Antibacterial peptide QS18 as well as preparation method and application thereof

Abstract

The invention provides an antibacterial peptide QS18 as well as a preparation method and application thereof, and belongs to the technical field of antibacterial peptides. Aiming at the public health problem of cryptococcus neoformans, the invention provides an antibacterial peptide QS18, and the amino acid sequence of the antibacterial peptide QS18 is shown as SEQ ID NO: 1. The antibacterial peptide QS18 has good inhibiting and killing effects on cryptococcus neoformans, is high in stability, has the advantages of low hemolytic activity and low cytotoxicity, is expected to become a novel candidate drug for resisting cryptococcus neoformans, and has a wide market application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and specifically relates to an antimicrobial peptide QS18 and a preparation method and application thereof. Background Art

[0002] Fungal infections and drug resistance pose a major challenge to public health and clinical treatment. In recent years, the morbidity and mortality of invasive fungal diseases have continued to rise, and the resistance of pathogenic fungi to antifungal drugs has also been increasing. Cryptococcus is one of the most common pathogens of invasive fungal infections in humans, mainly causing pneumonia and meningitis, and also causing skin, bone or internal organ infections. Cryptococcus neoformans is the most pathogenic of cryptococci. It is an opportunistic fungal pathogen that is widely distributed in the natural environment. Neoformal cryptococcal disease is prone to occur in immunocompromised people, and currently there are still very limited drugs for the clinical treatment of neoforman cryptococcal infections.

[0003] Antimicrobial peptides can effectively inhibit and kill invading pathogenic microorganisms and are important antimicrobial components in the animal immune system. At appropriate inhibitory concentrations, antimicrobial peptides can interact with a variety of microbial targets such as biofilm / wall components or intracellular organelles. The former destroys the integrity of the cytoplasmic membrane, and the latter interferes with the normal metabolic activities of the cells, ultimately leading to bacterial death. However, although antimicrobial peptides targeting Cryptococcus neoformans have been reported, the antimicrobial peptides are unstable in structure, resulting in poor elimination effects. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an antimicrobial peptide QS18, which contains two pairs of disulfide bonds and has high stability, so as to achieve good inhibitory and killing effects on Cryptococcus neoformans.

[0005] The present invention provides an antimicrobial peptide QS18, the amino acid sequence of the antimicrobial peptide QS18 is shown in SEQ ID NO:1.

[0006] The present invention provides a method for preparing the antimicrobial peptide QS18, comprising the following steps:

[0007] The crude antimicrobial peptide QS18 was synthesized by solid phase synthesis;

[0008] The crude antimicrobial peptide QS18 is folded and modified in a renaturation solution, and purified to obtain the antimicrobial peptide QS18.

[0009] Preferably, the refolding solution is an aqueous solution comprising the following components: 4-6 mM reduced glutathione, 0.4-0.6 mM oxidized glutathione, 0.08-0.12 mol / L NaCl and 0.08-0.12 mol / L Tris-HCl;

[0010] The mass percentage concentration of the antimicrobial peptide QS18 in the refolding solution is 8% to 12%.

[0011] Preferably, the purification method comprises desalting and purification by HPLC reverse phase column chromatography;

[0012] The conditions of the HPLC reverse phase column chromatography are as follows: mobile phase A is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.1%, mobile phase B is a trifluoroacetic acid acetonitrile solution with a volume percentage concentration of 0.1%, and sample loading is started after the baseline is stable, with a sample loading volume of 50 μL; the chromatographic column is a silica gel alkyl bonded phase C18 column with a specification of 4.6 mm×300 mm, a colloid particle size of 5 μm, and a pore size of 100 Å; a binary mobile phase gradient elution system is used for gradient elution, and within 30 minutes, the content of mobile phase B in the eluent increases linearly from 0% to 80% by volume, the flow rate is 1 mL / min, the detection wavelength is 215 nm, and the measurement temperature is 25°C.

[0013] The present invention provides an antibacterial agent, wherein the active ingredient comprises the antimicrobial peptide QS18, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by the preparation method.

[0014] Preferably, the concentration of the active ingredient is not less than 3.13 μg / mL.

[0015] The present invention provides an antimicrobial infection drug, the active ingredient of which comprises the antimicrobial peptide QS18, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by the preparation method.

[0016] The present invention provides use of the antimicrobial peptide QS18, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by the preparation method in preparing a product for inhibiting infection by Cryptococcus neoformans.

[0017] Preferably, the product comprises at least one of the following: medicine, preservative, feed, feed additive, food, food additive and cosmetic.

[0018] Preferably, the strain of Cryptococcus neoformans includes ATCC32045 and / or BNCC225501.

[0019] The present invention provides an antimicrobial peptide QS18, the amino acid sequence of which is shown in SEQ ID NO: 1. The antimicrobial peptide QS18 has good inhibitory and killing effects on Cryptococcus neoformans, high stability, low hemolytic activity and low cytotoxicity, and is expected to become a novel candidate drug for anti-Cryptococcus neoformans, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The mass spectrometry results of the unfolded antimicrobial peptide QS18;

[0021] Figure 2 The mass spectrometry results of the folded antimicrobial peptide QS18;

[0022] Figure 3 This is the bactericidal kinetics result diagram of the antimicrobial peptide QS18 against Cryptococcus neoformans;

[0023] Figure 4 This is the result of the hemolytic activity of the antimicrobial peptide QS18 on human erythrocytes;

[0024] Figure 5 This is the result of the cytotoxicity experiment of the antimicrobial peptide QS18;

[0025] Figure 6 This is the stability test result diagram of the antimicrobial peptide QS18;

[0026] Figure 7 The morphological changes of Cryptococcus neoformans after the interaction with the antimicrobial peptide QS18 were observed by scanning electron microscopy and transmission electron microscopy. The scale of the scanning electron microscopy image is 100 μm, and the scale of the transmission electron microscopy image is 200 nm.

[0027] Figure 8 This is the result diagram of the inhibition and clearance of Cryptococcus neoformans biofilm by the antimicrobial peptide QS18;

[0028] Fig. 9 This is a graph showing the results of in vivo efficacy testing of the antimicrobial peptide QS18 in a mouse model of systemic infection induced by Cryptococcus neoformans. DETAILED DESCRIPTION

[0029] The present invention provides an antimicrobial peptide QS18, the amino acid sequence of the antimicrobial peptide QS18 is shown in SEQ ID NO: 1 (QCFKVCFRKRCFTKCSRS).

[0030] In the present invention, the antimicrobial peptide QS18 is obtained from the transcript of spider, has a molecular weight of 2226.72 Da, and contains two pairs of disulfide bonds, so that it exhibits good enzyme resistance to trypsin and chymotrypsin, especially better resistance to enzymatic hydrolysis of chymotrypsin.

[0031] The present invention provides a method for preparing the antimicrobial peptide QS18, comprising the following steps:

[0032] The crude antimicrobial peptide QS18 was synthesized by solid phase synthesis;

[0033] The crude antimicrobial peptide QS18 is folded and modified in a renaturation solution, and purified to obtain the antimicrobial peptide QS18.

[0034] The invention adopts a solid phase synthesis method to synthesize a crude antimicrobial peptide QS18.

[0035] The present invention has no special limitation on the solid phase synthesis method, and the solid phase protein synthesis method known in the art can be used. In the embodiment of the present invention, the synthetic antimicrobial peptide QS18 was commissioned to be synthesized by Hangzhou Gutuo Biotechnology Co., Ltd.

[0036] The crude polypeptide is folded and modified in a refolding solution, and purified to obtain the antimicrobial peptide QS18.

[0037] In the present invention, the refolding solution is preferably an aqueous solution comprising the following components: 4-6 mM reduced glutathione, 0.4-0.6 mM oxidized glutathione, 0.08-0.12 mol / L NaCl and 0.08-0.12 mol / L Tris-HCl, with a pH value of 6.8-7.2; it can be 5 mM oxidized glutathione, 0.1 mol / L NaCl and 0.1 mol / L Tris-HCl, with a pH value of 7.0. The mass concentration of the antimicrobial peptide QS18 in the refolding solution is preferably 8%-12%, and can be 10%. The time for the folding modification is preferably 22-26 h, and can be 24 h. The temperature for the folding modification is preferably 23-27 ° C, and can be 25 ° C.

[0038] In the present invention, the purification method preferably includes desalting and purifying by HPLC reverse phase column chromatography. The conditions of the HPLC reverse phase column chromatography are: mobile phase A is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.1%, mobile phase B is a trifluoroacetic acid acetonitrile solution with a volume percentage concentration of 0.1%, and the sample is loaded after the baseline is stable, and the loading amount is 50 μL; the chromatographic column is a silica gel alkyl bonded phase C18 column with a specification of 4.6 mm × 300 mm, a colloid size of 5 μm, and a pore size of 100A; a binary mobile phase gradient elution system is used for gradient elution, and within 30 minutes, the content of mobile phase B in the eluent increases linearly from 0% to 80% by volume, the flow rate is 1 mL / min, the detection wavelength is 215 nm, and the measurement temperature is 25°C. After the above desalting and purification method, the antimicrobial peptide QS18 is obtained, and the purity is identified, and the purity is not less than 95%. Mass spectrometry detection of the antimicrobial peptide QS18 shows that there are two disulfide bonds in the antimicrobial peptide QS18.

[0039] The present invention provides an antibacterial agent, wherein the active ingredient comprises the antimicrobial peptide QS18, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by the preparation method.

[0040] In the present invention, the chemical modification type of the chemically modified antimicrobial peptide QS18 preferably includes at least one of the following: internal disulfide bond connection of the antimicrobial peptide QS18, acetylation modification of the N-terminus or side chain N group, polyethylene glycol modification, etc.

[0041] In the present invention, the concentration of the active ingredient is preferably not less than 3.13 μg / mL, and can be 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 15 μg / mL, 18 μg / mL, or 20 μg / mL.

[0042] The present invention provides an antimicrobial infection drug, the active ingredient of which comprises the antimicrobial peptide QS18, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by the preparation method.

[0043] The present invention provides the use of the antimicrobial peptide QS18, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by the preparation method in preparing a product for inhibiting neoformans infection.

[0044] In the present invention, the novel Cryptococcus infection preferably includes at least one of the following diseases: dermatophytosis, pneumonia, bronchitis, and the like.

[0045] In the present invention, the concentration of the antimicrobial peptide QS18 in the product is preferably not less than 3.13 μg / mL, and can be 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 15 μg / mL, 18 μg / mL, 20 μg / mL. The product preferably includes at least one of the following: medicine, preservative, feed, feed additive, food, food additive and cosmetics. The present invention has no special restrictions on the preparation method of the product, and the preparation method of the product known in the art can be used.

[0046] In one embodiment of the present invention, the antimicrobial peptide QS18 has a good inhibitory and killing effect on Cryptococcus neoformans. The antimicrobial peptide QS18 exhibits a strong effect of complete growth inhibition at 10×MIC within 60 minutes and 5×MIC after 120 minutes. In another embodiment, the minimum inhibitory concentrations of the antimicrobial peptide QS18 for Cryptococcus neoformans strain ATCC32045 and Cryptococcus neoformans strain BNCC225501 are 6.56 μg / mL and 3.13 μg / mL, respectively, which is better than the positive control drug fluconazole. At the same time, biofilm inhibition and removal experiments show that the ability of QS18 to inhibit biofilm formation is related to concentration, has a certain concentration dependence and strong biofilm inhibition, and has the potential to become a drug for inhibiting and eliminating Cryptococcus neoformans biofilm. In addition, the present invention also carried out animal experiments, using a mouse model of systemic infection induced by Cryptococcus neoformans as the object, and evaluated the anti-infection efficacy of the antimicrobial peptide QS18. The results showed that the administration of the antimicrobial peptide QS18 can significantly reduce the load of Cryptococcus neoformans in organs such as the lungs, liver, kidneys, and spleen, thereby effectively inhibiting the spread of Cryptococcus neoformans to target organs, and showing good in vivo anti-cryptococcus neoformans infection activity.

[0047] In another embodiment of the present invention, the antimicrobial peptide QS18 also has the advantages of low hemolytic activity and low cytotoxicity, which provides good safety for the preparation of in vivo ingestion products.

[0048] In the present invention, in order to explore the mechanism of the antimicrobial peptide QS18 inhibiting Cryptococcus neoformans, the morphological changes of the antimicrobial peptide QS18 before and after treating Cryptococcus neoformans were observed by scanning electron microscopy and transmission electron microscopy. The results showed that the antimicrobial peptide QS18 in fungal cells caused leakage of cytoplasmic contents and cell shrinkage, which indicates that the antimicrobial peptide QS18 directly acts on the cell membrane and cell wall of bacteria by interacting with the lipid bilayer, penetrating or dissolving the cell membrane and cell wall to achieve the purpose of killing bacteria.

[0049] The following is a detailed description of an antimicrobial peptide QS18 provided by the present invention, its preparation method and application, in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] (1) An antimicrobial peptide sequence was mined from the spider venom gland transcriptome, and the following antimicrobial peptide amino acid sequence was designed: QCFKVCFRKRCFTKCSRS (SEQ ID NO: 1), and the crude product QS18 was synthesized by solid phase synthesis;

[0052] (2) restoring the disulfide bonds of crude QS18;

[0053] Table 1. Renaturation solution (based on 50 mL system)

[0054] name Dosage Antimicrobial peptide QS18 5mg 0.1MTris-HCl 605.70mg 0.1molNaCl 292.20mg 5mM reduced glutathione 76.93mg 0.5 mM oxidized glutathione 15.32mg Deionized water 50mL

[0055] The polypeptide refolding solution was prepared according to the formula in Table 1, and concentrated hydrochloric acid was added to adjust the pH to 7, and placed in a constant temperature box at 25°C for 24 hours.

[0056] (3) desalting and purifying the renatured polypeptide by HPLC reverse phase column chromatography, and identifying its purity until the purity of the polypeptide is not less than 95%;

[0057] HPLC purification and identification method:

[0058] The crude QS18 solution and the polypeptide refolding solution were filtered with a 0.45 μm filter membrane, respectively. The mobile phase A was 0.1% trifluoroacetic acid-water, and the mobile phase B was 0.1% trifluoroacetic acid-acetonitrile. After the baseline was stable, the sample was loaded, and the loading volume was 50 μL. The chromatographic column was a silica gel alkyl bonded phase C18 column (4.6 mm×300 mm, colloid size 5 μm, pore size 100A). A binary mobile phase gradient elution system was used for gradient elution, that is, within 30 minutes, the content of mobile phase B in the eluent increased linearly from 0% to 80%, the flow rate was 1 mL / min, the detection wavelength was 215 nm, and the measurement was performed at 25°C.

[0059] (4) Determination by mass spectrometry (e.g. Figure 1 and Figure 2 ), its molecular weight was determined to be 2226.72Da. And the peak shape was consistent with that after disulfide bond renaturation.

[0060] Experimental Example 2

[0061] Antimicrobial activity test of antimicrobial peptide QS18

[0062] The minimum inhibitory concentration is the lowest drug concentration that can inhibit bacterial growth and reproduction. The experiment uses a two-fold gradient dilution method. The specific experimental operation is as follows: prepare fresh bacterial solution and use a UV spectrophotometer to detect the OD of the bacterial solution. 600 , according to 1OD 600 =1×10 9 CFU / mL, dilute the above bacterial solution with fresh LB liquid medium to adjust the concentration to 2×10 6 CFU / mL. Then, 100 μL of physiological saline was added to a sterile 96-well plate in advance, and the disulfide bond-containing antimicrobial peptide QS18 prepared in Example 1 was added to the first well. The samples to be tested were diluted two-fold in sequence, and 100 μL of a 2×10 6CFU / mL of bacterial solution was pipetted and mixed, and then placed in a 37°C constant temperature incubator for overnight culture. Finally, the light absorption value of the bacterial solution at 600nm was detected by an ELISA instrument, and the average value of the sample concentration of the well where no bacterial growth was detected and the adjacent wells was taken as the minimum inhibitory concentration, i.e., the MIC value. The antibacterial activity of the antimicrobial peptide QS18 is shown in Table 2.

[0063] Table 2 Minimum inhibitory concentration of antimicrobial peptide QS18 against standard strains

[0064]

[0065] From Table 2, the minimum inhibitory concentrations of the antimicrobial peptide QS18 against Cryptococcus neoformans strain ATCC32045 and Cryptococcus neoformans strain BNCC225501 are 6.56 μg / mL and 3.13 μg / mL, respectively. The above experimental results show that the antimicrobial peptide QS18 provided by the present invention has a good antibacterial effect on Cryptococcus neoformans.

[0066] Experimental Example 3

[0067] Bactericidal kinetics test of antimicrobial peptide QS18

[0068] Inoculate the new Cryptococcus strain BNCC225501 onto LB solid medium plates and incubate them upside down at 37°C until colonies grow. Pick a single colony with an inoculation loop and inoculate it into LB liquid medium, and incubate it in a 37°C shaking incubator until the logarithmic growth phase. Dilute the bacterial solution to 1×10 6 CFU / mL, add the sample to be tested (antimicrobial peptide GK37 or vancomycin) to the diluted bacterial solution to make the final concentration 1×MIC, 5×MIC and 10×MIC, and set the negative control to add the corresponding volume of normal saline (NC). The bacterial solution with the sample to be tested was quickly placed in a 37°C shaking incubator and cultured at 150rpm. Take 10μL of the bacterial solution at 0min, 15min, 30min, 60min, 120min, and 240min and dilute it with sterile normal saline to 1×10 3 Take 50 μL of the solution and spread it on the LB solid medium plate, then put it in a 37℃ incubator and invert it for 16 hours, and count the colonies. Do 3 parallels for each group, and take the average value of the results.

[0069] The results are as follows Figure 3 The antimicrobial peptide QS18 showed a potent effect of complete growth inhibition at 10×MIC within 60 min and 5×MIC after 120 min, indicating rapid bactericidal activity at higher concentrations.

[0070] Experimental Example 4

[0071] Hemolytic activity and cytotoxicity assays

[0072] Dilute the washed red blood cells with saline to adjust the density to 10 7 ~10 8 / mL, and the samples to be tested were prepared into different gradient concentrations. The two were placed at 37°C for 30 minutes, then centrifuged at 1000rpm for 5 minutes, and the light absorption value of the supernatant at 540nm was detected by an enzyme marker. In this experiment, physiological saline was used as a negative control, and the same volume of TritonX-100 (10%) was used as a positive control. The hemolytic activity was proportional to the light absorption value at 540nm. The hemolytic activity is shown in the attached Figure 4 As shown, the antimicrobial peptide QS18 has low hemolytic activity.

[0073] Cytotoxicity assay was performed using HaCaT keratinocytes. When the cells grew well and the density reached 80% of the bottom of the bottle, the culture medium was discarded and washed three times with sterile PBS. The adherent cells were then digested with trypsin. After termination, fresh DMEM culture medium containing 10% FBS and penicillin-streptomycin (100 μg / mL) was added and mixed by pipetting, and the cell suspension concentration was adjusted to 5×10 5 / mL, the experiment used a sterile 96-well plate, added 200μL of the above cell suspension to each well, and placed in a cell culture incubator for overnight culture. The next day, different concentrations of the antimicrobial peptide QS18 and colistin (denoted as Colistin) were added to the test samples, and the concentration gradient was set to 100μg / mL, 50μg / mL, 25μg / mL, 12.5μg / mL, 6.25μg / mL, 3.13μg / mL, 1.56μg / mL, and 3 replicates for each concentration. The control group used the same volume of sterile PBS, and then placed in a constant temperature incubator at 37°C and 5% CO2 for continued culture for 24h. Add 10μL of CCK-8 solution to each well, place it in an incubator under light-proof conditions and continue to culture for 2h, and use an enzyme marker to detect the absorbance of the colored solution at 450nm in each well.

[0074] The test results are as follows Figure 5 As shown, the results indicate that the cell cytotoxicity of the antimicrobial peptide QS18 is extremely low.

[0075] Experimental Example 5

[0076] Stability test of antimicrobial peptide QS18

[0077] 1 mg / mL antimicrobial peptide QS18 (linear antimicrobial peptide QS18 and disulfide bond folded antimicrobial peptide QS18) was incubated with trypsin or chymotrypsin at a mass ratio of 250:1 (peptide: enzyme) in a digestion buffer (5 mM CaCl2, 50 mM Tris-HCl, pH 7.4) at 37 °C. After incubation, the enzyme reaction was stopped by heating at 95 °C for 10 min. 80 μL of the reaction product was added to 20 μL SDS-PAGE loading buffer (5×), mixed by vortexing, and heated at 95 °C for 5 min. The heat-treated products of incubation for 0, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 12 h were respectively taken for OmniPAGE. TM Precast Hepes Tris gel (4% to 20%, 15 wells) was used for SDS-PAGE analysis. LL-37 and colistin (Colisitn) were used as controls. Figure 5 "L" stands for protein ladder and "S" stands for protease-free peptides. Figure 5 It can be seen that linear QS18 is easily affected by proteolysis; folded QS18 containing two pairs of disulfide bonds can be stable in trypsin for 1 hour, and after incubation with chymotrypsin for 12 hours, it still shows resistance to chymotrypsin. This shows that the antimicrobial peptide QS18 has a certain degree of enzyme resistance.

[0078] In order to determine that protease does not affect the antibacterial activity of QS18, the longest time that QS18 can still exist stably under trypsin or chymotrypsin co-incubation, that is, 2 hours, was selected to explore its antibacterial activity. First, QS18 was incubated with chymotrypsin or trypsin (the mass ratio of QS18 to enzyme was 250:1) at 37°C for 2 hours. After heat treatment at 95°C for 10 minutes to stop the enzymatic reaction, 10 μL of the heat-inactivated protease sample was added to 90 μL of diluted Cryptococcus neoformans cell suspension (1×10 6 CFU / mL) at a final concentration of 4×MIC QS18, and a positive control was added, whether digested or not, and the mixture was incubated at 37°C for 3 h. 100 μL of fresh YPDA broth medium was added to the mixture, which was then incubated for another 16 h. As a negative control, a sample containing heat-inactivated protease was included, without peptide, and containing only culture medium. Inhibition of fungal growth was detected by measuring the absorbance at 595 nm using a Synergy H1 microplate reader (Bio-Tek). The experiment was repeated at least three times. The test results are shown in Figure 6 As shown, the antimicrobial activity of the antimicrobial peptide QS18 was still detectable after incubation with trypsin and chymotrypsin for 2 h.

[0079] Experimental Example 6

[0080] Scanning and transmission electron microscopy observations of the morphology of Cryptococcus neoformans treated with antimicrobial peptide QS18

[0081] Scanning electron microscopy and transmission electron microscopy were used to observe the morphological changes of Cryptococcus neoformans BNCC225501 after interaction with 1×MIC and 10×MIC concentrations of antimicrobial peptide QS18. Cryptococcus neoformans were cultured in a 37°C constant temperature shaker until the logarithmic growth phase, then mixed with 40-50 mL of LB liquid culture medium and cultured until it became obviously turbid. A 50 mL centrifuge tube was centrifuged at 3500 rpm for 5 min, the supernatant was discarded, the bottom precipitate was washed 3 times with saline, and then the bacterial solution was resuspended with a small amount of sterile saline. The resuspended bacterial solution was taken out and added to a 1.5 mL centrifuge tube and mixed with the polypeptide to make the final concentration of the polypeptide 1×MIC and 10×MIC. An equal volume of saline and bacterial solution was added to the negative control group. Incubate in a 37°C constant temperature incubator for 2 hours until it became obviously turbid, then take it out, centrifuge it at 3500 rpm for 5 minutes, aspirate and discard the supernatant, add 2.5% glutaraldehyde to fix the bottom precipitate, ensure that the precipitate is the size of a mung bean, and refrigerate at 4°C overnight. Prepare two samples, one of which is treated with agar to precipitate the bottom, then sectioned and sprayed with gold, and the morphological changes of bacteria are observed under a scanning electron microscope, and the appropriate observation magnification is selected to take photos and record. The other is embedded in resin, sectioned and sprayed with gold, and photographed and recorded using a transmission electron microscope.

[0082] The results are as follows Figure 7 As shown. Scanning electron microscopy and transmission electron microscopy showed that the antimicrobial peptide QS18-treated fungal cells caused leakage of cytoplasmic contents and cell shrinkage, indicating that they interacted with the lipid bilayer, thereby destroying the integrity of the cell membrane. In contrast, untreated cells were round or oval, with smooth cell surfaces and clear cell boundaries. The above results indicate that the antimicrobial peptide GK37 can directly act on the bacterial cell membrane and cell wall, penetrate or dissolve, and then kill bacteria.

[0083] Experimental Example 7

[0084] Biofilm inhibition and removal experiments

[0085] This experiment tested the ability of the antimicrobial peptide QS18 at different concentrations (0.5×MIC, 1×MIC, 2×MIC, 4×MIC, 8×MIC) to inhibit the biofilm formation and biofilm removal of Cryptococcus neoformans BNCC225501. The specific experimental process is as follows: The biofilm inhibition experiment mainly observes the bacterial biofilm formed by the test sample and Cryptococcus neoformans after co-incubation for a period of time, and then evaluates its effect of inhibiting the formation of biofilm by comparing it with the negative control group. The biofilm removal experiment is to first culture the bacteria alone for a period of time, and then add a certain concentration of the test sample and co-incubate the biofilm to evaluate its removal effect on the biofilm that has been formed. The specific experimental method is as follows.

[0086] Biofilm inhibition: First, pick a single colony of Cryptococcus neoformans and culture it in a constant temperature shaker at 180 rpm and 37°C until the logarithmic growth phase. Centrifuge at 3500 rpm for 5 minutes at room temperature, remove the supernatant, wash three times with saline and resuspend. After measuring the concentration with a multifunctional microplate reader, dilute it to 1×10 with 1640 liquid culture medium containing 10% fetal bovine serum. 6 CFU / mL. The sample to be tested was diluted with saline to 8×MIC concentration, 200μL was taken and placed in the first column, and 100μL of saline was added to the other 3 columns. The double gradient method was used for equal dilution, and 5 gradient concentrations were set. Mix by blowing and beating, and the last column was discarded. Each concentration was repeated three times. Set up a column of negative control group and add 100μL of saline. Set up two columns as fluconazole (FCZ) positive control group, dilute FCZ with saline to 2×MIC, that is, 6.25μg / ml (20.4μM), and dilute the other column to 1×MIC. In each of the two positive controls, take 100μL of the corresponding dilution multiple of the sample and add it to the corresponding column. After all the samples in the experimental group and the control group have been added, use a spray gun to add 100μL of bacterial solution to each well and mix by blowing and beating. Put it in a 37℃ incubator and incubate for 24h. After incubation, use a pipette to remove the bacterial solution, then gently rinse three times with 200 μL of sterile PBS culture medium, 1 min each time, remove the washing solution and wait for air drying, then add 100 μL of 99% methanol to fix for 15 min, remove and wait for air drying again, use 100 μL of 0.1% crystal violet stain for 5 min, then fill a basin with deionized water, gently tap on the water surface to ensure that the stain is evenly washed off and less residue is left, add 100 μL of 95% ethanol after air drying and place in a constant temperature shaker for 30 min, and detect the absorbance at 600 nm with an enzyme reader.

[0087] Biofilm removal: Fresh bacterial solution was prepared in the same way as above. Dilute to 1×10 6CFU / mL, then add 200μL of bacterial solution to each well of the 96-well plate, 3 replicate groups, 5 concentration gradient groups, one NC group, and two positive control gradient groups, and incubate in a 37℃ constant temperature incubator for 24h. After incubation, use a pipette to remove the bacterial solution, wash with sterile PBS 3 times, and let it stand for 1min each time before aspirating. The sample to be tested is diluted to 0.5×MIC concentration, 1×MIC concentration, 2×MIC concentration, 4×MIC concentration, and 8×MIC concentration with 1640 liquid culture medium containing 10% fetal bovine serum, and 200μL of sample is added to the wells of the 96-well plate with bacterial solution, and the negative control NC group is added with the same volume of 1640 liquid culture medium, and the positive control group FCZ is diluted to 2×MIC concentration and 1×MIC concentration with 1640 liquid culture medium containing 10% fetal bovine serum. Incubate in a 37℃ constant temperature incubator for 24h. Use a pipette to remove the bacterial solution again, rinse gently three times with 200 μL of sterile PBS culture medium, 1 min each time, remove the washing solution and wait for air-drying, then add 100 μL of 99% methanol to fix for 15 min, remove the solution and wait for air-drying again, stain with 100 μL of 0.1% crystal violet stain for 5 min, then fill a basin with deionized water, gently tap on the water surface to ensure that the stain is evenly washed away and less residue is left, add 100 μL of 95% ethanol after air-drying, place in a constant temperature shaker for 30 min, and detect the absorbance at 600 nm with an enzyme reader.

[0088] The results are as follows Figure 8 As shown. In the biofilm inhibition experiment, the inhibition rate of the antimicrobial peptide QS18 on the biofilm of Cryptococcus neoformans can reach 60% at a concentration of 0.5×MIC, and the inhibition rate increases with the increase of concentration. It shows that the ability of QS18 to inhibit biofilm formation is related to concentration, and has a certain concentration dependence and strong biofilm inhibition. In the biofilm removal experiment, the antimicrobial peptide QS18 effectively eradicated the preformed biofilm to 65% at 1×MIC, and its ability to remove biofilm is also concentration-dependent. This shows that the antimicrobial peptide QS18 has the potential to become a drug for inhibiting and eliminating the biofilm of Cryptococcus neoformans.

[0089] Experimental Example 8

[0090] In vivo efficacy of QS18 in a mouse model of Cryptococcus neoformans-induced systemic infection.

[0091] Four days before infection, 20 ± 2 g C57BL / 6J female mice were injected intraperitoneally with 150 mg / kg cyclophosphamide to make them immunocompromised. Cryptococcus neoformans BNCC225501 was cultured in YPDA medium to mid-logarithmic phase, and fungal cells were harvested by centrifugation at 3500 rpm for 5 min and washed three times in saline. The fungal pellet was resuspended in the same buffer and the OD value was adjusted to 4 × 107 CFU / mL. 200 μL of fungal solution was intraperitoneally injected into immunocompromised mice, with a total of 8×10 6 CFU. Twelve hours after intraperitoneal inoculation of fungi, mice were treated with intraperitoneal injection of QS18 (1, 2, 4 mg / kg), fluconazole (2, 4 mg / kg, denoted as FCZ), and saline. Mice were killed 12 hours later, and blood was collected by orbital bleeding. Organs (lungs, liver, kidneys, and spleen) were also collected and divided into two parts: one part was placed in a grinding tube containing 1 mL of saline and then ground in a tissue grinder to assess the fungal load. After serial dilution in saline for 5 times, 100 μL of the grinding solution was inoculated on a YPDA agar plate, incubated at 37°C for 48 hours, and then the colonies were counted; at the same time, another part of the organs was subjected to histopathological examination. The liver, spleen, kidney, and lung sections were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histopathological analysis.

[0092] The results are as follows Fig. 9 Analysis of Cryptococcus neoformans BNCC225501 and CFU counts (y-axis) in organs (lungs, liver, kidneys and spleen) was performed, as shown in Fig. 9 As shown in Figures A, C, E, and G, the results showed that the fungal burden (colony forming units per organ) was significantly reduced after QS18 peptide treatment, thereby significantly inhibiting the spread of Cryptococcus neoformans to target organs. Both the antimicrobial peptide QS18 and the positive control showed a dose-dependent reduction in fungal burden. At a dose of 2 mg / kg, the antimicrobial peptide QS18 showed potent in vivo antifungal activity comparable to that of the positive control. The results of histopathological analysis are shown in Figures A, B, C, E, and G. Fig. 9 As shown in B, D, F and H, tissue damage was observed, characterized by extensive necrosis, dilated capillaries filled with red blood cells and infiltration of inflammatory cells (mainly neutrophils). Compared with the control and 1 mg / kg dose concentration groups, the 2 mg / kg or 4 mg / kg dose concentration groups showed reduced cell necrosis and reduced infiltrating neutrophils, indicating that QS18 treatment has a positive effect on controlling Cryptococcus neoformans infection. Both the antimicrobial peptide QS18 and the positive control showed a dose-dependent reduction in fungal burden.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antimicrobial peptide QS18, characterized in that: The amino acid sequence of the antimicrobial peptide QS18 is shown in SEQ ID NO:

1.

2. A method for preparing the antimicrobial peptide QS18 according to claim 1, characterized in that: The following steps are involved: The crude antimicrobial peptide QS18 was synthesized by solid phase synthesis; The crude antimicrobial peptide QS18 is folded and modified in a renaturation solution, and purified to obtain the antimicrobial peptide QS18.

3. The preparation method according to claim 2, characterized in that: The refolding solution is an aqueous solution comprising the following components: 4-6 mM reduced glutathione, 0.4-0.6 mM oxidized glutathione, 0.08-0.12 mol / L NaCl and 0.08-0.12 mol / L Tris-HCl; The mass percentage concentration of the antimicrobial peptide QS18 in the refolding solution is 8% to 12%.

4. The preparation method according to claim 2, characterized in that: The purification method comprises using HPLC reverse phase column chromatography for desalting and purification; The conditions of the HPLC reverse phase column chromatography are as follows: mobile phase A is a trifluoroacetic acid aqueous solution with a volume percentage concentration of 0.1%, mobile phase B is a trifluoroacetic acid acetonitrile solution with a volume percentage concentration of 0.1%, and sample loading is started after the baseline is stable, with a sample loading volume of 50 μL; the chromatographic column is a silica gel alkyl bonded phase C18 column with a specification of 4.6 mm×300 mm, a colloid particle size of 5 μm, and a pore size of 100 Å; a binary mobile phase gradient elution system is used for gradient elution, and within 30 minutes, the content of mobile phase B in the eluent increases linearly from 0% to 80% by volume, the flow rate is 1 mL / min, the detection wavelength is 215 nm, and the measurement temperature is 25°C.

5. An antibacterial agent, characterized in that The active ingredient comprises the antimicrobial peptide QS18 according to claim 1, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by any one of the preparation methods described in claims 2 to 4.

6. The novel cryptococcal antibacterial agent according to claim 5, characterized in that: The concentration of the active ingredient is not less than 3.13 μg / mL.

7. An antibacterial infection drug, characterized in that: The active ingredient comprises the antimicrobial peptide QS18 according to claim 1, the chemically modified antimicrobial peptide QS18 or the antimicrobial peptide QS18 prepared by any one of the preparation methods described in claims 2 to 4.

8. Use of the antimicrobial peptide QS18 according to claim 1, the chemically modified antimicrobial peptide QS18, or the antimicrobial peptide QS18 prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a product for inhibiting infection by Cryptococcus neoformans.

9. The use according to claim 8, characterized in that: The product comprises at least one of the following: medicine, preservative, feed, feed additive, food, food additive and cosmetic.

10. The use according to claim 8 or 9, characterized in that: The strains of Cryptococcus neoformans include ATCC32045 and / or BNCC225501.

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