Anti-pseudomonas aeruginosa antibacterial peptide and application thereof
The existing antimicrobial peptides with specific amino acid sequences have solved the problem of insufficient activity and poor stability of Pseudomonas aeruginosa, effectively inhibiting Pseudomonas aeruginosa, and applied in drugs, feed additives and food preservatives, with the advantages of high efficiency and low cost.
Patent Information
- Application Number
- CN202510500328.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-21
- Publication Date
- 2025-07-04
AI Technical Summary
The existing antimicrobial peptides have insufficient antimicrobial activity, poor stability and potential toxicity to Pseudomonas aeruginosa, which limits its promotion in clinical and practical applications.
A new anti-Pseudomonas aeruginosa antibacterial peptide was designed, with its amino acid sequence VKTGNRAKVKVIKGLKKASKLHAGQAKTLKKVLGNGKKRSKSRN, a molecular weight of 5012.062 Da, an isoelectric point of 12.811, and a net charge number of 17.509. It was prepared by the solid phase synthesis method of polypeptide, and has good antibacterial effect and stability.
This antibacterial peptide has significant antibacterial activity against Pseudomonas aeruginosa, is stable, is not easy to cause drug resistance, is highly synthetic and has low production cost, and is suitable for the preparation of drugs, feed additives and food preservatives.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to an antibacterial peptide against Pseudomonas aeruginosa and its application. Background Art
[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) is a Gram-negative bacillus that widely exists in water, soil, plants and human skin. It is an important opportunistic pathogen, especially likely to cause infections in immunocompromised individuals. This bacterium has various virulence factors, such as endotoxin, exotoxin, polysaccharide capsule, etc., which can lead to serious nosocomial infections, including respiratory tract infections, wound infections, urinary tract infections, etc. In recent years, the problem of drug resistance of Pseudomonas aeruginosa has become increasingly prominent, and the emergence of multi-drug resistant strains has brought great challenges to clinical treatment. Its resistance mechanisms mainly include: (1) Reduced outer membrane permeability: Pseudomonas aeruginosa can reduce the expression of outer membrane channel proteins (such as OprD) through gene mutation, thereby reducing the outer membrane permeability and hindering the entry of antibiotics into the bacterial cell; (2) Overexpression of active efflux systems: This bacterium contains various drug-resistant efflux pump systems (such as MexAB-OprM, MexCD-OprJ, etc.), which can pump out the antibiotics that enter the bacterial cell, reducing their effective concentration; (3) Biofilm formation: Pseudomonas aeruginosa can form biofilms, and this structure can protect the bacterial cells from the attack of antibiotics, increasing drug resistance; (4) Production of antibiotic-inactivating enzymes: This bacterium can produce various enzymes (such as β-lactamase, aminoglycoside-modifying enzymes, etc.) to directly inactivate antibiotics.
[0003] Due to the increasingly serious problem of drug resistance of Pseudomonas aeruginosa, the development of new antibacterial agents has become an urgent need. Antimicrobial peptides (AMPs) have become a research hotspot due to their broad-spectrum antibacterial activity and unique mechanism of action. Antimicrobial peptides can effectively kill Pseudomonas aeruginosa by disrupting the bacterial cell membrane, inhibiting biofilm formation, etc. However, natural antimicrobial peptides have problems such as poor stability and high toxicity. Novel antimicrobial peptides through modification or design are expected to overcome these disadvantages and provide new solutions for the treatment of Pseudomonas aeruginosa infections.
[0004] Antimicrobial peptides are a class of small molecule peptides that are widely present in nature and have attracted much attention for their broad-spectrum antimicrobial properties. They can quickly kill a variety of pathogens, including bacteria, fungi, viruses, and parasites. With the widespread use of antibiotics, the emergence of drug-resistant bacteria has become a global challenge, which has prompted scientists to look for new antimicrobial agents. Antimicrobial peptides have shown significant advantages in combating drug-resistant bacteria due to their unique biological activity and different mechanisms of action from traditional antibiotics. Therefore, antimicrobial peptides are regarded as highly promising alternatives to antibiotics and have been widely used in food preservation, drug development, skin care products, and cosmetic preservatives, showing broad development prospects.
[0005] Although antimicrobial peptides have significant advantages in the field of antibacterial drugs, natural antimicrobial peptides still have some limitations, such as insufficient antimicrobial activity, poor stability, and potential toxicity of some antimicrobial peptides, which limit the widespread promotion of antimicrobial peptides in practical applications. Summary of the invention
[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide an antimicrobial peptide against Pseudomonas aeruginosa.
[0007] Another object of the present invention is to provide the application of the above anti-Pseudomonas aeruginosa antimicrobial peptide.
[0008] The object of the present invention is achieved through the following technical scheme: an antibacterial peptide against Pseudomonas aeruginosa, whose amino acid sequence is as follows: VKTGNRAKVKKVIKGLKKASKLHAGQAKTLKKVLGNGKKKRSKSRN.
[0009] The anti-Pseudomonas aeruginosa antimicrobial peptide has a molecular weight of 5012.062 Da, an isoelectric point of 12.811, and a net charge number of 17.509.
[0010] Application of the anti-Pseudomonas aeruginosa antimicrobial peptide in inhibiting the activity of Pseudomonas aeruginosa.
[0011] Application of the anti-Pseudomonas aeruginosa antimicrobial peptide in the preparation of microbial inhibitors.
[0012] The microbial antibacterial agent is an antibacterial drug, a feed additive or a food preservative.
[0013] The microbial antibacterial agent is used for inhibiting and / or killing Pseudomonas aeruginosa.
[0014] The microorganism is Pseudomonas aeruginosa; preferably Pseudomonas aeruginosa ( Pseudomonas aeruginosa )PAO1.
[0015] Implementing the present invention has the following beneficial effects The present invention provides an antibacterial peptide against Pseudomonas aeruginosa with a brand-new amino acid sequence. Experimental results show that the antibacterial peptide has good inhibitory effects on Pseudomonas aeruginosa, and has advantages such as good stability, not easily causing drug resistance, high synthesis efficiency and low production cost, and can be used to prepare drugs for preventing diseases caused by Pseudomonas aeruginosa, or feed additives or food preservatives. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 It is a HPLC report result diagram of chemically synthesized antibacterial peptide.
[0017] Figure 2 It is a MS report result diagram of chemically synthesized antibacterial peptide.
[0018] Figure 3 It is a cell morphology result diagram of Pseudomonas aeruginosa without treatment with antibacterial peptide.
[0019] Figure 4 It is a cell morphology result diagram of Pseudomonas aeruginosa after the action of antibacterial peptide. DETAILED DESCRIPTION OF THE INVENTION
[0020] The following further illustrates the specific implementation of the present invention in conjunction with examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. Reagents or instruments not indicating the manufacturer are regarded as conventional products that can be obtained through commercial purchase.
[0021] Example 1: Synthesis of antibacterial peptide Through the previous research of this research group, based on the prediction of the deep learning model and using bioinformatics to analyze antibacterial peptides, a polypeptide with the following amino acid sequence was predicted: VKTGNRAKVKKVIKGLKKASKLHAGQAKTLKKVLGNGKKKRSKSRN.
[0022] The antibacterial peptide of the present invention is synthesized by conventional solid-phase peptide synthesis.
[0023] Sequence characteristics: The sequence type is an amino acid sequence, containing 46 amino acid residues, with a molecular weight of 5012.062 Da and a net charge number of 17.509.
[0024] The specific synthesis steps are as follows: 1) Resin swelling: Weigh 2-Chlorotrityl Chloride Resin with a substitution degree of 0.1 mmol (SUNRESIN Company, China, product number LXSS03-1-1204). Put the resin into a reaction tube, add DCM (dichloromethane) solvent. The addition amount of DCM is calculated according to 15 mL / g of resin, and shake for 40 min.
[0025] 2) Connect the first amino acid: Filter out the DCM solvent through a sintered glass filter, add Fmoc-Arg(Pbf)-OH amino acid with a mass three times that of the resin, then add 2,4,6-trimethylpyridine with a mass equal to that of the resin, and finally add DCM (15 mL / g, that is, the addition amount is calculated according to 15 mL / g of resin) to dissolve. After shaking for 12 h, wash with DMF (dimethylformamide) three times.
[0026] 3) Deprotection: Add 10 mL of 20% v / v piperidine DMF solution. The addition amount of the piperidine DMF solution is calculated according to 15 mL / g of resin. After shaking for 6 min, filter out the piperidine DMF solution, then add another 10 mL of 20% v / v piperidine DMF solution. The addition amount of the piperidine DMF solution is calculated according to 15 mL / g of resin, and shake for 20 min.
[0027] 4) Detection: Filter out the piperidine DMF solution, take a dozen or so resin particles, wash them three times with ethanol, add one drop of ninhydrin and one drop of phenol solution respectively, and heat at 105 °C - 110 °C for 3 min. A dark blue color change indicates a positive reaction.
[0028] 5) First cleaning: Wash with DMF three times in sequence. The dosage of DMF each time is calculated according to 10 mL / g of resin.
[0029] 6) Blocking: Add methanol (the addition amount is calculated according to 10 mL / g of resin) and DIEA (N,N-diisopropylethylamine) with a mass three times that of the resin, shake for 20 min, and then wash with DMF three times.
[0030] 7) Condensation (generating the second amino acid Lysine in the sequence): Add Fmoc-Lys(boc)-OH amino acid with a mass three times that of the resin, HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate) with a mass 2.7 times that of the resin, and DIEA (N,N-diisopropylethylamine) with a mass 10 times that of the resin into the reaction tube, dissolve with 5 mL (as little as possible) of DMF, and react for 1 h.
[0031] 8) Second cleaning: Wash three times in sequence with DMF (the addition amount is calculated according to 10 mL / g of resin).
[0032] 9) Repeat the above operation and connect the amino acids in the sequence from left to right in turn.
[0033] 10) After the last amino acid is connected, deprotect and wash the resin as follows: DMF (10 mL / g resin) twice, DCM (10 mL / g resin) twice, and dry by suction for 8 min.
[0034] 11) Cleave the polypeptide from the resin: The amount of cleavage solution is calculated as 10 mL / g resin, and shake at a constant temperature for 150 min. (The cleavage solution is prepared by volume percentage and consists of the following: TFA (trifluoroacetic acid) 95% v / v, water 2.5% v / v, TIS (triisopropylsilane) 2.5% v / v.
[0035] 12) Blow-dry and wash: Blow-dry the cleavage solution as much as possible with nitrogen, chromatograph it with ether, wash it with ether six times, and then volatilize it at room temperature to obtain the crude peptide.
[0036] 13) Analyze and identify the polypeptide by HPLC-MS: A. Prepare a solution with a concentration of 1 mg / mL from the crude peptide; B. Filter the dissolution solution with a 0.45 μm filter membrane; C. Analysis: Take 10 μL for HPLC-MS analysis. The mobile phase is water and acetonitrile, for 25 min, isocratic elution. First, equilibrate the HPLC with an isocratic gradient for 5 min and then inject the sample. The gradient is 40% v / v water and 60% v / v acetonitrile.
[0037] 14) Lyophilize the purified solution to obtain the finished product.
[0038] 15) Seal and package the white powdery polypeptide and store it at -20 °C.
[0039] The report of HPLC is as Figure 1 shown, and the detection report of MS is as Figure 2 shown. It can be seen that the target polypeptide with a purity greater than 90% is prepared.
[0040] Example 2: Determination of antibacterial spectrum Determine the antibacterial activity of the antibacterial peptide in Example 1 and evaluate the antibacterial activities against Gram-positive bacteria (such as Staphylococcus aureus) and Gram-negative bacteria (such as Escherichia coli).
[0041] Inoculate each test strain by streaking on an antibiotic-free MH solid medium and incubate it in an incubator at 37 °C for 16 h. Select 3 - 5 colonies and inoculate them into 3 mL of MH liquid medium. Incubate overnight at 37 °C and 200 r / min. The next morning, measure the OD 600 value of the bacterial solution with a spectrophotometer and dilute it to 5×10 5CFU / mL. Take 90 μL of the diluted bacterial solution and add it to a 96-well plate. According to the two-fold dilution method, after dissolving and diluting the antimicrobial peptide with ddH2O, take 10 μL each and add them successively to the 96-well plate containing the bacterial suspension, with a final concentration of 128 μg / mL. Place the 96-well plate in an incubator at 37 °C for 16 h, and measure the OD of each well. 600 This experiment was repeated 3 times, with 3 parallels each time.
[0042] The results are shown in Table 1. The antimicrobial peptide showed antibacterial activity against only Pseudomonas aeruginosa among the 14 tested strains, including 8 Gram-positive bacteria (such as Bacillus cereus, Staphylococcus aureus, etc.) and 6 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, etc.), but was ineffective against other common bacterial strains.
[0043] Table 1 Determination of the antibacterial spectrum of the antimicrobial peptide against 14 strains
[0044] "+" indicates that the antimicrobial peptide has antibacterial activity against the strain at 128 μg / mL; "-" indicates that the antimicrobial peptide has no antibacterial activity against the strain at 128 μg / mL; For the strains in the table with ATCC numbers, they can be purchased from the American Type Culture Collection; Pseudomonas aeruginosa PAO1 is an international standard strain and can be purchased from Beina Biotechnology; the other numbered strains were isolated and preserved in this laboratory, and the species were determined based on molecular biology and Bergey's Manual of Determinative Bacteriology during the isolation process.
[0045] Example 3: Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Perform antibacterial activity determination on the synthesized antimicrobial peptide in Example 1 to test its activity against Pseudomonas aeruginosa.
[0046] In this example, Pseudomonas aeruginosa was selected as the test strain. Streak-inoculate Pseudomonas aeruginosa on an antibiotic-free MH solid medium and place it in an incubator at 37 °C for 16 h. Select 3 - 5 colonies and inoculate them into 3 mL of MH liquid medium and culture overnight. The next morning, measure the OD value of the bacterial solution using a spectrophotometer and dilute it to 5×10 600 value, and 5CFU / mL. Take 90 μL of the diluted bacterial solution and add it to a 96-well plate. After diluting the antimicrobial peptide by the two-fold dilution method, take 10 μL in turn and add it to the 96-well plate containing the bacterial suspension. The final concentrations are 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL respectively. Ofloxacin is used as a control. Place the 96-well plate in an incubator at 37 °C for 16 h and measure the OD of each well 600 . The minimum inhibitory concentration is the lowest concentration of the antimicrobial peptide at which no bacterial growth is visible. To determine the minimum bactericidal concentration (MBC), 10 μL of the suspension is taken from each well and spotted on an agar plate. The lowest peptide concentration that results in no bacterial colony growth is recorded as the MBC. This experiment is repeated 3 times, with 3 parallels each time
[0047] As shown in Table 2, the MIC value of the antimicrobial peptide against Pseudomonas aeruginosa is 4 μg / mL, and the antibacterial ability is significant
[0048] Further determination of the minimum bactericidal concentration (MBC) of the above antimicrobial peptide shows that, as shown in Table 2, the MBC value of the antimicrobial peptide is consistent with its MIC value. The experiment shows that the above antimicrobial peptide has good antibacterial activity against Pseudomonas aeruginosa
[0049] Table 2 MIC and MBC of the antimicrobial peptide against Pseudomonas aeruginosa (μg / mL)
[0050] Example 4: Observation of the cell morphology of Pseudomonas aeruginosa before and after the action of the antimicrobial peptide Inoculate Pseudomonas aeruginosa into fresh MH broth liquid medium and culture it overnight at 37 °C and 200 r / min. The next morning, measure the OD value of the bacterial solution with an ultraviolet spectrophotometer 600 value and dilute it to 1×10 8 CFU / mL, add the antimicrobial peptide to make its final concentration 2×MIC, use ddH2O as a negative control, incubate at 37 °C for 1 h, then centrifuge at 8000 rpm for 3 min, discard the supernatant, wash the bacterial cells 3 times with PBS buffer, add 2.5% (v / v) glutaraldehyde, and fix overnight at 4 °C. After sufficient fixation, centrifuge at 8000 rpm for 5 min, wash 3 times with PBS buffer, and then dehydrate successively with ethanol solutions with volume fractions of 30%, 50%, 70%, 80%, and 90% for 15 min each time, and dehydrate with 100% ethanol 2 times. Finally, transfer to tert-butanol, then perform critical point drying, sputter gold with an ion sputtering instrument, and observe the bacterial cell morphology under a scanning electron microscope
[0051] The results of scanning electron microscopy showed that the cell morphology of Pseudomonas aeruginosa without peptide treatment was normal, the cell membrane was intact, and the surface was smooth (as shown in Figure 3 ); however, after treatment with the antimicrobial peptide, obvious changes occurred on the membrane surface of the bacteria, becoming shrunken, rough, with obvious membrane rupture and exudation of cytoplasmic components (as shown in Figure 4 ). The experiment showed that the antimicrobial peptide could disrupt the cell membrane of Pseudomonas aeruginosa, change the permeability of the cell membrane, and ultimately lead to the death of the bacteria.
[0052] Example 5: Determination of the stability of the antimicrobial peptide Thermal stability determination: The antimicrobial peptide was placed at 25, 50, 75, and 100 °C for 1 h respectively, and then restored to room temperature, and its MIC against Pseudomonas aeruginosa was determined; the untreated antimicrobial peptide (4 °C) was used as a control. This experiment was repeated 3 times, with 3 parallels each time.
[0053] Acid-base stability determination: Buffer solutions with pH values of 2 (50 mM, glycine-HCl buffer), 6 (50 mM, sodium phosphate buffer), 10 (50 mM, glycine-NaOH buffer), and 12 (50 mM, glycine-NaOH buffer) were prepared. The above antimicrobial peptide was dissolved in the buffer solution and incubated at 37 °C for 1 h, and its MIC against Pseudomonas aeruginosa was determined; the antimicrobial peptide dissolved in ddH2O was used as a control. This experiment was repeated 3 times, with 3 parallels each time.
[0054] Protease stability determination: Pepsin (Aladdin), trypsin (Sangon Biotech), and proteinase-K (Aladdin) were dissolved in ddH2O respectively, with a final concentration of 20 μg / mL. The above antimicrobial peptide was mixed with the above proteases at a volume ratio of 1:1 at 37 °C for 1 h. Then, the peptide-protease mixture was heated at 60 °C for 15 minutes to inactivate the protease activity. Its MIC against Pseudomonas aeruginosa was determined; the antimicrobial peptide mixed with ddH2O was used as a control. This experiment was repeated 3 times, with 3 parallels each time.
[0055] Table 3 shows the results of the thermal stability determination. It can be found from the table that the activities of the above antimicrobial peptides remained unchanged after treatment at a temperature as high as 100 °C, indicating that the above antimicrobial peptides have good thermal stability and can withstand the heat treatment in the food processing process.
[0056] Table 3 Thermal stability determination of the antimicrobial peptide
[0057] Table 4 shows the results of the acid-base stability determination. It can be seen that under the condition of pH 2, the antimicrobial peptide may reduce its antibacterial activity due to changes in charge state, weakened membrane interaction, and decreased structural stability. Under alkaline conditions such as pH 10 and 12, adverse changes may occur in the charge state and structural stability of the antimicrobial peptide, affecting its interaction with the bacterial membrane and resulting in a decrease in activity.
[0058] Table 4 Acid-base Stability Determination of Antimicrobial Peptide
[0059] Table 5 shows the results of the protease stability determination. The antimicrobial peptide has relatively small changes in activity and is relatively stable under the influence of pepsin and proteinase K. However, under the influence of trypsin, the activity of the above-mentioned antimicrobial peptide decreases and no antibacterial activity is shown at 128 μg / mL, indicating that the above-mentioned antimicrobial peptide maintains good stability in the presence of pepsin and proteinase K and poor stability in the presence of trypsin.
[0060] Table 5 Protease Stability Determination of Antimicrobial Peptide
[0061] The above embodiments are only the preferred embodiments of the present invention, which are only used to explain the present invention rather than limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit and essence of the present invention shall fall within the protection scope of the present invention.
Claims
1. An antibacterial peptide against Pseudomonas aeruginosa, characterized in that: The amino acid sequence of the described Pseudomonas aeruginosa antibacterial peptide is as shown in SEQ ID NO:
1.
2. The antibacterial peptide against Pseudomonas aeruginosa according to claim 1, characterized in that: The molecular weight of the described Pseudomonas aeruginosa antibacterial peptide is 5012.062 Da, the isoelectric point is 12.811, and the net charge number is 17.
509.
3. Use of the Pseudomonas aeruginosa antibacterial peptide according to claim 1 or 2 in inhibiting the activity of Pseudomonas aeruginosa.
4. Use of the Pseudomonas aeruginosa antibacterial peptide according to claim 1 or 2 in the preparation of a microbial inhibitor.
5. The application according to claim 4, characterized in that: The described microbial inhibitor is an antibacterial drug, a feed additive or a food preservative.
6. The application according to claim 4, wherein: The described microorganism is Pseudomonas aeruginosa.
7. The application according to claim 6, characterized in that: The microorganism described is Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) PAO1.