Antibacterial peptide AI-SC-4 and application thereof

Abstract

The application relates to the technical field of biological pharmacy, in particular to an antibacterial peptide AI-SC-4 and application thereof. The antibacterial peptide AI-SC-4 is an artificial synthetic non-natural 22-peptide, the amino acid sequence is shown in SEQ ID NO:1, the theoretical molecular weight is 2700.26 Da, and the antibacterial peptide is a cationic amphiphilic polypeptide rich in basic amino acids and hydrophobic amino acids. The antibacterial peptide has the advantages of clear structure, small molecular weight and convenient artificial synthesis, and shows strong in-vitro antibacterial activity on gram-positive bacteria and gram-negative bacteria, and can be used for drug for resisting drug-resistant bacteria infection, a disinfectant, a skin external preparation and an oral care product, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to an antimicrobial peptide AI-SC-4 and its applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The advent of antibiotics has not only saved patients' lives but also played a crucial role in significant advancements in medicine and surgery. However, increased antibiotic use has led to the emergence of drug-resistant bacteria, and antibiotic resistance has become one of the greatest threats to global public health. Antimicrobial peptides, as a novel anti-infective agent, have gradually come into focus. As a core component of the innate immune system, antimicrobial peptides are key defensive substances generated in response to the invasion of pathogenic microorganisms. These short peptides possess both antimicrobial and anti-malignant proliferating cell activities, effectively inhibiting bacteria, fungi, parasites, viruses, and tumor cells. Antimicrobial peptides not only have broad-spectrum antibacterial activity, effectively killing various bacteria / fungi, but also induce resistance in pathogens at a much slower rate than with traditional antibiotics. These characteristics make antimicrobial peptides a promising addition to the existing antibiotic system.

[0004] Antimicrobial peptides are widely distributed in various organisms, having been found in insects, fish, mammals, amphibians, and plants. Immune cells, phagocytes, and mucosal epithelial cells in both animals and plants are important sites of their presence. The antimicrobial mechanism of antimicrobial peptides is complex. One core pathway is their efficient binding to the negatively charged cell membrane, composed of amphipathic molecules. This characteristic constitutes the structural prerequisite for the interaction between antimicrobial peptides and the cell membrane, subsequently inducing changes in membrane permeability or the formation of pores in lipid membranes, leading to the outflow of essential intracellular substances and ultimately cell death. Furthermore, current research has demonstrated that in addition to killing bacteria by altering bacterial cell membrane permeability, antimicrobial peptides can also bind to bacterial target proteins, downregulating the expression levels of genes related to bacterial biofilms and virulence factors. This inhibits bacterial biofilm formation and reduces inflammatory responses induced by virulence factors, exerting antimicrobial efficacy through multiple pathways, resulting in significantly higher bactericidal efficiency than traditional antibiotics. Summary of the Invention

[0005] In view of this, the present invention provides an antimicrobial peptide AI-SC-4 and its application.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides an antimicrobial peptide AI-SC-4, the amino acid sequence of which is:

[0008] a1) SEQ ID NO: 1; or

[0009] a2) An amino acid sequence of SEQ ID NO: 1 that has undergone substitution and / or deletion and / or addition of one or more amino acid sequences and has the same function as SEQ ID NO: 1; or

[0010] a3) An amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology with SEQ ID NO: 1 and has the same function as SEQ ID NO: 1.

[0011] SEQ ID NO: 1

[0012] Met-Phe-Gln-Phe-Phe-Val-Thr-Val-Thr-Lys-His-Leu-Ala-Arg-Arg-Ile-Ser-Lys-Thr-Phe-Arg-Ser, single-letter sequence: MFQFFVTVTKHLARRISKTFRS.

[0013] Furthermore, the antimicrobial peptide AI-SC-4 contains 22 amino acid residues and has a theoretical molecular weight of 2700.26 Da, making it a positively charged cationic amphiphilic polypeptide.

[0014] The antimicrobial peptide AI-SC-4 provided by this invention is a synthetic non-natural polypeptide, rich in basic amino acids such as arginine and lysine, as well as hydrophobic amino acids such as phenylalanine and valine. It has a typical cationic amphiphilic structure, small molecular weight, and is easy to synthesize and purify in solid phase.

[0015] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antimicrobial peptide AI-SC-4 described in the first aspect.

[0016] Thirdly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0017] Fourthly, the present invention provides the application of the antimicrobial peptide AI-SC-4 described in the first aspect, the nucleic acid molecule described in the second aspect, or the recombinant expression vector described in the third aspect in the preparation of antimicrobial products.

[0018] Furthermore, the bacteria include Gram-positive bacteria or Gram-negative bacteria.

[0019] Furthermore, the bacteria are Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, or Streptococcus.

[0020] Fifthly, the present invention provides an antibacterial product comprising the antimicrobial peptide AI-SC-4 described in the first aspect, the nucleic acid molecule described in the second aspect, or the recombinant expression vector described in the third aspect.

[0021] Furthermore, the bacteria include Gram-positive bacteria or Gram-negative bacteria.

[0022] Furthermore, the bacteria are Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, or Streptococcus.

[0023] Furthermore, antimicrobial products include antimicrobial drugs or care products.

[0024] Furthermore, the antibacterial drug is a topical preparation, which is selected from at least one of solutions, gels, creams, sprays, or wound dressing preparations.

[0025] Furthermore, the care product is an oral care product, including mouthwash. The care product can be used to inhibit bacteria, reduce skin infections or improve acne, and inhibit the formation of bacteria and biofilms in the oral cavity.

[0026] In a sixth aspect, the present invention provides a mouthwash comprising the antimicrobial peptide AI-SC-4 described in the first aspect.

[0027] Furthermore, the mouthwash includes citric acid, sodium dihydrogen phosphate, glycerin, Tween, antimicrobial peptide AI-SC-4, menthol, and deionized water.

[0028] The method for preparing the mouthwash includes the following steps: (1) preparing a citrate-sodium dihydrogen phosphate buffer solution; (2) adding glycerol and Tween to the citrate-sodium dihydrogen phosphate buffer solution to prepare a premix; (3) adding antimicrobial peptide AI-SC-4 to the premix, stirring evenly, then adding menthol, and adding deionized water and stirring to obtain the final product.

[0029] In a seventh aspect, the present invention provides a medical dressing comprising the antimicrobial peptide AI-SC-4 described in the first aspect.

[0030] Furthermore, the medical dressing includes lithium magnesium silicate, sodium hyaluronate, bovine lactoferrin, antimicrobial peptide AI-SC-4, and deionized water.

[0031] The preparation method of the medical dressing includes the following steps: (1) dissolving lithium magnesium silicate and sodium hyaluronate in deionized water and stirring to form a uniform gel matrix solution; (2) dissolving bovine lactoferrin and antimicrobial peptide AI-SC-4 in deionized water to prepare an active ingredient solution; (3) adding the active ingredient solution to the gel matrix solution and stirring to obtain the dressing.

[0032] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0033] (1) The antimicrobial peptide AI-SC-4 provided by the present invention is a non-natural synthetic polypeptide, rich in basic amino acids such as arginine and lysine, as well as hydrophobic amino acids such as phenylalanine and valine. It has a typical cationic amphiphilic structure, small molecular weight, and is easy to synthesize and purify in solid phase.

[0034] (2) The antimicrobial peptide AI-SC-4 provided by the present invention has a purity of over 98%, a molecular weight that matches the theoretical value, low endotoxin and trifluoroacetic acid residues, and a net peptide content of about 84.45%, meeting the pharmaceutical research grade standard.

[0035] (3) The antimicrobial peptide AI-SC-4 provided by the present invention has excellent broad-spectrum in vitro antibacterial ability against Gram-positive and Gram-negative bacteria, with a minimum MIC of 2 μg / mL. It can be prepared into solutions, gels, creams, sprays or mouthwashes, etc., and is suitable for the prevention and adjuvant treatment of skin and oral infections, with good application prospects. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1 This is the HPLC chromatogram of the antimicrobial peptide AI-SC-4 in Example 1;

[0038] Figure 2 The image shows the ESI-MS mass spectrum of the antimicrobial peptide AI-SC-4 in Example 1.

[0039] Figure 3 This is a chromatogram showing the trifluoroacetic acid residue analysis of the antimicrobial peptide AI-SC-4 in Example 1. Detailed Implementation

[0040] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0041] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0042] Example 1:

[0043] The antimicrobial peptide AI-SC-4 has the following amino acid sequence: SEQ ID NO: 1

[0044] Met-Phe-Gln-Phe-Phe-Val-Thr-Val-Thr-Lys-His-Leu-Ala-Arg-Arg-Ile-Ser-Lys-Thr-Phe-Arg-Ser.

[0045] The antimicrobial peptide AI-SC-4 was synthesized by Nanjing Genscript Biotech Co., Ltd. The HPLC chromatogram of the antimicrobial peptide AI-SC-4 is shown below. Figure 1 As shown, from Figure 1 As can be seen, the purified peptide exhibits a single main peak at 9.883 min, with an area percentage of 98.22%, indicating high purity. The ESI-MS mass spectrum of the antimicrobial peptide AI-SC-4 is shown below. Figure 2 As shown, from Figure 2 As can be seen, the measured molecular weight of the antimicrobial peptide AI-SC-4 is 2700.0 Da, consistent with the theoretical molecular weight of 2700.26 Da. The residual trifluoroacetic acid analysis chromatogram of the antimicrobial peptide AI-SC-4 is shown below. Figure 3 As shown, the nitrogen content was determined using an elemental analyzer, and the net peptide content was calculated to be 84.45%. Ion chromatography showed that the residual trifluoroacetic acid was approximately 0.40%; the endotoxin content was less than 10 EU / mg as determined by the Limulus Amebocyte Lysate (LAL) gel electrophoresis; solubility tests showed that AI-SC-4 was insoluble in ultrapure water and 0.1 M phosphate buffer solution (PBS, pH 7.4), but completely soluble in DMSO.

[0046] Example 2: In vitro antibacterial experiment of antimicrobial peptide AI-SC-4

[0047] 1. Test strain and control drug

[0048] Selected standard strains:

[0049] (1) Gram-positive bacteria: Staphylococcus aureus ATCC 6538; Bacillus subtilis ATCC 6633;

[0050] (2) Gram-negative bacteria: Pseudomonas aeruginosa ATCC 27853; Escherichia coli ATCC 25922.

[0051] Positive control drug:

[0052] (1) Cefazolin, used for Staphylococcus aureus, with a control concentration range of 0.06–128 μg / mL; (2) Ceftazidime, used for Pseudomonas aeruginosa, with a control concentration range of 0.125–256 μg / mL.

[0053] 2. Determination of Minimum Inhibitory Concentration (MIC)

[0054] The assay was performed according to GB / T 39101-2020 "Determination of Antibacterial Activity of Polypeptides - Inhibition Zone Method" and the micro-broth dilution method. The test strains were revived in a suitable culture medium and cultured to the logarithmic growth phase, with the bacterial suspension concentration adjusted to approximately 5 × 10⁻⁶. 5 CFU / mL. The AI-SC-4 antimicrobial peptide samples were prepared into gradient concentrations, mixed with bacterial suspension at a 1:1 volume ratio, and dispensed into 96-well plates. The plates were incubated at 37°C for 16-20 h. The lowest drug concentration at which no visible growth was observed was determined by visual inspection or by measuring absorbance at 600 nm, and this was taken as the MIC.

[0055] Each strain was tested in three independent replicates, and the results are shown in Table 1. Table 1 presents the antimicrobial activity data of the antimicrobial peptide AI-SC-4. The test results are the values ​​determined in three independent replicates, and the MICs of the three replicates were completely consistent.

[0056] Table 1. Antimicrobial activity test data of antimicrobial peptide AI-SC-4

[0057]

[0058] Judgment criteria: Staphylococcus aureus MIC ≤ 32 μg / mL and Pseudomonas aeruginosa MIC ≤ 128 μg / mL are considered effective.

[0059] As shown in Table 1, the MICs of AI-SC-4 against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli were 2 μg / mL, 3 μg / mL, 8 μg / mL, and 6 μg / mL, respectively, indicating strong antibacterial activity.

[0060] 3. Inhibition zone test

[0061] The inhibition zone method was performed according to GB / T 39101-2020. The bacterial suspension was evenly spread on the surface of a solid culture medium. Holes were punched or tablets were placed on the plate, and a solution containing a certain amount of the sample was added. The plate was incubated at 37 ℃ for 24 h, and the diameter of the inhibition zone was measured. Each sample and strain combination was repeated three times. The results are shown in Table 2.

[0062] Table 2 Results of AI-SC-4 inhibition zone test

[0063]

[0064] Judgment criteria: The inhibition zone diameter is ≥ 10 mm to be effective.

[0065] As shown in Table 2, the inhibition zone diameters of AI-SC-4 against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli were 20.5 mm, 19.4 mm, 16.8 mm, and 18.1 mm, respectively, all of which were greater than 10 mm, indicating strong antibacterial activity.

[0066] 4. Positive control results

[0067] Table 3 shows the MIC and inhibition zone diameter of the positive control drugs cefazolin and ceftazidime against two strains of Staphylococcus aureus and Pseudomonas aeruginosa under the same conditions.

[0068] Table 3 Antibacterial activity of positive control drugs

[0069]

[0070] Table 3 shows the antibacterial activities of cefazolin and ceftazidime against Staphylococcus aureus and Pseudomonas aeruginosa. Combined with Tables 1 and 2, it can be seen that the antibacterial activity of antimicrobial peptide AI-SC-4 is comparable to that of cefazolin and ceftazidime. In summary, under the same experimental conditions, antimicrobial peptide AI-SC-4 exhibits strong in vitro antibacterial activity against both Staphylococcus aureus ATCC6538 and Pseudomonas aeruginosa ATCC27853, with its MIC value significantly lower than the set judgment threshold, demonstrating good development potential.

[0071] Example 3 Antimicrobial peptide AI-SC-4 mouthwash

[0072] First, prepare the buffer solution: Take 2.1 g of citric acid and 3.8 g of sodium dihydrogen phosphate, add 50 mL of deionized water and stir to dissolve. Adjust the pH to 6.8 to obtain a citric acid-sodium dihydrogen phosphate buffer solution. Next, prepare the premix solution by adding 2.0 g of glycerol and 0.2 g of Tween 80 to the above buffer solution and stirring in a 35 ℃ constant temperature water bath for 15 min until completely dissolved to form a premix solution. Then, add the active ingredient by adding 0.03 g of antimicrobial peptide AI-SC-4 to the premix solution and stirring magnetically for 20 min until uniformly dispersed. Finally, complete the final product preparation by adding 0.1 g of menthol, adding deionized water to 100 g, stirring for 10 min, filtering through a 0.22 μm filter membrane for sterilization, and then dispensing to obtain the final product.

[0073] Performance verification results

[0074] The product exhibits excellent performance. In terms of antibacterial effect, it achieves a 99.2% inhibition rate against Streptococcus mutans within 24 hours and a 98.7% inhibition rate against Staphylococcus aureus, demonstrating superior overall antibacterial efficacy compared to traditional mouthwashes containing 0.1% chlorhexidine. Regarding stability, after storage at 4 ℃ and room temperature for 6 months, the antimicrobial peptide retention rate still reaches over 92%. Safety was verified through rabbit oral mucosal irritation experiments, with no redness, swelling, ulceration, or other irritation reactions observed during the experiments, indicating good biocompatibility.

[0075] Example 4: Antimicrobial peptide AI-SC-4 medical dressing

[0076] First, the matrix solution was prepared by adding 1.0 g of lithium magnesium silicate and 0.5 g of sodium hyaluronate to 60 mL of sterile deionized water, stirring at room temperature for 30 min, and then ultrasonically dispersing for 15 min to form a homogeneous gel matrix solution. Next, the active ingredient solution was prepared by dissolving 4.0 g of bovine lactoferrin in 20 mL of sterile deionized water and stirring at 37 ℃, while simultaneously adding 0.08 g of antimicrobial peptide AI-SC-4 to 10 mL of sterile deionized water and ultrasonically dissolving. Then, the two active ingredient solutions were slowly added to the gel matrix solution, and the mixture was magnetically stirred at room temperature for 25 min to form a homogeneous hydrogel. Finally, the hydrogel was dispensed into sterile containers, sterilized with ethylene oxide, and sealed to obtain the wound dressing.

[0077] Performance verification results

[0078] This wound dressing exhibits excellent comprehensive performance. The antibacterial layer shows an inhibition zone diameter of 18.6 mm against Staphylococcus aureus, with an inhibition rate of up to 99.5% after 24 hours. In terms of physical properties, the gel tensile strength is 120 kPa, the self-healing efficiency within 30 minutes after cutting reaches 92%, and the water retention rate also reaches 85%. The healing effect was verified by a rat infected wound model. After 7 days of use, the wound healing rate reached 82%, which is significantly better than the 45% of Vaseline dressing alone. In terms of biosafety, cytotoxicity experiments showed that the cell survival rate was over 95%, and there was no skin sensitization reaction.

[0079] The above description is merely a further embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An antibacterial peptide AI-SC-4, characterized in that, The amino acid sequence of the antibacterial peptide AI-SC-4 is SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the antibacterial peptide AI-SC-4 of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 2.

4. Use of the antibacterial peptide AI-SC-4 of claim 1, the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3 in the preparation of an antibacterial product.

5. An antibacterial product, characterized by The antibacterial product comprises the antibacterial peptide AI-SC-4 of claim 1, the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3.

6. The antimicrobial product of claim 5, wherein, The bacteria comprise gram-positive bacteria or gram-negative bacteria; or, the antibacterial product comprises an antibacterial medicament or a care product.

7. The antimicrobial product of claim 6, wherein, The bacteria are Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli or Streptococcus.

8. The antimicrobial product of claim 6, wherein, The antibacterial medicament is a topical preparation, the topical preparation being selected from at least one of a solution, a gel, a cream, a spray or a wound dressing; or, the care product is an oral care product, the oral care product comprising a mouthwash.

9. A mouthwash, characterized in that, The mouthwash comprises the antibacterial peptide AI-SC-4 of claim 1.

10. A medical dressing, characterized in that, The medical dressing comprises the antibacterial peptide AI-SC-4 of claim 1.

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