Antimicrobial peptide Ple-AB, its preparation method and application

Abstract

This invention relates to the field of antimicrobial peptide technology, and particularly to the antimicrobial peptide Ple-AB, its preparation method, and its applications. Using the fungal defensin Plectasin as a template, this invention designs and modifies the antimicrobial peptide Ple-AB. Compared with Plectasin, this antimicrobial peptide exhibits significantly higher antimicrobial activity and better bactericidal effects against Gram-positive bacteria; it also shows higher stability, particularly excellent resistance to trypsin; and its fermentation expression level is significantly improved. Furthermore, this antimicrobial peptide has very low hemolytic activity and cytotoxicity, making it suitable for development in antimicrobial drugs, additives, cosmetics, health products, and other fields, with broad application value and market prospects.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, and in particular to the antimicrobial peptide Ple-AB, its preparation method, and its application. Background Technology

[0002] The increasing resistance to traditional antibiotics has promoted the development and application of antimicrobial peptides (AMPs). AMPs are widely found in animals, plants and microorganisms and are an important component of the host's innate immune system. They have inhibitory effects on microorganisms, parasites and even tumors (Liu Y, Shi J, Tong Z, Jia Y, Yang B, Wang Z. The revitalization of antimicrobial peptides in the resistanceera. Pharmacol Res. 2021(07);163:105276.). The multi-target mechanism of action of AMPs makes them less prone to drug resistance. However, natural antimicrobial peptides have drawbacks such as host cell toxicity, low expression levels, and protease sensitivity. Some antimicrobial peptides also exhibit insufficient bactericidal activity. Therefore, improving the yield and activity of antimicrobial peptides and minimizing their toxicity has become a major challenge in the development of antimicrobial peptide drugs (Jiang Y, Chen Y, Song Z, Tan Z, Cheng J. Recent advances in design of antimicrobial peptides and polypeptides toward clinical translation. Adv Drug Deliv Rev. 2021(03); 170:261-280.). Targeted modification of natural antimicrobial peptides to overcome these problems is of great significance for the development of antimicrobial peptide drugs.

[0003] Plectasin, the first fungal defensin isolated from saprophytic ascomycetes, is a polypeptide composed of 40 amino acid residues. It belongs to the cysteine-stabilized α-helix and β-sheet superfamily and exhibits broad-spectrum antibacterial activity against Gram-positive bacteria (Mygind PH, Fischer RL, Schnorr KM, Hansen MT). CP, Ludvigsen S, et al. Plectasin is a peptide antibiotic with therapeutic potential from asaprophytic fungus. Nature. 2005; 437(7061):975-80. However, plectasin suffers from poor stability and strong cytotoxicity (Li Z, Wang X, Wang X, et al. Research advances on plectasin and its derivatives as new potential antimicrobial candidates. Process Biochemistry. 2017; 62-70.), and its expression level is low during fermentation, which increases its production cost and limits its clinical application. Summary of the Invention

[0004] This invention provides the antimicrobial peptide Ple-AB, its preparation method, and its applications.

[0005] This invention designs and modifies the fungal defensin Plectasin. Through continuous screening, a Plectasin-derived peptide, Ple-AB, was obtained by amino acid truncation and combination modification. Its amino acid sequence is shown in SEQ ID NO.1. This peptide has significantly improved antibacterial activity compared to Plectasin, and also has higher stability and expression level.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a polypeptide, wherein the amino acid sequence of the polypeptide is any one of the following (1) to (3):

[0008] (1) The amino acid sequence as shown in SEQ ID NO.1;

[0009] (2) A sequence obtained by linking one or more of protein tags, restriction enzyme sites, and linker peptides to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1;

[0010] (3) An amino acid sequence with the same function obtained by substituting, deleting and / or inserting one or more amino acids into the amino acid sequence shown in SEQ ID NO.1.

[0011] The amino acid sequence shown in SEQ ID NO.1 is as follows: GFGCPWDEMQCHNHCKSIKGYKGGYCAKGGFVCKCY.

[0012] In (2) above, adding a protein tag, restriction enzyme site, or linker peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.1 generally does not affect the structure and function of the protein with the sequence shown in SEQ ID NO.1. Therefore, polypeptides having the sequence shown in (2) are also within the scope of protection of this invention.

[0013] Based on the above-mentioned amino acid sequence, those skilled in the art can covalently or non-covalently connect modifying groups to the C-terminus and / or N-terminus of the polypeptide. The modifying groups include acetyl, amide, aldehyde, methyl, ester, alkyl, etc., and the polypeptides obtained therefrom are also within the scope of protection of this invention.

[0014] Secondly, the present invention provides a nucleic acid molecule encoding the polypeptides described above.

[0015] Based on the amino acid sequence of the polypeptide described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the polypeptide. Due to the degeneracy of codons, the nucleotide sequence of the above nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the above polypeptide are within the protection scope of this invention.

[0016] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2. The nucleic acid molecule with the sequence shown in SEQ ID NO.2 can be efficiently expressed in Pichia pastoris, increasing the expression level of the polypeptide.

[0017] Thirdly, the present invention provides biological materials comprising the nucleic acid molecules described above, wherein the biological materials are expression cassettes, vectors, or host cells.

[0018] The expression cassette described above can be obtained by linking a promoter, terminator, stop codon, and / or restriction enzyme site to a nucleic acid molecule encoding the polypeptide. The expression cassette may also contain other transcriptional and translational regulatory elements.

[0019] In some embodiments of the present invention, an XhoI restriction site and a Kex2 restriction site are added to the 5' end of the nucleic acid molecule, and TAA and TAG stop codon sequences and a NotI restriction site are added to the 3' end to obtain the expression cassette, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0020] The vectors mentioned above include, but are not limited to, plasmid vectors, viral vectors, transposons, etc., among which plasmid vectors include replicating vectors and non-replicating vectors.

[0021] In some embodiments of the present invention, the nucleic acid molecule or its expression cassette is linked to the vector pPICZαA to obtain a recombinant yeast expression vector.

[0022] The host cells mentioned above include microbial cells or animal cells, wherein microorganisms include bacteria (e.g., Escherichia coli) or fungi (e.g., Pichia pastoris).

[0023] Fourthly, the present invention provides a recombinant Pichia pastoris, wherein the recombinant Pichia pastoris expresses the polypeptides described above, or contains the nucleic acid molecules described above, or contains an expression cassette or vector containing the nucleic acid molecules.

[0024] In some embodiments of the present invention, the expression vector containing the nucleic acid molecule is linearized and then transformed into Pichia pastoris X-33 to obtain recombinant Pichia pastoris.

[0025] Fifthly, the present invention provides a method for preparing the above-described polypeptide, the method comprising: expressing the polypeptide using Pichia pastoris as a host cell.

[0026] Specifically, the method includes: fermenting and culturing the recombinant Pichia pastoris to secrete the polypeptide.

[0027] Sixthly, the present invention provides the application of the above-described polypeptides, nucleic acid molecules, or biological materials in the preparation of antibacterial products.

[0028] Preferably, the antibacterial product is a preservative, bactericide, disinfectant, antiseptic, feed additive, food additive, or drug.

[0029] Preferably, the bacteria targeted by the antibacterial agent described in this invention are Gram-positive bacteria. These Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus dysgalactiae, and Streptococcus agalactiae, among others.

[0030] In a seventh aspect, the present invention provides the application of the above-described polypeptides, nucleic acid molecules, or biological materials in food preservation, antibacterial and preservative treatment of cosmetics, or environmental disinfection.

[0031] Eighthly, the present invention provides a medicament comprising the polypeptides described above.

[0032] Ninthly, the present invention provides a feed additive comprising the polypeptides described above.

[0033] In a tenth aspect, the present invention provides a bactericide or preservative comprising the polypeptides described above.

[0034] The beneficial effects of this invention are as follows: Using the fungal defensin Plectasin as a template, the antimicrobial peptide Ple-AB was designed and modified. Compared with Plectasin, this antimicrobial peptide exhibits significantly higher antimicrobial activity and better bactericidal effect against Gram-positive bacteria; it also has higher stability, especially showing excellent resistance to trypsin; and its fermentation expression level is significantly improved. Furthermore, this antimicrobial peptide exhibits very low hemolytic activity against mouse erythrocytes and low cytotoxicity against mouse macrophages, demonstrating the advantage of low toxicity.

[0035] This invention optimizes the gene sequence of the antimicrobial peptide Ple-AB, constructs a specific expression vector, achieves efficient expression of the antimicrobial peptide Ple-AB in Pichia pastoris, and establishes a complete purification system, enabling large-scale production of the antimicrobial peptide Ple-AB. It can be developed for use in antimicrobial drugs, additives, cosmetics, health products and other fields, and has broad application value and market prospects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 The above is the electrophoresis result of linearization of the recombinant Ple-AB vector in Example 3 of the present invention; wherein, M: Trans5K DNA marker; 1: recombinant vector Ple-AB that has not been linearized; 2: recombinant vector Ple-AB that has been linearized.

[0038] Figure 2 This is the induction curve of changes in wet weight and total protein concentration of Ple-AB fermenter expressed in Example 5 of the present invention over time.

[0039] Figure 3 The results show the antibacterial activity of the fermentation supernatant after 120 hours of induction of the Ple-AB recombinant yeast strain in Example 5 of this invention.

[0040] Figure 4 The results of Tricine-SDS-PAGE electrophoresis of the fermentation supernatant from the Ple-AB recombinant yeast strain at different induction times in Example 5 of this invention are shown. M: ultra-low molecular weight protein marker; 1-6: represent the electrophoretic bands of the fermentation supernatant after induction for 120h, 96h, 72h, 48h, 24h, and 0h, respectively.

[0041] Figure 5The results of Tricine-SDS-PAGE for the antimicrobial peptide Ple-AB in Example 6 of this invention are shown; where M: ultra-low molecular weight protein marker, 1: unpurified fermentation sample; 3-4: purified samples; 5-6: purified samples, loading volume 10 μL; M: 5 μL ultra-low molecular weight protein marker.

[0042] Figure 6 This is the mass spectrometry identification result of the antimicrobial peptide Ple-AB in Example 6 of the present invention.

[0043] Figure 7 The results of the hemolytic activity test of the antimicrobial peptide Ple-AB in Example 9 of this invention are shown.

[0044] Figure 8 The results of the cytotoxicity experiment of the antimicrobial peptide Ple-AB in Example 10 of this invention are shown. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] The culture medium and buffer formulations involved in the following examples are as follows:

[0047] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl; solid LB medium is supplemented with 2% agarose.

[0048] Low-salt LB medium: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl; solid low-salt LB medium is supplemented with 2% agar powder.

[0049] MHB medium: Weigh 2.4g of powder and dissolve it in 90mL of distilled water. Stir with a magnetic stirrer and bring the volume up to 100mL.

[0050] MHA culture medium: Weigh 3.65g of powder and dissolve it in 90mL of distilled water. Stir with a magnetic stirrer and bring the volume up to 100mL.

[0051] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose; solid YPD medium is supplemented with 2% agar powder.

[0052] YPDS medium: peptone 20 g / L, yeast extract 10 g / L, sorbitol 182.2 g / L, glucose 20 g / L, agar powder 20 g / L.

[0053] BMGY medium ( / L): 10g yeast extract, 20g peptone, 10mL glycerol, 100mL 13.4% amino acid-free yeast nitrogen source (YNB), 2mL 0.02% biotin, 1mol / L phosphate buffer, pH 6.0, 100mL.

[0054] For instructions on using LB medium, low-salt LB, MH, YPD, YPDS, etc., please refer to the Invitrogen Pichia pastoris manual.

[0055] 50mM phosphate buffer (Solution A): 7.786g Na2HPO4, 4.407g NaH2PO4, add deionized water to 950mL, place in a magnetic stirrer until completely dissolved, adjust pH to 7.0, and bring the volume to 1000mL.

[0056] 1M NaCl 50mM Phosphate Buffer (Solution B): 7.786g Na2HPO4, 4.407g NaH2PO4, 58.46g NaCl, add deionized water to 950mL, place in a magnetic stirrer until completely dissolved, adjust pH to 7.0, and bring the volume to 1000mL.

[0057] The basic salt culture medium formula is: 100g NH4H2PO4, 40g K2SO4, 30g MgSO4·7H2O, 12g KH2PO4, 0.8g CaSO4 and 3g KOH, add water to make up to 1.8L, and mix with 200mL 45% glucose.

[0058] The gene amplification and transformant identification methods involved in the following examples are PCR and DNA sequencing.

[0059] The protein detection method involved in the following examples is Tricine-SDS-PAGE.

[0060] The protein concentration determination method used in the following examples is the Bradford method.

[0061] The protein molecular weights used in the following examples were determined using the MALDI-TOF MS method.

[0062] The protein purification methods described in the following examples are based on ion chromatography.

[0063] The fermentation method involved in the following examples is a high-density fermentation method.

[0064] The bacterial strains and plasmids involved in the following examples are shown in Table 1.

[0065] Table 1. Tested bacterial strains and plasmids

[0066]

[0067]

[0068] Example 1: Design of the antimicrobial peptide Ple-AB

[0069] Based on the fungal defensin Plectasin, multiple defensins homologous to mycomycin were compared and analyzed using bioinformatics. Amino acid sequences that had changed during evolution were truncated and deleted to alter the peptide's length, charge, isoelectric point, and other physicochemical properties. Finally, Asn5, Gly6, Asp11, and Asp12 were truncated to obtain the novel fungal defensin Plectasin-derived peptide Ple-AB, the amino acid sequence of which is shown in SEQ ID NO.1.

[0070] Example 2: Obtaining the Ple-AB gene fragment of the antimicrobial peptide

[0071] Based on the Pichia pastoris preferred codon table, the gene sequence encoding the antimicrobial peptide Ple-AB was optimized. A gene sequence encoding the Kex2 signal peptide cleavage site was inserted at the 5' end of the optimized gene sequence (SEQ ID NO.2), and stop codons (TAA and TAG) were added to the 3' end to terminate translation. Restriction endonuclease sites XhoI and NotI were added to both ends of the gene sequence, resulting in the nucleotide sequence of the gene expression cassette shown in SEQ ID NO.3. The designed gene sequence was synthesized by Sangon Biotech (Shanghai) Technology Service Co., Ltd. The DNA sequence shown in SEQ ID NO.3 and the vector pPICZαA were digested and ligated with XhoI and NotI to obtain the recombinant yeast expression vector, which was then stored in E. coli DH5α.

[0072] Example 3 Construction of yeast recombinant expression vector

[0073] 1. Plasmid extraction

[0074] The recombinant expression vector pPICZαA-Ple-AB was extracted from E. coli according to the plasmid extraction kit instructions, and the results are as follows: Figure 1 As shown.

[0075] 2. Linearization of the recombinant vector pPICZαA-Ple-AB

[0076] The constitutive recombinant expression vector pPICZαA-Ple-AB was digested with PmeI, and the digestion system is shown in Table 2.

[0077] Table 2

[0078]

[0079] After the above enzyme digestion system was added, the mixture was placed in a 37℃ water bath for 4 hours. The results were then detected by 1.5% agarose gel electrophoresis under the following conditions: 135-140V, 35 min. (Electrophoresis results...) Figure 1 The results show that the pPICZαA-Ple-AB recombinant vector is fully linearized.

[0080] 3. Purification and recovery of linearized recombinant plasmid vectors

[0081] The linearized recombinant plasmid was recovered using a standard DNA product purification kit, following the kit's instructions. The recovered product was detected by 1.5% agarose gel electrophoresis, lyophilized and concentrated, and stored at 4°C for later use.

[0082] Example 4: Construction of a recombinant yeast strain containing the Ple-AB gene

[0083] 1. Preparation of Pichia pastoris X-33 competent cells

[0084] 1) Pick a single colony of X-33 from a YPD plate and inoculate it into 10 mL of YPD liquid medium. Incubate overnight at 29°C and 250 rpm.

[0085] 2) Take 1% of the overnight culture medium of Pichia pastoris X-33 and inoculate it into 100 mL of YPD liquid medium. Incubate at 29°C and 250 rpm until OD reaches 100 mL. 600nm The absorbance value is 1.1-1.3;

[0086] 3) Centrifuge 50 mL of culture at 4℃, 4000 rpm for 5 min, then resuspend in 50 mL of pre-cooled sterile water;

[0087] 4) After centrifugation at 4℃, 4000rpm, and 5min, remove the supernatant and resuspend in 25mL of pre-cooled sterile water;

[0088] 5) After centrifugation at 4℃, 4000rpm, and 5min, remove the supernatant and resuspend in 2mL of pre-cooled 1M sorbitol;

[0089] 6) After centrifugation at 4℃, 4000rpm, and 5min, remove the supernatant, add 200μL of pre-cooled 1M sorbitol to resuspend the cells, and the resulting cells are X-33 competent cells.

[0090] 2. Electroconversion

[0091] Add 100 μL of competent yeast cells to the lyophilized linearized recombinant plasmid powder, mix gently, and transfer to an ice-cold electroporation cuvette. Incubate on ice for 5 min and perform electroporation at 1.2 kV, 25 μF, and 400 Ω. Immediately after electroporation, add 1 mL of ice-cold 1 M sorbitol solution, mix well, and transfer to a 2 mL centrifuge tube. Incubate at 29 °C for 2 h. Spread 200 μL of the recovered bacterial culture onto a YPDS plate containing 100 μg / mL zeocin antibiotic and incubate upside down at 29 °C until a single colony grows.

[0092] 3. Screening of positive transformants by induction in 48-well plates

[0093] Add 500 μL of BMGY medium to each well of a 48-well plate, and pick a single colony and place it into the plate. Set up blank control wells (no bacteria), negative control wells (pPICZαA empty plasmid), and positive control wells to confirm induced colonies. Incubate at 29℃ and 250 rpm for 24 h with shaking. Add 2.5 μL of methanol (final methanol concentration 0.5%) to each well, marking this as 0 h. Add methanol every 24 h, marking these values ​​as 0 h, 24 h, 48 h, and 72 h. After 96 h of induction, collect the fermentation broth from each well into a 1.5 mL centrifuge tube, centrifuge, and collect the supernatant for antibacterial activity testing.

[0094] Example 5: High-density fermentation of recombinant yeast strains

[0095] Single colonies of transformants were picked from YPD plates and inoculated into 50 mL shake flasks containing 10 mL of YPD liquid medium (containing 100 μg / mL zeocin). The culture was incubated at 29°C and 250 rpm for 18–24 h. Then, 1% of the transformants were inoculated into 1 L shake flasks containing 200 mL of YPD seed culture medium and incubated at 29°C and 250 rpm for 16–18 h. OD 600nm Approximately 6, reserved as a high-density fermentation seed liquid.

[0096] High-density fermentation was carried out in a 5L fermenter. The fermentation process was divided into three stages: (1) Cell growth stage: 2L of basic salt culture medium was added, sterilized at 121℃ for 20min, cooled to 29℃, pH adjusted to 5.0, and 9.6mL of culture medium was added. (1) PTM1, inoculate 200mL of bacterial solution (1:10), maintain aeration rate at 8vvm, rotation speed at 600rpm, and maintain dissolved oxygen at 20% or higher; (2) Glucose feeding growth stage: observe that the dissolved oxygen value starts to decrease slowly and then suddenly rises to over 80%, start feeding 50% glucose solution (12‰ PTM1), feed rate at 24mL / L / min, feed continuously for 6h, increase rotation speed to 1000rpm, and keep other fermentation conditions unchanged; (3) Methanol transition induction stage: fermentation conditions change, feed glucose for 6h, starve for half an hour, start feeding 100% methanol, gradually increase the flow rate from 1mL / L / min in the first hour to 6mL / L / min in the sixth hour, increase rotation speed to 1100rpm, increase pH to 5.5, control dissolved oxygen at 20% or higher, keep other fermentation conditions unchanged until fermentation ends.

[0097] Starting from the transition induction, samples were taken every 24 hours for protein expression analysis and antibacterial activity analysis. Figure 2 The curves showing the changes in wet weight and total protein concentration of Ple-AB cells expressed in a fermenter over time are shown. Figure 3 To assess the antibacterial effect of high-density fermentation supernatant from recombinant yeast strains, Figure 4 Electrophoresis image of fermentation supernatant proteins.

[0098] Example 6: Purification of the antimicrobial peptide Ple-AB

[0099] 1. Cation exchange chromatography purification:

[0100] After equilibrating the HiPrep SPFF cation exchange column (16 mm in length, 10 mm in inner diameter, GE Healthcare) with solution A for 3-5 column volumes, the sample was loaded. After injection, elution was first performed with 50 mM phosphate elution buffer (solution A) at pH 7.0. After the breakthrough peak was eluted, elution was performed with 50 mM phosphate elution buffer (solution B) at pH 7.0 containing 1 M NaCl. The elution peak was collected, and the elution status was monitored at UV 280 nm. Figure 5 To purify Ple-AB Tricine-SDS-PAGE, Figure 6 Purification of Ple-AB mass spectrometry detection.

[0101] 2. 1kDa dialysis bag for desalting

[0102] The collected elution peaks were dialyzed using a 1 kDa molecular weight cutoff dialysis bag at 4°C, with water changed every 2 hours for a total of 6 times. The dialysate was collected and freeze-dried in a low-temperature vacuum freeze dryer (-54°C, 0.016 mba) to obtain the antimicrobial peptide Ple-AB freeze-dried powder product.

[0103] Example 7: Detection of the antimicrobial activity of the antimicrobial peptide Ple-AB

[0104] The lyophilized antimicrobial peptide Ple-AB obtained in Example 6 was used to prepare a 1280 μg / mL solution of antimicrobial peptide Ple-AB and vancomycin using sterile physiological water. This solution was serially diluted 2-fold to a final concentration of 10 μg / mL. Different concentrations of Ple-AB and vancomycin solutions were added to sterile 96-well cell culture plates, 10 μL per well, with three replicates per sample. An equal volume (10 μL) of sterile physiological saline was used as a negative control to prepare MIC plates. Single colonies of the test strain were picked and activated overnight, then 1% transferred and cultured at 37°C to the logarithmic growth phase. The bacterial culture was then diluted to 1×10⁻⁶ with culture medium. 5 CFU / mL. Add 90 μL of bacterial suspension to each well of the prepared MIC plate, incubate at 37℃ for 16-18 h, and observe and record the experimental results. The minimum inhibitory concentration (MIC) of Ple-AB against pathogens was determined using the microbroth dilution method. The results are shown in Table 3. The antimicrobial peptide showed 2-8 times higher antimicrobial activity against most Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, and Streptococcus dysgalactiae strains compared to the parent peptide Plectasin.

[0105] Table 3. Antibacterial activity of Ple-AB against Gram-positive bacteria

[0106]

[0107] Example 8: Stability test of antimicrobial peptide Ple-AB

[0108] 1. Temperature stability test

[0109] The lyophilized antimicrobial peptide Ple-AB obtained in Example 6 was dissolved in sterile physiological saline to prepare a stock solution with a concentration of 128 μg / mL. The stock solution was placed in a water bath at 20, 40, 60, 80, and 100 °C for 1 h, and serially diluted 2 times to obtain final concentrations of 1, 2, 4, 8, 16, 32, and 64 μg / mL. S. aureus ATCC43300 was used as the test strain. PBS and culture medium were used as negative control and blank control groups, respectively. Each treatment group was set up in triplicate. The 96-well plates were incubated at 37℃ for 12-18 hours until visibly turbidity appeared in the negative control wells. The changes in the MIC of Ple-AB after different temperature treatments were observed and recorded (Liu H, Yang N, Mao R, Teng D, Hao Y, Wang X, Wang JA new high-yielding antimicrobialpeptide NZX and its antibacterial activity against Staphylococcus hyicus invitro / vivo. Appl Microbiol Biotechnol. 2020; 104(4):1555-1568). The results are shown in Table 4. The antimicrobial peptide Ple-AB was stable in activity and had no change in MIC at 20-80℃, but its activity decreased by 2-fold at 100℃. The parent peptide Plectasin was stable in activity and had no change in MIC at 20-60℃, but its activity decreased by 2-fold at 80-100℃.

[0110] Table 4 Temperature stability of Ple-AB

[0111]

[0112] 2. pH stability test

[0113] The lyophilized antimicrobial peptide Ple-AB was dissolved in different pH buffers and incubated at 37°C for 4 h. The effect of pH on the activity of the antimicrobial peptide was evaluated by the MIC assay. The pH buffers used were glycine-hydrochloric acid buffer (pH 2.0), citrate-sodium acetate buffer (pH 4.0), sodium phosphate buffer (pH 6.0), Tris-hydrochloric acid buffer (pH 8.0), and glycine-sodium hydroxide buffer (pH 10.0). The results are shown in Table 5. The antimicrobial peptide Ple-AB was stable in activity and had no change in MIC in pH 2–8, but its activity decreased by 2-fold in pH 10. The parent peptide Plectasin was stable in activity and had no change in MIC in pH 4–8, but its activity decreased by 2-fold in pH 4 and 10.

[0114] Table 5 pH stability of Ple-AB

[0115]

[0116] 3. Stability in gastrointestinal fluids

[0117] The lyophilized antimicrobial peptide Ple-AB was dissolved in sterile physiological saline and mixed thoroughly with simulated gastric and intestinal fluids. The mixtures were incubated at 37°C for 15 min, 30 min, 1 h, and 2 h. The effect of gastrointestinal fluids on the activity of the antimicrobial peptide was evaluated using the MIC assay. The results are shown in Table 6. Ple-AB showed stable activity and no change in MIC after 15 to 2 hours of treatment in simulated gastric fluid. Its activity remained unchanged for the first 30 minutes after treatment with simulated intestinal fluid, decreased by 2-fold after 1 hour, and decreased by more than 8-fold after 2 hours. The parent peptide Plectasin showed stable activity and no change in MIC after 15 to 30 minutes of treatment in simulated gastric fluid, decreased by 2-fold after 1 to 2 hours. Its activity remained unchanged for the first 15 minutes after treatment with simulated intestinal fluid, decreased by 2-fold after 30 minutes, and decreased by more than 8-fold after 1-2 hours.

[0118] Table 6. Stability of Ple-AB in gastrointestinal fluids

[0119]

[0120] Example 9: Hemolytic activity test of antimicrobial peptide Ple-AB

[0121] The lyophilized antimicrobial peptide Ple-AB was dissolved in sterile physiological saline to prepare a stock solution with a concentration of 512 μg / mL, which was then serially diluted 2-fold to a final concentration of 1 μg / mL. Blood was collected from the eyeballs of 6-week-old SPF-grade ICR female mice using heparin sodium anticoagulant tubes. The collected blood was centrifuged at 1500 rpm for 10 min at 4°C, and the red blood cells were washed three times with 10 mM PBS (pH 7.3) until the supernatant was colorless and clear, preparing an 8% red blood cell suspension. 100 μL of the red blood cell suspension and the antimicrobial peptide Ple-AB solution were added to a 96-well plate, incubated at 37°C for 1 h, centrifuged at 1500 rpm for 5 min, and the supernatant was transferred to an ELISA plate for UV absorbance at 540 nm. Physiological saline and 0.1% Triton X-100 were used as 0% and 100% hemolysis controls, respectively. The formula for calculating the degree of hemolysis is as follows (Yang N, Liu X, Teng D, Li Z, Wang X, Mao R, Wang X, Hao Y, Wang J. Antibacterial and detoxifying activity of NZ17074 analogs with multi-layers of selective antimicrobial actions against Escherichia coli and Salmonella enteritidis. Sci Rep. 2017 Jun 13; 7(1):3392): Hemolysis (%) = [(Abs540nm antimicrobial peptide Ple-AB - Abs540nm saline) / (Abs540nm 0.1% Triton X-100 - Abs540nm saline)] × 100%. The results are as follows Figure 7 As shown.

[0122] Example 10 Cytotoxicity test of antimicrobial peptide Ple-AB

[0123] RAW264.7 cells were cultured in DMEM complete medium at 37°C, 5% CO2, and saturated humidity. Cells were resuspended in DMEM complete medium by pipetting, with a concentration of 2.5 × 10⁻⁶ cells / mL. 5Cells / mL were seeded in 96-well plates at a density of 100 μL per well, with three replicates. After 24 h, the culture medium was removed, and 100 μL of the antimicrobial peptide Ple-AB at concentrations of 2, 4, 8, 16, 32, 64, 128, and 256 μg / mL were added to each well in a gradient manner. An equal volume of PBS was added to the control wells. After incubation for another 24 h, the culture medium was removed, and the plates were washed twice with PBS. 20 μL of 5 mg / mL MTT was added to each well (in the dark). The 96-well plates were transferred to an incubator and incubated for another 4 h. After discarding the MTT solution, 150 μL of DMSO was added to each well, and the plates were shaken for 10 min until the crystals at the bottom of the wells were completely dissolved. The absorbance of each well was measured at 570 nm. Cell viability was calculated using the following formula: Viability (%) = OD value of treatment group / OD value of control group × 100% (Yang N, Liu X, Teng D, Li Z, Wang X, Mao R, Wang X, Hao Y, Wang J. Antibacteria and detoxifying activity of NZ17074 analogs with multi-layers of selective antimicrobial actions against Escherichia coli and Salmonella enteritidis. SciRep. 2017 Jun 13; 7(1):3392). The results are as follows: Figure 8 As shown.

[0124] In summary, this invention uses the fungal defensin Plectasin as a template to design and modify the antimicrobial peptide Ple-AB. The gene encoding the antimicrobial peptide Ple-AB was successfully optimized, and the pPICZαA-Ple-AB recombinant expression vector was constructed. After linearization with PmeI, it was successfully transformed into Pichia pastoris X-33 to obtain a recombinant yeast strain. The antimicrobial activity of the purified antimicrobial peptide Ple-AB was tested, and the results showed that Ple-AB has good antimicrobial activity against Gram-positive bacteria. The stability of Ple-AB was tested using the MIC method. The results showed that Ple-AB maintained its antibacterial activity well under different temperatures, pH levels, and simulated gastric fluid environments, especially in simulated intestinal fluid environments, thus overcoming the trypsin resistance defect of most antimicrobial peptides (Yang N, Teng D, Mao RY, et al. A recombinant fungal defensin-like peptide-P2 combats multidrug-resistant Staphylococcus aureus and biofilms. Applied Microbiology and Biotechnology, 2019). Hemolytic assays showed that Ple-AB at concentrations ranging from 1 to 256 μg / mL produced almost no hemolysis of erythrocytes. Cytotoxicity assays showed that cell viability was above 80% at concentrations of 2-256 μg / mL. These experiments demonstrate that Ple-AB exhibits good antibacterial activity, high stability, high expression levels, and low toxicity, significantly increasing the potential for further clinical applications of antimicrobial peptides.

[0125] 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; and 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. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is any one of the following (1) to (2): (1) The amino acid sequence as shown in SEQ ID NO.1; (2) The sequence obtained by attaching a protein tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid molecule as described in claim 2 or 3; The biomaterial is an expression cassette, vector, or host cell.

5. A recombinant Pichia pastoris, characterized in that, The recombinant Pichia pastoris expresses the polypeptide of claim 1, or contains the nucleic acid molecule of claim 2 or 3, or an expression cassette or vector containing the nucleic acid molecule of claim 2 or 3.

6. The method for preparing the polypeptide according to claim 1, characterized in that, The method includes: using Pichia pastoris as a host cell to express the polypeptide of claim 1.

7. The application of the polypeptide of claim 1, the nucleic acid molecule of claim 2 or 3, or the biomaterial of claim 4 in the preparation of antibacterial products; in, The bacteria is Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus Staphylococcus epidermidis ( Staphylococcus epidermidis Streptococcus pyogenes ( ), Streptococcus dysgalactiae ) or alactamase-free streptococci ( Streptococcus agalactiae ).

8. The application according to claim 7, characterized in that, The antibacterial products are preservatives, bactericides, disinfectants, antiseptics, feed additives, food additives, or drugs.

9. The application of the polypeptide of claim 1, the nucleic acid molecule of claim 2 or 3, or the biomaterial of claim 4 in food preservation, antibacterial and preservative treatment of cosmetics, or environmental disinfection.

10. A drug, characterized in that, The drug comprises the polypeptide of claim 1.

11. A feed additive, characterized in that, The feed additive comprises the polypeptide as described in claim 1.

12. A bactericide or preservative, characterized in that, The bactericide or preservative comprises the polypeptide of claim 1.

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