibacterium acnes is a relatively slow-growing, anaerobic, lipophilic, gram-positive bacterium. In healthy adult skin, Cutibacterium acnes colonizes the pilosebaceous units of the face and upper body. These bacteria reside primarily in the deeper regions of hair follicles and pores. They use sebum, cellular debris, and metabolic by-products from the surrounding skin tissue as their primary source of energy and nutrients. Under normal circumstances, Cutibacterium acnes coexists harmoniously with skin cells. However, when the sebaceous glands within hair follicles are overactive (sebaceous hyperplasia) or the hair follicles are blocked, sebum secretion increases, potentially leading to excessive growth of Cutibacterium acnes. Cutibacterium acnes can produce secreted or surface adhesins that can interact with the host, such as Christie-Atkins-Munch-Petersen (CAMP) factor with hemolytic activity, dermatan sulfate-binding adhesive (DsA) with fibrinogen-binding activity, porphyrins with pro-inflammatory activity, short-chain fatty acids, and host tissue component-degrading enzymes (lipase, sialidase, hyaluronidase, etc.). In summary, an important cause of acne is the overgrowth of Cutibacterium acnes in the skin and the production of virulence factors, proteases, adhesion molecules, etc., which trigger the host's immune-inflammatory response.

[0005] According to the Guidelines for Primary Treatment of Acne Vulgaris (2023 edition), current acne treatment methods primarily include topical medications, systemic medications, and physical and chemical therapies. Topical medications primarily consist of tretinoin and antioxidants, which can kill Cutibacterium acnes, improve keratinization of pilosebaceous ducts, and dissolve microcomedones and comedones. However, because topical medications are applied locally, it is difficult to fully treat all acne lesions. Oral medications primarily consist of antibiotics and hormonal drugs. Antibacterial and anti-inflammatory drugs selected for Cutibacterium acnes and inflammatory responses can lead to drug resistance. Tretinoin can also be administered orally, but should be limited to severe acne. An increased risk of suicide has been reported six months after isotretinoin treatment. Hormonal drugs cannot be administered orally in large amounts for a long period of time, and they can cause drug-induced acne and other adverse reactions.Physical and chemical treatments mainly include photodynamic therapy and red-blue light therapy, laser light and intense pulsed light therapy, high frequency therapy, and chemical peeling therapy.Such treatments can also cause physical or chemical damage to otherwise normal skin.Therefore, there is still a need for better treatment methods for acne.

[0006] CAMP2 is closely related to the pathogenesis of acne associated with Cutibacterium acnes. Knocking out the Cutibacterium acnes CAMP2 gene significantly reduced Cutibacterium acnes colonization and reduced local immune cell infiltration and inflammatory cytokine expression levels in lesional skin tissue in a mouse acne model. Therefore, CAMP2 is considered an important candidate target for acne treatment. Designing a vaccine against Cutibacterium acnes as a key component could overcome the limitations of existing clinical treatments, thereby reducing acne severity, suppressing the production of inflammatory factors, and reducing the likelihood of scar formation. Summary of the Invention [Problem to be solved by the invention]

[0007] The anaerobic bacterium Cutibacterium acnes naturally lives in harmony with skin cells. However, excessive sebum secretion on the face can clog hair follicles, creating an anaerobic environment in which Cutibacterium acnes overgrows and subsequently secretes the virulence factor Christie-Atkins-Munch-Peterson factor (CAMP factor).

[0008] To solve the problem of the continuing lack of effective drugs for preventing and treating acne diseases, the present invention provides a recombinant protein vaccine targeting the CAMP pathogenic factor of Cutibacterium acnes. The recombinant protein vaccine neutralizes the CAMP pathogenic factor by inducing immune responses, such as antibody production, in vivo, and inhibits skin inflammatory responses, thereby exerting the effects of preventing and treating acne.

[0009] Therefore, it is an object of the present invention to provide a protein for preventing and / or treating acne, wherein the protein is a wild-type Cutibacterium acnes CAMP protein or a variant thereof. Another object of the present invention is to provide a vaccine comprising a protein for preventing and / or treating acne induced by Cutibacterium acnes. [Means for solving the problem]

[0010] To achieve the above-mentioned objects, the present invention first provides a protein for preventing and / or treating acne, wherein the protein is a wild-type Cutibacterium acnes CAMP protein (CAMP factor 2) having the amino acid sequence set forth in SEQ ID NO: 1, or the protein is a protein variant having sequence identity with SEQ ID NO: 1 and the same or substantially equivalent biological activity as SEQ ID NO: 1.

[0011] Preferably, the protein variant has at least 70%, 80%, 90%, 95%, 97%, 98%, 99% or more sequence identity with SEQ ID NO:1 and the same or substantially equivalent biological activity as SEQ ID NO:1.

[0012] More preferably, the protein variant comprises the following mutation in SEQ ID NO: 1: (a) glycine (G), threonine (T), serine (S), and tyrosine (Y) at position 82; (b) glycine (G), serine (S), threonine (T), and tyrosine (Y) at position 153; (c) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 154; (d) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 158; (e) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 160; (f) containing at least one of glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 161;

[0013] Most preferably, the amino acid sequence of the protein variant is obtained by substituting methionine (M) at position 82 of Cutibacterium acnes CAMP factor 2 protein with glycine (G), and the amino acid sequence of the protein variant is as set forth in SEQ ID NO:3.

[0014] The nucleotide sequence encoding the Cutibacterium acnes CAMP protein variant is as shown in SEQ ID NO:4.

[0015] The present invention provides a protein precursor for preventing and / or treating acne, the protein precursor comprising a protein for preventing and / or treating acne, wherein the protein is fused to a signal peptide and / or a protein tag, preferably the protein tag is selected from at least one of the following: a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modified protein tag, a maltose-binding protein tag, a c-Myc protein tag, an Avi-tag protein tag, and a nitrogen utilization substance A protein tag.

[0016] The protein for preventing and / or treating acne is further fused to a protease recognition site for excising the protein tag. Preferably, the protease is selected from at least one of enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

[0017] The present invention further provides the use of a protein or protein precursor in the preparation of a medicament for the prevention and / or treatment of acne, or acne induced or triggered by Cutibacterium acnes. Preferably, the acne is induced by infection with Cutibacterium acnes.

[0018] The present invention further provides a recombinant protein vaccine for preventing and / or treating acne, which comprises a Cutibacterium acnes CAMP protein or protein precursor and a pharmaceutically acceptable excipient or adjuvant component.

[0019] The adjuvant component is an immunoadjuvant, preferably selected from at least one of the following: aluminum salts, calcium salts, phytosaponin, phytopolysaccharide, monophosphate lipid A (MPL), muramyl dipeptide, muramyl tripeptide, squalene-based oil-in-water emulsion (MF59), recombinant cholera toxin (rCTB), GM-CSF cytokine, lipid, cationic liposome material, CpG ODN (synthetic CpG containing a nucleotide sequence with unmethylated cytosine and guanine dinucleotides as a core sequence), or TLR3 (Toll-like receptor 3 ligand).

[0020] The aluminum salt is selected from at least one of aluminum hydroxide and alum.

[0021] The calcium salt is tricalcium phosphate.

[0022] The phytosaponin is either QS-21 or ISCOM.

[0023] The phytopolysaccharide is astragalus polysaccharide (APS).

[0024] The lipid is selected from at least one of the following: phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), and dioleoylphosphatidylethanolamine (DOPE).

[0025] Cationic liposome materials include (2,3-dioleoyloxypropyl)trimethyl ammonium chloride (DOTAP), N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylamine chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamido)ethyl ammonium trifluoroacetate (DOSPA), and trimethyldodecylammonium bromide. bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), and dimethyldioctadecylammonium bromide (DDAB).

[0026] Preferably, the immune adjuvant is selected from at least one of an aluminum hydroxide adjuvant or a WGa01 adjuvant.

[0027] The vaccines are formulated as intradermal or subcutaneous injections, intramuscular injections, intravenous injections, oral preparations, or nasal inhalation preparations.

[0028] Preferably, the vaccine is formulated as an intramuscular injection.

[0029] The present invention further provides the use of the vaccine in the preparation of a medicament for preventing and / or treating an infection induced by infection with Cutibacterium acnes.

[0030] The present invention also provides polynucleotides that encode the proteins or protein precursors.

[0031] Further, the nucleotide sequence of the polynucleotide is selected from at least one of SEQ ID NO:2 or SEQ ID NO:4.

[0032] The present invention also provides a recombinant vector comprising the polynucleotide.

[0033] Further, the recombinant vector is selected from at least one of an Escherichia coli expression vector, an insect baculovirus expression vector, a mammalian cell expression vector, and a yeast expression vector.

[0034] Preferably, the Escherichia coli expression vector is pET-30a.

[0035] Preferably, the insect baculovirus expression vector is pFastBac1.

[0036] Preferably, the mammalian cell expression vector is a CHO cell expression vector.

[0037] More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0038] The present invention provides a host cell comprising the recombinant vector.

[0039] Furthermore, the host cell is Escherichia coli.

[0040] The present invention further provides a method for preparing a protein for preventing and / or treating acne, comprising the steps of constructing a recombinant vector containing the Cutibacterium acnes CAMP protein or protein variant, transforming a host cell, expressing the protein, and purifying the protein.

[0041] Preferably, in this preparation method, the gene for Cutibacterium acnes CAMP protein or protein variant is inserted into a pET-30a(+) vector to construct an expression recombinant plasmid.

[0042] Preferably, this preparation method employs a prokaryotic expression system, more preferably an Escherichia coli expression vector system.

[0043] Preferably, in this preparation method, the inducer for inducing protein expression is isopropyl-β-D thiogalactoside, sucrose, and galactose, and more preferably, the inducer is isopropyl-β-D thiogalactoside (IPTG).

[0044] Preferably, in this preparation method, the protein of interest is purified by an ultrafiltration membrane and an ion exchange process. [Effects of the Invention]

[0045] The present invention demonstrates the beneficial effect of expressing soluble Cutibacterium acnes CAMP protein in Escherichia coli using a prokaryotic expression system. After non-reducing electrophoresis and reverse-phase high-performance liquid chromatography analysis, the purity exceeded 95%. Electrophoretic analysis showed that the CAMP protein migrated at 25.3 kDa, consistent with its theoretical molecular weight. In the present invention, a recombinant protein vaccine was prepared by co-immunization of purified CAMP protein with an adjuvant. The prepared recombinant protein vaccine exhibited high antigenicity and provided effective resistance to inflammation induced by Cutibacterium acnes. Furthermore, the recombinant protein vaccine can also be used to treat or prevent inflammation induced by Cutibacterium acnes. The vaccine effectively induces the organism to generate cellular and humoral immunity, preventing acne symptoms induced by Cutibacterium acnes. In the present invention, the preparation method of recombinant protein vaccine is simple, and the quality of recombinant protein vaccine is easy to control and large-scale production, so recombinant protein vaccine is an alternative vaccine with potential clinical application value. [Brief explanation of the drawings]

[0046] [Figure 1] 1 shows an SDS-PAGE electrophoresis diagram of a mutant Cutibacterium acnes CAMP (CAMP Mut) protein during the preparation process in embodiment 1. [Figure 2]1 shows the molecular weight profile of mutant Cutibacterium acnes CAMP (CAMP Mut) protein detected by SDS-PAGE electrophoresis in embodiment 1 (Figure A shows the molecular weight detection diagram of mutant CAMP protein, Figure B shows the purity by non-reducing SDS-PAGE electrophoresis, and Figure C shows the spectrum of mutant Cutibacterium acnes CAMP (CAMP Mut) protein obtained by reverse-phase high-performance liquid chromatography analysis). [Figure 3] 10 shows a diagram comparing the hydrophobicity and hemolytic activity of CAMP protein before and after mutation in embodiment 2. FIG. [Figure 4] FIG. 10 shows a diagram illustrating the establishment of a mouse acne model using high, medium, and low doses of Cutibacterium acnes in embodiment 5. [Figure 5] 10 shows a trend graph of binding antibody titers in a long-term immunogenicity experiment of NIH mice vaccinated with the CAMP recombinant protein vaccine of embodiment 6. [Figure 6] 10 shows a graph showing the results of binding antibody titers and HE scores in a preventive experiment of CAMP recombinant protein vaccine against infection with Cutibacterium acnes in embodiment 7. FIG. [Figure 7] 10 shows a graph of acne diameter measurements and appearance scores in a treatment experiment on CAMP recombinant protein vaccine against infection with Cutibacterium acnes in embodiment 8. [Figure 8] 1 shows the appearance and histopathology of acne lesions in a therapeutic experiment on CAMP recombinant protein vaccine against infection with Cutibacterium acnes in Example 8, as well as a chart showing the results of pathological HE scores. [Figure 9] 10 shows a diagram showing the results of prophylactic administration of wild-type CAMP recombinant protein vaccine and CAMP recombinant protein vaccine in embodiment 9. Detailed Description of the Invention

[0047] Cutibacterium acnes CAMP factor is a secreted protein and pore-forming toxin that participates in the co-hemolytic activity of acid sphingomyelinase (ASMase) from other bacteria (e.g., Staphylococcus aureus or host bacteria). Cutibacterium acnes CAMP2 factor also enhances hemolytic and cytolytic activity when acting together with Staphylococcus aureus hemolysin. CAMP2 factor exhibits potent cytotoxicity against HaCaT keratinocytes and RAW264.7 macrophages, and can induce necrosis of sebocytes in sebaceous glands and induce inflammation. Vaccination of mice with CAMP factor significantly reduces Cutibacterium acnes growth and MIP-2 production. The expression levels of Cutibacterium acnes CAMP factors and two pro-inflammatory factors (IL-8 and IL-1β) in acne lesions are higher than those in normal skin. Cutibacterium acnes CAMP factors are an important cause of acne inflammation.

[0048] In other words, CAMP factor is a cytotoxic secreted protein that acts mainly on keratinocytes and macrophages and is an important factor promoting inflammatory responses. Cutibacterium acnes CAMP factor can bind to immunoglobulin G and M and act as a pore-forming toxin. Studies have shown that CAMP factor and sphingomyelinase exert co-hemolytic activity, which can confer cytotoxicity to keratinocytes and macrophages and enhance pathogenicity through the degradation and invasion of host cells. Furthermore, Cutibacterium acnes CAMP factor can induce necrosis of sebaceous gland cells and induce inflammation. Furthermore, some studies have shown that Cutibacterium acnes specifically targets skin cells, i.e., phagocytes such as keratinocytes and macrophages, and induces the production of pro-inflammatory cytokines, including IL-8, IL-1β, IL-12, and tumor necrosis factor-α, which causes inflammation in acne vulgaris.

[0049] Therefore, the present invention provides an anti-acne recombinant protein vaccine, its preparation method and its use. The anti-acne recombinant protein vaccine is prepared by Cutibacterium acnes CAMP factor 2 protein or protein variant and a compound adjuvant.

[0050] In the present invention, a recombinant protein is expressed in Escherichia coli using a prokaryotic expression vector, and a soluble recombinant protein is obtained from the supernatant after bacterial cell lysis. According to the prokaryotic expression system and the physical and chemical properties of the recombinant protein, the supernatant is obtained after high-pressure lysis of the bacterial cells, and then subjected to ultrafiltration and both anion exchange chromatography and cation exchange chromatography to obtain the target protein. The purified CAMP protein is administered for immunization together with an adjuvant.

[0051] Specifically, the applicant synthesized the gene sequence of the Cutibacterium acnes CAMP protein or protein variant and cloned the CAMP gene into the pET-30a(+) vector. The recombinant plasmid was transformed into BL21(DE3) competent cells, and positive monoclonal colonies were picked and expanded in culture. Subsequently, the inducer isopropyl-β-D-thiogalactoside (IPTG) was added to induce target protein expression. The harvested bacterial cells were lysed under high pressure, and the target protein was isolated from the supernatant after bacterial cell lysis. After removing impurities by ultrafiltration, the target protein was subjected to anion exchange chromatography and cation exchange chromatography to obtain a highly purified target protein. The acne genetically engineered recombinant subunit vaccine was prepared by characterizing the protein, determining its content, formulating it with adjuvants, and using other preparation methods. The Cutibacterium acnes CAMP factor 2 protein (NCBI reference sequence: WP_002518322.1) was selected as the base recombinant protein for optimization. To improve the expression efficiency of the protein, the applicant removed the signal peptide region and selected the mature peptide segment (29-267) as the antigen.

[0052] In some embodiments of the present invention, polynucleotides having at least 60% sequence identity with SEQ ID NO: 2 and having the function of the polynucleotide shown in SEQ ID NO: 2 are also within the scope of protection of the present invention. Specifically, other polynucleotides having sequence identity include those obtained from the nucleotide sequence shown in SEQ ID NO: 2 by substituting, deleting, or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) nucleotides, or those having one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) nucleotides added to the N-terminus and / or C-terminus and having the function of the polynucleotide shown in SEQ ID NO: 2. Alternatively, the other polynucleotide having sequence identity may be a polynucleotide having 70%, 80%, 90%, 95%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2.

[0053] In some embodiments of the present invention, amino acids having at least 60% sequence identity with SEQ ID NO: 1 and having the function of the amino acid shown in SEQ ID NO: 1 are also within the scope of protection of the present invention. Specifically, other amino acids having sequence identity include those obtained from the amino acid sequence shown in SEQ ID NO: 1 by substituting, deleting, or adding one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3), or those having the function of the amino acid shown in SEQ ID NO: 2 to which one or more amino acids (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, 1, 2, or 3) have been added to the N-terminus and / or C-terminus. Alternatively, other amino acids having sequence identity may be amino acids having 70%, 80%, 90%, 95%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 2.

[0054] In some embodiments of the invention, the protein variant comprises the following mutation in SEQ ID NO:1: (a) glycine (G), threonine (T), serine (S), and tyrosine (Y) at position 82; (b) glycine (G), serine (S), threonine (T), and tyrosine (Y) at position 153; (c) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 154; (d) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 158; (e) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 160; (f) containing at least one of glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 161;

[0055] In the most preferred embodiment of the present invention, an artificial modification has been made to SEQ ID NO: 1, i.e., methionine at position 82 has been mutated to glycine, and a gene and vector designed based on the modified amino acid sequence shown in SEQ ID NO: 3 can achieve higher antigen expression levels and reduce the hemolytic activity of sheep red blood cells.

[0056] The amino acid sequence of positions 29 to 267 of the wild-type Cutibacterium acnes CAMP factor 2 protein is shown as SEQ ID NO:1.

[0057] SEQ ID NO: 1 MVEPTTTISATSTHELSASDARNSIQLLNAHIATLQSVQKSVPGSDYSDQIRDLLKAAFDLRGLIETLAHGGIPFYDPSTIMPRIKLVATTIDTIHTATTTLQNKVRPAHVELGLEVTKA VLLTANPASTAKELDAEGAALKARLEKVSQYPDLTPNDVATVYVRTNFSKTIWQVRANRDRYILGHKSAAVYKTLNHAITKAVGVRLNPKTTVGNIQAARTELLAAYQTAFNSPDVKKAA

[0058] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 is SEQ ID NO:2. ATGGTTGAACCGACCACCACCATCAGCGCGACCAGCACCCACGAACTGAGCGCGAGCGACGCGCGTAACAGCATCCAGCTGCTGAACGCGCACATCGCGACCCTGCAGAGCGTTCAGAAATCCGTTCCGGGCAGCGATTACTCCGACCAGATCCGTGACCTGCTGAAAGCGGCGTTCGAC CTGCGTGGTCTGATCGAAACCCTGGCGCACGGTGGCATCCGTTTCTACGACCCGTCCACCATCGGCCCGCGTATCAAACTGGTGGCGACCACCATCGATACCATCCACACCGCGACCACCACCCTGCAGAACAAAGTGCGTCCGGCGCACGTGGAACTGGGTCTGGAAGTGACCAAAGCGG TGCTGCTGACCGCGAACCCGGCGTCCACCGCGAAAGAACTGGACGCGGAAGGTGCGGCGCTGAAAGCGCGCCTGGAAAAAAGTTAGCCAGTACCCGGATCTGACCCCGAACGATGTTGCGACCGTTTACGTTCGTACCAACTTCTCTAAAACCATCTGGCAGGTTCGTGCTAACCGTGATCG TTACATCCTGGGCCACAAATCTGCGGCGGTGTACAAAACCCTGAACCACGCGATCACCAAAGCGGTTGGTGTTCGCCTGAACCCGAAAACCACCGTGGGTAACATCCAGGCGGCGCGTACCGAACTGCTGCGGCGTACCAGACCGCGTTCAACTCTCCGGATGTTAAAAAAGCGGCGTAA

[0059] The sequence of the CAMP protein (mutant protein CAMP Mut) is shown in SEQ ID NO: 3: MVEPTTTISATSTHELSASDARNSIQLLNAHIATLQSVQKSVPGSDYSDQIRDLLKAAFDLRGLIETLAHGGIPFYDPSTIGPRIKLVATTIDTIHTATTTLQNKVRPAHVELGLEVTKA VLLTANPASTAKELDAEGAALKARLEKVSQYPDLTPNDVATVYVRTNFSKTIWQVRANRDRYILGHKSAAVYKTLNHAITKAVGVRLNPKTTVGNIQAARTELLAAYQTAFNSPDVKKAA

[0060] The nucleotide sequence encoding SEQ ID NO:3 is shown in SEQ ID NO:4: ATGGTTGAACCGACCACCACCATCAGCGCGACCAGCACCCACGAACTGAGCGCGAGCGACGCGCGTAACAGCATCCAGCTGCTGAACGCGCACATCGCGACCCTGCAGAGCGTTCAGAAATCCGTTCCGGGCAGCGATTACTCCGACCAGATCCGTGACCTGCTGAAAGCGGCGTTCGAC CTGCGTGGTCTGATCGAAACCCTGGCGCACGGTGGCATCCGTTTCTACGACCCGTCCACCATCGGCCCGCGTATCAAACTGGTGGCGACCACCATCGATACCATCCACACCGCGACCACCACCCTGCAGAACAAAGTGCGTCCGGCGCACGTGGAACTGGGTCTGGAAGTGACCAAAGCGG TGCTGCTGACCGCGAACCCGGCGTCCACCGCGAAAGAACTGGACGCGGAAGGTGCGGCGCTGAAAGCGCGCCTGGAAAAAAGTTAGCCAGTACCCGGATCTGACCCCGAACGATGTTGCGACCGTTTACGTTCGTACCAACTTCTCTAAAACCATCTGGCAGGTTCGTGCTAACCGTGATCG TTACATCCTGGGCCACAAATCTGCGGCGGTGTACAAAACCCTGAACCACGCGATCACCAAAGCGGTTGGTGTTCGCCTGAACCCGAAAACCACCGTGGGTAACATCCAGGCGGCGCGTACCGAACTGCTGCGGCGTACCAGACCGCGTTCAACTCTCCGGATGTTAAAAAAGCGGCGTAA

[0061] The solution of the present invention will be described with reference to various embodiments. Those skilled in the art will understand that the following embodiments do not limit the scope of the present invention, but are intended to merely illustrate the present invention. If specific techniques or conditions are not specified in these embodiments, they should be in accordance with the techniques or conditions described in the literature or product specifications of the technical field. If the manufacturer of the reagents or equipment used is not indicated, they are all conventional products that can be obtained by commercial purchase.

[0062] Embodiment 1 Preparation of Cutibacterium acnes CAMP protein

[0063] Based on NCBI database analysis and literature search, the Cutibacterium acnes CAMP factor 2 protein (NCBI reference sequence: WP5002518322.1) was selected as the base recombinant protein for optimization. To improve protein expression efficiency, the signal peptide region was removed, and the mature peptide segment (29–267) was selected as the antigen. A methionine (M) initiation codon was added to the beginning of the mature peptide segment, and an artificial modification was made, i.e., the methionine (M) at position 82 was mutated to a glycine (G). The gene and vector designed based on the modified amino acid sequence were expected to achieve higher antigen expression levels and reduce hemolytic activity against sheep red blood cells.

[0064] The sequence of the mutant protein (CAMP protein) containing a full length of 240 amino acids is shown as SEQ ID NO:3. MVEPTTTISATSTHELSASDARNSIQLLNAHIATLQSVQKSVPGSDYSDQIRDLLKAAFDLRGLIETLAHGGIPFYDPSTIGPRIKLVATTIDTIHTATTTLQNKVRPAHVELGLEVTKA VLLTANPASTAKELDAEGAALKARLEKVSQYPDLTPNDVATVYVRTNFSKTIWQVRANRDRYILGHKSAAVYKTLNHAITKAVGVRLNPKTTVGNIQAARTELLAAYQTAFNSPDVKKAA

[0065] The corresponding nucleotide sequence is SEQ ID NO:4: ATGGTTGAACCGACCACCACCATCAGCGCGACCAGCACCCACGAACTGAGCGCGAGCGACGCGCGTAACAGCATCCAGCTGCTGAACGCGCACATCGCGACCCTGCAGAGCGTTCAGAAATCCGTTCCGGGCAGCGATTACTCCGACCAGATCCGTGACCTGCTGAAAGCGGCGTTCGACCTGCGTGGTCTGATCGAAACCCTGGCGCACGGTGGCATCCCGTTCTACGACCCGTCCACCATCGGCCCGCGTATCAAACTGGTGGCGACCACCATCGATACCATCCACACCGCGACCACCACCCTGCAGAACAAAGTGCGTCCGGCGCACGTGGAACTGGGTCTGGAAGTGACCAAAGCGGTGCTGCTGACCGCGAACCCGGCGTCCACCGCGAAAGAACTGGACGCGGAAGGTGCGGCGCTGAAAGCGCGCCTGGAAAAAGTTAGCCAGTACCCGGATCTGACCCCGAACGATGTTGCGACCGTTTACGTTCGTACCAACTTCTCTAAAACCATCTGGCAGGTTCGTGCTAACCGTGATCGTTACATCCTGGGCCACAAATCTGCGGCGGTGTACAAAACCCTGAACCACGCGATCACCAAAGCGGTTGGTGTTCGCCTGAACCCGAAAACCACCGTGGGTAACATCCAGGCGGCGCGTACCGAACTGCTGGCGGCGTACCAGACCGCGTTCAACTCTCCGGATGTTAAAAAAGCGGCGTAA

[0066] The cAMP protein gene (restriction enzyme cleavage sites: NdeI / XhoI) was cloned into the pET-30a(+) vector to construct the plasmid pET-30a-CAMP. The plasmid was verified by restriction enzyme digestion and sequenced using an automated sequencer. After successful vector construction, it was transformed into competent Escherichia coli (E. coli) BL2(DE3) cells. After culturing the positive bacteria, the inducer IPTG was added for inducible expression, and the culture was harvested by centrifugation to collect wet bacteria. The bacteria were suspended in a 1:10 volume ratio of wet bacteria to buffer (20 mM PB, pH 7.5) and subjected to ultrasonic lysis or high-pressure lysis. After cell lysis, the supernatant was collected by centrifugation or membrane filtration. The supernatant was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to 300 kDa, then purified by anion exchange chromatography, and finally by cation exchange. The molecular weight and purity of the supernatant were analyzed by SDS-polyacrylamide gel electrophoresis, and the purity was analyzed by reversed-phase high-performance liquid chromatography (R-HPLC).

[0067] The target protein was expressed and purified using an expression vector constructed by gene cloning, resulting in a CAMP protein with high stability and high expression level. Throughout the purification process, the purity of the target protein could be improved at each purification step, and the protein was relatively stable (Figure 1: M: protein molecular weight (catalog number: 26614, Thermo Scientific™); Lane 1: uninduced whole cell lysate; Lane 2: induced whole cell lysate; Lane 3: lysate supernatant; Lane 4: 300 kDa ultrafiltration permeate / filtrate; Lane 5: flow-through fraction from anion exchange chromatography; Lane 6: elution pool from cation exchange chromatography).

[0068] Reducing SDS-polyacrylamide gel electrophoresis showed that the calculated relative molecular weight of the target protein was 25,300 daltons, which essentially matched its theoretical molecular weight (Figure 2A). Figures 2B-2C show the purity of the cAMP protein detected by non-reducing SDS-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography (R-HPLC), respectively. The detection results show that the purity is greater than 95%.

[0069] The method for preparing wild-type CAMP protein (CAMP WT) is the same as the method for preparing CAMP protein, except that the amino acid sequence of the gene cloned into the pET-30a(+) vector is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 2.

[0070] Embodiment 2 Hemolytic activity of Cutibacterium acnes CAMP protein

[0071] Freshly purchased sheep blood was centrifuged at 1000 × g for 5 min at 4 °C to collect red blood cells. The sheep red blood cells were washed three times with PBS (0.01 mol / L Na3PO4, 0.015 mol / L NaCl, pH 7.0), followed by centrifugation at 1000 × g for 5 min at 4 °C and removal of the supernatant. A total of 1 mL of sheep red blood cells was washed three times with phosphate buffer (0.01 mol / L Na3PO4, 0.015 mol / L NaCl, pH 7.0, PBS), followed by centrifugation at 1000 × g for 5 min at 4 °C and removal of the supernatant. Finally, a 2% (v / v) red blood cell suspension was prepared in PBS and pretreated with neurophospholipase (neurophospholipase) at a final concentration of 50 mU / L for 30 min. Then, cAMP protein and wild-type cAMP protein were diluted to various concentrations. A total 2% sheep red blood cell suspension was mixed with an equal volume of diluted protein solution (200 μl of reaction mixture), incubated at 37°C for 1 hour, then placed at 4°C for 1 hour and centrifuged at low speed. Then, 100 μl of the supernatant was collected. The optical density at 405 nm was measured using an ELISA instrument, and the hemolysis rate was calculated. The results showed that the hemolysis rate of cAMP was reduced and statistically significantly different from that before the mutation (Figure 3B). Figure 3A shows the structural simulation diagram of cAMP protein and wild-type cAMP protein. The results show that the hydrophobicity of the N-terminal region of cAMP protein is reduced compared to that of wild-type cAMP protein. Blue represents hydrophobicity, and a larger blue area indicates stronger hydrophobicity.

[0072] Embodiment 3 Preparation of an anti-acne recombinant protein (SEQ ID NO: 3) vaccine

[0073] The concentration of the purified CAMP protein was detected by ultraviolet spectrophotometry, diluted to 0.5 mg / mL with PBS, and sterile filtered for subsequent use. Sterility testing was performed according to the current Pharmacopoeia of the People's Republic of China, and endotoxin was detected by the gel method. The protein could only be used if the endotoxin content was 10 EU / mL or less. The CAMP protein (SEQ ID NO: 3) was thoroughly mixed and adsorbed into the adjuvant.

[0074] Preparation of aluminum hydroxide adjuvanted vaccine: Phosphate buffer (10 mM PB, pH 7.0): 0.866 g of disodium hydrogen phosphate and 0.538 g of sodium dihydrogen phosphate monohydrate were dissolved in 800 mL of ultrapure water. After complete dissolution, the solution was brought to a final volume of 1 L with ultrapure water. The solution was filtered through a 0.22 μm filter membrane. Phosphate buffer was added to the aluminum hydroxide adjuvant to prepare a mixture with an aluminum content of 0.84 mg / mL, and the mixture was stirred at room temperature for 10 minutes. Antigen dilution: The protein stock solution was diluted to 500 μg / mL with phosphate buffer. The aluminum content of the prepared vaccine was 0.42 mg / mL, and the antigen content was 250 μg / mL. The mixture was brought to the final volume and stirred at room temperature for 1 hour to obtain a vaccine formulated with aluminum hydroxide adjuvant.

[0075] Preparation of WGa01-adjuvanted vaccine: The protein stock solution was diluted to 500 μg / mL with 20 mM PB and 250 mM NaCl, pH 7.5. Equal volumes of WGa01 adjuvant and antigen solution (500 μg / mL) were mixed to obtain the WGa01-adjuvanted vaccine.

[0076] Embodiment 4 Cultivation of Cutibacterium acnes

[0077] Cutibacterium acnes was purchased from BNCC and cultured according to the manufacturer's instructions. The liquid thioglycollate medium and plates were incubated in an anaerobic incubator for 24 hours to remove oxygen. Approximately 0.5 mL of the liquid medium was aspirated and transferred to a cryotube. The mixture was then completely dissolved and added to a test tube containing 10 mL of medium, followed by thorough mixing. A total of 0.2 mL of bacterial suspension was aspirated and transferred to the plate, thoroughly coating it. This procedure was repeated twice to obtain two plates. After three days, a single bacterial colony was picked from the plate and cultured anaerobically in 100 mL of liquid medium at 37°C. The colony morphology was found to be normal. Culture was continued until the broth became turbid, indicating that the bacteria had entered the logarithmic growth phase.

[0078] Embodiment 5: Establishment of a mouse acne model caused by Cutibacterium acnes

[0079] The cultured Cutibacterium acnes was transferred to a centrifuge tube and centrifuged at 3000 × g for 5 minutes. After removing the supernatant, the cells were washed twice with deoxygenated PBS and suspended in deoxygenated PBS. The suspension was prepared using live Cutibacterium acnes cells at a concentration of 1 × 10 8 cells / mL, 5*10 8 cells / mL and 1*10 9 The cells were prepared at low, medium, and high doses of cells / mL. Referring to the non-patent document Photochem Photobiol Sci. 2006 Jan; 5(1): 66-72, the OD measured at 550 nm by a spectrophotometer was 0.98 to 1.02. 8 The viable cell count of Cutibacterium acnes was calculated based on the assumption that the number of viable cells / mL was equivalent to 50 μL of the bacterial suspension. A total of 50 μL of the bacterial suspension was injected intradermally into the abdominal skin of male ICR mice. On day 7, the animals were dissected, and the skin tissue was fixed in 4% paraformaldehyde and paraffin sections were prepared.

[0080] Dewaxing paraffin sections in water: Paraffin sections were sequentially placed in xylene I for 20 minutes, xylene II for 20 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, and then 75% alcohol for 5 minutes for gradient dewaxing, followed by washing with water. Hematoxylin staining: Paraffin sections were stained with hematoxylin for 3 to 5 minutes, and excess staining solution was then washed off with tap water. Paraffin sections were fractionated in 1% hydrochloric acid solution for several seconds, rinsed with tap water, stained blue with 0.6% to 0.7% ammonia solution, and finally rinsed with running water for several seconds. Eosin staining: Paraffin sections were dehydrated in gradient alcohol (85% ethanol → 95% ethanol) and stained with eosin staining solution for 5 minutes. Dehydration and mounting: Paraffin sections were sequentially placed in absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, absolute ethanol III for 5 minutes, n-butanol for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes until clear. The paraffin sections were then removed from xylene, slightly dried, and mounted with neutral gum. Interpretation of paraffin sections: Cell nuclei are blue, and cytoplasm is red.

[0081] The results showed that after injection of high, medium, and low doses of Cutibacterium acnes, localized acanthosis, hyperkeratosis, and acanthosis were observed. The pilosebaceous units were obviously enlarged, and keratinized material was observed. The infundibulum of the hair follicle was filled with keratinized material and expanded into an ampulla-like structure. The capillaries in the upper dermis were obviously dilated and infiltrated with inflammatory cells, accompanied by subcutaneous tissue edema. An abscess developed in the dermis, and the blood vessels around the abscess were dilated and congested, and numerous inflammatory cells were infiltrated (see Figure 4).

[0082] Embodiment 6: CAMP recombinant protein vaccine can induce long-term immunity in NIH mice

[0083] Six-week-old NIH mice were selected, and the CAMP protein vaccine according to Example 3 was prepared using WGa01 adjuvant and aluminum hydroxide adjuvant separately. Each mouse received three intramuscular injections into the thigh. On days 7, 21, 35, 90, and 180 after immunization, blood was collected from the mouse orbit or retro-orbital venous plexus of the mouse eyeball without anticoagulation. The blood was left at room temperature for 1 to 2 hours and then centrifuged at 1800 × g for 10 minutes at 4°C to separate the serum. The serum was stored at -20°C for later use. The steps of the experimental procedure are shown in Figure 5. Detection of binding antibody titer: The antigen was diluted to a concentration of 1 μg / mL in coating solution (50 mm carbonate coating solution), and 100 μL of the diluted antigen was added to an ELISA plate at each well. The plate was covered with a membrane and incubated overnight at 2°C to 8°C. Plate washing: The plate was washed three times with washing solution (0.05% PBST), and the washing solution was added to the plate at 300 μL / well, and the residual solution was removed. Blocking: Blocking solution (1% BSA PBST) was added to the plate at 100 μL / well, and the plate was covered with a membrane and incubated at 37°C for 1 hour. Sample dilution: Each sample was diluted with 1% BSA according to the corresponding dilution ratio, and the diluted solution was added to the plate. Plate washing: The plate was washed once with washing solution (0.05% PBST), and the washing solution was added to the plate at 300 μL / well, and the residual solution was removed. Sample addition: Both quality control samples and test samples were added to the ELISA plate at 100 μL / well, and the plate was covered with a membrane and incubated at 37°C for 1 hour. Plate washing: The plate was washed three times with washing solution (0.05% PBST), and the washing solution was added to the plate at 300 μL / well, and the residual solution was removed. Addition of test reagent: The test reagent, goat anti-mouse IgG secondary antibody HRP, was diluted 10,000-fold with 1% BSA, and the diluted solution was added to the plate at 100 μL / well. The plate was covered with a membrane and incubated at 37°C for 1 hour. Plate washing: The plate was washed five times with washing solution (0.05% PBST), and the washing solution was added to the plate at 300 μL / well, and the remaining solution was removed. Color development: TMB substrate color development solution was added to the plate at 100 μL / well, and the plate was allowed to develop at room temperature in the dark for 5 to 10 minutes. Termination: Stop solution was added to the plate at 100 μL / well to terminate the reaction.Plate reading: The ELISA instrument was set to a detection wavelength of 450 nm (reference wavelength 630 nm) and the OD values ​​were read.

[0084] The results are shown in Figure 5A. When Al(OH)3 adjuvant was used, 21 days after the first immunization, the serum specific binding antibody levels of mice in the 1 μg, 5 μg, 10 μg, and 25 μg CAMP dose groups all increased rapidly and peaked on day 35. On days 90 and 180, the specific binding antibody levels remained high.

[0085] The results are shown in Figure 5B. When WGa01 adjuvant was used, specific binding antibodies were produced in the 1 μg, 5 μg, 10 μg, and 25 μg dose groups on day 7 after the initial immunization of mice with CAMP recombinant protein. 21 days after the initial immunization, the serum levels of specific binding antibodies in mice in each dose group rapidly increased and peaked on day 35. 90 days after the initial immunization, antibody levels were still high, although no statistically significant decline was detected. On day 180, the specific binding antibodies remained high.

[0086] Conclusion: NIH mice are sensitive to the CAMP recombinant protein vaccine, which exhibits good immunogenicity and can induce strong functional immune antibodies in mice. Intramuscular injection of the CAMP recombinant protein vaccine can induce long-term immunity in NIH mice for more than 180 days.

[0087] Embodiment 7: Preventive effect of CAMP recombinant protein vaccine against infection by Cutibacterium acnes

[0088] Six-week-old ICR mice were selected and vaccines were prepared using the WGa01 adjuvant according to embodiment 3. Each mouse received three intramuscular injections in the thigh. 14 days after the last immunization, blood was collected from the retro-orbital plexus of the mouse's orbit without anticoagulation, left at room temperature for 1-2 hours, centrifuged at 1800 × g for 10 minutes at 4 °C to separate the serum, and stored at -20 °C for later use. The method for detecting the binding antibody titer was the same as that in embodiment 6. One week after blood collection, 50 μL of bacterial suspension (5 × 10 8 Cells / mL) were intradermally injected into the abdominal skin of male ICR mice. After 7 days, the skin tissue of the mice was dissected and fixed with 4% paraformaldehyde in the same manner as in Example 5, followed by HE staining experiments.

[0089] The results are shown in Figure 6. After two intramuscular injections into the thigh of each mouse, the titers of CAMP-specific binding antibodies detected in the serum of ICR mice were relatively high in all CAMP dose groups (1 μg, 5 μg, and 25 μg) (Figure 6A). Compared with the model group, inflammation levels were significantly reduced in the low-, medium-, and high-dose groups (P<0.001), demonstrating statistical significance (Figure 6B). The CAMP recombinant protein vaccine exerts a good anti-inflammatory effect against inflammation induced by Cutibacterium acnes and can provide some degree of skin protection.

[0090] Embodiment 8: Therapeutic effect of CAMP recombinant protein vaccine on infection with Cutibacterium acnes

[0091] Six-week-old ICR mice were selected and injected with 50 μL of bacterial suspension (5 × 10 8The vaccine was prepared using aluminum hydroxide and WGa01 adjuvants, respectively, according to Example 3, and administered intramuscularly to the thigh of each mouse three times on days 0, 21, and 42. The diameter of the acne lesions was measured and an appearance score was assigned at weeks 2, 4, 6, and 8. On day 56, the mice were dissected and the mouse skin tissues were fixed with 4% paraformaldehyde in the same manner as in Example 5, followed by HE staining experiments.

[0092] The results are shown in Figure 7. At week 6, compared with the model group, the diameter of acne lesions and the appearance score were significantly reduced in the positive drug group (fusidic acid cream and tretinoin cream as the positive drug, a combination drug applied daily) and the 10 μg CAMP protein (aluminum hydroxide adjuvant group and WGa01 adjuvant vaccine group), demonstrating a statistically significant difference (P<0.05). At week 8, compared with the model group, the diameter of acne lesions and the appearance score were significantly reduced in the positive drug group, the 5 μg and 10 μg aluminum hydroxide adjuvant recombinant protein vaccine groups, and the 10 μg WGa01 adjuvant recombinant protein vaccine group, demonstrating a statistically significant difference (P<0.05) (Figure 7).

[0093] Compared with the model group, the histopathological scores of the positive drug group, the various doses of recombinant protein vaccine group with aluminum hydroxide adjuvant, and the 10 μg recombinant protein vaccine group with WGa01 adjuvant were significantly reduced, showing statistically significant differences (P<0.0001) (Figure 8).

[0094] Embodiment 9: CAMP recombinant protein vaccine and wild-type CAMP recombinant protein vaccine exert anti-inflammatory effects against infection with Cutibacterium acnes.

[0095] Six-week-old ICR mice were selected, and vaccines were prepared using WGa01 adjuvant and aluminum hydroxide adjuvant, respectively, according to Example 3. Each mouse was intramuscularly injected (10 μg / mouse) three times into the thigh. After immunization, 50 μL of bacterial suspension (5*10 8 A total of 1000 cells / mL of the antibody was intradermally injected into the abdominal skin of male ICR mice. After 7 days, the eyeballs were dissected and removed, and blood was collected. After leaving the eyeballs at room temperature for 1 to 2 hours, the blood was centrifuged at 1800 × g for 10 minutes at 4°C to separate the serum, and the method for detecting the bound antibody titer was the same as in Example 6. The mouse skin tissue was fixed with 4% paraformaldehyde in the same manner as in Example 5, and a subsequent HE staining experiment was performed.

[0096] The results are shown in Figure 9. The specific binding antibody titers of the CAMP recombinant protein vaccine and wild-type CAMP recombinant protein vaccine were high when adjuvanted with WGa01 or aluminum hydroxide (Figure 9A). Compared with the model group, the use of WGa01 adjuvant, aluminum hydroxide adjuvant, CAMP recombinant protein vaccine, and wild-type CAMP recombinant protein vaccine all significantly reduced the level of inflammation induced by Cutibacterium acnes infection (P<0.05), demonstrating a statistically significant difference (Figure 9B).

Claims

1. 1. A protein for preventing and / or treating acne, wherein the protein is a wild-type Cutibacterium acnes CAMP protein having the amino acid sequence shown in SEQ ID NO: 1, or the protein is a CAMP protein variant having sequence identity with SEQ ID NO: 1 and the same or substantially equivalent biological activity as SEQ ID NO:

1.

2. 2. The protein for preventing and / or treating acne according to claim 1, wherein the protein variant has at least 70%, 80%, 90%, 95%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 1 and the same or substantially equivalent biological activity as SEQ ID NO:

1.

3. The protein variant comprises the following mutation in SEQ ID NO: 1: (a) glycine (G), threonine (T), serine (S), and tyrosine (Y) at position 82; (b) glycine (G), serine (S), threonine (T) and tyrosine (Y) at position 153; (c) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 154; (d) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 158; (e) glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 160; and (f) Glycine (G), glutamine (Q), threonine (T), serine (S), and tyrosine (Y) at position 161 The protein for preventing and / or treating acne according to claim 1, comprising at least one of:

4. 4. The protein for preventing and / or treating acne according to claim 3, wherein the amino acid sequence of the protein variant is obtained by substituting methionine at position 82 of wild-type Cutibacterium acnes CAMP factor 2 protein with glycine, and the amino acid sequence is the amino acid sequence shown in SEQ ID NO:

3.

5. The protein for preventing and / or treating acne according to claim 4, wherein the nucleotide sequence encoding the Cutibacterium acnes CAMP protein variant is the sequence shown in SEQ ID NO:

4.

6. 6. A protein precursor comprising a protein for preventing and / or treating acne according to any one of claims 1 to 5, wherein the protein is fused to a signal peptide and / or a protein tag, preferably the protein tag is selected from at least one of the following: a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modified protein tag, a maltose binding protein tag, a c-Myc protein tag, an Avi tag, and a nitrogen utilization substance A protein tag.

7. 7. The protein precursor of claim 6, wherein the protein for preventing and / or treating acne is fused to a protease recognition site for excising the protein tag, and preferably the protease is selected from at least one of enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

8. Use of a protein according to any one of claims 1 to 5 or a protein precursor according to any one of claims 6 and 7 in the preparation of a medicament for preventing and / or treating acne or acne induced or triggered by Cutibacterium acnes.

9. 9. The use according to claim 8, wherein the acne is induced by infection with Cutibacterium acnes.

10. A recombinant protein vaccine for preventing and / or treating acne, comprising a protein according to any one of claims 1 to 5 or a protein precursor according to any one of claims 6 and 7, and a pharmaceutically acceptable excipient or adjuvant component.

11. 11. The recombinant protein vaccine of claim 10, wherein the adjuvant component is an immunoadjuvant, preferably the immunoadjuvant is selected from at least one of an aluminum salt, a calcium salt, a phytosaponin, a phytopolysaccharide, a monophosphate lipid A, a muramyl dipeptide, a muramyl tripeptide, a squalene-based oil-in-water emulsion, a recombinant cholera toxin, a GM-CSF cytokine, a lipid, a cationic liposome material, a CpG ODN, or a TLR3.

12. the recombinant protein vaccine is one of the following: the aluminum salt is selected from at least one of aluminum hydroxide and alum; the calcium salt is tricalcium phosphate; the phytosaponin is either QS-21 or ISCOM; the phytopolysaccharide is Astragalus polysaccharide; the lipid is selected from at least one of phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine; and the cationic liposome material is one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3- 12. The recombinant protein vaccine according to claim 11, wherein the immunoadjuvant is selected from at least one of: [2-(2-spermidinecarboxamido)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-spermidinecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, and dimethyldioctadecylammonium bromide; and preferably, the immunoadjuvant is selected from at least one of aluminum hydroxide adjuvant or WGa01 adjuvant.

13. The recombinant protein vaccine according to any one of claims 10 to 12, wherein the vaccine is formulated as an intradermal or subcutaneous injection, an intramuscular injection, an intravenous injection, an oral preparation or a nasal inhalation preparation, preferably the vaccine is formulated as an intramuscular injection.

14. Use of a vaccine according to any one of claims 10 to 13 in the preparation of a medicament for the prevention and / or treatment of infectious diseases induced by infection with Cutibacterium acnes.

15. A polynucleotide encoding a protein according to any one of claims 1 to 5 or a protein precursor according to any one of claims 6 and 7, wherein the nucleotide sequence of said polynucleotide is preferably selected from at least one of SEQ ID NO: 2 or SEQ ID NO:

4.

16. A recombinant vector comprising the polynucleotide of claim 15.

17. The recombinant vector according to claim 16, wherein the recombinant vector is selected from at least one of an Escherichia coli expression vector, an insect baculovirus expression vector, a mammalian cell expression vector, and a yeast expression vector, preferably the Escherichia coli expression vector is pET-30a, preferably the insect baculovirus expression vector is pFastBac1, preferably the mammalian cell expression vector is a CHO cell expression vector, more preferably the CHO cell expression vector is pTT5 or FTP-002.

18. 18. A host cell comprising the recombinant vector of claim 16 or 17, further wherein the host cell is an Escherichia coli cell.

19. A method for preparing a protein for preventing and / or treating acne, comprising the following steps: constructing a recombinant vector containing the protein of any one of claims 1 to 5 or the protein precursor of any one of claims 6 and 7, transforming a host cell, expressing the protein, and purifying the protein.

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