Antibacterial peptide for inhibiting gram-negative bacteria as well as preparation method and application of antibacterial peptide

Abstract

The invention discloses an antibacterial peptide sequence for inhibiting gram-negative bacteria and application of the antibacterial peptide sequence. A plurality of antibacterial peptides are designed for gram-negative bacteria, and antibacterial test results show that the amino acid sequence shown in SEQ ID No.4 has an exact inhibition effect on two gram-negative bacteria and also has a certain inhibition effect on staphylococcus aureus; in addition, the amino acid sequence as shown in SEQ ID No.2 has an exact inhibition effect on gram-positive bacterium micrococcus luteus and fungus candida albicans, and the inhibition rates are 99.32% and 93.42% respectively. The invention further provides application of the antibacterial peptide in preparation of drugs for resisting bacterial or fungal infection.

Description

Technical Field

[0001] The invention relates to antimicrobial peptides, in particular to the application of antimicrobial peptides for inhibiting Gram-negative bacteria, belonging to the field of antimicrobial peptides and their application. Background Art

[0002] Gram-negative bacteria appear red during the staining process. Their cell walls are thinner and contain less peptidoglycan than Gram-positive bacteria, but their outer membrane provides additional protection. This structure enhances Gram-negative bacteria's resistance to many antimicrobial drugs and also increases their ability to survive in the environment. The presence of the outer membrane not only increases the difficulty of drug penetration, but also limits the effectiveness of existing antimicrobial peptides. Therefore, designing and screening antimicrobial peptides against Gram-negative bacteria can effectively improve treatment efficiency, reduce drug resistance and side effects, and provide more options for clinical treatment. Summary of the invention

[0003] One of the purposes of the present invention is to provide an antimicrobial peptide that inhibits pathogenic bacteria;

[0004] The second purpose of the present invention is to apply the antimicrobial peptide to prepare drugs for resisting bacterial or fungal infections.

[0005] The above-mentioned object of the present invention is mainly achieved through the following technical solutions:

[0006] One aspect of the present invention is to provide an antimicrobial peptide for inhibiting pathogenic bacteria, which is selected from the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 4.

[0007] The present invention designs a plurality of antimicrobial peptides (AMPs) for Gram-negative bacteria. The results of the antibacterial test show that the amino acid sequence (B4) shown in SEQ ID No. 4 has a definite inhibitory effect on two strains of Gram-negative bacteria, wherein the inhibition rate on Escherichia coli is 51.32% and the inhibition rate on Klebsiella pneumoniae is 74.69%. In addition, B4 also has a certain inhibitory effect on Staphylococcus aureus, and its inhibition rate is 45.79%. In addition, the amino acid sequence (B2) shown in SEQ ID No. 2 has a definite inhibitory effect on Gram-positive bacteria Micrococcus luteus and fungus Candida albicans, and its inhibition rates are 99.32% and 93.42% respectively.

[0008] Another aspect of the present invention is to use the antimicrobial peptide in the preparation of a drug for inhibiting bacterial or fungal infection; wherein the bacteria are selected from Gram-negative bacteria or Gram-positive bacteria, and the Gram-negative bacteria are preferably Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium; the Gram-positive bacteria are preferably Staphylococcus aureus or Micrococcus luteus; and the fungus is preferably Candida albicans.

[0009] The present invention provides a pharmaceutical composition for inhibiting pathogenic bacteria, wherein the pharmaceutical composition contains the antimicrobial peptide of the present invention and a carrier or excipient acceptable for pharmaceutical preparations; wherein the bacteria are selected from Gram-negative bacteria or Gram-positive bacteria, and the Gram-negative bacteria are preferably Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium; the Gram-positive bacteria are preferably Staphylococcus aureus or Micrococcus luteus; and the fungus is preferably Candida albicans.

[0010] Definitions of terms used in this invention

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods, devices and materials are now described.

[0012] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides apply equally to describing peptides and describing proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the result of hemolytic activity detection of candidate AMP.

[0014] Figure 2 The results of the antibacterial test of candidate AMPs on bacteria or fungi. DETAILED DESCRIPTION

[0015] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0016] Experimental Example 1 Design and screening of antimicrobial peptides against Gram-negative bacteria

[0017] 1. Design of antimicrobial peptides against Gram-negative bacteria

[0018] Four antimicrobial peptides (AMPs) were designed to inhibit Gram-negative bacteria, and their amino acid sequences are as follows:

[0019] Antimicrobial peptides (B1):

[0020] GGGKAKALAKLAG (SEQ ID No. 1)

[0021] Antimicrobial peptides (B2):

[0022] LKGLAAKLLGLKK (SEQ ID No. 2)

[0023] Antimicrobial peptide (B3):

[0024] GKGAKKGLKGLKG (SEQ ID No. 3)

[0025] Antimicrobial peptides (B4):

[0026] LGGLAKKKAAAKL (SEQ ID No. 4)

[0027] 2. Test methods

[0028] (I) Hemolytic activity detection

[0029] The hemolytic activity of candidate AMPs against eukaryotic cells was tested using 2% rabbit erythrocytes.

[0030] (1) In a total system of 400 μL, 390 μL of 2% rabbit red blood cells and 10 μL of candidate AMPs solution were added. The final concentration of the candidate AMPs in the total system was 128 μM.

[0031] (2) The PBS group was used as a negative control, and the distilled water group was used as a positive control.

[0032] (3) Incubate the mixed solution at 37°C for 3 h.

[0033] (4) The sample was centrifuged at 5000 rpm for 10 min, and then the supernatant was aspirated and its absorbance at 540 nm was measured.

[0034] (5) The hemolysis rate is calculated as follows:

[0035]

[0036] Among them, OD S is the absorbance value of the experimental group at 540 nm; OD N is the absorbance value of the negative control group at 540 nm; OD P It is the absorbance value of the positive control group at 540 nm.

[0037] (II) Determination of bacterial inhibition rate by broth microdilution method

[0038] At a fixed concentration of 128 μM, the inhibitory activities of the four candidate AMPs against three Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium) and three Gram-positive bacteria (Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus) were qualitatively tested.

[0039] (1) Pick a single colony and culture it in antibiotic-free CaMHB liquid culture medium at 37°C and 200 rpm overnight.

[0040] (2) Transfer the inoculum to fresh culture medium at 1% of the inoculum volume and culture at 37°C and 200 rpm until the logarithmic growth phase (Micrococcus luteus: OD 450 =0.4-0.6, other strains: OD 600 =0.4-0.6). Then, the cell concentration was adjusted to about 5×10 5 CFU / mL.

[0041] (3) Add 100 μL of the diluted bacterial solution into a 96-well plate, with each well containing 100 μL of a different candidate AMPs solution, and the final concentration of AMPs in each well is 128 μM.

[0042] (4) The 100 μL double distilled water + 100 μL CaMHB liquid culture medium group was the blank control group; the 100 μL bacterial liquid + 100 μL double distilled water group was the negative control group.

[0043] (5) The whole system was incubated at 37°C for 16 h. After the reaction was completed, the OD value was measured.

[0044] (6) Finally, the antibacterial activity of different candidate AMPs against different bacteria was obtained, and the calculation formula of bacterial inhibition rate was as follows:

[0045]

[0046] Among them, OD S is the OD value of the experimental group at 16h. is the initial OD value of the experimental group; OD P is the OD value of the negative control group at 16h. is the initial OD value of the negative control group.

[0047] (III) Determination of fungal inhibition rate by broth microdilution method

[0048] The inhibitory activity against fungi (Candida albicans) was qualitatively detected at a fixed concentration of 128 μM.

[0049] (1) Pick a single colony and culture it in antibiotic-free PDB liquid medium at 30°C and 200 rpm overnight;

[0050] (2) 1% of the inoculum was transferred to fresh culture medium and cultured at 30°C and 200 rpm until the logarithmic growth phase (OD 600 =0.4-0.6). Then, the cell concentration was adjusted to about 5×10 5 CFU / mL.

[0051] (3) Add 100 μL of the diluted bacterial solution into a 96-well plate, with each well containing 100 μL of a different candidate AMPs solution, and the final concentration of AMPs in each well is 128 μM.

[0052] (4) The 100 μL double distilled water + 100 μL PDB liquid culture medium group was the blank control group; the 100 μL bacterial liquid + 100 μL double distilled water group was the negative control group.

[0053] (5) The whole system was incubated at 30°C for 32 h. The OD value was measured after the reaction was completed.

[0054] (6) Finally, the antibacterial activity of different candidate AMPs against Candida albicans was obtained, and the calculation formula of the fungal inhibition rate was the same as that of the bacterial inhibition rate.

[0055] 3. Test results

[0056] (I) Hemolytic test results of candidate AMPs

[0057] At a concentration of 128 μM, the hemolytic activity of the four candidate AMPs on eukaryotic cells was evaluated. We observed that the hemolytic rates of the four candidate AMPs were low, among which B1, B3, and B4 were completely non-hemolytic with a hemolytic rate of 0.00%; the hemolytic rate of B2 was 6.57%, which was also within a safe range ( Figure 1 ). The above illustrates the safety of clinical application of these four AMPs.

[0058] (II) Inhibitory effects of candidate AMPs on bacteria and fungi

[0059] Among the four candidate peptides verified, B4 was effective against two strains of Gram-negative bacteria, with an inhibition rate of 51.32% against Escherichia coli and 74.69% against Klebsiella pneumoniae. B4 also had a certain inhibitory effect on Staphylococcus aureus, with an inhibition rate of 45.79%. In addition, the inhibition rates of B2 against Gram-positive bacteria Micrococcus luteus and fungus Candida albicans were 99.32% and 93.42%, respectively; in summary, B4 can inhibit the growth of two strains of Gram-negative bacteria ( Figure 2 ).

[0060] In summary, the hemolytic assay results showed that B1, B3, and B4 were completely non-hemolytic, and the hemolytic rate of B2 was 6.57%, proving that the four peptides were safe for clinical application. The antibacterial test results showed that B2 and B4 of the four candidate AMPs had an inhibitory effect on at least one pathogen, among which B2 had a significant inhibitory effect on Micrococcus luteus and Candida albicans, and B4 had a significant inhibitory effect on two strains of Gram-negative bacteria.

Claims

1. An antimicrobial peptide for inhibiting pathogenic bacteria, characterized in that: It is selected from the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No.

4.

2. The antimicrobial peptide according to claim 1, characterized in that The pathogenic bacteria are bacteria or fungi; wherein the bacteria are Gram-negative bacteria or Gram-positive bacteria.

3. The antimicrobial peptide according to claim 2, characterized in that The Gram-negative bacteria are Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium; the Gram-positive bacteria are Staphylococcus aureus or Micrococcus luteus; and the fungus is Candida albicans.

4. Use of the antimicrobial peptide according to claim 1 in the preparation of a medicament for inhibiting bacterial or fungal infection.

5. The use according to claim 4, characterized in that The bacteria are selected from Gram-negative bacteria or Gram-positive bacteria, wherein the Gram-negative bacteria are preferably Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium; the Gram-positive bacteria are preferably Staphylococcus aureus or Micrococcus luteus; and the fungus is Candida albicans.

6. A pharmaceutical composition for inhibiting pathogenic bacteria, characterized in that: The pharmaceutical composition contains the antimicrobial peptide according to claim 1 and a carrier or excipient acceptable for pharmaceutical preparations.

7. The pharmaceutical composition according to claim 6, characterized in that The pathogenic bacteria are bacteria or fungi; wherein the bacteria are selected from Gram-negative bacteria or Gram-positive bacteria, and the Gram-negative bacteria are preferably Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium; the Gram-positive bacteria are preferably Staphylococcus aureus or Micrococcus luteus; and the fungi are preferably Candida albicans.

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