Antifungal peptide MAF-1A derived peptide, derived peptide incubation and application thereof

Abstract

The present invention provides an antifungal peptide MAF-1A derivative peptide, a derivative peptide incubation and an application thereof, which belongs to the field of biotechnology and can have higher antibacterial activity and better stability in an actual medicinal environment, so that it can be applied to clinical fungal infection treatment and drug development. The antifungal peptide MAF-1A derivative peptide includes any one of the following: 8KGF7, 9GIF6, 10KLF6, 10GIF6, 3GGH0, 3VIH0, 12GGH5, 12VIH5; the derivative peptide incubation includes any one of the following: 10KLF6-Cu 2+ 、3GGH0‑Cu 2+ 、3VIH0‑Cu 2+ 、12GGH5‑Cu 2+ 、12VIH5‑Cu 2+ The applications include the use of the derivative peptides or derivative peptide incubations in inhibiting fungi, the use of the derivative peptides in preparing antifungal polypeptide drugs, and the use of the derivative peptides in preparing drugs for improving and treating invasive candidiasis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an antifungal peptide MAF-1A derivative peptide and application thereof. Background Art

[0002] Antifungal peptide MAF-1A (Musca domestica Antifungal Peptide-1A) is a cationic antifungal peptide isolated from housefly larvae. It is a 26-amino acid linear α-helical antimicrobial peptide with certain antifungal activity. Antifungal peptide MAF-1A is effective against a variety of Candida fungi, including Candida albicans (the most common pathogen of invasive candidiasis).

[0003] In the process of drug application of antifungal peptide MAF-1A, the antibacterial activity of antifungal peptide MAF-1A is not high and the stability is poor in the actual pharmaceutical environment. The above characteristics make the antifungal peptide MAF-1A have certain limitations in the treatment of clinical fungal infections and drug development. Summary of the invention

[0004] The present invention proposes an antifungal peptide MAF-1A derivative peptide, a derivative peptide incubation and an application thereof, which can have higher antibacterial activity and better stability in an actual medicinal environment, and thus can be applied to clinical fungal infection treatment and drug development.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides an antifungal peptide MAF-1A derivative peptide, wherein the amino acid sequence of the antifungal peptide MAF-1A derivative peptide comprises at least any one of the following: 8KGF7, as shown in SEQ ID NO: 1. 9GIF6, as shown in SEQ ID NO: 2. 10KLF6, as shown in SEQ ID NO: 3. 10GIF6, as shown in SEQ ID NO: 4. 3GGH0, as shown in SEQ ID NO: 5. 3VIH0, as shown in SEQ ID NO: 6.

[0007] In an implementation of the first aspect, the 10KLF6-derived peptide comprises at least any one of the following: 12GGH5, as shown in SEQ ID NO: 7; 12VIH5, as shown in SEQ ID NO: 8.

[0008] In a second aspect, the present invention provides an antifungal peptide MAF-1A derived peptide incubation product, the antifungal peptide MAF-1A derived peptide incubation product comprises at least any one of the following: 3GGH0-Cu 2+ , the amino acid sequence of 3GGH0 is shown in SEQ ID NO: 5. 3VIH0-Cu2+ The amino acid sequence of 3VIH0 is shown in SEQ ID NO: 6. 2+ , the amino acid sequence of 10KLF6 is shown in SEQ ID NO: 3. 12GGH5-Cu 2+ The amino acid sequence of 12GGH5 is shown in SEQ ID NO: 7. 2 , the amino acid sequence of 12VIH5 is shown in SEQ ID NO:8.

[0009] In a third aspect, the present invention provides use of the antifungal peptide MAF-1A derivative peptide described in the first aspect or the antifungal peptide MAF-1A derivative peptide incubated product described in the second aspect in inhibiting fungi.

[0010] In one implementation of the third aspect, the fungus includes Candida parapsilosis.

[0011] In a fourth aspect, use of the antifungal peptide MAF-1A derivative peptide described in the first aspect and / or the antifungal peptide MAF-1A derivative peptide incubated product described in the second aspect in the preparation of an antifungal polypeptide drug.

[0012] In an implementation of the fourth aspect, the antifungal polypeptide drug is administered in vitro or in vivo.

[0013] In a fifth aspect, the present invention provides use of the antifungal peptide MAF-1A derivative peptide described in the first aspect and / or the antifungal peptide MAF-1A derivative peptide incubated product described in the second aspect in the preparation of drugs related to improving and treating invasive fungal diseases.

[0014] The beneficial effects of the present invention are:

[0015] The present invention provides antifungal peptide MAF-1A derivative peptides 10KLF6, 8KGF7, 9GIF6, 10GIF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 based on the antifungal peptide MAF-1A as a template peptide. The derivative peptides 8KGF7, 9GIF6, 10KLF6, 10GIF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 have higher antibacterial activity and better stability than the antifungal peptide MAF-1A; Cu 2+ - Antifungal Peptide Incubation 10KLF6-Cu 2+ 、3GGH0-Cu 2+ 、3VIH0-Cu 2+ 、12GGH5-Cu 2+ and 12VIH5-Cu 2 , the above Cu 2+-The antifungal peptide incubation product can maintain antibacterial activity under different temperatures, pH, metal salt ions, fetal bovine serum and trypsin environments, and can reduce the hemolytic toxicity to human red blood cells, so it can be used in clinical fungal infection treatment and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the spiral wheel diagram of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6 provided in the examples of the present application;

[0017] Figure 2 It is the secondary structure prediction diagram of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6 provided in the examples of the present application;

[0018] Figure 3 It is the transmembrane information prediction map of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6 provided in the examples of the present application;

[0019] Figure 4 It is a schematic diagram of the spatial structure of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6 provided in the examples of the present application;

[0020] Figure 5 It is a schematic diagram of the membrane binding localization evaluation results of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6 provided in the examples of the present application;

[0021] Figure 6 It is a schematic diagram of the antibacterial rate of the derivative peptide 10KLF6 after trypsin treatment provided in the examples of the present application;

[0022] Figure 7 It is the spiral wheel diagram of MAF-1A, 10KLF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 provided in the examples of the present application;

[0023] Figure 8 It is the secondary structure prediction diagram of MAF-1A, 10KLF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 provided in the examples of the present application;

[0024] Fig. 9 It is a transmembrane information prediction map of MAF-1A, 10KLF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 provided in the examples of the present application;

[0025] Fig.10It is a schematic diagram of the spatial structure of MAF-1A, 10KLF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 provided in the embodiments of the present application;

[0026] Fig.11 It is a schematic diagram of the membrane binding localization evaluation results of MAF-1A, 10KLF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 provided in the examples of the present application;

[0027] Fig.12 This is a graph showing the results of a hemolysis experiment of MAF-1A, 10KLF6, 12GGH5, 12VIH5 and their derivative peptides incubated in the examples of the present application;

[0028] Fig.13 Schematic diagram of the antibacterial activity of MAF-1A, 12GGH5, 12VIH5 and their derivative peptide incubations at different temperatures provided in the examples of the present application;

[0029] Fig.14 Schematic diagram of the antibacterial activity of MAF-1A, 12GGH5, 12VIH5 and their derivative peptide incubations at different pH values ​​provided in the examples of the present application;

[0030] Fig.15 It is a schematic diagram of the antibacterial activity of MAF-1A, 12GGH5, 12VIH5 and their derivative peptide incubations at different NaCl concentrations provided in the examples of the present application;

[0031] Fig.16 It is a schematic diagram of the antibacterial activity of MAF-1A, 12GGH5, 12VIH5 and their derivative peptide incubations at different fetal bovine serum concentrations provided in the examples of the present application;

[0032] Fig.17 Schematic diagram of the antibacterial activity of MAF-1A, 12GGH5, 12VIH5 and their derivative peptide incubations at different trypsin concentrations provided in the examples of the present application. DETAILED DESCRIPTION

[0033] The terms "first" and "second" etc. in the description and claims of the present invention are used to distinguish different objects rather than to describe a specific order of the objects.

[0034] The “and / or” in the embodiments of the present application indicates the relationship between objects. For example, A and / or B may indicate the following three situations: A exists alone, B exists alone, and A and B exist at the same time.

[0035] In the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0036] In the examples of the present application, the antifungal peptide MAF-1A (Musca domestica Antifungal Peptide-1A) is a cationic antifungal peptide isolated from housefly larvae, a 26-amino acid linear α-helical antimicrobial peptide, and has certain antifungal activity. MAF-1A is effective against a variety of Candida fungi, including Candida albicans (the most common pathogen of invasive candidiasis).

[0037] The method and device provided in the embodiments of the present application relate to the field of biotechnology, and can design an antifungal peptide MAF-1A derivative peptide based on the antifungal peptide MAF-1A, which can further improve the antibacterial activity and stability of the antifungal peptide.

[0038] In the prior art, the antifungal peptide MAF-1A has low antibacterial activity and poor stability in an actual medicinal environment. The above characteristics make the antifungal peptide MAF-1A have certain limitations in clinical fungal infection treatment and drug development. In order to solve the above problems, the embodiments of the present application provide antifungal peptide MAF-1A derivative peptides, derivative peptide incubations and applications thereof, which can have higher antibacterial activity and better stability in an actual medicinal environment, so that they can be applied to clinical fungal infection treatment and drug development.

[0039] Example 1: This example describes an antifungal peptide MAF-1A derivative peptide, the amino acid sequence of which includes at least one of the following:

[0040] 8KGF7, as shown in SEQ ID NO: 1 (KGFKATALKLIWSAKWSLKSLAKAMK);

[0041] 9GIF6, as shown in SEQ ID NO: 2 (GIFKATALKLIWSAKWSLKSLAKAMK);

[0042] 10KLF6, as shown in SEQ ID NO: 3 (KLFKHTAHWLIWSAKKSLKSLAKAMC);

[0043] 10GIF6, as shown in SEQ ID NO: 4 (GIFKATALKLIWSAKWSHRSLAKAMK);

[0044] 3GGH0, as shown in SEQ ID NO: 5 (GGHKETADKLIESAKQQLESLAKEMK);

[0045] 3VIH0, as shown in SEQ ID NO: 6 (VIHKETADKLIESAKQQLESLAKEMK);

[0046] 10KLF6-derived peptides.

[0047] Among them, the 10KLF6-derived peptides include at least any one of the following:

[0048] 12GGH5, as shown in SEQ ID NO: 7 (GGHKHTAHWLIWSAKKSLKSLAKAMC);

[0049] 12VIH5, as shown in SEQ ID NO: 8 (VIHKHTAHWLIWSAKKSLKSLAKAMC).

[0050] 1. Design, preparation and verification of 8KGF7, 9GIF6, 10KLF6 and 10GIF6

[0051] In the embodiments of the present application, 8KGF7, 9GIF6, 10KLF6 and 10GIF6 in the above-mentioned antifungal peptide MAF-1A derivative peptides are designed by amino acid substitution method based on the antifungal peptide MAF-1A and prepared by FMOC solid phase synthesis method. The amino acid sequences of the antifungal peptide MAF-1A and the above-mentioned antifungal peptide MAF-1A derivative peptides are shown in the following Table 1.

[0052] Table 1: Amino acid sequence table of antifungal peptide MAF-1A and peptides derived from the antifungal peptide MAF-1A

[0053]

[0054] Note: The shaded areas in Table 1 are the positions where amino acid substitutions occurred in the polypeptide sequence.

[0055] As shown in Table 1, the amino acid sequence of 8KGF7 (i.e., SEQ ID NO: 1) is obtained by replacing lysine (Lys, K) at position 2 in the amino acid sequence with glycine (Gly, G), glutamic acid (Glu, E) at positions 5 and 24 with alanine (Ala, A), aspartic acid (Asp, D) at position 8 with leucine (Leu, L), Glu at position 12 and Glutamine (Gln, Q) at position 16 with the aromatic amino acid tryptophan (Trp, W), Gln at position 17 with serine (Ser, S), and Glu at position 19 with Lys.

[0056] The amino acid sequence of 9GIF6 (i.e., SEQ ID NO: 2) is based on the amino acid sequence of the antifungal peptide MAF-1A, by replacing Lys at position 1 in the amino acid sequence with Gly, replacing Lys at position 2 with isoleucine (Iie, I), replacing Glu at positions 5 and 24 in the amino acid sequence with Ala, replacing Asp at position 8 with Leu, replacing Glu at position 12 and Gln at position 16 with Trp, replacing Gln at position 17 with Ser, and replacing Glu at position 19 with Lys.

[0057] The amino acid sequence of 10KLF6 (i.e., SEQ ID NO: 3) is based on the amino acid sequence of the antifungal peptide MAF-1A, by replacing Lys at position 2 in the amino acid sequence with Leu, replacing Glu at position 5 and Asp at position 8 in the sequence with histidine (His, H), replacing Lys at position 9 and Glu at position 12 with Trp, replacing Gln at position 16 and Glu at position 19 with Lys, replacing Gln at position 17 with serine (Ser, S), replacing Glu at position 24 with Ala, and replacing Lys at position 26 with cysteine ​​(Cys, C).

[0058] The amino acid sequence of 10GIF6 (i.e., SEQ ID NO: 4) is based on the amino acid sequence of the antifungal peptide MAF-1A, by replacing Lys at position 1 in the amino acid sequence with Gly, replacing Lys at position 2 in the amino acid sequence with Leu, replacing Glu at positions 5 and 24 in the amino acid sequence with Ala, replacing Asp at position 8 with Leu, replacing Glu at position 12 and Gln at position 16 with Trp, replacing Gln at position 17 with Ser, replacing Leu at position 18 with His, and replacing Glu at position 19 with arginine (Arg, R).

[0059] The design and preparation process of the above-mentioned 8KGF7, 9GIF6, 10KLF6 and 10GIF6 are described in detail below.

[0060] Specifically, the design strategies of the above-mentioned 8KGF7, 9GIF6, 10KLF6 and 10GIF6 are: ① Increase the positive charge, replace the negatively charged amino acids in the sequence with positively charged amino acids, and control the number of positive charges in the sequence to about 6; ② Add tryptophan (Trp, W); ③ Increase the hydrophobicity value and increase the number of hydrophobic amino acids in the sequence (40% to 60%).

[0061] Specifically, the process of preparing the above 8KGF7, 9GIF6, 10KLF6 and 10GIF6 by FMOC solid phase synthesis method is as follows.

[0062] The FMOC solid phase synthesis method can be divided into the following steps of the cycle: 1. Deprotection: The Fmoc protected column and monomer must be treated with an alkaline solvent (piperidine) to remove the amino protecting group. 2. Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activating agent. The activated monomer reacts with the free amino group to cross-link and form a peptide bond. In this step, a large amount of superconcentrated reagents are used to drive the reaction to completion. Cycle: These two steps of reaction are repeated until the synthesis is completed. 3. Elution and deprotection: The polypeptide is eluted from the column, and its protecting group is eluted and deprotected by a deprotecting agent (TFA).

[0063] The synthesized peptides were purified by HPLC and verified by LC-MS, and the purity of the peptide synthesis was ≥95%.

[0064] The physicochemical properties, antibacterial activity and hemolytic ability of the above-mentioned 8KGF7, 9GIF6, 10KLF6 and 10GIF6 were verified below.

[0065] (1) Prediction of physical and chemical properties

[0066] Experimental methods: The protein structure prediction software ProtParam (https: / / web.expasy.org / protparam / ) from the protein biology information website ExPASy was used to analyze and predict the physicochemical parameters of antifungal peptides and derived peptides, including molecular weight, theoretical isoelectric point, amino acid composition ratio, charge number, instability index, fat index, and hydrophilicity index (Grand average of hydropathicity, GRAVY), in order to predict the charge number, cationicity, hydrophilicity and stability of MAF-1A and its derived peptides (8KGF7, 9GIF6, 10KLF6 and 10GIF6).

[0067] Experimental results: As shown in Table 2, the derivative peptides 8KGF7, 9GIF6, 10KLF6, 10GIF6 and the template peptide MAF-1A all contain 26 amino acid residues, and the net charge numbers of the four derivative peptides are higher than those of the template peptide MAF-1A, of which 8KGF7 carries a charge number of +7, and the charge numbers of the other three derivative peptides are +6. The theoretical isoelectric point, fat index and GRAVY index of the four derivative peptides are all higher than those of MAF-1A, and the instability index of the four derivative peptides is lower than that of MAF-1A.

[0068] Table 2: Physical and chemical properties of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6

[0069]

[0070] Note: Proteins or peptides with an instability index prediction value of less than 40 are predicted to be stable, and those with a prediction value greater than 40 may be unstable; GRAVY (Grand Average of Hydropathy) is the hydropathicity index, which is the sum of the hydropathic values ​​of all amino acids divided by the residues in the sequence. The larger the positive value of the GRAVY index, the stronger the hydrophobicity.

[0071] (2) Hydrophobicity and hydrophobic moment analysis

[0072] Experimental method: The Protean 3D tool in DNAstar software was used to predict the hydrophobicity of MAF-1A and its derivative peptides (8KGF7, 9GIF6, 10KLF6 and 10GIF6) and draw the helical wheels.

[0073] Experimental results: The results are shown in Table 3. The number of hydrophobic residues in the four derived peptide sequences increased. The number of hydrophobic residues in 8KGF7, 9GIF6, 10KLF6 and 10GIF6 were 15 (57.69%), 16 (61.54%), 14 (53.85%) and 15 (57.69%), respectively. The hydrophobic values ​​of the four derived peptides were higher than that of the template peptide MAF-1A, which were 0.418, 0.526, 0.514 and 0.465, respectively. The hydrophobic moments of the four derived peptides were smaller than that of MAF-1A, which were 0.276, 0.224, 0.272 and 0.203, respectively. The spiral wheel diagram is shown in Figure 1 As shown, Figure 1 (Pink, blue, orange, and red represent hydrophilic amino acids; green and black represent hydrophobic amino acids) It shows that the hydrophobic and hydrophilic amino acids of the four derived peptides are evenly distributed on both sides of the helix.

[0074] Table 3: Hydrophobicity of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6

[0075]

[0076]

[0077] (3) Secondary structure prediction

[0078] Experimental method: The secondary structure prediction tool HNN in NPS@ was used to predict the secondary structure of antifungal peptide MAF-1A and its derivative peptides. Nps@ is a protein sequence analysis tool based on artificial neural network operation mode on the PBEL server in Lyon, France. The HNN software uses periodic connection, global weight and other algorithms to accurately predict the secondary structure of proteins.

[0079] Experimental results: The prediction results are shown in Table 4 and Figure 2 As shown in Table 4 and Figure 2 ( Figure 2 In the peptide chain of 10GIF6, 21 amino acids (accounting for 80.77% of the total amino acids) form an α-helix structure, and the remaining 7 amino acids form a random coil, accounting for 26.92% of the total amino acids.

[0080] Table 4: Secondary structures of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6

[0081]

[0082] (4) Transmembrane information prediction

[0083] Experimental method: The TMHMM method (http: / / www.cbs.dtu.dk / services / TMHMM / ) was used to predict the transmembrane information of the antifungal peptide MAF-1A and its derivative peptides. TMHMM can accurately predict the transmembrane region and the inner and outer regions of the membrane based on the hydrophobicity, charge bias, helical length and other properties of the transmembrane region of the protein or polypeptide, using the hidden Markov model, and can be used to determine whether the detection object is a membrane protein.

[0084] Experimental results: Figure 3As shown, the derived peptides 8KGF7, 9GIF6, 10KLF6 and 10GIF6 did not form a transmembrane region, which was consistent with the template peptide MAF-1A. 8KGF7 and 9GIF6 were predicted to be located inside the cell membrane, and 10KLF6 and 10GIF6 were predicted to be located outside the cell membrane.

[0085] (5) Spatial structure simulation

[0086] Experimental method: PEP-FOLD3 online software was used to simulate the spatial structure of the antifungal peptide MAF-1A and its derivative peptides.

[0087] Experimental results: The results are as follows Figure 4 As shown, 8KGF7, 9GIF6, 10KLF6 and 10GIF6 are all linear peptides mainly composed of α-helices, and their structures are consistent with the template peptide MAF-1A.

[0088] (6) Membrane binding localization assessment

[0089] Experimental method: The PPM web server was used to predict the membrane binding localization information of the antifungal peptide MAF-1A and its derivative peptides.

[0090] Experimental results: As shown in Table 5 and Figure 5 As shown, the depth of the four derivative peptides inserted into the membrane is All of them are greater than the template peptide MAF-1A, among which 8KGF7 has the largest penetration depth of The number of residues embedded in the membrane is 16, and the minimum penetration depth of 9GIF6 is Only 9 residues are embedded in the membrane, while all 26 amino acids of 10KLF6 and 10GIF6 are embedded in the membrane.

[0091] Table 5: Membrane binding localization evaluation table of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6

[0092]

[0093] Note: The shaded areas in the table are amino acids embedded in the membrane in the polypeptide sequence

[0094] (7) Prediction of antifungal activity and hemolytic activity

[0095] Experimental methods: The Database of Antimicrobial Activity and Structure of Peptides (DBAASP) v3.0 web was used to predict the antifungal activity and hemolytic activity of the antifungal peptide MAF-1A and its derivative peptides.

[0096] Experimental results: As shown in Table 6, it was predicted that the template peptide MAF-1A had no activity against Candida albicans and human erythrocytes, and the four derived peptides (8KGF7, 9GIF6, 10KLF6 and 10GIF6) all had anti-Candida albicans activity and hemolytic activity.

[0097] Table 5: Prediction results of antifungal activity and hemolytic activity of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6

[0098]

[0099] Note: Candida albicans: Candida albicans; Human erythrocyte: Human erythrocyte. The MIC of peptides predicted to have antifungal activity is <25μg / mL, and the MIC of inactive peptides is >100μg / mL. Peptides predicted to have hemolytic activity are defined as active peptides (peptide length does not exceed 30 amino acids) if their concentration is <40μg / mL and if they induce hemolysis >40%.

[0100] (8) In vitro antifungal activity assay

[0101] Experimental methods:

[0102] ① Detection of MIC values ​​of MAF-1A and its derivative peptides against Candida parapsilosis

[0103] Pick a single colony of Candida parapsilosis ATCC 22019 to prepare a concentration of 1×10 3 ~5×10 3cfu / mL of bacterial suspension, 100 μL of bacterial suspension was added to each well of a sterile 96-well plate, and then 100 μL of antimicrobial peptide solutions of different concentrations were added and mixed. The concentration gradient of MAF-1A was set to 1.2 mg / mL, 1.1 mg / mL, 1.0 mg / mL, 0.9 mg / mL, 0.8 mg / mL, 0.7 mg / mL, 0.6 mg / mL, and 0.5 mg / mL, and the concentration gradient of the derived peptides 8KGF7, 9GIF6, 10KLF6, and 10GIF6 was set to 0.64 mg / mL, 0.32 mg / mL, 0.16 mg / mL, 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL, so that the volume of each well was 200 μL. The experiment used bacterial solution not treated with antimicrobial peptides as the growth control group (200 μL bacterial solution not treated with antimicrobial peptides) and a blank control group of 200 μL SDB medium, with 3 replicates in each group. The 96-well plate was placed in a 35°C constant temperature incubator, and after culturing for 24 hours, it was taken out and mixed by pipetting, and the absorbance value OD490nm was determined by an ELISA instrument. The percentage of fungal growth inhibition was calculated according to formula (1-1), and the lowest drug concentration with a percentage of fungal growth inhibition greater than 80% was taken as the MIC value in accordance with the CLSI M27-A3 standard.

[0104] Fungal growth percentage = (OD of each well 490nm - OD of blank control group wells 490nm ) / (OD of growth control well 490nm - OD of blank control group wells 490nm )×100%

[0105] Fungal growth inhibition percentage = (1-fungal growth percentage) × 100% formula (1-1)

[0106] ② MFC detection of MAF-1A and its derived peptides against Candida parapsilosis

[0107] In the above MIC test, 10 μL of culture was taken from each well of the 96-well plate cultured for 24 h, diluted 100 times in sterile saline, and 100 μL was evenly spread on the SDA plate. The plate was cultured at 35°C for 24 h, and the colonies were counted. The minimum drug concentration at which the number of colonies growing on the plate was less than 5 was taken as the minimum fungicidal concentration (MFC) value, with 3 replicates per group.

[0108] Experimental results: Table 6 shows that the MIC values ​​of 8KGF7, 9GIF6, 10KLF6 and 10GIF6 against Candida parapsilosis are 0.08 mg / mL, 0.16 mg / mL, 0.05 mg / mL and 0.16 mg / mL, respectively. Compared with the MIC value of the template peptide MAF-1A (0.6 mg / mL), the activities of the derivative peptides are increased by 7.5 times, 3.75 times, 12 times and 3.75 times, respectively. The activity of 10KLF6 is the largest increase compared with the template peptide MAF-1A, and it has obvious antifungal activity.

[0109] In the MFC determination experiment, the MFC values ​​of the derivative peptides 8KGF7, 9GIF6, 10KLF6 and 10GIF6 against Candida parapsilosis were 0.16 mg / mL, 0.32 mg / mL, 0.07 mg / mL and 0.16 mg / mL, respectively, which were lower than the MFC value of 0.70 mg / mL of the template peptide MAF-1A. Among them, the MFC value of the derivative peptide 10KLF6 was significantly reduced by 10 times.

[0110] Table 6: MFC measurement results of MAF-1A, 8KGF7, 9GIF6, 10KLF6 and 10GIF6

[0111]

[0112] (9) Determination of hemolytic activity of MAF-1A and its derivative peptide 10KLF6

[0113] The template peptide MAF-1A and the derivative peptide 10KLF6 with the highest antibacterial activity were selected for in vitro hemolysis experiments. The specific methods and results are as follows:

[0114] Experimental methods:

[0115] ① Use anticoagulant tubes to collect fresh blood from healthy people, transfer the blood to a 15mL centrifuge tube, and after stratification, aspirate the upper serum and the middle white blood cells and platelets and discard them. Aspirate the red blood cells at the bottom and transfer them to a 1.5mL EP tube, centrifuge at 3000rpm for 5min, discard the upper serum, and repeatedly rinse the red blood cell sediment three times with PBS buffer until there is no obvious light yellow color in the supernatant.

[0116] ② The washed erythrocytes were diluted to 8% (v / v) with PBS buffer and added to a sterile 96-well plate, 100 μL per well. Different gradients of MAF-1A and derivative peptide 10KLF6 solutions were prepared and added to the erythrocytes, 100 μL per well, and 3 replicates were set for each concentration, so that the final concentrations of MAF-1A were 1 / 3MIC, 1 / 2MIC, MIC, 2MIC, 3MIC (0.2 mg / mL, 0.3 mg / mL, 0.6 mg / mL, 1.2 mg / mL, 1.8 mg / mL), and the final concentrations of derivative peptide 10KLF6 were 1 / 3MIC, 1 / 2MIC, MIC, 2MIC, 3MIC (0.0167 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL), and the total volume of the liquid in each well was 200 μL. The experiment set up the positive control group as the reaction wells in which 100 μL 1% TritonX-100 solution replaced the peptide solution, and the negative control group as the reaction wells in which 100 μL PBS buffer replaced the peptide solution. After the 96-well plate was placed in a constant temperature incubator at 37°C for 1 hour, the liquid in each well was aspirated and transferred to a 1.5mL EP tube, centrifuged at 3000rpm for 5 minutes, the red blood cells were deposited at the bottom of the tube, 100 μL of the upper solution was aspirated and transferred to a new 96-well plate, and the absorbance value OD540nm was measured by an enzyme marker, and the hemolysis rate was calculated according to formula 1-2.

[0117] Hemolysis rate = (OD 540nm -PBS control group OD 540nm ) / (OD of 0.1% Triton X treated group 540nm PBS control group OD 540nm )×100% formula (1-2)

[0118] Experimental results: In vitro hemolysis experiments were conducted on the template peptide MAF-1A and its derivative peptides with the strongest antibacterial activity, 10KLF6. The results showed that MAF-1A caused hemolysis at a concentration of 1 / 2MIC, with a hemolysis rate of 0.56%. As the concentration increased, its hemolytic activity increased. The derivative peptide 10KLF6 caused hemolysis at a concentration of 1 / 3MIC, with a hemolysis rate of 10KLF6 of 19.11% at the MIC concentration, and as the concentration of 10KLF6 increased, its hemolytic activity became stronger (see Table 7 below).

[0119] Table 7: In vitro hemolysis test results of MAF-1A and 10KLF6

[0120]

[0121] (10) Enzymatic stability test of derivative peptide 10KLF6

[0122] Experimental method: The derivative peptide 10KLF6 with the strongest antibacterial activity in Experiment 2.10 was selected for the following experiment. Candida parapsilosis ATCC 22019 cultured to the logarithmic growth phase was cultured in SDB culture medium and the bacterial solution concentration was adjusted to 1.0×10 6 ~5.0×10 6 cfu / mL. 50 μL of the derivative peptide 10KLF6 solution with a final reaction concentration of MIC, 2MIC, 4MIC (0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL) and 50 μL of trypsin with a final reaction concentration of 1 mg / mL were mixed in a 37°C water bath and acted for 0 min, 20 min, 40 min, 60 min, 80 min, and 100 min. After the action time, 100 uL of the mixed solution of the derivative peptide and trypsin was taken into a sterile 96-well plate, and 100 uL of the test bacterial solution was added. The total volume of the liquid in each well was 200 μL. After culturing at 37°C for 24 hours, the 96-well plate was placed in a 35°C constant temperature incubator. After culturing for 24 hours, the absorbance value OD490nm was determined by an enzyme marker, and the fungal growth inhibition percentage was calculated according to formula (1-1).

[0123] Experimental results: Trypsin treatment affects the stability of the antibacterial activity of the derivative peptide 10KLF6. Figure 6 It can be seen that the antibacterial rates of 10KLF6 at MIC, 2MIC, and 4MIC concentrations were significantly reduced after trypsin treatment. After trypsin acted on 10KLF6 at different concentrations, as the treatment time increased, the antibacterial activity of 10KLF6 at different concentrations had no significant difference (P>0.05).

[0124] Summary: Based on the antifungal peptide MAF-1A, four derivative peptides were obtained by optimizing relevant structural parameters and combining bioinformatics software with the amino acid replacement method to design the charge number, hydrophobicity, α-helix, amphipathicity and other aspects of MAF-1A: 8KGF7, 9GIF6, 10KLF6, and 10GIF6. The results of the antibacterial activity experiment showed that the minimum concentration of the four peptides used to inhibit the growth of Candida parapsilosis was significantly lower than that of MAF-1A, and the antibacterial activity was significantly better than that of the template peptide MAF-1A, and they had the potential to be further developed into antifungal drugs. Among them, the MIC value of 10KLF6 was 12 times higher than that of the template peptide MAF-1A. The experimental results showed that the molecular modification of the antifungal peptide MAF-1A was effective in improving the antifungal activity of the peptide; however, the hemolytic activity of the derivative peptide 10KLF6 was increased compared with that of MAF-1A, and it increased with the increase of peptide concentration. It is speculated that the hydrophobicity and charge of the optimized derivative peptide increased significantly, which significantly improved the antibacterial activity of 10KLF6. The increased hydrophobicity would cause the peptide to aggregate itself, thereby preventing its transport and increasing the hemolytic activity.

[0125] 2. Design, preparation and verification of 3GGH0, 3VIH0, 12GGH5 and 12VIH5

[0126] In the embodiments of the present application, (1) 3GGH0 and 3VIH0 in the above-mentioned antifungal peptide MAF-1A derivative peptides are prepared by the FMOC solid phase synthesis method using the antifungal peptide MAF-1A as a template peptide, and adding copper-nickel binding motifs (ATCUN)-GGH and (ATCUN)-VIH to the N-terminus of the antifungal peptide MAF-1A, respectively; (2) 12GGH5 and 12VIH5 in the above-mentioned antifungal peptide MAF-1A derivative peptides are prepared by the FMOC solid phase synthesis method using 10KLF6 as a template peptide, and adding copper-nickel binding motifs (ATCUN)-GGH and (ATCUN)-VIH to the N-terminus of 10KLF6, respectively.

[0127] The amino acid sequence of 3GGH0 (i.e., SEQ ID NO: 5), the amino acid sequence of 3VIH0 (i.e., SEQ ID NO: 6), the amino acid sequence of 12GGH5 (i.e., SEQ ID NO: 7), and the amino acid sequence of 12VIH5 (i.e., SEQ ID NO: 8) are shown in Table 8 below.

[0128] Table 8: Amino acid sequence listing of MAF-1A, 3GGH0, 3VIH0, 10KLF6, 12GGH5 and 12VIH5

[0129]

[0130]

[0131] The preparation processes of the above-mentioned 3GGH0, 3VIH0, 12GGH5 and 12VIH5 are described in detail below.

[0132] MAF-1A and its derivative peptides 3GGH0, 3VIH0, antifungal peptide 10KLF6 and its derivative peptides 12GGH5, 12VIH5 are all linear peptides composed of 26 amino acid sequences: the amino acid sequence is as above. The company synthesized them using the FMOC solid phase synthesis method. The purity was determined to be greater than 95% by analytical high performance liquid chromatography (HPLC), and ESI mass spectrometry was performed to ensure the correctness of the peptide sequence. MAF-1A was dissolved to 4 mg / mL using sterile ultrapure water, and the remaining antifungal peptides were dissolved to 2 mg / mL and stored at -20°C.

[0133] The antibacterial activity and hemolytic ability of the above-mentioned 3GGH0, 3VIH0, 12GGH5 and 12VIH5 were verified below.

[0134] (1) Bioinformatics prediction analysis

[0135] As shown in Table 9 below, the charge number, relative molecular mass, and theoretical isoelectric point of the derivative peptides 3GGH0 and 3VIH0 are lower than those of the template peptide, and the instability index and hydrophilicity index are higher than those of the template peptide MAF-1A. The charge number, relative molecular mass, and theoretical isoelectric point of the derivative peptides 12GGH5 and 12VIH5 are lower than those of the template peptide 10KLF6, and the instability index is higher than that of the template peptide 10KLF6; the hydrophilicity index of 12GGHT5 is the same as that of the template peptide, and the hydrophilicity index of 12VIH5 is higher than that of the template peptide 10KLF6.

[0136] Table 9: Bioinformatics of MAF-1A, 3GGH0, 3VIH0, 10KLF6, 12GGH5 and 12VIH5

[0137]

[0138] Note: Proteins or peptides with an instability index prediction value of less than 40 are predicted to be stable, and those with a prediction value greater than 40 may be unstable; the hydrophilicity index is the sum of the hydrophilicity values ​​of all amino acids divided by the residues in the sequence. The larger the positive value of the hydrophilicity index, the stronger the hydrophobicity.

[0139] (2) Prediction of physical and chemical properties

[0140] Experimental method: According to the above methods in the examples of this application, the antimicrobial peptides MAF-1A, 10KLF6 and their derivative peptides were subjected to physicochemical properties, hydrophobicity and hydrophobic moment analysis, secondary structure prediction, transmembrane information prediction, spatial structure simulation and membrane binding localization evaluation.

[0141] Experimental results:

[0142] ① Hydrophobicity and helical wheel analysis results of antifungal peptides MAF-1A, 10KLF6 and derived peptides 3GGH0, 3VIH0, 12GGH5, 12VIH5:

[0143] The Protean 3D tool in DNASTAR software was used to analyze the hydrophobicity and hydrophobic moment of the derived peptides, and the helical wheel diagram was drawn. The results are shown in Table 10. The number of hydrophobic residues in the derived peptides 3GGH0 and 3VIH0 designed and modified with MAF-1A as the template peptide was 8 (30.77%) and 10 (38.46%), respectively; the number of hydrophobic residues in the derived peptides 12GGH5 and 12VIH5 designed and modified with 10KLF6 as the template peptide was 12 (46.15%) and 14 (53.85%), respectively. The hydrophobic residues of the derived peptide 12VIH5 were the same as those of the template peptide 10KLF6, and the hydrophobic residues of 12GGH5 were lower than those of the template peptide 10KLF6. The hydrophobic values ​​of the derivative peptides 3GGH0 and 3VIH0 were higher than those of the template peptide MAF-1A, which were 0.025 and 0.141, respectively. The hydrophobic value of the derivative peptide 12VIH5 was 0.539, which was higher than that of the template peptide 10KLF6, and the hydrophobic value of 12GGH5 was lower than that of 10KLF6. The hydrophobic moments of the derivative peptides 3GGH0 and 3VIH0 were lower than those of the template peptide MAF-1A, which were 0.442 and 0.376, respectively. The hydrophobic moments of the derivative peptides 12GGH5 and 12VIH5 were lower than those of the template peptide 10KLF6, which were 0.193 and 0.128, respectively. The helical wheel diagram showed that the hydrophobic and hydrophilic amino acids of the four derivative peptides were evenly distributed on both sides of the helix (e.g. Figure 7 As shown, pink, blue, orange, and red represent hydrophilic amino acids; green and black represent hydrophobic amino acids).

[0144] Table 10: Hydrophobic properties of MAF-1A, 3GGH0, 3VIH0, 10KLF6, 12GGH5 and 12VIH5

[0145]

[0146] ② Secondary structure prediction results of antifungal peptides MAF-1A, 10KLF6 and their derivative peptides 3GGH0, 3VIH0, 12GGH5, 12VIH5:

[0147] The secondary structure prediction tool HNN in NPS@ was used to analyze the secondary structures of antifungal peptide MAF-1A and its derivative peptides 3GGH0 and 3VIH0, antifungal peptide 10KLF6 and its derivative peptides 12GGH5 and 12VIH5. The prediction results showed that (see Tables 11 and Figure 8), the secondary structures of the antifungal peptide MAF-1A derived peptides 3GGH0, 3VIH0 and the antifungal peptide 10KLF6 derived peptides 12GGH5, 12VIH5 are mainly α-helical structures, and the remaining small areas are random coils, which are consistent with the template peptides MAF-1A and 10KLF6. The derived peptides 3GGH0, 3VIH0 and 12VIH5 all form α-helical structures at positions 3 to 23 in the peptide chain, accounting for 80.77% of the total number of amino acids; the derived peptide 12GGH5 forms an α-helical structure at positions 5 to 23 in the peptide chain, accounting for 73.08% of the total number of amino acids, and the rest is random coils.

[0148] Table 11: Secondary structures of MAF-1A, 3GGH0, 3VIH0, 10KLF6, 12GGH5 and 12VIH5

[0149]

[0150] ③ Prediction results of transmembrane information of antifungal peptides MAF-1A, 10KLF6 and their derivative peptides 3GGH0, 3VIH0, 12GGH5, 12VIH5:

[0151] The transmembrane information of MAF-1A and its derivative peptides 3GGH0 and 3VIH0, antifungal peptide 10KLF6 and its derivative peptides 12GGH5 and 12VIH5 were predicted by TMHMM Server v.20. The results are as follows Fig. 9 As shown, the derived peptides 3GGH0, 3VIH0, 12GGH5 and 12VIH5 did not form a transmembrane region, which was consistent with the template peptides MAF-1A and 10KLF6. 3GGH0, 3VIH0 and 12VIH5 were predicted to be located in the cell membrane, and 12GGH5 was predicted to be located outside the cell membrane.

[0152] ④ Spatial structure simulation results of antifungal peptides MAF-1A, 10KLF6 and their derivative peptides 3GGH0, 3VIH0, 12GGH5, 12VIH5:

[0153] The spatial structures of the antifungal peptide MAF-1A and its derivative peptides 3GGH0 and 3VIH0, the antifungal peptide 10KLF6 and its derivative peptides 12GGH5 and 12VIH5 were simulated using PEP-FOLD3 software. Fig.10 As shown, 3GGH0 and 3VIH0 are both linear peptides mainly composed of α-helix, and their structures are consistent with the template peptide MAF-1A; 12GGH5 and 12VIH5 are both linear peptides mainly composed of α-helix, and their structures are consistent with the template peptide 10KLF6.

[0154] ⑤ Evaluation results of membrane binding localization of antifungal peptides MAF-1A, 10KLF6 and their derivative peptides 3GGH0, 3VIH0, 12GGH5, 12VIH5:

[0155] The membrane binding localization of the antifungal peptide MAF-1A and its derivative peptides 3GGH0 and 3VIH0, and the antifungal peptide 10KLF6 and its derivative peptides 12GGH5 and 12VIH5 were evaluated by PPM web server. The results are shown in Tables 12 and Fig.11 As shown, the depth of MAF-1A derived peptides 3GGH0 and 3VIH0 inserted into the membrane is The depths of 10KLF6-derived peptides 12GGH0 and 12VIH0 inserted into the membrane are smaller than those of the template peptide MAF-1A. The maximum penetration depth of 12VIH5 is The number of residues embedded in the membrane is 19, and the minimum penetration depth of 3GGH0 is Only 9 residues are embedded in the membrane.

[0156] Table 12: Membrane binding localization evaluation results of MAF-1A, 3GGH0, 3VIH0, 10KLF6, 12GGH5 and 12VIH5

[0157]

[0158]

[0159] Note: The shaded areas in the table are the amino acids in the polypeptide sequence that are embedded in the membrane.

[0160] (3) Determination of antifungal activity against Candida parapsilosis

[0161] Experimental methods:

[0162] C. parapsilosis strain culture: Use a disposable inoculation loop to pick a single colony of C. parapsilosis ATCC22019 in the logarithmic growth phase and fully suspend it in 1 mL of SDB medium. Use a hemocytometer to prepare a concentration of 1×10 6 ~5×10 6 CFU / mL cell suspension was used for standby use. When used, the bacterial suspension was diluted 200 times with SDB medium and then diluted 10 times to adjust the concentration of the bacterial suspension to 0.5×10 3 ~2.5×10 3 CFU / mL.

[0163] The concentration was 0.5×10 3 ~2.5×10 3CFU / mL of Candida parapsilosis ATCC22019 bacterial suspension was added to a sterile 96-well plate, 100 μL per well, and then 100 μL of different concentrations of antifungal peptides MAF-1A, 10KLF6, 3GGH0, 3VIH0, 12GGH5, and 12VIH5 were added and mixed. The concentration gradient of MAF-1A was set to 1.0 mg / mL, 0.9 mg / mL, 0.8 mg / mL, 0.7 mg / mL, 0.6 mg / mL, 0.5 mg / mL, 0.4 mg / mL, 0. The concentration gradient of antifungal peptide 10KLF6 and derivative peptides 3GGH0, 3VIH0, 12GGH5, and 12VIH5 was set to 0.10 mg / mL, 0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.04 mg / mL, 0.03 mg / mL, 0.02 mg / mL, and 0.01 mg / mL, so that the final volume of each well was 200 μL.

[0164] The experiment used bacterial solution not treated with antimicrobial peptides as the growth control group (200 μL bacterial solution not treated with antimicrobial peptides) and blank control group as 200 μL SDB medium, with 3 replicates in each group. The 96-well plate was placed in a 35°C constant temperature incubator, cultured for 24 hours, taken out and mixed by pipetting, and the absorbance value OD490nm was measured by an ELISA instrument to determine the result. The percentage of fungal growth inhibition was calculated according to formula (3-1), and the lowest drug concentration at which the percentage of fungal growth inhibition was greater than 80% was the MIC value.

[0165] Fungal growth percentage = (OD of each well 490nm - OD of blank control group wells 490nm ) / (OD of growth control well 490nm - OD of blank control group wells 490nm )×100%

[0166] Fungal growth inhibition percentage = (1-fungal growth percentage) × 100% Formula (3-1)

[0167] In the above MIC test, 10 μL of culture was taken from each well of the 96-well plate cultured for 24 h, diluted 100 times in sterile saline, and 100 μL was evenly spread on the SDA plate. The plate was cultured at 35°C for 24 h, and the colonies were counted. The minimum drug concentration at which the number of colonies growing on the plate was less than 5 was taken as the minimum fungicidal concentration (MFC) value, with 3 replicates per group.

[0168] Experimental results: As shown in Table 13: The MIC values ​​of the derivative peptides 12GGH5 and 12VIH5 against Candida parapsilosis were 0.02 mg / mL and 0.03 mg / mL, respectively, which were 2.5 and 1.67 times higher than the antibacterial activity of 10KLF6. The MIC values ​​of the derivative peptides 3GGH0 and 3VIH0 against Candida parapsilosis were 0.7 mg / mL. 2+ In the presence of 5-nitropropene, the MIC values ​​of MAF-1A derived peptides 3GGH0 and 3VIH0 were both 0.1 mg / mL, which was 7 times higher than the antibacterial activity of the antimicrobial peptide alone. The MIC values ​​of 10KLF6 derived peptides 12GGH5 and 12VIH5 were 0.015 mg / mL and 0.02 mg / mL, respectively, and the antibacterial activity was 1.3 and 1.5 times higher than that of the antifungal peptide alone. In the MFC test, the drug concentration at which the number of colonies growing on the plate was less than 5 was the MFC value. The results showed that the MFC values ​​of MAF-1A derived peptides 3GGH0 and 3VIH0 were 1.6 mg / mL and 1.4 mg / mL, respectively; the MFC values ​​of 10KLF6 derived peptides 12GGH5 and 12VIH5 were both 0.04 mg / mL, which was 1.75 times higher than the antibacterial activity of the template peptide; in the presence of metal Cu 2+ In the presence of , the MFC values ​​of the derived peptides 3GGH0, 3VIH0, 12GGH5, and 12VIH5 were 0.2 mg / mL, 0.2 mg / mL, 0.03 mg / mL, and 0.04 mg / mL, respectively, which were lower than the antifungal peptide acting alone.

[0169] Table 13: Antifungal activity assay results of MAF-1A, 3GGH0, 3VIH0, 10KLF6, 12GGH5 and 12VIH5

[0170]

[0171] (4) Determination of the antifungal activity of the derivative peptide 12GGH5 against drug-resistant Candida albicans

[0172] Experimental methods: Four strains of Candida albicans resistant to multiple azole antifungal drugs, B420, B422, B1294, and B2294, were isolated from non-repeated specimens sent by Guizhou Provincial People's Hospital from March 2018 to August 2021. A single colony of Candida albicans in the logarithmic growth phase was picked up using a disposable inoculation loop and fully suspended in 1 mL of SDB medium. A blood cell counting plate was used to prepare a concentration of 0.5×10 3 ~2.5×10 3CFU / mL of cell suspension for later use, 100 μL of the prepared bacterial suspension was added to each well in a sterile 96-well plate, and then 100 μL of different concentrations of antifungal peptide 12GGH5 solution was added and mixed, and the concentration gradient of the derivative peptide 12GGH5 was set to 0.15 mg / mL, 0.14 mg / mL, 0.13 mg / mL, 0.12 mg / mL, 0.11 mg / mL, 0.10 mg / mL, 0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.04 mg / mL, 0.03 mg / mL, 0.02 mg / mL, 0.01 mg / mL, so that the final volume of each well was 200 μL. The experiment used the bacterial solution not treated with antimicrobial peptides as the growth control group (200 μL of bacterial solution not treated with antimicrobial peptides) and the blank control group as 200 μL SDB medium, with 3 replicates per group. Place the 96-well plate in a 35°C constant temperature incubator. After culturing for 24 hours, take it out and mix it by blowing. Measure the absorbance value OD with an enzyme-labeled instrument. 490nm The results were determined and the percentage of fungal growth inhibition was calculated according to formula (3-1), with the lowest drug concentration at which the percentage of fungal growth inhibition was greater than 80% being the MIC value.

[0173] In the above MIC test, 10 μL of culture was taken from each well of the 96-well plate cultured for 24 h, diluted 100 times in sterile saline, and 100 μL was evenly spread on the SDA plate. The plate was cultured at 35°C for 24 h, and the colonies were counted. The minimum drug concentration at which the number of colonies growing on the plate was less than 5 was taken as the minimum fungicidal concentration (MFC) value, with 3 replicates per group.

[0174] Experimental results: As shown in Table 14: The MIC values ​​of the derivative peptide 12GGH5 against Candida albicans B420, B422, B1294, and B2294 resistant to multiple azole antifungal drugs were 0.06 mg / mL, 0.06 mg / mL, 0.06 mg / mL, and 0.07 mg / mL, respectively. In the MFC test, the drug concentration at which the number of colony growth on the plate was less than 5 was the MFC value. The results showed that the MFC values ​​of the derivative peptide 12GGH5 against drug-resistant Candida albicans B420, B422, B1294, and B2294 were 0.13 mg / mL, 0.15 mg / mL, 0.15 mg / mL, and 0.15 mg / mL, respectively.

[0175] Table 14: MIC values ​​of 12GGH5 against Candida albicans B420, B422, B1294, and B2294 resistant to multiple azole antifungal drugs

[0176]

[0177] Summary: In the antifungal activity test, the antibacterial activity of the derivative peptides 12GGH5 and 12VIH5 was significantly improved. The MIC values ​​of the derivative peptides 12GGH5 and 12VIH5 against Candida parapsilosis were 0.02 mg / mL and 0.03 mg / mL, respectively, which were 2.5 and 1.67 times higher than the antibacterial activity of 10KLF6, and the MFC values ​​were both 0.04 mg / mL, which was 1.75 times higher than the antibacterial activity of the template peptide.

[0178] (5) Hemolytic activity assay

[0179] It should be noted that, although the above-mentioned 8KGF7, 9GIF6, 10KLF6, 10GIF6, 3GGH0 and 3VIH0 are derivative peptides designed based on the antifungal peptide MAF-1A, and the above-mentioned 12GGH5 and 12VIH5 are designed based on 10KLF6, since 10KLF6 is also a derivative peptide of the antifungal peptide MAF-1A, the above-mentioned 8KGF7, 9GIF6, 10KLF6, 10GIF6, 3GGH0, 3VIH0, 12GGH5 and 12VIH5 can all be called derivative peptides of the antifungal peptide MAF-1A.

[0180] Experimental methods

[0181] ① Collect red blood cells: Use anticoagulant tubes to collect fresh blood from healthy people, transfer the blood to a 15mL centrifuge tube, and after stratification, aspirate the upper serum and the middle white blood cells and platelets and discard them. Aspirate the red blood cells at the bottom layer and transfer them to a 1.5mL EP tube, centrifuge at 3000rpm for 5min, discard the upper serum, and repeatedly rinse the red blood cell sediment with PBS buffer three times until there is no obvious light yellow color in the supernatant.

[0182] ② Antifungal peptides act on erythrocytes: The rinsed erythrocytes were diluted to 8% (v / v) with PBS buffer and added to a sterile 96-well plate, 100 μL per well. MAF-1A, 10KLF6, 12GGH5 and 12VIH5 solutions with final reaction concentrations of 1 / 3MIC, 1 / 2MIC, MIC, 2MIC, and 3MIC were prepared respectively, and 3 replicates were set for each concentration. The experiment set up a positive control group (100 μL of 0.1% TritonX-100 + 100 μL erythrocytes) and a negative control group (100 μL sterile PBS solution + 100 μL erythrocytes).

[0183] ③ ELISA detection: Place the 96-well plate in a constant temperature incubator at 37°C for 1 hour, centrifuge at 3000 rpm for 5 minutes, and measure the absorbance value OD on a multifunctional ELISA reader. 540nm The hemolysis rate was calculated according to the following formula.

[0184] Hemolysis rate = (OD of antifungal peptide treated group540nm -PBS control group OD 540nm ) / (OD of 0.1% Triton X treated group 540nm -PBS control group OD 540nm )×100%

[0185] Experimental results: In the test of antifungal activity, the antibacterial activity of the derivative peptides 12GGH5 and 12VIH5 was significantly improved. On this basis, the antifungal peptides MAF-1A, 10KLF6, 12GGH5, and 12VIH5 were further tested for their hemolytic activity on healthy human erythrocytes. As shown in Table 15, when the antifungal peptide concentration was 1 / 3MIC, MAF-1A (0.2 mg / mL) did not produce hemolysis, and the derivative peptides all produced hemolysis, and as the peptide concentration increased, the hemolysis increased. At a concentration of 2MIC, the antifungal peptide 10KLF6 (0.1 mg / mL) caused 51.5% hemolysis, and 12GGH5 (0.04 mg / mL) caused 42.4% hemolysis. As the antibacterial activity of the MAF-1A derivative peptide increased, the hemolytic activity of the peptide on human erythrocytes also increased.

[0186] Table 15: Hemolytic activity assay results of MAF-1A, 10KLF6, 12GGH5 and 12VIH5

[0187]

[0188] In summary, compared with the antifungal peptide MAF-1A, the minimum inhibitory concentration (MIC) of the antifungal peptide MAF-1A derivative peptides against Candida parapsilosis increased by 3.75 to 12 times, and the minimum bactericidal concentration (MFC) increased by 2.18-10 times. Among them, 10KLF6 had the highest antibacterial activity, with a MIC value of 0.05 mg / mL and an MFC of 0.07 mg / mL, which were 12 and 10 times higher than the MIC value (0.6 mg / mL) and MFC value (0.7 mg / mL) of the template peptide MAF-1A, respectively.

[0189] Example 2: This example describes an antifungal peptide MAF-1A derived peptide incubation product, wherein the antifungal peptide MAF-1A derived peptide incubation product comprises any one of the following:

[0190] 10KLF6-Cu 2+ , the amino acid sequence of 10KLF6 is shown in SEQ ID NO:3.

[0191] 12GGH5-Cu 2+ , the amino acid sequence of 12GGH5 is shown in SEQ ID NO:7.

[0192] 12VIH5-Cu 2+, the amino acid sequence of 12VIH5 is shown in SEQ ID NO:8.

[0193] 3GGH0-Cu 2+ , the amino acid sequence of 3GGH0 is shown in SEQ ID NO:5.

[0194] 3VIH0-Cu 2+ , the amino acid sequence of 3VIH0 is shown in SEQ ID NO:6.

[0195] In the present application embodiment, the above 10KLF6-Cu 2+ Based on the 10KLF6 provided in Example 1, 10KLF6 and Cu 2+ The above 3GGH0-Cu 2+ Based on the 3GGH0 provided in Example 1, 3GGH0 and Cu 2+ The above 3VIH0-Cu 2+ Based on the 3VIHO provided in Example 1, 3VIHO and Cu 2+ The above 12GGH5-Cu 2+ Based on the 12GGH5 provided in Example 1, 12GGH5 and Cu 2+ The above 12VIH5-Cu 2 Based on the 12VIH5 provided in Example 1, 12VIH5 and Cu 2+ Combined obtained.

[0196] The following is the 10KLF6-Cu 2+ 、3GGH0-Cu 2+ 、3VIH0-Cu 2+ 、12GGH5-Cu 2+ 、12VIH5-Cu 2+ The preparation process is described in detail.

[0197] Use sterile ultrapure water to prepare 10mM Tris-HCl buffer (pH 7.0) to dissolve CuSO4 powder into 1mM CuSO4 solution, and adjust the pH to pH 7.0 for use. Use sterile ultrapure water to dissolve the antibacterial peptide powder, prepare the antifungal peptide MAF-1A into a 4mg / mL stock solution, and prepare the other antifungal peptides into a 2mg / mL stock solution for use. Mix the prepared CuSO4 solution with the antifungal peptide MAF-1A and its derivative peptides 3GGH0, 3VIH0, antifungal peptide 10KLF6 and its derivative peptides 12GGH5, 12VIH5 derivative peptide solutions, and make Cu 2+-The final concentration of the antifungal peptide incubator was MIC, and incubated for 12 h at 35°C in a constant temperature incubator to obtain Cu 2+ - Antifungal peptide incubations.

[0198] Since the design and preparation methods of 10KLF6, 3GGH0, 3VIH0, 12GGH5, and 12VIH5 have been given, they will not be described in detail in the embodiments of the present application.

[0199] The following is the 10KLF6-Cu 2+ 、3GGH0-Cu 2+ 、3VIH0-Cu 2+ 、12GGH5-Cu 2+ 、12VIH5-Cu 2+ The stability, antibacterial activity and hemolytic ability of the peptide were verified.

[0200] (1) Determination of antifungal activity against Candida parapsilosis

[0201] Experimental method: This application example is for 3GGH0-Cu 2+ 、3VIH0-Cu 2+ 、10KLF6-Cu 2+ 、12GGH5-Cu 2+ 、12VIH5-Cu 2+ The antifungal activity against Candida parapsilosis was determined by referring to the experimental method for 10KLF6, 3GGH0, 3VIH0, 12GGH5, and 12VIH5 in Example 1, which will not be described in detail here.

[0202] The experimental results are shown in Table 16.

[0203] Table 16: MAF-1A-Cu 2+ 、3GGH0-Cu 2+ 、3VIH0-Cu 2+ 、10KLF6-Cu 2+ 、12GGH5-Cu 2+ and 12VIH5-Cu 2+ Table of antifungal activity test results against Candida parapsilosis

[0204]

[0205] (2) Hemolytic activity assay

[0206] Experimental method: This application example is for MAF-1A-Cu 2+ 、10KLF6-Cu 2+ 、12GGH5-Cu 2+ 、12VIH5-Cu 2+The hemolytic activity of MAF-1A-Cu was determined in reference to Example 1. 2+ 、10KLF6-Cu 2+ The experimental methods of 12GGH5, 12VIH5 are not described here.

[0207] The experimental results are shown in Table 17 and Fig.12 The results are shown in (wells 1 to 5 and wells 8 to 12 are respectively single peptide treatment with concentrations of 1 / 3MIC, 1 / 2MIC, MIC, and 2MIC and Cu 2+ -peptide treatment, the positive control group was 0.1% TritionX-100, and the negative control group was PBS treatment group). Fig.12 As shown, Cu 2+ In the presence of 5-nitropropene, the hemolysis rates of the antifungal peptides 12GGH5 and 12VIH5 were reduced to varying degrees; at the MIC concentration, the hemolysis rates of the derivative peptides 12GGH5 and 12VIH5 (<5%) were both reduced, among which the hemolysis rate of the derivative peptide 12GGH5 decreased from 19.42% to 1.95%, and the hemolysis rate of the derivative peptide 12VIH5 decreased from 16.63% to 0.00%.

[0208] Table 17: MAF-1A-Cu 2+ 、10KLF6-Cu 2+ 、12GGH5-Cu 2+ and 12VIH5-Cu 2+ Hemolytic activity assay results

[0209]

[0210] (3) Physicochemical factors against fungal peptide MAF-1A, derivative peptides 12GGH5, 12VIH5 and their Cu 2+ - Effect of antifungal peptide incubation on antibacterial activity

[0211] I. Temperature

[0212] Experimental method: Take Candida parapsilosis ATCC22019 in the logarithmic growth phase and prepare a bacterial suspension of 1.0×10 3 ~5.0×10 3 CFU / mL for later use. 2+ 、12GGH5、12GGH5-Cu 2+ 、12VIH5、Cu 2+ -12VIH5 (final reaction concentration MIC) was treated at -80℃, -20℃, 4℃, 25℃, 37℃, and 100℃ for 30min, and then quickly cooled to room temperature. The mixture and an equal volume of the test bacterial solution were added to a 96-well plate and incubated at 37℃ for 24h before measuring the OD490nm The untreated antifungal peptide solution was used as the experimental control, the same volume of Candida parapsilosis solution was used as the growth control group, and the same volume of SDB medium was used as the blank control group. The antifungal peptides and their Cu at different temperatures were determined according to the MIC determination method. 2 + - Changes in the antimicrobial activity of antifungal peptide incubations.

[0213] Experimental results: The effect of temperature on the antibacterial activity of antifungal peptides was determined. The results showed that the antibacterial activity of antifungal peptides MAF-1A, 12GGH5, and 12VIH5 at MIC concentrations decreased at temperatures of -80°C, -20°C, 4°C, 60°C, and 100°C, and the antibacterial activity decreased to 47.29-73.75%. At 37°C, the antibacterial performance of MAF-1A-derived peptides remained above 80%, which was similar to the antibacterial activity of antifungal peptides in the control group. Cu 2+ In the presence of temperature, the effects of different structures on antifungal peptides are different, among which MAF-1A-Cu 2+ The antifungal activity of 12GGH5 was maintained above 80% when the temperature was -80℃, -20℃, 4℃, 60℃, and 100℃, and the antifungal activity of 12GGH5 was less than 70%, while the antifungal activity of 12VIH5 was less than 50% after treatment at -60℃ and 100℃ ( Fig.13 , A: MAF-1A and MAF-1A-Cu 2+ , B: 12GGH5 and 12GGH5-Cu 2+ , C: 12VIH5 and 12VIH5-Cu 2+ ).

[0214] II.PH

[0215] Experimental method: Sterile hydrochloric acid stock solution (1 mol / L HCl solution) and sodium hydroxide solution (1 mol / L NaOH solution) were diluted with sterile double distilled water, and the pH was adjusted to 3, 5, 7, 9, and 11 as reserve solutions. Different pH reserve solutions were prepared to dissolve the antifungal peptides MAF-1A and MAF-1A-Cu 2+ 、12GGH5、12GGH5-Cu 2+ , 12VIH5, 12VIH5-Cu 2+ After standing at room temperature for 1 hour, adjust the pH back to 7.0 (the purpose is to eliminate the interference of over-acidic and over-alkaline environments on bacterial growth), take 100 μL of the above mixed solution and 100 μL of the Candida parapsilosis liquid to be tested (concentration of 1.0×10 3 ~5.0×10 3 CFU / mL) were added to a 96-well plate and cultured in a 35°C incubator for 24 h before measuring OD 490nmThe untreated antifungal peptide solution was used as the experimental control, the same volume of bacterial solution was used as the growth control group, and the same volume of SDB medium was used as the blank control group. The changes in antibacterial activity were determined according to the MIC determination method. 2+ - Changes in the antimicrobial activity of antifungal peptide incubations.

[0216] Experimental results: The effect of pH on the antibacterial activity of antifungal peptides was determined. The results showed that compared with the control group, the antibacterial activity of antifungal peptides MAF-1A, 12GGH5, and 12VIH5 decreased at pH 3, 5, 9, and 11, among which the antibacterial activity of MAF-1A and 12VIH5 decreased to 56.31% to 74.92%, and significantly decreased at pH 3 (P < 0.01). The antibacterial activity of 12GGH5 was 76.54% to 88.31%, and there was no significant decrease in antibacterial activity (P > 0.05). Cu 2+ In the presence of MAF-1A-Cu 2 + The antibacterial activity of 12GGH5-Cu 2+ 、12VIH5-Cu 2+ The antibacterial activity remained above 75%, and there was no significant decrease in antibacterial activity (P>0.05) ( Fig.14 ).

[0217] III. Metal Salt Ions

[0218] Experimental methods:

[0219] ① NaCl against fungal peptides and Cu 2+ -Effect of antifungal peptides: Take the logarithmic growth phase culture of Candida parapsilosis ATCC22019 and adjust the bacterial solution concentration to 1.0×10 3 ~5.0×10 3 CFU / mL. Antifungal peptides MAF-1A and MAF-1A-Cu were diluted with sterile ultrapure water. 2+ 、12GGH5、12GGH5-Cu 2+ 、12VIH5、12VIH5-Cu 2+ , so that the final reaction concentration is MIC, then add NaCl solutions with concentrations of 50mM, 100mM and 150mM, and mix well; take 100μL of the mixed solution and mix it with 100μL of the Candida parapsilosis culture to be tested, use the antifungal peptide not treated with NaCl solution as the experimental control, and set up a growth control (200μL culture without any treatment) and a blank control (200μL SDB culture medium), and each group is placed in a 35℃ incubator for 24h and then the OD is measured 490nmThe MIC of antifungal peptides and their Cu in different NaCl concentrations was determined according to the MIC determination method. 2+ - Changes in the antimicrobial activity of antifungal peptide incubations.

[0220] ②CuSO4 against fungal peptides and its Cu 2+ - Effect of antifungal peptides: Antifungal peptides MAF-1A and MAF-1A-Cu were diluted in sterile ultrapure water 2+ 、12GGH5、12GGH5-Cu 2+ 、12VIH5、12VIH5-Cu 2+ , so that the final concentration of the reaction is MIC, add 0.5mM, 1mM, 1.5mM and 2mM CuSO4, mix well, take 100μL of the mixture and 100μL of the Candida parapsilosis liquid to be tested (1.0×10 3 ~5.0×10 3 CFU / mL) were added to a 96-well plate and mixed. The antifungal peptides not treated with CuSO4 solution were used as experimental controls, and growth controls (200 μL bacterial solution without any treatment) and blank controls (200 μL SDB culture medium) were set up. Each group was placed in a 35°C incubator for 24 hours and then the OD was measured. 490nm The MIC determination method was used to determine the antifungal peptides and their Cu 2+ - Changes in the antimicrobial activity of antifungal peptide incubations.

[0221] Experimental results: The results showed that compared with the peptide control group without NaCl treatment, when the NaCl concentration was 0.45%, the antibacterial activity of MAF-1A and 12VIH5 decreased to 55.29% and 59.47%, respectively. The activity of MAF-1A and 12VIH5 decreased significantly (P < 0.01), and the antibacterial activity of 12GGH5 was 72.63%, which was not significantly reduced (P > 0.05). 2+ In the presence of CuSO4, the antibacterial activities of 12GGH5 and 12VIH5 were 81.29% and 78.75%, respectively, with no significant difference (P>0.05), and the antibacterial activity of MAF-1A was 47.34% (P<0.01). The effects of different concentrations of CuSO4 on the antibacterial activities of 12GGH5 and 12VIH5 were determined. The results showed that with the increase of CuSO4 solution at a certain concentration (0.5mM-1mM), the antibacterial activity of antifungal peptides increased. When the concentration of CuSO4 was 1.5mM, the antibacterial activities of MAF-1A, 12GGH5, and 12VIH5 were 83.60%, 97.11%, and 95.18%, respectively. When the concentration was 2mM, the antibacterial activities of MAF-1A, 12GGH5, and 12VIH5 decreased to 71.85%, 91.74%, and 94.58%, respectively ( Fig.15, A, B, C represent the effects of NaCl on MAF-1A and MAF-1A-Cu 2+ , 12GGH5 and 12GGH5-Cu 2+ , 12VIH5 and 12VIH5-Cu 2+ D, E, and F represent the effects of CuSO4 on MAF-1A, 12GGH5, and 12VIH5, respectively; * indicates P < 0.05 compared with Control, and ** indicates P < 0.01 compared with Control).

[0222] IV. Fetal bovine serum

[0223] Experimental method: Take the culture of Candida parapsilosis ATCC22019 in the logarithmic growth phase and adjust the bacterial solution concentration to 1.0×10 3 ~5.0×10 3 CFU / mL. Antifungal peptides MAF-1A and MAF-1A-Cu were diluted with sterile ultrapure water. 2+ 、12GGH5、12GGH5-Cu 2+ , 12VIH5, 12VIH5-Cu 2+ The antifungal peptide was incubated with fetal bovine serum at a final concentration of 12.5%, 25%, and 50% for 4 h, and then 100 μL of the mixture was mixed with 100 μL of the test bacterial solution (1×10 3 ~5×10 3 CFU / mL) were added to a 96-well plate, gently blown to mix, and placed in a 35°C incubator for 24 hours. The antifungal peptides not treated with fetal bovine serum were used as experimental controls, and growth controls (200 μL bacterial solution without any treatment) and blank controls (200 μL SDB culture medium) were set up. The MIC determination method was used to determine the antifungal peptides and their Cu in different concentrations of fetal bovine serum. 2 + - Changes in the antimicrobial activity of antifungal peptide incubations.

[0224] Experimental results: Fig.16 (A: MAF-1A and MAF-1A-Cu 2+ , B: 12GGH5 and 12GGH5-Cu 2+ , C: 12VIH5 and 12VIH5-Cu 2+ ; * indicates P < 0.05 compared with Control,

[0225] **P<0.01), as shown in the figure, with the increase of fetal bovine serum concentration, the antibacterial activity of antifungal peptides decreased; compared with the peptide control group without fetal bovine serum, the antibacterial activity of MAF-1A, 12GGH5, and 12VIH5 decreased significantly (P<0.05) when the serum concentration was 50%, and the antibacterial activity decreased to 28.81%, 29.97%, and 7.64%, respectively. Cu 2+ In the presence of fetal bovine serum, the antibacterial activities of the antifungal peptides MAF-1A, 12GGH5, and 12VIH5 were similar to those of the peptides used alone, with antibacterial activities of 37.46%, 29.97%, and 7.93%, respectively.

[0226] V. Trypsin

[0227] Experimental method: 2 mg / mL trypsin solution was prepared with sterile PBS buffer; the antifungal peptides MAF-1A and MAF-1A-Cu 2+ 、12GGH5、12GGH5-Cu 2+ 、12VIH5、12VIH5-Cu 2+ Mix with an equal volume of trypsin solution and incubate in a 37°C incubator for 1 h. After the reaction is completed, take out the sample and place it in a 60°C oven for 15 min to inactivate the trypsin. Add 100 μL / well of the inactivated mixture to a 96-well plate, and add an equal volume of diluted Candida parapsilosis ATCC22019 bacterial solution (1.0×10 3 ~5.0×10 3 CFU / mL), gently blow and mix, and place three parallel wells in each group in a 35°C incubator for 24 hours. The control group was treated with antifungal peptide without trypsin treatment, and a growth control (200 μL bacterial solution without any treatment) and a blank control (200 μL SDB culture medium) were set up. The culture OD was measured using a multifunctional microplate reader 490nm According to the MIC determination method, trypsin was tested for its antifungal effects on peptides and their Cu 2+ - Changes in the antimicrobial activity of antifungal peptide incubations.

[0228] Experimental results: MAF-1A, 12GGH5, 12VIH5 and their Cu 2+ -After the antifungal peptide incubation interacted with 2mg / mL trypsin, the effect of trypsin on the antibacterial activity of the antifungal peptides was observed. The results showed that compared with the peptides not treated with trypsin, the antibacterial activities of MAF-1A, 12GGH5, and 12VIH5 after trypsin treatment were reduced to 36.24%, 43.97%, and 45.81%, respectively (P<0.01); Cu 2+ In the presence of MAF-1A-Cu 2+、12GGH5-Cu 2+ 、12VIH5-Cu 2+ The antibacterial activities of the derivative peptide 12GGH5-Cu 2+ The antibacterial activity was higher than that of 12GGH5 used alone and the difference was significant (P<0.05)( Fig.17 , A: MAF-1A and MAF-1A-Cu 2+ , B: 12GGH5 and 12GGH5-Cu 2+ , C: 12VIH5 and 12VIH5-Cu 2+ , *P<0.05 compared with the control group without trypsin treatment, **P<0.01 compared with the control group without trypsin treatment, # indicates the difference between single peptide and antifungal peptide-Cu 2+ treatment P<0.05).

[0229] In summary, by introducing Cu 2+ Interact with antimicrobial peptides, thereby reducing the hemolytic activity of antimicrobial peptides and improving stability: The antimicrobial activity research group previously analyzed the transcriptomics and proteomics data of Candida parapsilosis treated with MAF-1A and found that the zinc finger protein of the clinical strain of Candida parapsilosis was downregulated by 1.66 times after peptide treatment, suggesting that the protein may be closely related to the pathogenicity of Candida parapsilosis. 2+ It can replace the zinc ions in the zinc finger structure and destroy the structure of the zinc finger protein. 2+ In the presence of , the MIC values ​​of MAF-1A derived peptides 3GGH0 and 3VIH0 were both 0.1 mg / mL, which was 7 times higher than the antimicrobial peptide alone (0.7 mg / mL). The MIC values ​​of 10KLF6 derived peptides 12GGH5 and 12VIH5 were 0.015 mg / mL and 0.02 mg / mL, respectively, which were 1.3 and 1.5 times higher than the antifungal peptide alone. 2+ In the presence of MAF-1A, the MFC values ​​of MAF-1A-derived peptides 3GGH0 and 3VIH0 were both 0.2 mg / mL, and their antibacterial activities were 8 and 7 times higher, respectively, than those of the antifungal peptides acting alone; the MFC value of the derived peptide 12GGH5 was 0.03 mg / mL, and its antibacterial activity was 1.3 times higher than that of the antifungal peptides acting alone; the MFC value of 12VIH5 was 0.04 mg / mL, and its antibacterial activity was the same as that of the antifungal peptides acting alone.

[0230] Example 3: This example describes the use of the antifungal peptide MAF-1A derivative peptide provided in Example 1 in inhibiting fungi. The fungi include Candida parapsilosis.

[0231] Example 4: This example describes the use of the antifungal peptide MAF-1A derivative peptide incubation provided in Example 2 in inhibiting fungi. The fungi include Candida parapsilosis.

[0232] Example 5: This example describes the use of the antifungal peptide MAF-1A derivative peptide provided in Example 1 in the preparation of an antifungal polypeptide drug. The antifungal polypeptide drug is for in vitro or in vivo administration.

[0233] Example 6: This example describes the use of the antifungal peptide MAF-1A derivative peptide incubation provided in Example 2 in the preparation of an antifungal polypeptide drug. The antifungal polypeptide drug is for in vitro or in vivo use.

[0234] Example 7: This example describes the use of the antifungal peptide MAF-1A derivative peptide provided in Example 1 in the preparation of drugs related to improving and treating invasive fungal diseases.

[0235] Example 8: This example describes the use of the antifungal peptide MAF-1A derivative peptide incubation provided in Example 2 in the preparation of drugs related to improving and treating invasive fungal diseases.

[0236] Example 9: This example describes the use of the antifungal peptide MAF-1A derivative peptide provided in Example 1 in the preparation of drugs for improving and treating superficial fungal infections.

[0237] Example 10: This example describes the use of the antifungal peptide MAF-1A derivative peptide incubation provided in Example 2 in the preparation of drugs related to improving and treating superficial fungal infections.

[0238] Example 11: This example describes the use of the antifungal peptide MAF-1A derivative peptide provided in Example 1 and the antifungal peptide MAF-1A derivative peptide incubation provided in Example 2 in the preparation of antifungal polypeptide drugs.

[0239] Example 12: This example describes the use of the antifungal peptide MAF-1A derivative peptide provided in Example 1 and the antifungal peptide MAF-1A derivative peptide incubation provided in Example 2 in the preparation of drugs related to improving and treating invasive fungal diseases.

[0240] Example 13: This example describes the use of the antifungal peptide MAF-1A derivative peptide provided in Example 1 and the antifungal peptide MAF-1A derivative peptide incubation provided in Example 2 in the preparation of drugs related to improving and treating superficial fungal infections.

[0241] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0242] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. Antifungal peptide MAF-1A derivative peptide, characterized in that The amino acid sequence of the antifungal peptide MAF-1A derived peptide includes at least any one of the following: 8KGF7, as shown in SEQ ID NO: 1; 9GIF6, as shown in SEQ ID NO: 2; 10KLF6, as shown in SEQ ID NO: 3; 10GIF6, as shown in SEQ ID NO: 4; 3GGH0, as shown in SEQ ID NO: 5; 3VIH0, as shown in SEQ ID NO: 6; 10KLF6-derived peptides.

2. The antifungal peptide MAF-1A derivative peptide according to claim 1, characterized in that The 10KLF6 derivative peptide includes at least any one of the following: 12GGH5, as shown in SEQ ID NO: 7; 12VIH5, as shown in SEQ ID NO:

8.

3. Antifungal peptide MAF-1A derived peptide incubation, characterized in that, The antifungal peptide MAF-1A derived peptide incubation material comprises at least any one of the following: 3GGH0-Cu 2+ , the amino acid sequence of 3GGH0 is shown in SEQ ID NO: 5; 3VIH0-Cu 2+ , the amino acid sequence of 3VIH0 is shown in SEQ ID NO: 6; 10KLF6-Cu 2+ , the amino acid sequence of 10KLF6 is shown in SEQ ID NO: 3; 12GGH5-Cu 2+ , the amino acid sequence of 12GGH5 is shown in SEQ ID NO: 7; 12VIH5-Cu 2 , the amino acid sequence of 12VIH5 is shown in SEQ ID NO:

8.

4. Use of the antifungal peptide MAF-1A derivative peptide according to claim 1 or 2 or the antifungal peptide MAF-1A derivative peptide incubation according to claim 3 in inhibiting fungi.

5. The use according to claim 4, characterized in that The fungi include Candida parapsilosis.

6. Use of the antifungal peptide MAF-1A derivative peptide according to claim 1 or 2 and / or the antifungal peptide MAF-1A derivative peptide incubation product according to claim 3 in the preparation of antifungal polypeptide drugs.

7. The use according to claim 6, characterized in that The antifungal polypeptide drug is for in vitro or in vivo administration.

8. Use of the antifungal peptide MAF-1A derivative peptide according to claim 1 or 2 and / or the antifungal peptide MAF-1A derivative peptide incubation product according to claim 3 in the preparation of drugs related to improving and treating invasive fungal diseases.

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