Antibacterial peptide MAP34B-2 and application thereof

Abstract

The application belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide MAP34B-2 and application thereof. The amino acid sequence of the antibacterial peptide MAP34B-2 provided by the application is shown as SEQ ID NO:1, and the C-terminal end can be subjected to amidation modification. The antibacterial peptide MAP34B-2 provided by the application has a small molecular weight, can change the permeability of cell membranes of Escherichia coli and Acinetobacter baumannii and inhibit the generation of the cell membranes, and thus plays a bacteriostatic and bactericidal role. Moreover, the antibacterial peptide MAP34B-2 provided by the application has low hemolytic activity and no cytotoxicity, and can be applied to the preparation of products with a bacteriostatic and bactericidal role, developed into antibacterial peptide bacteriostatic and bactericidal agents, and used for the preparation of medicines for preventing and / or treating bacterial infectious diseases.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, specifically relating to an antimicrobial peptide MAP34B-2 and its applications. Background Technology

[0002] Escherichia coli ( Escherichia coli Bacterium tumefaciens (BLT) is a Gram-negative, flagellated, motile facultative anaerobic bacterium. It is also an opportunistic pathogen, primarily transmitted through human-to-human and animal-to-human contact, fecal-oral transmission, and contaminated water and food, causing peritonitis, appendicitis, and pneumonia. The advent of antibiotics has improved the prognosis of patients with bacterial infections and reduced mortality from bacterial infections. However, with the overuse of antibiotics, coupled with bacterial evolution and selection, bacteria have gradually developed antibiotic resistance. To address the increasingly serious problem of bacterial resistance, the development of new antibiotics or alternative drugs is necessary.

[0003] Antimicrobial peptides (AMPs) are a class of small-molecule polypeptides that inhibit the growth of bacteria, fungi, and viruses. They are widely distributed in organisms and are an important component of the innate immune system. Antimicrobial peptides typically possess a net positive charge and an amphiphilic α-helix structure. This structure allows them to target and bind to the negatively charged bacterial plasma membrane. Furthermore, the amphiphilic nature promotes the interaction between the antimicrobial peptide and the bacterial plasma membrane, facilitating penetration and disruption of the bacterial cell membrane, ultimately leading to bacterial death. Antimicrobial peptides not only do not readily induce bacterial resistance but also exhibit broad-spectrum antibacterial activity, high efficacy with minimal dosage, and immunomodulatory effects. However, not all small-molecule polypeptides possess antimicrobial activity, and even those with antimicrobial activity face numerous limitations in practical production and application. For example, instability in practical applications, unknown hemolytic activity, and cytotoxicity severely restrict the development of antimicrobial peptides. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide MAP34B-2 and its application, which has good bactericidal and bacteriostatic effects on Gram-negative bacteria, including Escherichia coli and Acinetobacter baumannii, thereby damaging bacterial growth and cell membrane synthesis.

[0005] This invention provides an antimicrobial peptide MAP34B-2, the amino acid sequence of which is shown in SEQ ID NO:1.

[0006] Preferably, the C-terminus of the antimicrobial peptide MAP34B-2 is modified by amidation.

[0007] This invention provides the application of the antimicrobial peptide MAP34B-2 described above in the preparation of bactericides and / or bacteriostatic agents.

[0008] Preferably, the bactericidal and / or bacteriostatic agent is directed against Gram-negative bacteria.

[0009] Preferably, the Gram-negative bacteria include Escherichia coli (E. coli) and / or Acinetobacter baumannii. Escherichia coli Acinetobacter baumnnii

[0010] The application provides application of the antibacterial peptide MAP34B-2 in the preparation of a medicine for preventing and / or treating a bacterial infectious disease.

[0011] Preferably, the bacteria include Gram-negative bacteria.

[0012] Preferably, the Gram-negative bacteria include Escherichia coli and / or Acinetobacter baumannii.

[0013] The application provides a bactericidal and / or bacteriostatic product, and an active ingredient includes the antibacterial peptide MAP34B-2.

[0014] Preferably, the active ingredient further includes other bacteriostatic and bactericidal components; the other bacteriostatic and bactericidal components include antibiotics.

[0015] Beneficial effects:

[0016] The antibacterial peptide MAP34B-2 provided by the application has a small molecular weight, can change the permeability of cell membranes of Escherichia coli and Acinetobacter baumannii and inhibit the generation of the cell membranes, and plays a bacteriostatic and bactericidal role. Moreover, the antibacterial peptide MAP34B-2 provided by the application has low hemolytic activity and no cytotoxicity, can be applied to the preparation of a product with a bacteriostatic and bactericidal role, developed as an antibacterial peptide bacteriostatic and bactericidal agent, and prepared into a medicine for preventing and / or treating a bacterial infectious disease. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments.

[0018] Figure 1 The cell toxicity detection result of the antibacterial peptide MAP34B-2 on human immortalized keratinocytes Hacat;

[0019] Figure 2 The cell toxicity detection result of the antibacterial peptide MAP34B-2 on mouse fibroblasts L929;

[0020] Figure 3 The hemolytic activity of the antibacterial peptide MAP34B-2 on human red blood cells;

[0021] Figure 4 The influence of the antibacterial peptide MAP34B-2 on the outer membrane permeability of Escherichia coli.​​

[0022] Figure 5 Effect of antibacterial peptide MAP34B-2 on the permeability of outer membrane of Acinetobacter baumannii cells;

[0023] Figure 6 Effect of antibacterial peptide MAP34B-2 on the permeability of inner membrane of Escherichia coli cells;

[0024] wherein, represents P <0.001; represents P ＜ 0.0001. DETAILED DESCRIPTION

[0025] The present application provides an antibacterial peptide MAP34B-2, and the amino acid sequence is shown as SEQ ID NO: 1.

[0026] As an embodiment, the C-terminal of the antibacterial peptide MAP34B-2 is modified by amidation. Compared with the unmodified antibacterial peptide, the antibacterial peptide MAP34B-2 modified by amidation has the advantage of improving the stability of the antibacterial peptide.

[0027] The present application provides the use of the antibacterial peptide MAP34B-2 in the preparation of a bactericidal agent and / or a bacteriostatic agent.

[0028] As an embodiment, the bactericidal agent and / or the bacteriostatic agent of the present application is directed against gram-negative bacteria. As an embodiment, the gram-negative bacteria of the present application includes Escherichia coli and / or Acinetobacter baumannii.

[0029] The present application provides the use of the antibacterial peptide MAP34B-2 in the preparation of a drug for preventing and / or treating bacterial infectious diseases.

[0030] As an embodiment, the bacteria of the present application includes gram-negative bacteria. As an embodiment, the gram-negative bacteria of the present application includes Escherichia coli and / or Acinetobacter baumannii. As an embodiment, the Escherichia coli of the present application is Escherichia coli ATCC 25922. As an embodiment, the Acinetobacter baumannii of the present application is Acinetobacter baumannii ATCC 19606.

[0031] The present application provides a bactericidal and / or bacteriostatic product, and the active ingredient includes the antibacterial peptide MAP34B-2 described in the above scheme.

[0032] As an embodiment, the active ingredient of the present application further includes other bacteriostatic and bactericidal ingredients; the other bacteriostatic and bactericidal ingredients include antibiotics. As an embodiment, the antibiotics of the present application include ampicillin.

[0033] As an embodiment, the product of the present application includes a medicine or a skin care product. As an embodiment, the product of the present application further includes an excipient. The present application does not have strict requirements on the type of the excipient, which can be selected according to the type of the product. When the product is a medicine, the excipient is a pharmaceutically acceptable excipient; when the product is a skin care product, the excipient is a cosmetically acceptable excipient.

[0034] In order to further illustrate the present application, an antibacterial peptide MAP34B-2 and its application provided by the present application are described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0035] Example 1

[0036] The polypeptide sequence (RWGWRWKRWFR) described in SEQ ID NO: 1 is synthesized by an artificial synthesis method, and the carboxyl terminal is amidated, which is denoted as MAP34B-2, the molecular weight is 1719.01 Da, the isoelectric point (pI) is 12.48, and the secondary structure is an alpha helix.

[0037] Example 2

[0038] Minimum inhibitory concentration (MIC) determination

[0039] 1. Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC 19606 were used as test strains, and the activated strains were inoculated in LB liquid medium and placed in a constant temperature shaker for culture to the logarithmic growth phase.

[0040] 2. The concentration of the bacterial solution was detected, and fresh sterile liquid medium was used for dilution to 2×10 6 cfu / mL for standby.

[0041] 3. A sterile transparent 96-well plate was taken, 180 μL of fresh sterile liquid medium was added to the first column of holes, and 100 μL of fresh sterile liquid medium was added to the remaining holes. 20 μL of the test sample solution diluted to a certain concentration and filtered through a 0.22 μm filter membrane was added to the first column of holes (the test sample of rows A, B, and C is the polypeptide MAP34B-2 obtained in Example 1, and the test sample of rows D, E, and F is ampicillin).

[0042] 4. After uniform blowing with a pipette, 100 μL was taken and added to the second column of holes, and sequentially diluted, and when the last hole was taken, the 100 μL of sterile liquid was discarded.

[0043] 5. Add 100 μL of the pre-diluted bacterial solution to each well, at this time the sample concentration of the first column is the highest (the sample concentration of the first well is 100 μg / mL), and the rest is half of the concentration of the previous column.

[0044] 6. Place the 96-well plate in a 37°C constant temperature incubator for incubation, and take the average of the sample concentration of the previous well as the minimum inhibitory concentration. The results are shown in Table 1, and the minimum inhibitory concentration of the polypeptide MAP34B-2 to Escherichia coli ATCC 25922 is 18.75 μg / mL, and the minimum inhibitory concentration to Acinetobacter baumannii ATCC 19606 is >100.00 μg / mL.

[0045] Table 1 MIC of drugs to strains (μg / mL)

[0046]

[0047] Example 3

[0048] Minimum bactericidal concentration (MBC) determination

[0049] 1. Take 50 μL of the culture solution from the culture wells (1 / 2 MIC, MIC, 2xMIC, 4xMIC concentration wells) of Example 2 for Escherichia coli ATCC 25922 and Acinetobacter baumannii ATCC 19606 as the test strains.

[0050] 2. Spread the above culture solution on fresh LB plates without drugs, and incubate the spread plates at 37°C for 24 h.

[0051] 3. Observe the number of bacteria on the plates. The minimum drug concentration that kills 99.9% of bacteria is the MBC. The results are shown in Table 2, and the minimum bactericidal concentration of the polypeptide MAP34B-2 to Escherichia coli ATCC 25922 is 2xMIC, and the minimum bactericidal concentration to Acinetobacter baumannii ATCC 19606 is >4xMIC.

[0052] Table 2 MBC of drugs to strains

[0053]

[0054] Example 4

[0055] Cytotoxicity experiment

[0056] The toxicity of the polypeptide MAP34B-2 obtained in Example 1 to human immortalized keratinocytes HaCat and mouse fibroblasts L929 was determined by CCK-8 colorimetry, and the specific steps were as follows: prepare a cell suspension, count the cells using a counting plate and dilute with DMEM cell culture solution to a final concentration of 2x104 The MAP34B-2 was diluted to different concentrations (0.5xMIC, lxMIC, 2xMIC, 4xMIC, 8xMIC for E. coli ATCC 25922) with 5 replicates in each group. After the cells were adhered, the MAP34B-2 dilutions of different concentrations were added. After the end of the reaction with the cells, 110 μL of CCK8 solution with a concentration of 10% (i.e. the volume ratio of CCK8 to culture medium was 1:10) was added to each well in the 96-well plate, and the plate was incubated in the incubator for 4 h. The absorbance value at OD 450 nm was measured using an enzyme labeler.

[0057] The results are shown in Table 1. Figure 1 and Figure 2 As shown in Table 1, at 2xMIC, the survival rates of the two cells were both above 50%, indicating that the antibacterial peptide MAP34B-2 has a certain safety.

[0058] Example 5

[0059] Hemolysis experiment

[0060] A human red blood cell sample was used, and the red blood cells were washed with PBS until the supernatant was clear and colorless. The red blood cells were resuspended to a 4% (v / v) suspension with PBS. 500 μL of the red blood cell solution was taken into a 1.5 mL centrifuge tube, and 500 μL of the polypeptide MAP34B-2 solution of Example 1 of different concentrations (0.5xMIC, lxMIC, 2xMIC, 4xMIC, 8xMIC for E. coli ATCC 25922) was added. The negative control was PBS, and the positive control was 1% (v / v) Triton-100. After mixing, the mixture was incubated in a constant-temperature shaker at 37°C and 220 rpm for 1 h. The red blood cell precipitate was obtained by centrifugation. The supernatant was aspirated to detect the absorbance at 540 nm, and the hemolysis rate was calculated according to the formula: hemolysis rate (%) = (experimental group OD540-negative control group OD540) / (positive control group OD540-negative control group OD540) x 100%.

[0061] The results are shown in Table 2. Figure 3 As shown in Table 2, at 16xMIC, the hemolysis was always less than 5%, indicating that the antibacterial peptide MAP34B-2 has a certain safety.

[0062] Example 6

[0063] Bacterial outer membrane permeability experiment

[0064] Colonies of *Escherichia coli* ATCC 25922 and *Acinetobacter baumannii* ATCC 19606 were transferred to LB broth and cultured at 37°C with shaking until the logarithmic growth phase. The cultures were then washed three times with 5 mM HEPES buffer and diluted to OD600 = 0.5. The diluted bacterial solution was incubated with 1-phenylnaphthylamine (NPN) dissolved in anhydrous ethanol for 30 min to a final NPN concentration of 10 μM. Background fluorescence was recorded and subtracted using a full-wavelength microplate reader at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. In sterile black 96-well microtiter plates, the peptide MAP34B-2 from Example 1 was added to the bacterial suspension to final concentrations of 1×MIC, 2×MIC, 4×MIC, and 8×MIC (FI). peptide ), with PBS as the negative control (FI0) and 0.2 mg / mL polymyxin B as the positive control (FI). 100 ), and recorded the changes in bacterial outer membrane permeability over time by fluorescence intensity, according to the formula: Permeability (%) = (FI) peptide -FI0) / (FI 100 Calculate the penetration rate by multiplying (-FI0) by 100%.

[0065] The results are as follows Figure 4 and Figure 5 As shown, as the concentration gradually increases, the permeability of the antimicrobial peptide MAP34B-2 also gradually increases, approaching the result of the positive control. This indicates that MAP34B-2 uses the disruption of bacterial outer membrane permeability as one of its bactericidal mechanisms, and this effect is reflected in the concentration.

[0066] Example 7

[0067] Biomembrane inhibition function

[0068] Escherichia coli ATCC 25922 was cultured overnight at 37°C and 220 rpm. The overnight culture was then used for transfer. Once the bacteria reached the logarithmic growth phase, the culture was diluted with fresh medium to a concentration of 2 × 10⁻⁶. 7Cfu / mL. 100 μL of the bacterial solution was added to the wells of a 96-well plate containing 100 μL of an antibacterial substance solution (polypeptide MAP34B-2 and ampicillin of Example 1) at different concentrations (0.5xMIC, 1xMIC, 2xMIC, 4xMIC, 8xMIC), the positive control (PC) was the bacterial solution plus sterilized water, and the negative control (NC) was the culture medium plus sterilized water. Incubation was carried out at 37°C for 48 h. The bacterial solution was aspirated, and the floating bacteria were washed away with normal saline. After 24 h of incubation, the solution was aspirated, the bacterial film was dried and fixed, 20 min of treatment with crystal violet staining solution was carried out, the staining solution was aspirated and washed and dried, 200 μL of anhydrous ethanol was added and left to stand for 20 min to completely dissolve the dye, and the OD value was measured at 595 nm. The residual biofilm percentage (%) was calculated according to the formula: residual biofilm percentage (%) = (OD measured value-OD blank group) / (OD positive group-OD blank group) x 100%, and the residual biofilm percentage after treatment with the antibacterial peptide and the positive drug was calculated.

[0069] The results, as shown in Figure 6 With the gradual increase in the concentration, the degree of inhibition of the antibacterial peptide MAP34B-2 on the biofilm also gradually increased, which indicated that MAP34B-2 inhibited the formation of bacterial biofilm as one of the bactericidal mechanisms, and showed a concentration trend.

[0070] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments. Other embodiments can be obtained according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. An antimicrobial peptide MAP34B-2, characterized in that, The amino acid sequence is shown in SEQ ID NO:1; The C-terminus of the antimicrobial peptide MAP34B-2 is modified by amidation.

2. The use of the antimicrobial peptide MAP34B-2 according to claim 1 in the preparation of bactericides and / or bacteriostatic agents; The bactericide and / or bacteriostatic agent is targeted at Gram-negative bacteria; The Gram-negative bacteria mentioned are Escherichia coli (Escherichia coli) Escherichia coli ).

3. The use of the antimicrobial peptide MAP34B-2 according to claim 1 in the preparation of medicaments for the prevention and / or treatment of bacterial infectious diseases; The bacteria in question are Gram-negative bacteria; The Gram-negative bacterium mentioned is Escherichia coli.

4. A bactericidal and / or bacteriostatic product, characterized in that, The active ingredient includes the antimicrobial peptide MAP34B-2 as described in claim 1.

5. The bactericidal and / or bacteriostatic product according to claim 4, characterized in that, The active ingredients also include other antibacterial and bactericidal components; the other antibacterial and bactericidal components include antibiotics.

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