Fusarium oxysporum targeting antifungal peptide as well as preparation method and application thereof

Abstract

The invention discloses an antifungal peptide targeting fusarium oxysporum as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. According to the present invention, the polypeptide specifically bound with Fusarium oxysporum is obtained through screening, and the sequence of the polypeptide is WYHRSHPQWNHW; the antifungal peptide WY-66-10 is formed by hybridizing and fusing the targeted structural domain and the weak-activity antibacterial peptide 66-10, the antifungal peptide WY-66-10 can kill fusarium oxysporum in a targeted manner, the minimum inhibitory concentration is improved by 30 times or more compared with that of the original polypeptide 66-10, and the targeted antifungal peptide WY-66-10 still keeps relatively weak cytotoxicity and hemolytic activity at the concentration of 128 mu M, and can be used for preparing the antifungal peptide WY-66-10. The method has good application values of targeting specific microorganisms, saving antibacterial resources and maintaining micro-ecological balance.

Description

Technical Field

[0001] The present application relates to an antifungal peptide targeting Fusarium oxysporum and a preparation method and application thereof, belonging to the field of microbial technology. Background Art

[0002] Globally, the total number of deaths from fungal infections has risen to 3.75 million per year, double the previous estimate. Fungal diseases account for 10%-23% of crop losses each year. Agricultural productivity and global food security are affected by various plant pathogens, and filamentous fungi are a growing threat. Pathogenic fungi such as Fusarium and Aspergillus not only cause food mold but also produce mycotoxins such as aflatoxins, zearalenone, and ochratoxins, threatening food and feed safety. Fusarium oxysporum is a widespread soil-borne pathogen that severely affects more than 100 plant species, such as bananas, potatoes, cotton, and tomatoes, causing huge economic losses worldwide.

[0003] Currently, chemical fungicides remain the most effective weapon for controlling fungal contamination, but host toxicity, antifungal resistance, and environmental pollution limit their sustainable application. The escalating incidence of antifungal resistance also poses a huge threat and challenge to food safety and human health. In countries including China and the United States, the use of fungicides has increased significantly by 4 times. The degradation half-life of most fungicides is 45-120 days, which gives opportunistic fungal pathogens enough time to develop antifungal resistance. Among them, fungicide resistance has been reported in Fusarium species around the world, including triazoles, strobilurins, and benzimidazoles. The development of new, safe, efficient, and environmentally friendly alternatives to antifungal drugs has become a hot topic of current research.

[0004] Antimicrobial peptides (AMPs) are a class of short-chain polypeptides that can effectively fight against a variety of pathogens such as bacteria, fungi and viruses. They have many advantages such as safety, low toxicity, high efficiency and broad spectrum, and can be basically divided into two categories: natural and synthetic. Antimicrobial peptides have little impact on the evolution of drug resistance in pathogens and can be used as a new sustainable, safe and environmentally friendly alternative to traditional fungicides. At present, the research on antimicrobial peptides is mainly aimed at bacteria, and there is still room for improvement in the bactericidal effect on fungi, especially filamentous fungi, including antimicrobial activity, biological activity, toxicity, bioavailability and stability. In addition, broad-spectrum antimicrobial activity means indiscriminate inhibition or killing of beneficial and harmful microorganisms, which is very likely to destroy the food microbiome and ecological balance. Therefore, the development of targeted antimicrobial peptides that can effectively and accurately inhibit harmful fungi has important theoretical value for ensuring food security, saving antimicrobial resources and maintaining microecological balance. Summary of the Invention

[0005] To solve the above problems, the present invention provides a Fusarium oxysporum targeted binding peptide WYHRSHPQWNHW (named WY), and connects the targeted binding peptide WY with the antifungal peptide 66-10 (FRLKFH) to obtain the targeted antifungal peptide WYHRSHPQWNHWFRLKFH (named WY-66-10) with better antibacterial performance.

[0006] The first object of the present invention is to provide a targeting binding peptide of Fusarium oxysporum, wherein the amino acid sequence of the targeting binding peptide is shown as SEQ ID NO.1.

[0007] In one embodiment of the present invention, the amino acid sequence of the targeting binding peptide (WY) is: Trp-Tyr-His-Arg-Ser-His-Pro-Gln-Trp-Asn-His-Trp (i.e., WYHRSHPQWNHW, the amino acid sequence is shown in SEQ ID NO. 1).

[0008] In one embodiment of the present invention, the Fusarium oxysporum is Fusarium oxysporum CGMC 3.18025.

[0009] The second object of the present invention is to provide a targeted antifungal peptide, which is obtained by linking the above-mentioned targeting binding peptide (ie, WY) with an antifungal peptide.

[0010] In one embodiment of the present invention, the amino acid sequence of the antifungal peptide is shown as SEQ ID NO.2.

[0011] In one embodiment of the present invention, the amino acid sequence of the targeted antifungal peptide is as shown in SEQ ID NO. 3, namely WYHRSHPQWNHWFRLKFH (named WY-66-10).

[0012] In one embodiment of the present invention, the amino acid sequence of the targeted antifungal peptide is: Trp-Tyr-His-Arg-Ser-His-Pro-Gln-Trp-Asn-His-Trp-Phe-Arg-Leu-Lys-Phe-His (i.e., WYHRSHPQWNHWFRLKFH, the amino acid sequence is shown in SEQ ID NO. 3).

[0013] The third object of the present invention is to provide a recombinant microbial cell, which expresses any of the above-mentioned targeted binding peptides or targeted antifungal peptides.

[0014] In one embodiment, the recombinant microbial cell uses Escherichia coli, Bacillus subtilis, or yeast as a carrier.

[0015] The fourth object of the present invention is to provide the use of the above-mentioned targeted binding peptide or the above-mentioned targeted antifungal peptide or the above-mentioned microorganism in the preparation of a drug for inhibiting fungi.

[0016] In one embodiment of the present invention, the fungus is Fusarium oxysporum, Aspergillus fumigatus, Aspergillus ochraceus, specifically Fusarium oxysporum CGMCC3.18025, Aspergillus fumigatus, Aspergillus ochraceus CGMCC3.1364.

[0017] A fifth object of the present invention is to provide a fungus-inhibiting drug comprising the above-mentioned antifungal peptide or the above-mentioned recombinant microbial cell.

[0018] In one embodiment of the present invention, the fungus is Fusarium oxysporum, Aspergillus fumigatus, Aspergillus ochraceus, specifically Fusarium oxysporum CGMCC3.18025, Aspergillus fumigatus, Aspergillus ochraceus CGMCC3.1364.

[0019] In one embodiment of the present invention, the pharmaceutical dosage form is a liquid, a solid, or a spray.

[0020] The sixth object of the present invention is to provide a method for improving the antibacterial properties of a polypeptide, wherein the above-mentioned targeted binding peptide is linked to an antifungal peptide to obtain a targeted antifungal peptide;

[0021] The amino acid sequence of the antifungal peptide is shown in SEQ ID NO.2.

[0022] In one embodiment of the present invention, the antibacterial agent is the inhibition of Fusarium oxysporum, specifically Fusarium oxysporum CGMCC3.18025.

[0023] In one embodiment of the present invention, the targeted binding peptide WY can specifically recognize Fusarium oxysporum F. oxysporum (CGMCC3.18025) without causing a significant response to other species of fungi, and its affinity is 7.70 times that of the BSA (bovine serum albumin) control and 7.07 times that of the OVA (chicken ovalbumin) control.

[0024] In one embodiment, the targeted antifungal peptide WY-66-10 has specific recognition ability and increases the antibacterial activity of the original antibacterial short peptide by more than 30 times.

[0025] In one embodiment of the present invention, the targeted antifungal peptide WY-66-10 exhibits antifungal activity against F. oxysporum CGMCC3.18025 (MIC) without affecting other bacteria or fungi.

[0026] In one embodiment of the present invention, the targeted antifungal peptide WY-66-10 exhibited rapid bactericidal activity at a concentration of 4× the MIC, killing approximately 75% of fungal spores within 4 hours. The total number of spores decreased with prolonged exposure, reaching a plateau after 8 hours. WY-66-10 exhibited even greater bactericidal efficacy at a concentration of 8× the MIC, reaching 96% after 24 hours of exposure at 8× the MIC.

[0027] In one embodiment of the present invention, the targeted antifungal peptide WY-66-10 showed no toxicity to mouse macrophages RAW246.7 within the detection range, and the cell survival rate was still higher than 80% at a concentration of 128 μM.

[0028] In one embodiment of the present invention, the targeted antifungal peptide WY-66-10 has little effect on cell hemolysis.

[0029] In one embodiment of the present invention, physiological saline solutions with different concentrations of NaCl, KCl, MgCl2, ZnCl2 and FeCl3 have little effect on the antibacterial effect of the antifungal peptide WY-66-10.

[0030] In one embodiment of the present invention, different pH values ​​have little effect on the antibacterial effect of the targeted antifungal peptide WY-66-10.

[0031] The present invention provides a method for screening polypeptides specifically binding to F. oxysporum by utilizing a phage display library and preparing targeted antifungal peptides.

[0032] In one embodiment, the preparation method of the targeted antifungal peptide WY-66-10 is as follows:

[0033] (1) A short peptide WY with affinity for F. oxysporum was obtained by screening a phage display peptide library (Ph.D.TM.12Phage display peptide library), the sequence of which is shown in SEQ ID NO. 1. This short peptide has the function of binding to F. oxysporum but does not have bactericidal activity;

[0034] (2) Based on the structure-function relationship of broad-spectrum antimicrobial peptides, the recognition peptide WY was linked to a weakly active peptide 66-10, whose sequence is shown in SEQ ID NO. 2, to obtain a targeted antifungal peptide against F. oxysporum, whose sequence is shown in SEQ ID NO. 3. This method can synthesize antimicrobial peptides that can exert a targeted killing effect on F. oxysporum without affecting other bacteria and fungi.

[0035] (3) The peptide was prepared by solid-phase chemical synthesis, followed by reverse-phase high-performance liquid chromatography purification and mass spectrometry identification. The peptide was finally named WY-66-10 after testing and verifying its antimicrobial activity, biosafety, and stability.

[0036] Beneficial effects

[0037] (1) The present invention uses phage display biopanning technology to screen a phage 12-peptide ligand, WY (WYHRSHPQWNHW), for F. oxysporum from a phage library with high affinity and specificity. The screened WY (WYHRSHPQWNHW) showed no significant binding ability to a variety of bacteria and fungi, but exhibited an affinity for F. oxysporum that was 7.70 times that of the BSA control group and 7.07 times that of the OVA control group.

[0038] (2) The targeted antifungal peptide WY-66-10 modified by this method has high cell selectivity. Antifungal activity testing showed that WY-66-10 had a significant inhibitory effect on F.oxysporum and had very low cytotoxicity (cell survival rate was still higher than 80% at 128μM) and hemolytic activity (hemolysis rate of mouse erythrocytes at 128μM was ≤8.59%). Compared with the original peptide 66-10 (FRLKFH), the targeted antifungal peptide WY-66-10 not only changed the antibacterial spectrum and had no antibacterial activity against 8 fungi such as Aspergillus flavus, Aspergillus ochraceus, and Penicillium expansum, but also increased the antibacterial therapeutic index by more than 30 times. In summary, the antimicrobial peptide WY-66-10 is a targeted antifungal peptide with high application value.

[0039] (3) A targeted antifungal peptide against F. oxysporum was designed to ensure that it kills specific harmful fungi without affecting other beneficial bacteria and fungi, which is conducive to efficient sterilization while maintaining the balance of microbial ecology. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Figures 2 and 3 show peptide enrichment during four rounds of bio-panning and positive clones identified by ELISA for binding to F.oxysporum. (A) Peptide enrichment during four rounds of bio-panning; (B) Positive clones identified by ELISA for binding to F.oxysporum.

[0041] Figure 2 The specificity of 6 phage-displayed peptides to various bacteria and fungi; (A) fungal specificity; (B) bacterial specificity;

[0042] Figure 3 Chromatogram and ESI-MS spectrum of phage-displayed peptide pep1; (A) Chromatogram of phage-displayed peptide pep1; (B) ESI-MS spectrum of phage-displayed peptide pep1;

[0043] Figure 4 This is the specificity map of pep1 for 9 fungi;

[0044] Figure 5 The chromatogram and ESI-MS spectrum of antimicrobial peptide 66-10; (A) Chromatogram of antimicrobial peptide 66-10; (B) ESI-MS spectrum of antimicrobial peptide 66-10;

[0045] Figure 6 The chromatogram and ESI-MS spectrum of the targeted antifungal peptide WY-66-10; (A) Chromatogram of the targeted antifungal peptide WY-66-10; (B) ESI-MS spectrum of the targeted antifungal peptide WY-66-10;

[0046] Figure 7 Minimum inhibitory concentration (MIC) and bactericidal kinetics curve of the targeted antifungal peptide WY-66-10 against 9 fungi; (A) Minimum inhibitory concentration (MIC) of the targeted antifungal peptide WY-66-10 against 9 fungi; (B) Bactericidal kinetics curve;

[0047] Figure 8 is the cytotoxicity graph of the targeted antifungal peptide WY-66-10;

[0048] Figure 9 Figure 2 is the hemolytic activity of the targeted antifungal peptide WY-66-10;

[0049] Figure 10 Figure 2 shows the stability of the targeted antifungal peptide WY-66-10; (A) physiological salt stability of the targeted antifungal peptide WY-66-10; (B) acid-base stability. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples, where specific conditions are not specified, generally follow conventional conditions in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

[0051] In the following examples, Escherichia coli ATCC25922, Staphylococcus aureus ATCC43300, Salmonella Typhimurium ATCC14028, Aspergillus flavus CMCC3.2890, Aspergillus parasiticus CGMCC3.1364, Fusarium proliferatum CGMCC3.14301, and Fusarium oxysporum CGMCC3.18025 were obtained from the China Center for the Collection of General Microorganisms.

[0052] Aspergillus ochraceus, Aspergillus fumigatus, Penicillium expansum, Penicillium digitatum, and Fusarium graminearum are strains preserved in the laboratory of Jiangnan University.

[0053] The detection methods involved in the following embodiments are as follows:

[0054] 1. Indirect ELISA method

[0055] (1) Add 100 μL of 1×10 7 F.oxysporum spores were coated at 4°C for 12 h, and an equal amount of 3% BSA was used as a negative control;

[0056] (2) Remove the spore suspension and wash the plate three times with 0.05% PBST. Add 300 μL of 3% BSA to each well and incubate at room temperature for 1 h to block nonspecific binding to the plate.

[0057] (3) Remove the blocking solution, wash the plate six times with 0.05% PBST, add 100 μL of monoclonal phage, and incubate at room temperature for 2 h.

[0058] (4) The monoclonal phage suspension was discarded, and the plate was washed six times with 0.05% PBST. 100 μL of HRP-labeled anti-M13 diluted antibody (1:5000, Pharmacia #27-9411-01 / Sigma-Aldrich, Burlington, MA, USA) was added to each well. The plate was shaken at room temperature for 1 h and then washed six times with 0.05% PBST.

[0059] (5) Prepare HRP substrate solution as follows: ABTS stock solution can be prepared in advance: dissolve 22 mg of ABTS (Sigma #A1888) in 100 mL of 50 mM sodium citrate solution, filter sterilize, and store at 4°C. For each plate to be tested, add 36 μL of 30% H2O2 to 21 mL of ABTS stock solution before the test step. Add 200 μL of substrate solution to each well and incubate at room temperature for 15 minutes. Add H2SO4 stop solution and incubate at room temperature for 10-15 minutes. Record the absorbance at 405 nm using a microplate reader.

[0060] 2. Phage titer determination

[0061] (1) Inoculate a single colony of E. coli ER2738 into 5-10 mL of LB medium and culture on a shaker until mid-logarithmic phase (OD600 ~ 0.5). Prepare 7.5% top agar, divide into 3 mL aliquots in sterile test tubes, and store at 45°C until ready for use.

[0062] (2) Pre-warm LB / IPTG / Xgal plates at 37°C and prepare one plate for each phage dilution. Prepare 10-fold serial dilutions of phage in LB. Recommended dilution range: amplified phage culture supernatant: 10 8 -10 11 ; Unamplified panning eluate: 10 1 -10 4 .

[0063] (3) When the bacterial culture reaches mid-logarithmic phase, divide it into 200 μL aliquots in microcentrifuge tubes. Add 10 μL of phage of different dilutions to each tube, quickly shake to mix, and incubate at room temperature for 1-5 minutes. Add the infected cells to the upper agar culture tube pre-warmed at 45°C, quickly mix, and immediately pour onto the LB / IPTG / Xgal plate. After cooling, invert and incubate at 37°C for 12 hours. Calculate the plaque forming unit (pfu) titer of the phage.

[0064] Example 1: Phage display technology for targeted peptide selection and specificity verification

[0065] 1. Preparation of spore suspension

[0066] F. oxysporum CGMCC 3.18025 was inoculated into PDA solid medium and grown in a constant temperature incubator at 30°C for 5-7 days. The F. oxysporum mycelium was washed and collected with normal saline, and the mycelium was removed by gauze filtration. The liquid phase was removed by centrifugation at 6000 rpm for 10 min at 4°C, and the F. oxysporum spore suspension was resuspended to a concentration of 2 x 10 7 individuals / mL.

[0067] 2. Affinity panning of phage specific binding to F. oxysporum using Ph.D. TM -12 phage display peptide library.

[0068] (1) According to the instructions, 100 μL of the obtained spore suspension was added to a 96-well enzyme-labeled plate and coated at 4°C for 12 h. After blocking with 3% bovine serum albumin (BSA) for 1 h, the plate was washed 6 times with 300 μL of TBS containing 0.1% v / v Tween-20 (TBST). 100 μL of Ph.D. 9 -12 phage display peptide library at a final concentration of 1 x 10 TM PFU was added and incubated at 28°C for 1 h, and then washed with TBST buffer containing 0.05% Tween-20 (0.1% and 0.2% TBST were used in the second and third rounds, respectively, to increase the selection pressure). 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2) was added and incubated at 28°C for 8 min. The elution buffer was neutralized to pH 7 with 1 M Tris-HCl, pH 9.1.

[0069] (2) 10 μL of the first round of eluate was used for phage titer determination. The remaining elution buffer was amplified in 30 mL of Escherichia coli ER2738 at a concentration of 10 6 CFU / mL. The amplified phage titer was determined, and the supernatant was collected by centrifugation. The supernatant was precipitated with 1 / 6 volume of PEG / NaCl solution, and incubated at 4°C for 12 h. The supernatant was discarded by centrifugation at 10000 rpm for 15 min at 4°C, and the phage was collected and resuspended in 100 μL of TBS.

[0070] (3) The amplified phage obtained in the first round was used for the next round of biological screening, with OVA and BSA alternately used as blocking agents. Four rounds of biological panning were performed according to the "adsorption-washing-elution-amplification" cycle. The phage after elution was subjected to titer determination, and the results are shown in Figure 1 (A) The degree of enrichment of specific phage increased with the number of panning rounds.

[0071] (4) Randomly select 30 monoclonal phages on the last round of titer determination plate for amplification and identify positive phages by indirect ELISA. The OD value of the sample to be tested / the OD value of the negative control ≥ 2.1 is considered a positive clone. Figure 1 As shown in (B), a total of 25 phage clones were identified as positive. 405nm >0.5) for DNA sequencing, and the DNA sequence was analyzed using Snapgene Viewer software, and six different peptide sequences were finally obtained (Table 1).

[0072] Table 1 DNA sequencing to identify the peptide sequences displayed by phage monoclonal clones

[0073]

[0074] 3. Verification of peptide specificity displayed by phage monoclonal display

[0075] The specificity of the peptide sequences listed in Table 1 was identified by indirect ELISA using various typical bacteria and fungi, including Escherichia coli ATCC25922, Staphylococcus aureus ATCC43300, Salmonella Typhimurium ATCC14028, Aspergillus flavus CMCC3.2890, Aspergillusochraceus CGMCC3.1364, Penicillium digitatum, and Fusarium oxysporum CGMCC3.18025, as coating antigens. BSA and OVA were used as negative controls.

[0076] Specificity verification found that the peptide pep1 displayed by phage monoclonal clones only showed significant specific binding ability to F.oxysporum, while pep13, pep15, pep22, pep28, and pep29 did not induce significant reactions to various bacteria, fungi, BSA, and OVA ( Figure 2 A and Figure 2 B) Therefore, pep1 was considered to be a specific display peptide of F. oxysporum.

[0077] 4. Preparation of peptides

[0078] The selected WY (WYHRSHPQWNHW) was synthesized by Shanghai Bioengineering and purified to a purity of more than 95% using analytical reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular weight of the peptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS). The results showed that the molecular weight of each synthesized peptide was almost the same as its theoretical molecular weight. Figure 3 as shown.

[0079] 5. Specificity verification of WYHRSHPQWNHW binding peptide

[0080] (1) Preparation of spore suspension

[0081] The spore suspension of Fusarium oxysporum CGMCC 3.18025, Aspergillus flavus, Aspergillus ochraceus, Penicillium digitatum, Fusarium graminearum, Fusarium proliferatum CGMCC 3.14301, Aspergillus parasiticus CGMCC 3.1364, Aspergillus fumigatus, and Penicillium expansum was prepared according to the method of step 1.

[0082] (2) The spore suspension (1 x 10 7 spores / mL) of the nine fungi prepared in step (1) was used as the coating antigen, and the WY specificity was verified by indirect ELISA. BSA and OVA were used as negative controls. The specific implementation steps were the same as the indirect ELISA determination method, except that 100 μL of Ph.D. TM -12 phage display peptide library was replaced with 100 μg / mL of WY solution, and the other steps remained the same.

[0083] The specificity verification results are shown in Table 1. Figure 4 As shown in Table 1, the results showed that the WYHRSHPQWNHW binding peptide showed a certain degree of affinity to F. oxysporum, but did not cause significant reactions to other fungal species, and its affinity was 7.70 times that of the BSA control and 7.07 times that of the OVA control. Therefore, it was determined that the WYHRSHPQWNHW binding peptide was a specific binding polypeptide for F. oxysporum.

[0084] Example 2: Design and synthesis of targeted anti-fungal peptides

[0085] The WYHRSHPQWNHW binding peptide of the present invention can improve the antibacterial properties of antimicrobial peptides. In this example, a short peptide 66-10 (MIC: 512 μM) that has been reported to have weak antibacterial activity was selected, and its amino acid sequence is: FRLKFH; the WYHRSHPQWNHW binding peptide of the present invention and the short peptide 66-10 are directly connected to synthesize a new polypeptide WY-66-10 with targeted antibacterial activity against F. oxysporum. The polypeptide sequences and molecular weights of the three are shown in Table 2.

[0086] Table 2 Sequences and molecular weights of targeting peptides, antimicrobial peptides, and targeting antifungal peptides

[0087] Peptide number sequence MW theoretical value MW measured value WY WYHRSHPQWNHW 1733.856 1733.85 SEQ ID NO.1 66-10 FRLKFH 847.013 847.00 SEQ ID NO.2 WY-66-10 WYHRSHPQWNHWFRLKFH 2562.869 2563.25 SEQ ID NO.3

[0088] Peptides 66-10 and WY-66-10 were synthesized by Sangon Biotech (Shanghai, China) and purified to a purity of more than 95% using analytical reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular weight of the peptides was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Figure 5 、 Figure 6 As shown, the molecular weight of each synthetic peptide was consistent with the theoretical molecular weight, indicating that the three peptides were successfully synthesized.

[0089] Example 3: Determination of antibacterial activity, biosafety, and stability of targeted antifungal peptides

[0090] 1. Determination of antibacterial activity:

[0091] (1) Preparation of spore suspension

[0092] The steps are the same as in Example 1.

[0093] (2) Determination of minimum inhibitory concentration (MIC)

[0094] The MICs of the targeted peptide (WYHRSHPQWNHW), antimicrobial peptide (FRLKFH), and targeted antifungal peptide (WYHRSHPQWNHWFRLKFH) against nine fungi, including F. oxysporum, were determined using the standard two-fold dilution method. The specific steps are as follows:

[0095] Detection was performed using a 96-well cell culture plate. 50 μL of serially diluted peptide solutions of different concentrations were added to each well, and an equal volume of spore suspension was added to each well (the final concentration of the bacterial suspension in the cell culture plate was 5 × 10 4 The MIC of the targeted antifungal peptide against the fungus was determined by measuring the lowest concentration at which OD600 < 0.1 after 24 h of incubation at 30°C.

[0096] See the results Figure 7 (A) The targeted antifungal peptide WY-66-10 exhibits bactericidal activity against F. oxysporum with a minimum inhibitory concentration of 16 μM, representing a more than 30-fold increase in antifungal activity compared to the original antifungal peptide 66-10. WY-66-10 exhibited no bactericidal activity against other test fungi at a high concentration of 128 μM, or exhibited only weak inhibitory activity (64 μM). This demonstrates that the targeted antifungal peptide WY-66-10 exhibits targeted bactericidal activity against F. oxysporum.

[0097] (2) Fungicidal kinetics

[0098] The plate count method was used to determine the fungicidal kinetics of the targeted antifungal peptide against F.oxysporum. WY-66-10 was diluted with sterile water and incubated with F.oxysporum spores (concentration of 7×10 7 Spores were cultured in a 30°C incubator for 24 hours. A spore suspension without antimicrobial peptide was used as a negative control. Spores were diluted and plated onto PDA plates at intervals of 0, 4, 8, 12, and 24 hours. Spores were counted after 36 hours of incubation at 30°C.

[0099] See the results Figure 7 (B) At a concentration of 4× the MIC, WY-66-10 exhibited rapid bactericidal activity, killing approximately 75% of fungal spores within 4 hours. The total spore count decreased with prolonged exposure, reaching a plateau after 8 hours. WY-66-10 exhibited enhanced bactericidal efficacy at a concentration of 8× the MIC, reaching 96% after 24 hours of exposure at 8× the MIC.

[0100] 2. Biosafety Determination

[0101] (1) Cytotoxicity

[0102] The cytotoxicity of mouse macrophage RAW246.7 cells was detected using the CCK-8 assay. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were mixed at a ratio of 9:1, and 1% penicillin-streptomycin (double antibody) was added to prepare a complete culture medium. Mouse macrophage RAW246.7 cells were thawed in liquid nitrogen and cultured at 2×10 4 Cells were plated at a density of 100 cells / well in a 96-well cell culture plate and incubated overnight at 37°C, 5% CO₂, until the cells reached 80%-90% of the well bottom. The peptide solution was serially diluted in cell culture medium to 1, 2, 4, and 8× the MIC. Medium without peptide solution served as a negative control, and medium without cell treatment served as a blank control. After incubation of cells with the targeted antifungal peptide for 12 hours, 100 μL of CCK-8 dilution was added to each well. After incubation at 37°C, 5% CO₂ for 1 hour, absorbance was recorded at 450 nm using a microplate reader. Cell viability was calculated according to the following formula.

[0103]

[0104] See the results Figure 8 The targeted antifungal peptide WY-66-10 showed no toxicity to mouse macrophages RAW246.7 within the detection range, and the cell survival rate was still higher than 80% at a concentration of 128 μM.

[0105] (2) Hemolytic activity

[0106] Mouse erythrocytes were washed three times with phosphate buffered saline (PBS) and diluted to a 2% volume concentration. The peptide solution was serially diluted with sterile water to 1, 2, 4, and 8× the MIC, mixed evenly with the mouse erythrocytes, incubated at 37°C for 1 hour, and centrifuged at 3500 rpm for 5 minutes. Mouse erythrocytes were treated with 2% Triton X-100 as a negative control, and PBS was used as a blank control. The supernatant was carefully transferred to a new 96-well cell culture plate, and the absorbance at 570 nm was recorded using a microplate reader to calculate the hemolytic activity.

[0107] See the results Figure 9 The hemolytic rate of the targeted antifungal peptide WY-66-10 on mouse erythrocytes was less than 5% at each tested concentration, indicating that the targeted antifungal peptide WY-66-10 of the present invention had little effect on cell hemolysis.

[0108] 3. Stability determination

[0109] (1) Physiological salt stability

[0110] The MICs of the targeted antifungal peptides against F. oxysporum were determined using gradient dilutions of peptide solutions containing different concentrations of physiological salt medium (150 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 1 mM ZnCl2, and 2 mM FeCl3). A spore suspension without antimicrobial peptide was used as a negative control. The assay was performed in the same manner as in steps (1) and (2) of Example 3, except that physiological salt solutions with final concentrations of 150 mM NaCl, 4.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 1 mM ZnCl2, and 2 mM FeCl3 were added to the medium.

[0111] See the results Figure 10 (A) Except for the effect of CaCl2 on the antibacterial effect of the targeted antifungal peptide WY-66-10, the change in the MIC value measured in other physiological salt solutions with different concentrations did not exceed 2 times, and the antibacterial activity was slightly improved in 150mM NaCl solution.

[0112] (2) Acid-base stability

[0113] The MIC of the target antifungal peptide against F. oxysporum was determined using different concentrations of peptide solution gradiently diluted with different pH medium (4.5, 6, 8, 9.5). The spore suspension without the addition of the antibacterial peptide was used as a negative control. The determination method was the same as the steps (1) and (2) of the antibacterial activity determination in Example 3, except that the pH of the medium was 4.5, 6, 8, and 9.5, respectively.

[0114] The results are shown in Table 2. Figure 10 (B), the MIC value determined in the solution with different pH of acid and base did not change significantly or the change fold did not exceed 2 folds.

[0115] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A targeting binding peptide of Fusarium oxysporum, characterized in that The amino acid sequence of the targeting binding peptide is shown in SEQ ID NO.

1.

2. A targeted antifungal peptide against Fusarium oxysporum, characterized in that The targeted antifungal peptide is obtained by connecting the targeted binding peptide according to claim 1 with an antifungal peptide.

3. The targeted antifungal peptide according to claim 2, characterized in that The amino acid sequence of the antifungal peptide is shown in SEQ ID NO.

2.

4. A recombinant microbial cell, characterized in that The recombinant microbial cell expresses the targeted binding peptide or targeted antifungal peptide according to any one of claims 1 to 3.

5. Use of the targeted binding peptide according to claim 1, the targeted antifungal peptide according to claim 2, or the recombinant microbial cell according to claim 3 in the preparation of a fungus-inhibiting drug, characterized in that: The fungus is Fusarium oxysporum.

6. A fungus-inhibiting drug, characterized in that: Containing the antifungal peptide according to claim 2 or the recombinant microbial cell according to claim 3.

7. The drug according to claim 6, characterized in that The fungi are Fusarium oxysporum, Aspergillusfumigatus, Aspergillus ochraceus.

8. The drug according to claim 6, characterized in that Pharmaceutical dosage forms include liquid, solid, and spray.

9. A method for improving the antibacterial performance of an antifungal peptide, characterized in that: connecting the targeted binding peptide according to claim 1 with an antifungal peptide to obtain a targeted antifungal peptide; The amino acid sequence of the antifungal peptide is shown in SEQ ID NO.

2.

10. The method according to claim 9, wherein the antibacterial activity is inhibition of Fusarium oxysporum, Aspergillus fumigatus, and Aspergillus ochraceus.

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