A deer-derived antibacterial peptide against gram-negative bacteria and a preparation method and application thereof

Abstract

The application discloses a deer-derived antibacterial peptide against gram-negative bacteria and a preparation method and application thereof, belongs to the technical field of biology, and the amino acid sequence is shown as SEQ ID No. 1. It is tested that the antibacterial peptide S6 has obvious inhibiting effect on most of tested gram-negative bacteria, the average antibacterial activity reaches 8.00 muM, the hemolytic activity and cytotoxicity are relatively low, the cell selectivity index is 32, and the antibacterial peptide S6 has relatively strong anti-enzymatic ability. The antibacterial mechanism of the antibacterial peptide S6 is determined, it is judged that the antibacterial peptide S6 has recognition and damage effects on the cell membrane of gram-negative bacteria, and finally leads to the death of bacteria. Therefore, the antibacterial peptide S6 is an antibacterial peptide against gram-negative bacteria with high clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a deer-derived antimicrobial peptide against Gram-negative bacteria, its preparation method, and its application. Background Technology

[0002] Antimicrobial peptides are small molecule polypeptides that help plants and animals defend against external microbial invasion and eliminate mutated cells within the body. They possess numerous advantages, including a broad antimicrobial spectrum, a wide variety of types, and a low likelihood of inducing resistance mutations. Therefore, they are considered the most powerful alternative to antibiotics. However, natural antimicrobial peptides often suffer from weak activity, low cell selectivity, poor stability, and high synthesis costs, hindering their widespread application. Existing deer-derived peptides exhibit poor antimicrobial efficacy and toxic side effects, lacking clinical application value; therefore, their modification is necessary. Summary of the Invention

[0003] In view of the above shortcomings, the present invention provides a deer-derived antimicrobial peptide that resists Gram-negative bacteria, which solves the problems of poor biocompatibility, low resistance to enzymatic hydrolysis and weak antimicrobial activity of natural deer-derived antimicrobial peptides.

[0004] The technical solution adopted in this invention is as follows: The amino acid sequence of a deer-derived antimicrobial peptide S6, which is effective against Gram-negative bacteria, is shown in SEQ ID NO.1. Its C-terminus is amidated with -NH2, resulting in high biocompatibility and strong antimicrobial activity.

[0005] Furthermore, its molecular formula is shown in formula (I):

[0006]

[0007] Further, the preparation method of the deer-derived antimicrobial peptide S6 against Gram-negative bacteria as described above includes the following steps:

[0008] S1: Using natural deer-derived peptide as the template, its sequence is shown in SEQ ID NO.2. Starting from the 6th amino acid, the peptide chain is truncated, and the sequences II and RFFGG at the tail of the peptide chain are removed. The resulting core sequence is shown in SEQ ID NO.1. Its C-terminus is amidated with -NH2.

[0009] S2: Peptide resin was obtained by solid-phase chemical synthesis using a peptide synthesizer. The obtained peptide resin was then cleaved by TFA to obtain peptides.

[0010] S3: After purification by reversed-phase high-performance liquid chromatography and identification by mass spectrometry, the peptide was prepared by detecting its in vitro antibacterial activity, hemolytic activity, protease resistance, cytotoxicity and mechanism of action. Finally, it was named antimicrobial peptide S6.

[0011] The present invention also provides the use of the deer-derived antimicrobial peptide S6, as described above, against Gram-negative bacteria, in the preparation of a medicament for treating diseases caused by Gram-negative bacterial infections.

[0012] Furthermore, the Gram-negative bacteria mentioned above are Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, or Acinetobacter baumannii.

[0013] The present invention also provides a medicament suitable for treating and / or preventing Gram-negative bacterial infections, said medicament containing a deer-derived antimicrobial peptide S6 against Gram-negative bacteria as described above.

[0014] The beneficial effects and advantages of this invention are as follows: This invention yields an antimicrobial peptide S6 with high antibacterial activity, low toxicity, and low cost, specifically targeting Gram-negative bacteria. Antimicrobial activity and biocompatibility tests on the obtained antimicrobial peptide S6 revealed significant differences in inhibitory effects against various Gram-negative and Gram-positive bacteria, including *Escherichia coli*, *Salmonella typhimurium*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Staphylococcus epidermidis*. The average antimicrobial activity against Gram-negative bacteria reached 8.00 μM, while it showed no antimicrobial activity against Gram-positive bacteria. Meanwhile, it exhibits low hemolysis and cytotoxicity, showing no significant hemolysis even at a concentration of 128 μM, with a cell selectivity index as high as 32. Furthermore, antimicrobial peptide S6 maintains good antibacterial activity and stability even at high concentrations of pepsin and trypsin (16 mg / mL), demonstrating strong resistance to enzymatic degradation. Mechanistic studies have shown that antimicrobial peptide S6 binds to the LPS component of the outer membrane of Gram-negative bacteria, accumulating on the bacterial membrane surface, disrupting the outer membrane and disrupting the cytoplasmic membrane potential, leading to the outflow of bacterial contents and ultimately bacterial death. This membrane-breaking bactericidal mechanism, different from traditional antibiotics, makes it a potent alternative to antibiotics. Therefore, in summary, antimicrobial peptide S6 is an anti-Gram-negative bacterial peptide with high clinical application value. Attached Figure Description

[0015] Figure 1 The chromatogram of antimicrobial peptide S6;

[0016] Figure 2 This is the mass spectrum of the antimicrobial peptide S6;

[0017] Figure 3 This is a graph showing the hemolytic activity of the antimicrobial peptide S6.

[0018] Figure 4 This is a graph showing the cellular activity of the antimicrobial peptide S6.

[0019] Figure 5 This is a fluorescence microscopy image of the antimicrobial peptide S6.

[0020] Figure 6 The graph shows the LPS binding capacity of the antimicrobial peptide S6.

[0021] Figure 7 This is a competitive inhibition diagram of LPS against the antimicrobial peptide S6.

[0022] Figure 8 This is a graph showing the outer membrane permeability of antimicrobial peptide S6.

[0023] Figure 9 The graph shows the effect of antimicrobial peptide S6 on the plasma membrane potential of E. coli ATCC 25922. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0025] Example 1

[0026] Design of Antimicrobial Peptides: In the design and optimization of antimicrobial peptides, truncating the peptide chain while retaining its core active sequence is an effective strategy. This not only significantly reduces the synthesis cost of the peptide chain but also improves its stability and bioactivity. Using the sequence of a natural deer-derived peptide: RFIPPILRPPVRPPFRPPFRPPFRPPPIIRFFGG, truncating from position 6 (amino acid I) while removing the II and RFFGG sequences at the tail of the peptide chain yields the core sequence LRPPVRPPFRPPFRPPFRPP, with its C-terminus after amidation using -NH2. This truncation method effectively preserves the core functional structure of the antimicrobial peptide while reducing production and synthesis costs. Furthermore, shorter peptide chains generally exhibit higher stability in vivo because they are more easily penetrated and interact with target cells, reducing the likelihood of enzymatic degradation. Removing redundant sequences preserves the bioactivity of the peptide chain while mitigating immune responses and non-specific binding. Therefore, truncating the peptide chain optimizes antimicrobial activity, reduces costs, and enhances the application potential of antimicrobial peptides.

[0027] Table 1. Amino acid sequence and main structural parameters of antimicrobial peptide S6

[0028]

[0029] Example 2

[0030] The antimicrobial peptide S6 was synthesized using a solid-phase chemical synthesis method, and the specific steps are as follows:

[0031] 1. The preparation of peptides proceeds sequentially from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (where X is the first amino acid at the C-terminus of each antimicrobial peptide) is inscribed into Wang resin, and then the Fmoc group is removed to obtain X-Wang resin. Next, Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxylic acid-trimethyl-Y, where Y is the second amino acid at the C-terminus of each antimicrobial peptide) is added. This procedure is followed sequentially from the C-terminus to the N-terminus until the synthesis is complete, yielding a resin with side chains protected by the removed Fmoc group.

[0032] 2. Add the cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter. Wash the precipitate with TFA (trifluoroacetic acid), mix the washings with the above filtrate, concentrate using a rotary evaporator, add about 10 times the volume of pre-cooled anhydrous diethyl ether, precipitate at -20°C for 3 hours, and a white powder will precipitate. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous diethyl ether, and vacuum dry to obtain the peptide. The cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5.

[0033] 3. Equilibrate the column for 30 min using 0.2 mol / L sodium sulfate (adjusted to pH 7.5 with phosphoric acid). Dissolve the peptide in 90% acetonitrile aqueous solution, filter, and elute using a C18 reversed-phase atmospheric pressure column with gradient elution (eluent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), at a flow rate of 1 mL / min and a detection wavelength of 220 nm. Collect the main peak and freeze-dry. Further purify using a reversed-phase C18 column with eluent A being 0.1% TFA / aqueous solution and eluent B being 0.1% TFA / acetonitrile solution, with an elution concentration of 25%B to 40%B, an elution time of 12 min, and a flow rate of 1 mL / min. Collect the main peak again and freeze-dry.

[0034] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weights shown in the mass spectra were basically consistent with the theoretical molecular weights in Table 1 (see appendix). Figure 1 The purity of the antimicrobial peptides is greater than 95% (see appendix). Figure 2 ).

[0035] Example 3:

[0036] The in vitro antimicrobial activity, hemolysis, cell viability, and mechanism of action of the prepared antimicrobial peptide S6 were tested.

[0037] 1. Determination of antibacterial activity: The peptide was prepared into a stock solution for use. The minimum inhibitory concentration (MIC) of the antimicrobial peptide was determined using the micro-broth dilution method. Single colonies were inoculated into MHB and incubated at 220 rpm and 37°C until the logarithmic growth phase. The concentration was then adjusted to the OD value using MHB. 600nm=0.1, and finally further diluted 1000 times with MHB to 0.5-1×10 5 CFU / mL; Add 95 μL of 0.2% BSA diluent to row A of a 96-well plate, and 50 μL of 0.2% BSA diluent to the remaining wells. Add 5 μL of 2.56 mM peptide to the wells in row A, mix thoroughly, then add 50 μL to row B, and so on, serially diluting to row G. After mixing, discard 50 μL. Set up three replicates for each peptide assay; Add 50 μL of bacterial culture to the wells in rows A and G of the 96-well plate, and add 50 μL of bacterial culture to wells 1-6 in row H as a positive control. Add 50 μL of fresh MHB medium to wells 7-12 as a negative control. After mixing, incubate at 37°C for 24 h. Measure the absorbance at 492 nm using a microplate reader to determine the minimum inhibitory concentration. The results are shown in Table 2.

[0038] Table 2 Antibacterial activity of antimicrobial peptide S6

[0039]

[0040]

[0041] As shown in Table 2, antimicrobial peptide S6 has high antimicrobial activity against Gram-negative bacteria, but no antimicrobial activity against Gram-positive bacteria.

[0042] 2. Determination of hemolytic activity: 1 mL of fresh human blood was collected, anticoagulated with heparin, and dissolved in 2 mL of PBS solution. After centrifugation at 1000g for 5 min, red blood cells were collected. The blood cells were washed three times with PBS and resuspended in 10 mL of PBS. 50 μL of the red blood cell suspension was mixed with 50 μL of antimicrobial peptide S6 solution of different concentrations, incubated at 37℃ for 1 h, and then centrifuged at 4℃ for 5 min at 1000g. The supernatant was measured at 570 nm using an ELISA reader. The average value of each group was taken and compared. 50 μL of red blood cells with 50 μL of PBS served as a negative control; 50 μL of red blood cells with 50 μL of 0.1% Tritonx-100 served as a positive control. The minimum hemolytic concentration was the concentration of the antimicrobial peptide that caused a 10% hemolysis rate. The test results are shown below. Figure 3 And Table 3.

[0043] Table 3. Determination of hemolytic activity of antimicrobial peptide S6

[0044]

[0045] pass Figure 3As shown in Table 3, antimicrobial peptide S6 did not exhibit significant hemolytic activity within the detection range. To analyze the clinical application potential of antimicrobial peptide S6, a comprehensive evaluation can be conducted using the selectivity index (the ratio of hemolytic concentration to inhibitory concentration). Table 3 shows that antimicrobial peptide S6 possesses a high selectivity index, indicating that the designed antimicrobial peptide S6 has the potential to clinically replace antibiotics.

[0046] 3. Protease resistance: The peptide (2.56 mM) was mixed with equal volumes of pepsin solutions (4 mg / mL, 8 mg / mL, 16 mg / mL) and trypsin solutions (4 mg / mL, 8 mg / mL, 16 mg / mL) at different concentrations and incubated at 37°C for 4 h. After incubation, the mixture was boiled for 10 min. Then, the treated peptide and a group of untreated peptides were serially diluted in 0.2% BSA and mixed with an equal volume of bacterial culture. Finally, the mixture was incubated at 37°C for 24 h. The results are shown in Table 4.

[0047] Table 4. Minimum inhibitory concentration of S6 against E. coli 25922 after protease treatment.

[0048]

[0049]

[0050] Table 4 shows that antimicrobial peptide S6 exhibits stable antimicrobial activity in protease solutions of different concentrations, and the protease has little effect on its antimicrobial activity; that is, it maintains good antimicrobial activity even under high concentrations (16 mg / mL) of pepsin and trypsin. Therefore, antimicrobial peptide S6 possesses strong resistance to enzymatic degradation.

[0051] 4. Cell viability assay: RAW264.7 cells in logarithmic growth phase were divided into groups of 3 × 10⁻⁶ cells. 4 The cells were seeded at a density of 50 μL per well in 96-well plates, and after overnight adhesion, drug was administered. In the experimental groups, 50 μL of S6 diluted culture medium at different concentration gradients (0 μmol / L, 1 μmol / L, 10 μmol / L, 100 μmol / L, 1 mmol / L, 10 mmol / L) was added to each well, with 6 replicates per group. In the control group, 50 μL of pure culture medium was added to each well. After incubation in a cell culture incubator for 24 h, 50 μL of culture medium with a concentration of 5 mg / mL MTT was added to each well, and the cells were incubated at 37°C for 4 h. After centrifugation at 1000 rpm for 10 min, the supernatant was discarded, and 150 μL of DMSO was added to each well. The absorbance was measured at 570 nm. Results are shown below. Figure 4 As the concentration of antimicrobial peptides increases, cell viability first rises and then falls, and cells still maintain a high viability even at a concentration of 100 μmol / L.

[0052] 5. Fluorescence microscopy observation: To test the integrity of the bacterial cell membrane, the peptide-treated bacteria were observed using a fluorescence microscope. E. coli ATCC 25922 in the mid-growth stage was centrifuged, washed three times, and resuspended in PBS buffer (pH = 7.4, 10 mM) to OD0.05. 600nm =0.2, antimicrobial peptide S6 was added to the bacterial culture (final concentration: 4 μM), and then incubated at 37°C for 1 h. Then, SYTO 9 dye (final concentration: 1 μM) and PI dye (final concentration: 4 μM) were added, and incubation continued for another 30 min. Bacteria not incubated with the peptide were used as controls. Images were acquired using a fluorescence microscopy cell imaging system. Test results are shown below. Figure 5 The nucleic acid dye propidium iodide (PI) can only enter cells with damaged cell membranes and bind to DNA, releasing red fluorescence. For example... Figure 5 As shown, the introduction of SYTO 9 caused the bacteria to emit green fluorescence, while the red fluorescence emitted by PI was also detected. The two gradually merged, indicating that the antimicrobial peptide S6 induces membrane damage.

[0053] 6. LPS Binding Assay: LPS (E. coli O111:B4, 50 μg / mL) and BC (5 μg / mL) were incubated in Tris buffer (50 mM, pH 7.4) for 4 h. Then, in a 96-well plate, the peptide was serially diluted in Tris buffer (pH 7.4), and the BC-LPS mixture was added to each well. Fluorescence values ​​in each well were measured using a multi-mode microplate reader at excitation and emission wavelengths of 580 nm and 620 nm, respectively. Results are as follows: Figure 6 As shown, antimicrobial peptide S6 binds to LPS in a dose-dependent manner, and the binding effect on LPS becomes more pronounced with increasing concentration of antimicrobial peptide S6. Antimicrobial peptide S6 binds approximately 20% of LPS at 4 μM and more than 50% at 64 μM, demonstrating a strong LPS binding capacity.

[0054] 7. LPS Competitive Inhibition Assay: LPS (E. coli O111:B4) was prepared to a concentration of 1024 μg / mL–4 μg / mL. Equal volumes (50 μL) of LPS and antimicrobial peptide S6 were incubated in sterile 96-well plates at 37°C for 1 h. Then, 100 μL of bacterial solution (final concentration 0.8–1 × 10⁻⁶) was added to the antimicrobial peptide S6-LPS mixture. 5 The mixture was incubated at 37°C for 90 min (CFU / mL). 20 μL of the mixture was removed from each well, diluted with an appropriate amount of PBS, plated, and incubated overnight. The following day, the colony count was determined, and the initial CFU / well count was retrospectively calculated. This experiment was independently repeated three times. Results are as follows: Figure 7As shown, bacterial mortality is inversely proportional to LPS concentration. High concentrations of LPS significantly reduce the bactericidal activity of antimicrobial peptide S6, further indicating that antimicrobial peptide S6 binds to LPS on the bacterial outer membrane, thereby achieving its antibacterial effect.

[0055] 8. Extracellular membrane permeability assay: Prepare bacterial suspension according to the method described in step 1 of this embodiment (using HEPES buffer with pH=7.2 and 5mM glucose instead of PBS) and resuspend to OD600 nm=0.4. Add NPN probe (final concentration 10μM) and incubate at 37℃ in a shaker for 30 minutes under light-protected conditions. Dilute the peptides with outer membrane HEPES buffer in a 96-well plate. Mix equal volumes of bacterial suspension and peptides of different concentration gradients in the 96-well plate and detect fluorescence values ​​using a microplate reader (excitation wavelength 350nm, emission wavelength 420nm). Bacterial suspension treated with 10μg / mL polymyxin B serves as a positive control, and untreated bacterial suspension serves as a negative control. Outer membrane permeability = (fluorescence value of treated sample - fluorescence value of negative control) / (fluorescence value of positive control - fluorescence value of negative control) × 100%. Results are as follows: Figure 8 As shown, antimicrobial peptide S6 exhibits a strong concentration-dependent ability to disrupt the outer membrane. Specifically, at lower concentrations, antimicrobial peptide S6 significantly increased outer membrane permeability (>100%).

[0056] 9. Cell membrane depolarization assay: Prepare bacterial suspension according to the method described in step 1 of this embodiment (using HEPES buffer containing 20 mM glucose at pH 7.2 instead of PBS) and resuspend at OD. 600 nm =0.05. After adding DiSC3-5 dye (final concentration 0.4 μM), incubate in a 37°C constant temperature shaker for 1.5 hours under light-protected conditions, then add K. + (Final concentration 100mM) Continue incubation for 30 minutes. Add 2 mL of bacterial suspension to a sterile 24-well plate, add different concentrations of peptides, mix well, and record fluorescence values ​​using a microplate reader (excitation wavelength 622nm, emission wavelength 670nm). Gentamicin-treated bacterial suspension served as an antibiotic control, melitoxin-treated bacterial suspension as a positive control, and untreated bacterial suspension as a negative control. Results are as follows: Figure 9 As shown, the antimicrobial peptide S6 induces plasma membrane depolarization in a dose- and time-dependent manner, and the fluorescence intensity of DiSC3-5-treated bacterial cells increases within 2000 seconds. This disruption of membrane potential leads to uncontrolled ion movement and membrane potential dissipation, resulting in bacterial death.

Claims

1. A deer-derived antimicrobial peptide S6 against Gram-negative bacteria, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1, and its C-terminus is amidated with -NH2.

2. The deer-derived antimicrobial peptide S6 against Gram-negative bacteria according to claim 1, characterized in that, Its molecular formula is shown in formula (I): Formula (I).

3. The method for preparing the deer-derived antimicrobial peptide S6 against Gram-negative bacteria according to claim 1, characterized in that, The steps are as follows: S1: Using natural deer-derived peptide as the template, its sequence is shown in SEQ ID NO.

2. Starting from the 6th amino acid, the peptide chain is truncated, and the sequences II and RFFGG at the tail of the peptide chain are removed. The resulting core sequence is shown in SEQ ID NO.

1. Its C-terminus is amidated with -NH2. S2: Peptide resin was obtained by solid-phase chemical synthesis using a peptide synthesizer. The obtained peptide resin was then cleaved by TFA to obtain peptides. S3: After purification by reversed-phase high-performance liquid chromatography and identification by mass spectrometry, the peptide was prepared by detecting its in vitro antibacterial activity, hemolytic activity, protease resistance, cytotoxicity and mechanism of action. Finally, it was named antimicrobial peptide S6.

4. The application of the deer-derived antimicrobial peptide S6 against Gram-negative bacteria according to claim 1 in the preparation of a drug for treating diseases caused by Gram-negative bacteria, wherein the Gram-negative bacteria are Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, or Acinetobacter baumannii.

5. A drug suitable for treating and / or preventing Gram-negative bacterial infections, characterized in that, The drug contains a deer-derived antimicrobial peptide S6, which is effective against Gram-negative bacteria as described in claim 1.

Images