Broad-spectrum antibacterial peptide and application thereof

Abstract

This invention relates to the field of biomedical technology and discloses a broad-spectrum antimicrobial peptide and its applications. The invention obtains a broad-spectrum antimicrobial peptide H by mining non-classical open reading frames (ncORFs) in the human genome. Experimental verification shows that the broad-spectrum antimicrobial peptide H has significant inhibitory effects on both Gram-positive bacteria (Staphylococcus aureus ATCC 29213) and Gram-negative bacteria (Escherichia coli K88). Furthermore, the broad-spectrum antimicrobial peptide H can form an α-helix. The α-helix structure projection diagram shows that one side is rich in positively charged hydrophilic amino acid residues, and the other side is rich in hydrophobic amino acid residues, exhibiting good amphiphilicity. In addition, the broad-spectrum antimicrobial peptide H provided by this invention has low hemolytic activity and no cytotoxicity, ensuring in vivo safety while achieving highly efficient bactericidal activity. It also features a short synthetic sequence, small molecular weight, and is easy to chemically synthesize.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a novel antimicrobial peptide and its application in the preparation of broad-spectrum antimicrobial drugs, antimicrobial coatings for medical devices, or preservatives. Background Technology

[0002] The spread of antibiotic resistance has become a global public health crisis, urgently requiring the development of antimicrobial drugs with novel mechanisms of action. Antimicrobial peptides (AMPs), with their unique advantages such as membrane-targeting action and low likelihood of inducing resistance, represent an important strategy for replacing traditional antibiotics and provide a key direction for addressing the resistance crisis.

[0003] Currently reported antimicrobial peptides are mostly from microbial sources, which suffer from drawbacks such as low bioactivity, low heterologous expression efficiency, and insufficient stability. Antimicrobial potency is generally expressed using the minimum inhibitory concentration (MIC). If the MIC value of an antimicrobial peptide is too high, large doses are required to achieve effectiveness, rendering the product worthless. Furthermore, increasing drug concentration can lead to cytotoxicity and hemolysis. As polypeptides, antimicrobial peptides are not particularly resistant to high temperatures or acids and alkalis. Antimicrobial peptides are bioactive polypeptides, and during bio-fermentation, host bacteria often produce many proteases that degrade these peptides, causing them to be degraded and lost during fermentation, rendering them unproductive. Naturally occurring or genetically engineered antimicrobial peptides constitute a very small proportion of the separation and purification matrix, containing a large amount of extraneous proteins and other substances, resulting in high separation and purification costs. Therefore, designing and developing antimicrobial peptides with simple structures, high bioactivity, and synthesized using solid-phase chemical methods is currently a pressing need to address the emergence of drug-resistant bacteria. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a broad-spectrum antimicrobial peptide and its applications.

[0005] The specific technical solution of this invention is as follows:

[0006] As a first aspect of the present invention, a broad-spectrum antimicrobial peptide is provided, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] This invention discloses a broad-spectrum antimicrobial peptide H obtained by mining non-classical open reading frames (ncORFs) in the human genome, the amino acid sequence of which is shown in SEQ ID NO.1. Experimental verification shows that the broad-spectrum antimicrobial peptide H has significant inhibitory effects on both Gram-positive bacteria (Staphylococcus aureus ATCC 29213) and Gram-negative bacteria (Escherichia coli K88). Furthermore, the broad-spectrum antimicrobial peptide H can form an α-helix; the α-helix structure projection diagram shows that one side is rich in positively charged hydrophilic amino acid residues, while the other side is rich in hydrophobic amino acid residues, exhibiting good amphiphilicity. In addition, the broad-spectrum antimicrobial peptide H provided by this invention has low hemolytic activity and no cytotoxicity, ensuring in vivo safety while achieving highly efficient bactericidal activity. It also features a short synthetic sequence, small molecular weight, and is easy to synthesize chemically.

[0008] As a second aspect of the present invention, the application of a broad-spectrum antimicrobial peptide in antibacterial activity is provided.

[0009] Preferably, the antibacterial activity is the inhibition of Gram-negative bacteria and / or Gram-positive bacteria.

[0010] Further preferred, the Gram-positive bacterium is Staphylococcus aureus ATCC 29213, and the Gram-negative bacterium is Escherichia coli K88.

[0011] Further preferably, the minimum inhibitory concentration (MIC) of the broad-spectrum antimicrobial peptide against Staphylococcus aureus ATCC 29213 is 13 μg / ml, and the minimum inhibitory concentration (MIC) of the broad-spectrum antimicrobial peptide against Escherichia coli K88 is 26 μg / ml.

[0012] As a third aspect of the present invention, the use of a broad-spectrum antimicrobial peptide in the preparation of a broad-spectrum antimicrobial drug for treating infections caused by Gram-positive and Gram-negative bacteria is provided.

[0013] Compared with the prior art, the present invention has the following technical effects:

[0014] (1) This invention obtains a broad-spectrum antimicrobial peptide H by mining non-classical open reading frames (ncORFs) in the human genome, the amino acid sequence of which is shown in SEQ ID NO.1. Experimental verification shows that the broad-spectrum antimicrobial peptide H has a significant inhibitory effect on both Gram-positive bacteria (Staphylococcus aureus ATCC 29213) and Gram-negative bacteria (Escherichia coli K88). At the same time, the broad-spectrum antimicrobial peptide H can form an α-helix. The projection diagram of the α-helix structure shows that one side is rich in positively charged hydrophilic amino acid residues, and the other side is rich in hydrophobic amino acid residues, which has good amphiphilicity. In addition, the broad-spectrum antimicrobial peptide H provided by this invention has low hemolytic activity and no cytotoxicity, ensuring in vivo safety while achieving high bactericidal effect.

[0015] (2) The broad-spectrum antimicrobial peptide H provided by the present invention has the characteristics of short synthetic sequence and small molecular weight, and is easy to chemically synthesize. Attached Figure Description

[0016] Figure 1 Specific prediction results for five antimicrobial peptides;

[0017] Figure 2 The results show the MIC determination of five antimicrobial peptides against Staphylococcus aureus ATCC 29213;

[0018] Figure 3 The results show the MIC determination of five antimicrobial peptides against Escherichia coli K88;

[0019] Figure 4 Experimental diagrams showing the observation of hemolysis of five antimicrobial peptides before and after 0h and 3h;

[0020] Figure 5 This is a predicted structural diagram of antimicrobial peptide H;

[0021] Figure 6 This is a projection diagram of the α-helix structure of antimicrobial peptide H. Detailed Implementation

[0022] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0023] Example 1: Discovery and Sequence Design of Antimicrobial Peptide H

[0024] This embodiment involves the discovery and sequence design of antimicrobial peptides, following these steps:

[0025] 1. Data Acquisition: Based on publicly available, mass spectrometry-validated research data on functional translation of human non-classical open reading frames, obtain potential microprotein sequences encoded by stably expressed ncORFs.

[0026] 2. Activity Prediction: The experimentally validated sequence library was scanned using the antimicrobial peptide prediction tool AmPEP. A prediction score threshold was set to screen for peptide sequences with potential antimicrobial activity. Candidate peptides with scores higher than 0.9 were selected, and five sequences, including antimicrobial peptide H, were initially chosen and named antimicrobial peptides C, D, K, H, and 13, respectively. Detailed prediction results are shown below. Figure 1The scores for antimicrobial peptides C, D, K, H, and 13 were 0.93, 0.98, 0.97, 0.93, and 0.97, respectively.

[0027] 3. Structure Prediction: The AlphaFold3 server was used to predict the three-dimensional structure of the antimicrobial peptide H sequence to assess its folding stability. (See results below.) Figure 5 .

[0028] Example 2 Chemical Synthesis and Purification of Antimicrobial Peptides

[0029] The antimicrobial peptides were synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. using the standard solid-phase peptide synthesis method (Fmoc strategy). After synthesis, the peptides were purified using preparative high-performance liquid chromatography (HPLC), with purities all >95%. The amino acid sequences of antimicrobial peptides C, D, K, H, and 13 are shown in Table 1, as indicated by SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.1, and SEQ ID NO.2, respectively.

[0030] Table 1. Amino acid sequences of five antimicrobial peptides

[0031] Antimicrobial peptides amino acid sequence C LRRPSWLTGRRRGVCPLLRVLLARG D VALKCYCRNSFWVLKNYKRS K LAGARNWWRHCLSPLLLPR H LSHCLRSSGRLAPHTSRRYLEFLQHGRH 13 LQGVRISSKYRRIISFSLIA

[0032] Example 3: In vitro antibacterial activity assay

[0033] The MICs of peptide H against various pathogens were determined using the microbroth dilution method according to CLSI M07 standards. The minimum inhibitory concentration (MIC) of antimicrobial peptide H against Staphylococcus aureus ATCC 29213 and Escherichia coli K88 was determined using the microbroth dilution method. Overnight cultures of Staphylococcus aureus ATCC 29213 and Escherichia coli K88 were resuspended in sterile PBS and adjusted to 0.5 McFarland turbidity (5 × 10⁻⁶). 5 (CFU / mL). The antimicrobial peptide obtained in Example 2 was aliquoted into 1 mg of sterile MRS broth medium and dissolved. The stock solution dilution gradient was as follows: 75 μL of antimicrobial peptide H stock solution was dissolved in 75 μL of sterile MRS broth medium, and 150 μL was added to a 96-well plate. This was followed by 7 serial dilutions. Finally, 100 μL of bacterial suspension was added to each well, and after 12 h of static incubation, the optical density (OD) value of each well was measured at 600 nm. Wells containing MRS medium without the antimicrobial peptide served as the negative control group, and wells containing MRS medium with a chloramphenicol concentration of 30 μg / mL served as the positive control group. OD... 600nm OD value relative to blank control 600nm The concentration of antimicrobial peptides at which the value does not increase significantly is defined as the minimum inhibitory concentration (MIC). See the results below. Figure 2 , Figure 3 and the OD of antimicrobial peptide H 600nmThe measurement results are recorded in Tables 2 and 3.

[0034] Table 2. Optical density values ​​of Staphylococcus aureus ATCC 29213 after incubation with different concentrations of antimicrobial peptide H for 12 h.

[0035] concentration (μg / ml) <![CDATA[OD 600nm (0h)]]> <![CDATA[OD 600nm (12h)]]> 0 0.042 ± 0.004 0.389± 0.015 1.68 0.043 ± 0.012 0.344 ± 0.003 3.35 0.044 ± 0.007 0.357 ± 0.011 6.71 0.044 ± 0.009 0.371 ± 0.008 13.41 0.043 ± 0.005 0.370 ± 0.013 26.82 0.046 ± 0.007 0.388 ± 0.029 53.65 0.058 ± 0.006 0.347 ± 0.008 107.30 0.082 ± 0.002 0.320 ± 0.019 214.59 0.105 ± 0.009 0.361 ± 0.009

[0036] Table 3. Optical density values ​​of Escherichia coli K88 after incubation with different concentrations of antimicrobial peptide H for 12 hours.

[0037] concentration (μg / ml) <![CDATA[OD 600nm (0h)]]> <![CDATA[OD 600nm (12h)]]> 0 0.043 ± 0.004 0.484 ± 0.014 1.68 0.043 ± 0.003 0.476 ± 0.006 3.35 0.044 ± 0.005 0.435 ± 0.004 6.71 0.052 ± 0.009 0.473 ± 0.002 13.41 0.043 ± 0.009 0.384 ± 0.022 26.82 0.063 ± 0.006 0.446 ± 0.002 53.65 0.068 ± 0.009 0.406 ± 0.008 107.30 0.058 ± 0.003 0.423 ± 0.012 214.59 0.044 ± 0.008 0.393 ± 0.002

[0038] According to Figure 2 and Figure 3 Analysis of the experimental data showed that within the test concentration range (13~214 µg / ml), the tested antimicrobial peptides (including antipeptides K, C, D, H, and 13) all exhibited varying degrees of antibacterial activity. The minimum inhibitory concentrations (MICs) of antimicrobial peptide C against Staphylococcus aureus ATCC 29213 and Escherichia coli K88 were 13 μg / ml and 13 μg / ml, respectively; the MICs of antimicrobial peptide D against Staphylococcus aureus ATCC 29213 and Escherichia coli K88 were 13 μg / ml and 107 μg / ml, respectively; the MICs of antimicrobial peptide K against Staphylococcus aureus ATCC 29213 and Escherichia coli K88 were 13 μg / ml and 13 μg / ml, respectively; the MICs of antimicrobial peptide H against Staphylococcus aureus ATCC 29213 and Escherichia coli K88 were 13 μg / ml and 26 μg / ml, respectively; and the MIC of antimicrobial peptide 13 against Staphylococcus aureus ATCC 29213 was 26 μg / ml, but it had no significant inhibitory effect on Escherichia coli K88. In antibacterial tests against Staphylococcus aureus ATCC 29213 and Escherichia coli K88, the OD value of chloramphenicol in the positive control group was close to zero at a concentration of 30 µg / ml, confirming the effectiveness of the experimental system.

[0039] Example 4 Evaluation of hemolytic activity and cytotoxicity

[0040] 1. Hemolysis Assay: This assay aimed to evaluate the potential hemolytic toxicity of various antimicrobial peptides to erythrocytes at their minimum inhibitory concentration (MIC). First, fresh blood was defibrinated and repeatedly washed with physiological saline to prepare a 2% erythrocyte suspension. Then, an experimental group (an equal volume of the antimicrobial peptide MIC solution and the erythrocyte suspension), a positive control (distilled water), and a negative control (physiological saline) were set up. All mixed samples were incubated at 37°C for 3 hours and observed periodically. After incubation, the supernatant was collected by centrifugation, and its absorbance at 540 nm was measured. The hemolysis rate was calculated using a formula. If the hemolysis rate exceeded 5%, it was considered to have hemolytic activity and further statistical analysis was required. The hemolysis results are shown in Table 4 below. The experimental graphs showing the hemolysis of the five antimicrobial peptides before and after 0h and 3h are shown below. Figure 4 As shown.

[0041] Table 4. Results of the toxicity of five antimicrobial peptides to sheep erythrocytes.

[0042] Antimicrobial peptides Hemolysis rate (%) C <![CDATA[11.7% ± 0.216% b ]]> D <![CDATA[3.17% ± 0.26% b ]]> K <![CDATA[5.9% ± 0.082% b ]]> H <![CDATA[2.0% ± 0.3% b ]]> 13 <![CDATA[3.0% ± 0.245% b ]]> negative control <![CDATA[109%± 10.01% a ]]> Positive control <![CDATA[5.7%±1.52% b ]]>

[0043] 2. Cytotoxicity: This experiment aimed to investigate the potential toxicity of the antimicrobial peptide H to the RAW264.7 cell line. First, RAW264.7 cells were resuscitated and cultured in complete medium to the logarithmic growth phase. Then, the antimicrobial peptide stock solution was diluted to a series of working concentrations (including 1 / 4×, 1 / 2×, and 1×MIC) in complete medium. Cells were seeded into 96-well plates, and different concentrations of antimicrobial peptide solution were added for treatment. Blank wells (medium medium only) and control wells (cells plus medium) were also included. The treatment time was 24 hours. Finally, cell viability was assessed using the CCK-8 assay: CCK-8 solution was added to each well, and after culturing for another hour, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The cytotoxicity results are shown in Table 5 below.

[0044] Table 5. Toxicity results of five antimicrobial peptides against the RAW264.7 cell line.

[0045] Antimicrobial peptides Concentration (μg / ml) Cell viability C 1×MIC <![CDATA[0.9±0.03a b <!-- 4 -->]]> D 1×MIC <![CDATA[0.96±0.04 a ]]> K 1×MIC <![CDATA[0.61±0.03 c ]]> H 1×MIC <![CDATA[0.91±0.08a b ]]> 13 1×MIC <![CDATA[0.91±0.14 b ]]> Blank group 0 <![CDATA[0 d ]]> control group 0 <![CDATA[1.01±0.16 a ]]>

[0046] The results in Tables 4 and 5 show that antimicrobial peptide H has low hemolytic activity and no cytotoxicity.

[0047] Example 5: Structural Analysis of Antimicrobial Peptide H

[0048] The AlphaFold3 prediction model based on antimicrobial peptide H predicts the structure of antimicrobial peptide H. The three-dimensional structure of antimicrobial peptide H predicted by AlphaFold3 (70 < pIDDT < 90, high confidence) is shown below. Figure 5 ,like Figure 6As shown, the sequence of antimicrobial peptide H contains 28 amino acid residues, and its wheel diagram clearly shows the chemical distribution of each residue (red indicates basic, blue indicates acidic, green indicates polar and uncharged, and yellow indicates nonpolar). In particular, basic amino acids (such as R and H) are widely distributed in the sequence, while the 28th position at the C-terminus is the only acidic amino acid (E). Figure 6 The connecting lines reveal a potential network of interactions between residues, with the arginine (R) at position 27 adjacent to the glutamic acid (E) at position 28, suggesting the possible formation of a salt bridge crucial for structural stability. Furthermore, the clustered regions of nonpolar amino acids (such as L, F, and A) may constitute the hydrophobic core of the protein. The above illustrations provide a visual basis for understanding the structure-function relationship of antimicrobial peptide H and guiding subsequent site-directed mutagenesis studies. Results show that antimicrobial peptide H can form an amphiphilic α-helix structure in solution, with one side enriched with positively charged residues (R, H) and the other side composed of hydrophobic residues (L, F, Y, G, etc.). This indicates that antimicrobial peptide H possesses good amphiphilicity, which facilitates its interaction with bacterial cell membranes. Additionally, the helical projection shows a clear separation of hydrophilic and hydrophobic surfaces, with the carboxyl terminus forming a tightly packed parallel coiled helical dimer. This conformation provides a structural basis for its targeted disruption of bacterial cell membranes, achieving broad-spectrum antibacterial activity while maintaining low hemolysis / low cytotoxicity.

[0049] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A broad-spectrum antimicrobial peptide, characterized in that: The amino acid sequence of the broad-spectrum antimicrobial peptide is shown in SEQ ID NO.

1.

2. The broad-spectrum antimicrobial peptide as described in claim 1, characterized in that: The broad-spectrum antimicrobial peptide is used to inhibit Gram-negative bacteria and / or Gram-positive bacteria.

3. The broad-spectrum antimicrobial peptide as described in claim 1, characterized in that: The Gram-positive bacterium is Staphylococcus aureus ATCC 29213.

4. The broad-spectrum antimicrobial peptide as described in claim 3, characterized in that: The Gram-negative bacterium mentioned is Escherichia coli K88.

5. The application of the broad-spectrum antimicrobial peptide as described in any one of claims 1 to 4 in antibacterial activity.

6. The application as described in claim 5, characterized in that: The antibacterial effect refers to the inhibition of Gram-negative bacteria and / or Gram-positive bacteria.

7. The application according to claim 6, characterized in that: The Gram-positive bacteria is Staphylococcus aureus ATCC29213, and the Gram-negative bacteria is Escherichia coli K88.

8. The application according to claim 7, characterized in that: The minimum inhibitory concentration of the broad-spectrum antimicrobial peptide against Staphylococcus aureus ATCC29213 is 13 μg / ml.

9. The application according to claim 7, characterized in that: The minimum inhibitory concentration of the broad-spectrum antimicrobial peptide against Escherichia coli K88 is 26 μg / ml.

10. The use of the broad-spectrum antimicrobial peptide according to any one of claims 1 to 4 in the preparation of a broad-spectrum antimicrobial drug for treating infections caused by Gram-positive and Gram-negative bacteria.

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