An antibacterial fusion protein and a preparation method and application thereof

Abstract

The application discloses an antibacterial fusion protein and a preparation method and application thereof. The antibacterial fusion protein has an amino acid sequence with at least 90% homology with the amino acid sequence shown in SEQ ID NO. 1 and the same function, or an amino acid sequence obtained by modifying, substituting, deleting or adding one or more than one amino acid of the amino acid sequence and having the same function. The antibacterial fusion protein has the advantages of low toxicity, good biocompatibility, difficulty in inducing drug resistance, high antibacterial activity, high production efficiency, low cost and the like.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial fusion protein technology, and in particular relates to an antimicrobial fusion protein, its preparation method and application. Background Technology

[0002] Fungi, such as Malassezia furfur ( Malassezia Malassezia furfur (spp.) plays an important role in common scalp and skin conditions such as dandruff and seborrheic dermatitis. Malassezia furfur is a type of lipophilic yeast and a resident flora of the human body, commonly found in areas with high sebum secretion. Its excessive proliferation disrupts the skin's ecological balance, leading to scalp barrier damage and inflammatory responses. In its natural state, Malassezia furfur forms biofilms, exhibiting high resistance to antibiotics or antibacterial substances. These fungi break down triglycerides in sebum to produce free fatty acids, stimulating excessive proliferation of scalp keratinocytes and inflammatory responses, thus causing dandruff and dermatitis symptoms.

[0003] Currently, antifungal drugs mainly include azole antifungals, zinc pyrithione, and selenium sulfide. However, long-term use of chemical drugs has problems such as drug resistance, high recurrence rates, potential toxicity, and limited therapeutic effects. Therefore, there is an urgent need to develop a new antibacterial product. Summary of the Invention

[0004] To address at least some of the technical problems in the prior art, the present invention provides an antimicrobial fusion protein, its preparation method, and its applications. Specifically, the present invention includes the following:

[0005] In a first aspect, the present invention provides an antimicrobial fusion protein having the amino acid sequence shown in (I) or (II): (I) An amino acid sequence that has at least 90% homology with and has the same function as the amino acid sequence shown in SEQ ID NO.1; (II) An amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0006] In some embodiments, the antimicrobial fusion protein according to the present invention has the amino acid sequence shown in SEQ ID NO.1.

[0007] A second aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antimicrobial fusion protein according to a first aspect of the invention.

[0008] A third aspect of the invention provides a carrier molecule comprising the nucleic acid molecule described in the second aspect of the invention.

[0009] A fourth aspect of the invention provides a host cell comprising a nucleic acid molecule according to a second aspect of the invention or a carrier molecule according to a third aspect of the invention.

[0010] A fifth aspect of the present invention provides a method for preparing an antimicrobial fusion protein according to a first aspect of the present invention, wherein the antimicrobial fusion protein is prepared by artificial synthesis or genetic engineering.

[0011] A sixth aspect of the invention provides an antimicrobial composition comprising the antimicrobial fusion protein according to the first aspect of the invention and optional excipients.

[0012] A seventh aspect of the invention provides the use of the antimicrobial fusion protein according to the first aspect of the invention or the antimicrobial composition according to the sixth aspect of the invention in the preparation of antimicrobial products.

[0013] In some embodiments, according to the application described in the present invention, the antimicrobial product includes a drug, a shampoo and conditioner, or a skin care product.

[0014] An eighth aspect of the invention provides a method for inhibiting fungi, comprising the step of using an antimicrobial fusion protein according to a first aspect of the invention.

[0015] In gene-engineered expression systems, due to the characteristics of exogenous genes, protease degradation, protein folding errors, and incorrect protein modifications, the activity of exogenous proteins is often reduced, resulting in their inability to be expressed or secreted. This invention, through extensive optimization, achieves normal secretory expression of antibacterial fusion proteins in gene-engineered expression systems. Furthermore, it possesses advantages such as low toxicity, good biocompatibility, low likelihood of inducing drug resistance, environmental friendliness, high antibacterial activity, high production efficiency, and low cost. It can be used to prepare drugs or daily chemical products for the treatment or prevention of dandruff, seborrheic dermatitis, and related diseases caused by fungi including Malassezia furfur. Attached Figure Description

[0016] Figure 1 This study investigated the heterologous expression and activity assays of antimicrobial peptides in Pichia pastoris X33. A represents the expression results of antimicrobial peptide Satanin1 in Pichia pastoris, where M is the protein molecular weight standard, and 1, 2, 3, and 4 represent different clones. B represents the expression results of antimicrobial peptide hIL11-Satanin1 in Pichia pastoris, where M is the protein molecular weight standard, and 1, 2, 3, and 4 represent different clones. C represents the expression results of antimicrobial peptide Satanin1-hIL11 in Pichia pastoris, where M is the protein molecular weight standard, and 1, 2, 3, and 4 represent different clones. D represents the inhibitory effect of antimicrobial peptide hIL11-Satanin1 and its supernatant on Malassezia furfur after 48 h of treatment.

[0017] Figure 2 To optimize the expression conditions of the antimicrobial peptide Satanin1-hIL11 in Pichia pastoris X33, the following images are shown: A: SDS-PAGE electrophoresis of expression induced by gradient temperature (M: protein molecular weight standard, 1: 20℃, 2: 25℃, 3: 28℃, 5: 30℃); B: SDS-PAGE electrophoresis of expression induced by different concentrations of methanol for 24 h (M: protein molecular weight standard, 1: control group, 2: 0.5% methanol, 3: 1% methanol, 4: 1.5% methanol, 5: 2% methanol, 6: 2.5% methanol, 7: 3% methanol); C: SDS-PAGE electrophoresis of expression induced by different concentrations of methanol for 48 h (same as B); D: SDS-PAGE electrophoresis of expression induced by different concentrations of methanol for 72 h (same as B); E: SDS-PAGE electrophoresis of expression induced by different concentrations of methanol for 96 h (same as B); F: SDS-PAGE of expression induced by different concentrations of methanol for 120 h (same as B). SDS-PAGE electrophoresis images after h, numbered the same as Figure B.

[0018] Figure 3 To optimize the expression system of the antimicrobial peptide Satanin1-hIL11 in Pichia pastoris X33, the following diagrams are presented: A: SDS-PAGE electrophoresis of high-copy strains expressing the antimicrobial peptide Satanin1-hIL11, screened by LiquidPTVA method; M: protein molecular weight standard, 1: 100 µg / mL bleomycin, 2 and 3: 2000 µg / mL bleomycin, 4 and 5: 3000 µg / mL bleomycin, 6 and 7: 5000 µg / mL bleomycin; B: schematic diagram of single, double, triple, and quadruple copies of the expression element; C: SDS-PAGE electrophoresis of recombinant plasmids containing different copy numbers of expression elements expressed in Pichia pastoris; M: protein molecular weight standard, 1: single copy, 2: double copy, 3: triple copy, 4: quadruple copy; D: quantitative diagram of the protein bands in diagram A; E: quantitative diagram of the protein bands in diagram C.

[0019] Figure 4 The study investigated the inhibitory effect of the antimicrobial peptide Satanin1-hIL11 on Malassezia furfur. A represents the determination of the minimum inhibitory concentration (MIC) of Satanin1-hIL11 against Malassezia furfur; B represents the inhibition of biofilm formation by Satanin1-hIL11 against Malassezia furfur; and C represents the inhibitory effect of different concentrations of Satanin1-hIL11 on Malassezia furfur. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0023] Antimicrobial fusion protein In one aspect, the present invention provides an antimicrobial fusion protein (sometimes also referred to as an "antimicrobial peptide") having the amino acid sequence shown in (I) or (II): (I) An amino acid sequence that has at least 90% homology with and has the same function as the amino acid sequence shown in SEQ ID NO.1; (II) An amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0024] In a preferred embodiment, the sequence of the antimicrobial fusion protein of the present invention is shown in SEQ ID NO.1.

[0025] The "antibacterial" properties of this invention are the inhibition of fungal growth or activity, inhibition of fungal proliferation, or direct killing of fungi, wherein the fungus is preferably Malassezia furfur.

[0026] In this document, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0027] In this document, protein variant sequences obtained by modification, substitution, deletion, or addition of one or more amino acids are also within the scope of protection of this invention. The term "modification" refers to covalent chemical modification of the side chains of amino acid residues in the sequence (e.g., glycosylation, polyethylene glycolation, acetylation, etc.). The term "substitution" refers to replacing one or more residues in the original sequence with one or more different amino acid residues. The term "deletion" refers to removing one or more amino acid residues from the original sequence.

[0028] In this invention, conservative amino acid substitutions are preferred, and these conservative amino acids are preferably selected from the amino acids in Table 1.

[0029] Table 1 In this invention, the method for detecting the antibacterial effect is not particularly limited, and methods known in the art can be used, such as the Oxford cup method, filter paper disc method, inhibition zone method, minimum inhibitory concentration determination, time-sterilization curve method, etc.

[0030] In gene-engineered expression systems, the activity, expression, or secretion of the antimicrobial peptide Satanin1 can be reduced due to factors such as the characteristics of the exogenous gene, protease degradation, protein folding errors, and incorrect protein modifications. To achieve normal secretory expression of Satanin1 in gene-engineered expression systems, this invention attempts to link different proteins with Satanin1. Extensive screening experiments revealed that linking hIL11 to Satanin1 significantly increases the expression level of Satanin1 in gene-engineered expression systems. Specifically, while linking hIL11 to the N-terminus of Satanin1 results in high expression levels, the antimicrobial activity is poor. However, linking hIL11 to the C-terminus of Satanin1 not only significantly increases the expression level but also maintains excellent antimicrobial activity.

[0031] Based on this, the present invention also provides a method for improving the expression level and antibacterial activity of the antimicrobial peptide Satanin1 in Pichia pastoris, which includes the step of linking hIL11 to the C-terminus of the antimicrobial peptide Satanin1.

[0032] In a preferred embodiment, the method for increasing the expression level and antimicrobial activity of the antimicrobial peptide Satanin1 in Pichia pastoris includes: (1) Construct a recombinant vector containing the genes hIL11 and the antimicrobial peptide Satanin1; (2) The vector is transformed into host cells and cultured under conditions suitable for protein expression to obtain the fusion protein; (3) Collect fusion proteins.

[0033] Nucleic acid molecules In one aspect, a nucleic acid molecule is provided that comprises a nucleotide sequence encoding the antimicrobial fusion protein described in this invention.

[0034] As used in this invention, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0035] The nucleic acid molecule of this invention comprises a coding sequence obtained through codon optimization. A codon is a group of three adjacent nucleotides in a messenger RNA molecule that represents a specific amino acid during protein synthesis. "Codon optimization" refers to altering the codon composition of recombinant nucleic acids without changing the amino acid sequence.

[0036] In a preferred embodiment, the nucleotide sequence has the sequence shown in SEQ ID NO.2.

[0037] Once the coding sequence of the antimicrobial fusion protein described in this invention is obtained, recombinant technology can be used to obtain the antimicrobial fusion protein in large quantities. An exemplary method is to clone its coding gene into a vector, then transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0038] carrier molecules In one aspect, the present invention provides a carrier molecule comprising the nucleic acid molecule described herein.

[0039] The term "vector" in this invention refers to an artificial construct that can introduce a foreign gene or nucleic acid sequence into a host cell and guide its replication and / or expression. The vector of this invention is not limited and can be a cloning vector, expression vector, etc. In some embodiments, the vector contains a target gene encoding the antimicrobial fusion protein of this invention, a promoter, a terminator, or optionally, a marker gene. The vector can be a known vector or a self-constructed vector. Known vectors include phage vectors, plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0040] host cells In one aspect, the present invention provides a host cell comprising the nucleic acid molecule or the carrier molecule described in the present invention.

[0041] The host cell of this invention refers to any cell type suitable for transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector containing the nucleic acid molecules of this invention. Host cells may include bacterial, fungal, plant, or animal cells, wherein examples of bacterial host cells include, but are not limited to, *Escherichia coli*. Escherichia coli ),salmonella( Salmonella Bacillus subtilis ( Bacillus subtilis ), pneumococcus ( Pneumococcus Streptococcus ( Streptococcus Haemophilus influenzae ( ) Haemophilus influenzae Examples of fungal host cells include, but are not limited to, Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Pichia pastoris () Pichia pastoris Examples of plant host cells include, but are not limited to: tobacco Benzodiaceae cells, Arabidopsis thaliana cells, rice suspension cells, etc.; examples of animal host cells include, but are not limited to: CHO (Chinese hamster ovary cell line), NSO cells, etc.

[0042] Preparation method In one aspect, the present invention provides a method for preparing the antimicrobial fusion protein described herein. The preparation method is not particularly limited, and includes preparation via artificial synthesis or genetic engineering.

[0043] In some embodiments, the antimicrobial fusion protein of the present invention is obtained by artificial synthesis. Methods for artificially synthesizing antimicrobial fusion proteins are known in the art, for example, the antimicrobial fusion protein of the present invention is obtained by direct amino acid synthesis.

[0044] In some embodiments, the antimicrobial fusion protein of the present invention is obtained through genetic engineering expression. Genetic engineering expression systems for this purpose include, but are not limited to, prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include, but are not limited to, *E. coli* expression systems. Eukaryotic cell expression systems include, but are not limited to, enzyme expression systems, insect cell expression systems, and mammalian cell expression systems.

[0045] In a preferred embodiment, the antimicrobial fusion protein of the present invention can be prepared by the following steps: (1) Construct a recombinant vector containing an antimicrobial fusion protein expressing the amino acid sequence shown in SEQ ID NO.1; (2) The vector is transformed into host cells and cultured under conditions suitable for the expression of the antimicrobial fusion protein; (3) Collect antimicrobial fusion proteins.

[0046] Antibacterial Composition In one aspect, the present invention provides an antimicrobial composition comprising the antimicrobial fusion protein according to the present invention and optional excipients.

[0047] In a preferred embodiment, the antimicrobial composition is a pharmaceutical composition comprising pharmaceutically acceptable excipients. Each excipient must be "acceptable," meaning it is compatible with other components of the formulation (e.g., antimicrobial fusion proteins) and does not harm the patient. Pharmaceutically acceptable excipients include, but are not limited to, buffers, emulsifiers, colorants, diluents, fillers, wetting agents, binders, lubricants, sweeteners, and antioxidants. Examples of buffers include, but are not limited to, citrate, histidine, and succinate. Examples of emulsifiers include, but are not limited to, polysorbate. Examples of colorants include, but are not limited to, sodium copper chlorophyllin, betaine, curcumin, β-carotene, and anthocyanins. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media. Fillers include, but are not limited to, sucrose, trehalose, and xylitol. Wetting agents include, but are not limited to, water. Binders include, but are not limited to, hydroxypropyl methylcellulose and povidone. Lubricants include, but are not limited to, magnesium stearate and micronized silica. Sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, and aspartame. Antioxidants include, but are not limited to, ascorbic acid, sodium ascorbate, and tea polyphenols.

[0048] In a preferred embodiment, the antibacterial composition of the present invention is a cosmetic composition comprising a cosmetically acceptable carrier or excipient. "Cosmetically acceptable" means that the carrier or excipient is non-toxic, non-irritating, and non-allergenic to the human body, meets the requirements of cosmetic hygiene standards or regulations, is safe for the human body under reasonably foreseeable use, and possesses chemical stability and physical compatibility with the active ingredient (e.g., the antibacterial fusion protein described in this invention) and other components. This means that they will not undergo adverse chemical reactions that lead to the deactivation of the active ingredient, nor will they cause system stratification, precipitation, or spoilage.

[0049] In this invention, the carriers or excipients acceptable in cosmetics are not particularly limited, and examples include, but are not limited to, solvents, thickeners, emollients, humectants, and pH adjusters. Solvents include, but are not limited to, deionized water and purified water; thickeners include, but are not limited to, carbomer, hydroxyethyl cellulose, acrylate copolymers, and magnesium aluminum silicate; emollients include, but are not limited to, caprylic / capric triglycerides, squalane, jojoba oil, polydimethylsiloxane, and cyclopentasiloxane; humectants include, but are not limited to, sodium hyaluronate, sodium PCA, trehalose, and urea; pH adjusters include, but are not limited to, citric acid and sodium citrate; and antioxidants include, but are not limited to, ascorbic acid, sodium ascorbate, and tea polyphenols.

[0050] application One aspect of the present invention provides the use of the antimicrobial fusion protein or antimicrobial composition according to the present invention in the preparation of antimicrobial products.

[0051] In this invention, examples of antibacterial products include, but are not limited to, drugs, hair care products (such as, but not limited to, shampoos, hair lotions, hair gels, conditioners, etc.) or skin care products (such as facial cleansers, skin lotions, day creams, night creams, sunscreens, isolation creams, etc.).

[0052] Methods to inhibit fungi One aspect of the present invention provides a method for inhibiting fungi, comprising the step of using the antimicrobial fusion protein described herein. In some embodiments, the method for inhibiting fungi may be a therapeutic method, i.e., for therapeutic purposes. In other embodiments, the method is a non-therapeutic method, such as for hair care, skin care, equipment and environmental disinfection, experimental research, etc.

[0053] Example 1 This embodiment demonstrates the heterologous expression and activity determination of antimicrobial peptides in Pichia pastoris.

[0054] 1. Construction and expression of recombinant expression plasmid pPICZαA-Satanin1 (1) The amino acid sequence of the mature peptide of Satanin1 was codon optimized on the codon optimization website http: / / www.jcat.de / according to the preference of Pichia pastoris. The gene sequence obtained after optimization was synthesized by Beijing Ruiboxing Technology Co., Ltd. into the recombinant expression plasmid pPICZαA-Satanin1. The protein sequence and gene sequence are shown in Table 2 and Table 3.

[0055] Table 2 Satanin1 protein sequence Table 3. Satanin1 gene sequence (2) The recombinant expression plasmid pPICZαA-Satanin1 was linearized by digestion with the restriction endonuclease Sac I. The reaction mixture (total 50 μL) consisted of 5 μg of the recombinant expression plasmid pPICZαA-Satanin1, 5 μL of 10× buffer, 2 μL of Sac I (10 U / μL), and sterile water to a final volume of 50 μL. The reaction conditions were: digestion at 37°C for 3 h. After the reaction, the linearized plasmid was recovered according to the instructions of the Kangwei Century Agarose Gel Extraction Kit.

[0056] (3) Select a single colony of wild-type Pichia pastoris strain X33 and inoculate it into 10 mL of YPD medium (1% yeast extract, 2% peptone, 2% glucose, diluted to 1 L with water, autoclaved at 115℃). Incubate at 28℃ and 220 rpm for 24 h with shaking. Then, transfer 100 μL of the bacterial culture to 100 mL of YPD medium and incubate at 28℃ and 220 rpm with shaking until OD600≈1.3-1.6. Centrifuge the bacterial culture at 4℃ and 5,000 rpm for 10 min, discard the supernatant, and resuspend the bacterial culture in 50 mL of pre-cooled sterile water. Centrifuge the resuspended bacterial culture at 4℃ and 5,000 rpm for 10 min, discard the supernatant, and resuspend the bacterial culture in 30 mL of pre-cooled 1 M sorbitol. Repeat this step twice. The resuspended bacterial culture was centrifuged at 4°C and 5,000 rpm for 10 min. After discarding the supernatant, the bacterial culture was resuspended in 1 mL of pre-cooled 1 M sorbitol to prepare Pichia pastoris wild-type X33 strain electrotransfer competent cells.

[0057] (4) 5 μg of linearized plasmid was electroporated into 100 μL of X33 competent cells (electroporation conditions: 2.0 kV, 25 μF, 200 Ω, 5 ms). Immediately after electroporation, 1 mL of pre-cooled 1 M sorbitol was added, mixed thoroughly, and then added to a 50 mL centrifuge tube. The cells were incubated at 28°C for 3 h. The cultured cells were centrifuged at 6,000 rpm for 30 s, 900 μL of supernatant was discarded, and the cells were resuspended and plated on YPDS solid medium containing bleomycin (100 μg / mL) (1% yeast extract, 2% peptone, 2% glucose, 1 mol / L sorbitol, 20 g / L agarose, water added to a final volume of 1 L, autoclaved at 115°C). The cells were incubated upside down at 28°C for 2-3 days until clear colonies appeared. Pick a single colony and streak it onto YPD solid medium containing bleomycin (100 μg / mL). Incubate at 28°C upside down for 1-2 days until clear colonies grow.

[0058] (5) Pick a single colony and put it into 10 mL of YPD liquid medium containing bleomycin (100 μg / mL), and culture at 30℃ and 220 rpm for 24 h. 100 μL of bacterial culture was transferred to 50 mL of BMGY medium (1% yeast extract, 2% peptone, 100 mmol / L pH 6.0 potassium phosphate solution, 1.34% YNB, 1% glycerol, 0.2 mg / L biotin), and cultured at 30°C and 220 rpm until OD600≈2. After centrifugation at 4°C and 5,000 rpm for 10 min, the supernatant was discarded and the bacterial cells were collected. The bacterial cells were resuspended in 50 mL of BMMY medium (1% yeast extract, 2% peptone, 100 mmol / L pH 6.0 potassium phosphate solution, 1.34% YNB, 0.5% methanol, 0.2 mg / L biotin), and cultured with shaking at 30°C and 220 rpm. Anhydrous methanol was added to the culture every 24 h (maintaining a final concentration of 0.5%). After culturing for 96 h, the bacterial culture was centrifuged at 13,000 rpm for 15 min at 4°C, and the protein supernatant was collected and stored at 4°C. The expression of the antimicrobial peptide Satanin1 was detected using SDS-PAGE. Figure 1 As shown in Figure A, no obvious band was observed at the predicted protein size position (theoretical molecular weight approximately 4.3 kDa) corresponding to the protein marker, indicating that the antimicrobial peptide Satanin1 failed to be successfully expressed and secreted.

[0059] 2. Construction and expression of recombinant expression plasmids pPICZαA-hIL11-Satanin1 and pPICZαA-Satanin1-hIL11 (1) Following the aforementioned method, the gene sequence was optimized and recombinant expression plasmids pPICZαA-hIL11-Satanin1 and pPICZαA-Satanin1-hIL11 were synthesized. The protein sequences and gene sequences are shown in Tables 4 and 5.

[0060] Table 4 Amino acid sequences Table 5 Gene Sequences (2) Following the aforementioned method, the linear recombinant expression plasmid was transformed into X33 competent cells, and positive transposons were selected for protein expression. The expression of the antimicrobial peptides hIL11-Satanin1 and Satanin1-hIL11 was detected using SDS-PAGE, and the results are as follows: Figure 1Figures B and C are shown in Figure 1. The concentration of extracellular secreted proteins was determined using the Bradford assay, and the specific experimental procedures were performed according to the instructions of the Super-Bradford Protein Quantification Kit (CW0013) from Kangwei Century Company. Clear bands were observed at the predicted protein size positions (theoretical molecular weight approximately 25 kDa) corresponding to the protein markers. Both the antimicrobial peptides hIL11-Satanin1 and Satanin1-hIL11 were successfully expressed and secreted, with expression levels of approximately 25 mg / L and 50 mg / L, respectively.

[0061] 3. Antibacterial activity assay (1) Prepare olive oil Sabouraud solid medium: Add 0.5 g of peptone, 0.3 g of yeast extract, 0.3 g of malt extract, 1 g of glucose and 1.5 g of agar to 100 mL of water. After autoclaving, add 1 g of pre-filtered and sterilized olive oil.

[0062] (2) Preparation of pathogenic fungal suspension: Lyophilized Malassezia furfur ATCC12078 powder was suspended in culture medium and spread onto Sabouraud dextrose agar using a sterile spreader. The suspension was incubated at 30°C in a biological incubator, passaged every 48 h, and samples from generations 2-5 were used for testing. Single colonies were picked and transferred to 5 mL of Sabouraud dextrose liquid medium, and cultured at 30°C with shaking at 220 rpm for 24 h. 5 mL of the culture was collected after the incubation period, and the number of cells was counted using a hemocytometer. The concentration of the fungal suspension was then adjusted to 1×10⁻⁶. 6 CFU / mL available for use.

[0063] (3) The supernatant of the culture medium after antimicrobial peptide expression was collected by centrifugation at 4℃, and sterilized by filtration twice through a 0.22 μm filter membrane before storage. The sterilized olive oil Sabouraud broth cooled to approximately 50℃ was mixed with the antimicrobial peptide supernatant at a volume ratio of 1:1, and poured into plates after thorough mixing. After solidification, 100 μL of the fungal suspension was spread using a sterile spreader, and the plates were incubated at 30℃ for 48 h to observe the growth of the pathogenic fungi. A blank group (without bacterial suspension), a control group (mixed olive oil Sabouraud broth and induced empty supernatant), and a normal culture group (NT) on olive oil Sabouraud broth were also included. The results are as follows: Figure 1As shown in Figure D, the fermentation supernatant containing the antimicrobial peptide Satanin1-hIL11 completely inhibited the growth of Malassezia furfur within 48 h, but the fermentation supernatant containing the antimicrobial peptide hIL11-Satanin1 showed no inhibitory activity against Malassezia furfur. Introducing hIL11 into either the N-terminus or C-terminus of the antimicrobial peptide Satanin1 promoted its expression and secretion, but introducing hIL11 into the N-terminus affected its activity. Excessive proliferation of Malassezia furfur stimulates excessive proliferation and inflammatory responses of scalp keratinocytes, leading to dandruff and dermatitis symptoms. The activity of the antimicrobial peptide Satanin1-hIL11 makes it suitable for application in the daily chemical industry for the prevention and treatment of dandruff and seborrheic dermatitis caused by the proliferation of Malassezia furfur.

[0064] Example 2 This example demonstrates the optimization of expression conditions for the antimicrobial peptide Satanin1 in Pichia pastoris.

[0065] 1. Optimization of induction temperature, induction time, and methanol concentration Low-temperature induction during Pichia pastoris fermentation significantly affects cellular metabolism, increases intracellular alcohol oxidase activity and energy levels, reduces cell death, and inhibits protease activity. The aggregation and degradation of exogenous proteins during expression are also related to induction temperature. Methanol is an inducer in yeast expression systems; appropriately increasing methanol concentration can enhance protein expression, but excessively high concentrations can damage cells. Therefore, it is necessary to find the optimal methanol induction concentration.

[0066] (1) Following the method in Example 1, Pichia pastoris X33 cells transformed with the recombinant expression plasmid pPICZαA-Satanin1-hIL11 were cultured for 96 h at gradient induction temperatures (20℃, 25℃, 28℃, and 30℃) and 220 rpm. After expression, the supernatant was collected for detection. The expression of the antimicrobial peptide Satanin1-hIL11 was detected using SDS-PAGE, and the results are as follows: Figure 2 As shown in Figure A, based on the protein band size, the highest expression level of the antimicrobial peptide Satanin1-hIL11 was obtained by inducing expression at a temperature of 30°C.

[0067] (2) Following the method in Example 1, Pichia pastoris X33 transformed with the recombinant expression plasmid pPICZαA-Satanin1-hIL11 was induced for 120 h at 30℃ and 220 rpm using different concentrations of methanol (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%). A control group without methanol was also included. Supernatant was collected every 24 h for analysis. The expression of the antimicrobial peptide Satanin1-hIL11 was detected using SDS-PAGE. The results are as follows: Figure 2 Figures B, C, D, E, and F are shown. Based on the protein band size, the highest expression level of the antimicrobial peptide Satanin1-hIL11 was obtained after induction of expression for 96 h at a 1.5% methanol concentration.

[0068] 2. Screening of high-copy strains using the Liquid PTVA method Posttransformational vector amplification (PTVA) is a method of screening for multi-copy Pichia pastoris strains by increasing antibiotic concentration to increase the yield of recombinant protein expression. Traditional PTVA is relatively expensive and time-consuming. The improved Liquid PTVA allows cultures to be grown in liquid medium, with final selection performed only on agar plates, thereby reducing the overall amount of antibiotic used and increasing the speed of clonal amplification.

[0069] (1) Twenty-five positive clones were randomly selected from 100 µg / mL bleomycin resistance selective medium and cultured until mid-log phase. Then, 50 µL of bacterial suspension was centrifuged, the supernatant was removed, and the culture was sequentially screened using YPD liquid medium with progressively increasing concentrations (bleomycin concentrations of 100, 2000, 3000, and 5000 µg / mL). The bacterial suspension cultured to the exponential growth phase was adjusted to OD600≈1, and then diluted 10-fold to 10⁻⁶. -3 Take 100 µL and spread it evenly on YPD solid medium with increasing concentration gradient (bleomycin concentration 100, 2000, 3000, 5000 µg / mL). Incubate in an inverted incubator at 28℃ for 2-3 days until clear colonies grow.

[0070] (2) Following the method in Example 1, positive transposons were selected for protein expression. The expression of the antimicrobial peptide Satanin1-hIL11 was detected using SDS-PAGE, and the results are as follows: Figure 3 As shown in Figure A, the changes in extracellular protein production were obtained by grayscale analysis of protein bands using ImageJ software. The results are as follows: Figure 3As shown in Figure D. Electrophoresis results showed that the Liquid PTVA method significantly increased the expression level of the antimicrobial peptide Satanin1-hIL11. Among them, compared with the expression plasmid selected by 100 µg / mL bleomycin, the expression level of the expression plasmid selected by 5000 µg / mL bleomycin was the highest, at 100 mg / L, which was 1.23 times that of a single copy, and higher than that of the expression plasmid selected by 3000 µg / mL bleomycin (1.16 times) and 2000 µg / mL (1.08 times).

[0071] 3. Construct recombinant expression plasmids containing different copy numbers of expression elements. (1) Using the single-copy pPICZαA-Satanin1-hIL11 plasmid as a template plasmid, primers for constructing double-copy, triple-copy, and quadruple-copy plasmids were designed and constructed. The primer sequences are shown in Table 6, and the schematic diagram is shown below. Figure 3 As shown in B.

[0072] (2) Using the wild-type Pichia pastoris X33 genome as a template, PCR was performed using 2BsmBI-Paox1-F / 2NotI-Taox1-R primers. The recovered product yielded a 1910 bp expression element, Paox1-αfactor-Satanin1-hIL11. After digestion and recovery with restriction endonucleases BsmBI and NotI, the element was inserted between the single-copy plasmids BsmBI and NotI in pPICZαA-Satanin1-hIL11 to construct a double-copy plasmid. The expression element was then amplified by PCR using 3NotI-Paox1-F / 3XbaI-Taox1-R primers. After digestion and recovery with restriction endonucleases NotI and XbaI, the recovered product was inserted between the double-copy plasmids NotI and XbaI in pPICZαA-Satanin1-hIL11 to construct a triple-copy plasmid. The expression element was amplified by PCR using 4XbaI-Paox1-F / 4SalI-Taox1-R primers. The recovered product was digested with restriction endonucleases XbaI and SalI, and then inserted between the XbaI and SalI of the three-copy plasmid pPICZαA-Satanin1-hIL11 to construct a four-copy plasmid.

[0073] (3) Following the method in Example 1, recombinant expression plasmids containing different copy numbers of expression elements were transformed into X33 competent cells, and positive transposons were selected for protein expression. The expression of the antimicrobial peptide Satanin1-hIL11 was detected using SDS-PAGE, and the results are as follows: Figure 3 As shown in C, the changes in extracellular protein production were obtained by grayscale analysis of protein bands using ImageJ software. The results are as follows. Figure 3As shown in Figure E. SDS-PAGE electrophoresis results showed that increasing the copy number significantly improved the expression level of the antimicrobial peptide Satanin1-hIL11. Among them, the expression level of the three-copy expression plasmid was the highest, 1.36 times that of the single-copy plasmid, which was higher than that of the two-copy (1.04 times) and four-copy expression plasmids (1.25 times).

[0074] Table 6 Primer Sequences Example 3 This embodiment demonstrates the evaluation of the inhibitory effect of the antimicrobial peptide Satanin1-hIL11 on Malassezia furfur.

[0075] 1. Preparation of freeze-dried antimicrobial peptide Satanin1-hIL11 powder The supernatant of the antimicrobial peptide Satanin1-hIL11 expression after centrifugation was pre-frozen at -80℃ for 6 h until completely frozen. The completely frozen supernatant was then freeze-dried under vacuum at -51℃ and 20 Pa for 96 h until completely dry, yielding freeze-dried powder of the antimicrobial peptide Satanin1-hIL11.

[0076] 2. Detection of the inhibitory effect of the antimicrobial peptide Satanin1-hIL11 on the growth of Malassezia furfur using the micro-liquid dilution method. (1) Prepare olive oil Sabouraud liquid medium: Add 0.5 g of peptone, 0.3 g of yeast extract, 0.3 g of malt extract and 1 g of glucose to 100 mL of water. After autoclaving, add 1 g of pre-filtered and sterilized olive oil.

[0077] (2) The Satanin1-hIL11 sample powder was diluted with deionized water to prepare sample solutions with concentrations of 128, 96, 64, 48, 24 and 12 mg / L. Ketoconazole was prepared to 128 mg / L as a positive control, and 1×PBS solution was prepared with deionized water as a blank control.

[0078] (3) Adjust the concentration of the fungal suspension to 1×10 6CFU / mL. 100 μL of fungal suspension was added to a 96-well plate. The experimental group received 100 μL of different concentrations of Satanin1-hIL11 solution (final concentrations of 64, 48, 32, 24, 12, and 6 mg / L), while the control group received an equal volume of olive oil Sabouraud broth. A blank control group and a positive control group were also set up, with three parallel wells in each group. The culture conditions were 30℃ for 48 h. *Malassezia furfur* colonies appeared milky white and floated on the surface of the liquid medium, visible to the naked eye under natural light, and the colony growth results could be directly read. Using the positive control as the standard, the lowest concentration of antimicrobial peptide with no visible bacterial growth was taken as the MIC value against *Malassezia furfur*, as shown in Figure 4A. The micro-liquid dilution method was used to further test the inhibitory effect of the antimicrobial peptide Satanin1-hIL11 on *Malassezia furfur*, with a minimum bactericidal concentration of 64 mg / L.

[0079] 3. Inhibitory effect of antimicrobial peptide Satanin1-hIL11 on biofilm formation of Malassezia furfur. (1) Dilute the Satanin1-hIL11 sample powder with deionized water to prepare sample solutions with concentrations of 256, 192, 128, 96, 48 and 24 mg / L. In addition, ketoconazole was prepared to 128 mg / L as a positive control, and 1×PBS solution was prepared with deionized water as a blank control.

[0080] (2) Adjust the concentration of the fungal suspension to 1×10 6 CFU / mL. 100 μL of *Malassezia furfur* culture was added to each 96-well plate. The experimental groups were treated with 100 μL of different concentrations of Satanin 1-hIL11 solution (final concentrations of 128, 96, 64, 48, 24, and 12 mg / L), while the control group was treated with an equal volume of olive oil-based Sabouraud broth. A blank control group and a positive control group were also included, with three parallel wells in each group. The culture conditions were 30℃ for 24 h of static incubation.

[0081] (3) After cultivation, discard the liquid in the 96-well plate and wash three times with PBS; after discarding the PBS, add 4% paraformaldehyde for fixation for 15 min; after 15 min, remove the 4% paraformaldehyde and wash three times with PBS; add 100 μL of 0.1% crystal violet staining solution for staining for 15 min. After staining, wash three times with PBS and air dry. Finally, add 100 μL of 95% ethanol to each well to dissolve the crystal violet staining solution on the biofilm, let it stand in the dark for 40 min, and then measure the absorbance at 600 nm. Calculate the biofilm inhibition rate using the positive control as the standard. The calculation formula is: Inhibition rate (%) = 100 - (OD test group - OD blank group) / (OD fungal group - OD blank group) × 100%. The results are as follows: Figure 4As shown in B.

[0082] 4. Antimicrobial curve of antimicrobial peptide Satanin1-hIL11 against Malassezia furfur. (1) Dilute the Satanin1-hIL11 sample powder with deionized water to make sample solutions with final concentrations of MIC (64 mg / L), 2×MIC (128 mg / L), and 4×MIC (256 mg / L) in each tube. In addition, prepare ketoconazole at 128 mg / L as a positive control and prepare 1×PBS solution with deionized water as a blank control.

[0083] (2) Adjust the concentration of the fungal suspension to 1×10 6 CFU / mL. 200 μL of *Malassezia furfur* bacterial suspension was added to a 12 mL shake tube. The experimental group received 200 μL of Satanin 1-hIL11 solution at different concentrations (0.5×MIC, MIC, 2×MIC), while the control group received an equal volume of olive oil Sabouraud broth. A blank control group and a positive control group were also included, with three replicates for each group. Culture conditions were a 30℃ biological incubator with shaking at 180 rpm. At 0 h, 6 h, 12 h, and 24 h, 50 μL of bacterial suspension was serially diluted 10-10 times with sterile physiological saline. -6 Double the amount of each concentration by spreading 200 μL onto Sabouraud dextrose agar medium and incubating statically in a 30°C biological incubator for 2 days. Count the samples after incubation. Each concentration was repeated three times. Results are shown below. Figure 4 As shown in C, the bactericidal activity of Satanin1-hIL11 against Malassezia furfur is dose-time dependent, and the inhibition rate increases with increasing concentration and duration of action.

[0084] In summary, this invention, through genetic engineering, for the first time heterologously expresses the antimicrobial peptide Satanin1-hIL11 in Pichia pastoris, which can effectively inhibit the infection of Malassezia furfur. This invention further optimizes the expression conditions of the antimicrobial peptide Satanin1-hIL11, achieving an expression level as high as 100 mg / L under laboratory shake-flask culture conditions, providing a good foundation for subsequent large-scale fermentation production.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antimicrobial fusion protein, characterized in that, It has the amino acid sequence shown in (I) or (II): (I) An amino acid sequence that has at least 90% homology with and has the same function as the amino acid sequence shown in SEQ ID NO.1; (II) An amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.

1.

2. The antimicrobial fusion protein according to claim 1, characterized in that, It has the amino acid sequence shown in SEQ ID NO.

1.

3. A nucleic acid molecule, characterized in that, It includes a nucleotide sequence encoding the antimicrobial fusion protein according to claim 1 or 2.

4. A carrier molecule, characterized in that, It includes the nucleic acid molecule according to claim 3.

5. A host cell, characterized in that, It includes the nucleic acid molecule according to claim 3 or the carrier molecule according to claim 4.

6. The method for preparing the antimicrobial fusion protein according to claim 1 or 2, characterized in that, Prepared through artificial synthesis or genetic engineering.

7. An antibacterial composition, characterized in that, It includes the antimicrobial fusion protein according to claim 1 or 2.

8. The use of the antimicrobial fusion protein according to claim 1 or 2, or the antimicrobial composition according to claim 7, in the preparation of antimicrobial products.

9. The application according to claim 8, characterized in that, The antibacterial products include drugs, shampoos and conditioners, or skin care products.

10. A method for inhibiting fungi, characterized in that, It includes the step of using the antimicrobial fusion protein according to claim 1 or 2.

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