An antibacterial short peptide with high protease stability and its preparation method and application

Abstract

The present invention discloses an antibacterial short peptide with high protease stability, its preparation method and application, belonging to the field of bioengineering technology. The amino acid sequence of the antibacterial short peptide DabDab is: LWDabLDabWDab-NH2, and its C-terminus is amidated with -NH2, where Dab is 2,4-diaminobutyric acid. The antibacterial activity, hemolytic activity, cytotoxicity and protease stability of the antibacterial short peptide DabDab of the present invention were detected, and it was found that it has high antibacterial activity against several tested Gram-negative bacteria and certain antibacterial activity against several tested Gram-positive bacteria. The antibacterial short peptide DabDab has a high cell selectivity with TI = 16.00, and does not show obvious cytotoxicity at 128 μg / mL. The antibacterial short peptide DabDab still maintains its original MIC or the MIC only increases by 2 times after being incubated with pepsin or trypsin for 8 h, and has high protease stability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an antibacterial short peptide with high protease stability and a preparation method and application thereof. Background Art

[0002] The long-term uncontrolled use and abuse of antibiotics have led to antibiotic residues in livestock products, which are transmitted through the food chain. Bacterial resistance has evolved and spread rapidly in complex environments, and even threatens human health and causes environmental pollution. Therefore, it is particularly important to find a new type of antimicrobial agent to replace antibiotics. Antimicrobial peptides (AMPs) are an important part of the body's innate immune system. They have a wide range of inhibitory effects on bacteria, fungi and viruses. Because they have a special antimicrobial mechanism different from antibiotics, they are considered to be an ideal substitute for antibiotics. Although many advantages of antimicrobial peptides indicate that antimicrobial peptides are the most promising antibiotic substitutes to overcome bacterial resistance, at present, natural antimicrobial peptides still have certain limitations in development and application, such as the weak activity, short half-life, and poor protease stability of some antimicrobial peptides. Among them, the fact that antimicrobial peptides are easily degraded by various digestive enzymes in the gastrointestinal tract and lose their activity, resulting in poor protease stability, is a bottleneck problem that needs to be solved urgently. Summary of the invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide an antimicrobial short peptide with high protease stability to solve the problem of poor enzyme stability of existing antimicrobial peptides.

[0004] The technical scheme adopted by the present invention is as follows: an antibacterial short peptide DabDab with high protease stability, whose amino acid sequence is: LWDabLDabWDab-NH2, whose C-terminus is amidated with -NH2, and the Dab is 2,4-diaminobutyric acid.

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

[0006]

[0007] Another object of the present invention is to provide a method for preparing an antibacterial short peptide DabDab with high protease stability, the steps being as follows:

[0008] Step 1: The polypeptide adopts an α-helical structure, and leucine is selected to be placed at positions 1 and 4 to form a leucine zipper, and tryptophan is selected to be placed at positions 2 and 6 to form a tryptophan zipper, so as to stabilize the α-helical structure and provide hydrophobicity to the polypeptide, thereby improving the antibacterial activity, and 2,4-diaminobutyric acid is placed at positions 3, 5, and 7 to provide a positive charge to the polypeptide while improving the ability of the polypeptide to resist protease degradation, thereby obtaining an amino acid sequence of the polypeptide of LWDabLDabWDab, and the C-terminus of the polypeptide is amidated with -NH2, and finally a polypeptide DabDab with a net charge number of +4 is obtained;

[0009] Step 2: The polypeptide is synthesized by solid phase chemical synthesis, and after reverse phase high performance liquid chromatography purification and mass spectrometry identification, the antibacterial activity, hemolytic activity, cytotoxicity and protease stability of the polypeptide are tested, and finally named as the antibacterial short peptide DabDab.

[0010] Another object of the present invention is to provide the use of the antibacterial short peptide DabDab with high protease stability as described above in the preparation of a drug for treating Gram-positive and / or Gram-negative bacterial infectious diseases.

[0011] Furthermore, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis and Staphylococcus epidermidis.

[0012] Furthermore, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium.

[0013] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive bacteria and / or Gram-negative bacteria infection, wherein the drug contains the antibacterial short peptide DabDab with high protease stability as described above.

[0014] The present invention has the following advantages and beneficial effects: the present invention adopts non-natural amino acid modification to obtain an antibacterial short peptide DabDab with high protease stability. The antibacterial short peptide DabDab with high protease stability of the present invention is tested for antibacterial activity, hemolytic activity, cytotoxicity and protease stability, and it is found that the antibacterial short peptide DabDab has high antibacterial activity against several Gram-negative bacteria measured, such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and has certain antibacterial activity against several Gram-positive bacteria measured, such as Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis. The cell selectivity of the antibacterial short peptide DabDab is high (TI=16.00), and no obvious cytotoxicity is shown at 128μg / mL. In addition, the antibacterial short peptide DabDab still maintains the original MIC or the MIC is only increased by 2 times in the incubation with pepsin or trypsin for 8h, and has high protease stability. In summary, the antibacterial short peptide DabDab of the present invention has the potential to become a broad-spectrum antibacterial drug for treating Gram-positive and Gram-negative bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the reverse phase high performance liquid chromatogram of the antibacterial short peptide DabDab;

[0016] Figure 2 It is the reverse phase high performance liquid chromatogram of the antibacterial short peptide KK;

[0017] Figure 3 This is the mass spectrum of the antibacterial short peptide DabDab;

[0018] Figure 4 is the mass spectrum of the antibacterial short peptide KK;

[0019] Figure 5 This is the hemolytic activity diagram of the antimicrobial short peptides DabDab and KK;

[0020] Figure 6 This is the cytotoxicity diagram of the antibacterial short peptide DabDab. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the embodiments and drawings.

[0022] Example 1

[0023] Design of antimicrobial peptides

[0024] 1. Select Leu to be placed at positions 1 and 4 to form a Leu zipper, and Trp to be placed at positions 2 and 6 to form a Trp zipper, to provide hydrophobicity to the antimicrobial peptide and improve the antibacterial activity. Place Dab at positions 3, 5, and 7 to provide positive charge to the antimicrobial peptide while improving the ability of the antimicrobial peptide to resist protease degradation, and use -NH2 amidation at the C-terminus of the antimicrobial peptide to increase the number of positive charges. Finally, an antimicrobial peptide with seven amino acids was obtained, named the antimicrobial short peptide DabDab, and its net charge number was +4. Replace Dab at positions 3, 5, and 7 in the antimicrobial short peptide DabDab sequence with Lys, and further obtain its control polypeptide KK. The sequences, molecular weights, and charge numbers of the two are shown in Table 1.

[0025] Table 1 Sequence, molecular weight and charge number of antibacterial peptides DabDab and KK

[0026]

[0027] Example 2

[0028] Synthesis and identification of antimicrobial peptides

[0029] The designed antimicrobial peptide was synthesized by Nanjing Synpeptide Co Ltd. via solid phase synthesis and purified by reverse phase high performance liquid chromatography (RP-HPLC) to obtain the target compound for subsequent antibacterial activity, hemolytic activity, cytotoxicity and protease stability determination.

[0030] The reverse phase HPLC chromatograms of antimicrobial peptides DabDab and KK are attached. Figure 1 , 2 .

[0031] The mass spectra of antibacterial peptides DabDab and KK are attached. Figure 3 , 4 .

[0032] Example 3

[0033] Antibacterial activity of antimicrobial peptides

[0034] The antibacterial activity of antimicrobial peptides was determined by measuring the minimum inhibitory concentration (MIC) of antimicrobial peptides. The bacterial solution frozen at -40°C was inoculated into MHB medium and cultured overnight at 37°C and 220 rpm in a shaking incubator. The bacterial solution was then transferred to a new MHB medium and cultured until its logarithmic growth phase. The concentration of the bacterial solution was adjusted to OD using a UV-visible spectrophotometer. 600 nm= 0.38 or so for later use. Take a sterile 96-well plate and use the microbroth dilution method to determine the MIC of the antimicrobial peptide. Add the initial concentration of 5.12 mg / mL AMPs stock solution to the 96-well plate, use 0.2% BSA (containing 0.01% acetic acid) filtered through a 0.22μm water filter as the diluent, perform a 2-fold gradient dilution, and then add 50μL of the bacterial solution diluted 1000 times to each well. MHB + 0.2% BSA containing bacteria is used as a positive control, and sterile MHB medium + 0.2% BSA is used as a negative control. Place the 96-well plate in a 37°C incubator and incubate for 16-18h. After the incubation, all negative controls remain clear and transparent, indicating that the test process is pollution-free. Use an enzyme marker to measure the turbidity of each well at a wavelength of 492nm. The lowest peptide concentration at which the turbidity does not increase compared with the negative control is defined as the MIC of the antimicrobial peptide. Perform three independent repeated tests. The MICs of the antimicrobial peptides DabDab and KK are shown in Table 2.

[0035] Table 2 Antibacterial activity of antibacterial peptides DabDab and KK (μg / mL)

[0036]

[0037] As can be seen from Table 2, the MIC value of KK against P. aeruginosa PAO1 is greater than 64 μg / mL, the MIC values ​​against other Gram-negative bacteria tested are 8-32 μg / mL, and the MIC value against Gram-positive bacteria is 32 μg / mL. The antibacterial short peptide DabDab showed high antibacterial activity against all bacteria tested, with the lowest MIC value against Gram-negative bacteria reaching 4 μg / mL, and the MIC value against Gram-positive bacteria was 16-32 μg / mL.

[0038] Example 4

[0039] Hemolytic activity of antimicrobial peptides

[0040] Collect 2mL of fresh blood from healthy volunteers, centrifuge at 4℃, 3000r for 10min, discard the supernatant, wash 3 times with PBS, and finally resuspend with 10mL PBS. Use PBS as the diluent, add the AMPs storage solution to the diluent of the 96-well plate, perform a 2-fold gradient dilution, then add 50μL of red blood cell suspension to each well and incubate at 37℃ for 1h. After the incubation, centrifuge the 96-well plate at 4℃ and 1000×g for 10min; aspirate 50μL of supernatant from each well, transfer to a new 96-well plate, and measure the absorbance at 570nm. Use 50μL red blood cells + 50μL 0.1% Triton X-100 as the positive control, and 50μL red blood cells plus 50μL PBS buffer as the negative control. Repeat this test three times. See the attached manual for the test results. Figure 5The biocompatibility of the antimicrobial peptides was evaluated by the minimum hemolytic concentration (MHC) at which the antimicrobial peptides caused 5% hemolysis of human red blood cells, and their cell selectivity was further evaluated by calculating the therapeutic index (TI) of the antimicrobial peptides, as shown in Table 3.

[0041] Table 3 Biocompatibility of antimicrobial peptides DabDab and KK

[0042]

[0043] a The geometric mean (GM) of the minimum inhibitory concentration of AMPs against the assayed bacteria was 128 μg / mL, when no detectable antibacterial activity was observed at 64 μg / mL;

[0044] b MHC is the lowest concentration of AMPs that causes 5% hemolysis of human red blood cells (hRBCs), and when no detectable hemolytic activity is observed at 128 μg / mL, 256 μg / mL is used to calculate the therapeutic index;

[0045] c TI is the ratio of MHC to GM.

[0046] Instructions attached Figure 5 It can be seen that the antimicrobial peptides DabDab and KK did not cause hemolysis at 128 μg / mL, and their biosafety was relatively high. Further calculation of the therapeutic index of the antimicrobial peptides DabDab and KK revealed that the TI value of the antimicrobial peptide DabDab (16.00) was higher than that of KK (10.29). Overall, the antimicrobial peptide DabDab had the best cell selectivity.

[0047] Example 5

[0048] Cytotoxicity of antimicrobial peptides

[0049] The cytotoxicity of antimicrobial peptides was determined by MTT method. Porcine intestinal epithelial cells IPEC-J2 were selected as the test cells.

[0050] After the cells are subcultured, the complete medium in the cell culture bottle is poured out and the cells are washed 2 to 3 times with sterile PBS. Then 1 mL of 0.25% trypsin solution is added to digest the adherent cells. After digestion, add complete medium and blow to form a cell suspension, and add 50 μL of cell suspension to each well of the 1st to 11th columns of the sterile 96-well plate. Place the 96-well plate in a carbon dioxide incubator and culture for 8 to 12 hours. Take a new sterile 96-well plate, dilute the antimicrobial peptide in multiple ratios with the culture medium, and then add each concentration of antimicrobial peptide to the corresponding wells of the 96-well plate covered with cell suspension. The positive control is 50 μL of complete medium + 50 μL of cell suspension, and the negative control is 100 μL of complete medium. The 96-well plate is placed in a carbon dioxide incubator for 18 to 20 hours. After the incubation, add 25 μL of 5 mg / mL MTT solution to each well. Continue to incubate in the CO2 incubator for 3 hours, then discard all the liquid in each well, add 150 μL DMSO to each well, and measure the absorbance at 570 nm. This experiment was repeated three times independently. The test results are shown in Figure 6 .

[0051] Attached to the instruction manual Figure 6 It can be seen that the antimicrobial short peptides DabDab and KK did not show obvious cytotoxicity, and within the concentration range measured, the cell survival rate reached more than 80%.

[0052] Example 6

[0053] Protease stability of antimicrobial peptides

[0054] E. coli 25922 was used as a typical Gram-negative bacterium and S. aureus 29213 was used as a typical Gram-positive bacterium to determine the sensitivity of the antimicrobial peptides to pepsin and trypsin.

[0055] The antimicrobial peptide storage solution to be tested was incubated with trypsin solution or pepsin solution at 37°C for 8 hours, then boiled for 15 minutes and cooled to room temperature. The solution after pepsin and peptide were mixed was adjusted to pH = 2 with concentrated hydrochloric acid, and then the MIC of the antimicrobial peptide in different proteases was determined by microbroth dilution method. This experiment was repeated three times independently. The test results are shown in Table 4.

[0056] Table 4 Protease stability of antimicrobial peptides DabDab and KK

[0057]

[0058] a The control was the MIC value of AMPs against E. coli ATCC 29213 or S. aureus ATCC 29213 in the absence of protease.

[0059] b The initial concentrations of pepsin and trypsin were both 10 mg / mL.

[0060] As shown in Table 4, the short peptide KK was found to have poor protease stability. After incubation with trypsin for 8 h, the MIC against E. coli 25922 increased from 8 μg / mL to >64 μg / mL, and the MIC against S. aureus 29213 increased from 32 μg / mL to >64 μg / mL. The antimicrobial short peptide DabDab has high protease stability. After incubation with pepsin and trypsin for 8 h, the MIC remained unchanged or only increased by 2 times.

[0061] In summary, an antibacterial short peptide DabDab with high protease stability was obtained, which has good antibacterial activity and biocompatibility.

Claims

1. An antibacterial short peptide DabDab with high protease stability, characterized in that: Its amino acid sequence is: LWDabLDabWDab-NH2, its molecular formula is as shown in formula (I), its C-terminus is amidated with -NH2, and the Dab is 2,4-diaminobutyric acid, 2. The method for preparing an antibacterial short peptide DabDab with high protease stability according to claim 1, characterized in that: Here are the steps: Step 1: The polypeptide adopts an α-helical structure, leucine is placed at positions 1 and 4 to form a leucine zipper, tryptophan is placed at positions 2 and 6 to form a tryptophan zipper, and 2,4-diaminobutyric acid is placed at positions 3, 5, and 7. The amino acid sequence of the obtained polypeptide is: LWDabLDabWDab, and its C-terminus is amidated with -NH2, and finally a polypeptide DabDab with a net charge number of +4 is obtained; Step 2: The polypeptide is synthesized by solid phase chemical synthesis, and after reverse phase high performance liquid chromatography purification and mass spectrometry identification, the antibacterial activity, hemolytic activity, cytotoxicity and protease stability of the polypeptide are tested, and finally named as the antibacterial short peptide DabDab.

3. Use of the antibacterial short peptide DabDab with high protease stability as claimed in claim 1 in the preparation of a medicament for treating infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria; the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis; the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.

4. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The drug contains the antibacterial short peptide DabDab with high protease stability as claimed in claim 1.

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