A pH-responsive antimicrobial peptide LIH and its preparation method and application

Abstract

The present invention provides a pH-responsive antimicrobial peptide LIH, its preparation method, and application. The peptide comprises a main chain and a side chain, wherein the amino acid sequence of the main chain is shown in SEQ ID No. 1, and the amino acid sequence of the side chain is shown in SEQ ID No. 2, wherein the side chain is connected to the ε-amino group of a lysine residue at the end of the main chain. The pH-responsive antimicrobial peptide LIH of the present invention has no bactericidal activity against all tested pathogenic microorganisms at pH = 7.0, but has high bactericidal activity against Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis at pH = 6.0. It is also non-toxic to human red blood cells, making it a potential alternative to feed antibiotics.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular relates to a pH-responsive antimicrobial peptide LIH, a preparation method thereof, and an application thereof. Background Art

[0002] Antimicrobial peptides are a class of naturally occurring active polypeptides, typically composed of 10 to 30 amino acid residues, that exhibit broad-spectrum antimicrobial activity. Unlike antibiotics, which disrupt key microbial metabolic pathways, antimicrobial peptides typically eliminate pathogenic microorganisms by physically disrupting bacterial cell membranes. Therefore, compared to antibiotics, antimicrobial peptides are less likely to develop drug resistance and offer significant potential as an alternative to antibiotics in feed.

[0003] In recent years, the technology for creating antimicrobial peptides has matured. However, their indiscriminate killing effects, which often fail to distinguish friend from foe, have hindered their practical application in production. There is an urgent need to develop antimicrobial peptides with specific bactericidal activity tailored to the microenvironmental response within the organism. Existing antimicrobial peptides, such as LI, exhibit broad-spectrum antimicrobial activity, but their in vivo availability is poor, preventing effective release at specific infection sites. Therefore, a pH-responsive antimicrobial peptide is needed to provide a theoretical foundation and technical support for the development of feed-grade antimicrobial peptides. Summary of the Invention

[0004] Based on the above shortcomings, the purpose of the present invention is to provide a pH-responsive antimicrobial peptide LIH, which has high bactericidal ability in a weakly acidic environment and no bactericidal activity in a neutral environment, so that it has the potential to become a substitute for feed antibiotics.

[0005] The object of the present invention is achieved by the following technical solution: a pH-responsive antimicrobial peptide LIH, comprising a main chain and a side chain, wherein the amino acid sequence of the main chain is shown in SEQ ID No. 1, the amino acid sequence of the side chain is shown in SEQ ID No. 2, and the side chain is connected to the ε-amino group of the lysine residue at the end of the main chain.

[0006] Furthermore, its molecular formula is shown in formula (I):

[0007]

[0008] Another object of the present invention is to provide a method for preparing a pH-responsive antimicrobial peptide LIH, comprising: using antimicrobial peptide LI as a template, linking lysine K to the C-terminus of antimicrobial peptide LI, and linking two short peptides to the carboxyl terminus of lysine K and the ε-amino group of its residue, respectively. The amino acid sequence of the short peptides is: HHHH. Thus, the amino acid sequence of the obtained polypeptide is GKEFKRIVKWPWWPRRKHHHH (HHHH). The polypeptide is synthesized by solid-phase chemical synthesis, purified by reverse-phase high-performance liquid chromatography, identified by mass spectrometry, and then assayed for bactericidal activity and hemolytic activity. Finally, the polypeptide is named antimicrobial peptide LIH.

[0009] Another object of the present invention is to provide a use of a pH-responsive antimicrobial peptide LIH in the preparation of a medicament for treating Gram-positive and / or Gram-negative bacterial infectious diseases.

[0010] Furthermore, in the application described above, the Gram-negative bacteria is Escherichia coli.

[0011] Furthermore, in the application described above, the Gram-positive bacteria are Staphylococcus aureus or Enterococcus faecalis.

[0012] Furthermore, as described above, the antimicrobial peptide LIH has bactericidal activity against Escherichia coli, Staphylococcus aureus or Enterococcus faecalis at pH 6.0.

[0013] Another object of the present invention is to provide a drug suitable for treating and / or preventing Escherichia coli, Staphylococcus aureus or Enterococcus faecalis infection at pH = 6.0, wherein the drug contains the pH-responsive antimicrobial peptide LIH as described above.

[0014] The beneficial effects and advantages of the present invention are as follows: The antimicrobial peptide LIH of the present invention can switch the level of bactericidal activity according to environmental changes. Biological activity testing of the antimicrobial peptide LIH revealed that it had no bactericidal activity against all tested pathogenic microorganisms at pH 7.0, but had high bactericidal activity against Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis at pH 6.0. At a concentration of 128 μM, the antimicrobial peptide only caused 4.71% hemolysis of red blood cells, indicating that it has the potential to become a substitute for feed antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a high performance liquid chromatogram of the pH-responsive antimicrobial peptide of the present invention;

[0016] Figure 2 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry of the pH-responsive antimicrobial peptide of the present invention;

[0017] Figure 3This is a graph showing the hemolytic activity of the pH-responsive antimicrobial peptides and melittin ME of the present invention. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0019] Example 1

[0020] Design of pH-responsive antimicrobial peptide LIH

[0021] Using the antimicrobial peptide LI as a template, lysine K was connected to the N-terminus of the antimicrobial peptide LI. Taking advantage of the fact that the N-terminus and ε-amino group of lysine K can provide dehydration condensation sites for the molecular tail, two short peptides with the amino acid sequence of HHHH were connected to the carboxyl terminus of lysine K and the ε-amino group of its residue, respectively, to reduce the bactericidal activity of the antimicrobial peptide under neutral conditions. Since the eight-repeated histidine H provides a structural basis for the antimicrobial peptide to enhance its bactericidal activity in a weakly acidic environment, the pH-responsive antimicrobial peptide LIH was obtained, with a net charge of +6 and a hydrophobicity of 0.256, as shown in Table 1.

[0022] Table 1 Amino acid sequences of pH-responsive antimicrobial peptides and their pro-peptides

[0023]

[0024] The net charge of antimicrobial peptide LIH is +6 and the hydrophobicity value is 0.256.

[0025] Example 2

[0026] Synthesis of pH-responsive antimicrobial peptide LIH by solid-phase chemical synthesis

[0027] 1. The preparation of pH-responsive antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus using a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antimicrobial peptide) is connected to Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, the synthesis is carried out from the C-terminus to the N-terminus until the synthesis is completed, and the side chain protection resin with the Fmoc group removed is obtained;

[0028] 2. Add a cleavage reagent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with TFA (trifluoroacetic acid) to precipitate, mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, and then add about 10 times the volume of pre-cooled anhydrous ether. Precipitate at -20°C for 3 hours to precipitate a white powder. Centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain the polypeptide, wherein the cleavage reagent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0029] 3. Use 0.2M sodium sulfate (adjusted to pH 7.5 with phosphoric acid) to equilibrate the column for 30 minutes, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, and apply gradient elution (eluent: methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30) on a C18 reverse-phase atmospheric pressure column at a flow rate of 1 mL / min and a detection wave of 220 nm. Collect the main peak and freeze-dry. Further purification is performed using a reverse-phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, the elution concentration is 25% B to 40% B, the elution time is 12 minutes, the flow rate is 1 mL / min, and the main peak is collected as above and freeze-dried.

[0030] 4. Identification of antimicrobial peptide LIH: The antimicrobial peptide LIH obtained above was analyzed by electrospray mass spectrometry. The molecular weight (e.g. Figure 1 、 2 The molecular weight of the antimicrobial peptide LIH was basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide LIH was greater than 95%.

[0031] Example 3

[0032] 1. Determination of bactericidal activity: The pH-responsive antimicrobial peptide LIH was treated by gradient dilution method and the minimum bactericidal concentration was determined on LB solid agar. 2 mg / ml BSA (containing 0.01% acetic acid) was used as diluent, and then the pH value of the BSA dilution was adjusted to 6.0 using PBS solution as buffer solution. A series of gradient antimicrobial peptide solutions were prepared in sequence using the two-fold dilution method. 100 μL of the above solution was placed in a 96-well cell culture plate, and then an equal volume of the test bacterial solution (~10 5 A positive control (containing bacterial solution but no antimicrobial peptide) and a negative control (containing neither bacterial solution nor antimicrobial peptide) were set up. After incubation at 37°C for 2 hours, 10 μl of the culture was diluted to an appropriate concentration and evenly spread on LB solid agar. After incubation at 37°C for 14-18 hours, the number of colonies on the agar was counted. The antimicrobial peptide concentration that resulted in a bacterial kill rate greater than 99.9% was considered the minimum bactericidal concentration. The test results are shown in Table 2.

[0033] Table 2 Minimum bactericidal activity of pH-responsive antimicrobial peptide LIH against pathogenic microorganisms

[0034]

[0035] As can be seen from Table 2, at pH = 6.0, the antimicrobial peptide LIH exhibited high bactericidal activity against Gram-negative bacteria (E. coli 25922, E. coli K88 and E. coli UB1005) and Gram-positive bacteria (S. aureus 29213, S. aureus 25923 and E. faecalis 29212).

[0036] 2. Determination of hemolytic activity: Collect 1 mL of fresh human blood, dissolve it in 2 mL of PBS solution after anticoagulation with heparin, centrifuge at 3000 rpm for 10 minutes, collect red blood cells; wash with PBS solution 3 times, and resuspend with 10 mL of PBS solution; take 50 μL of red blood cell suspension and mix it evenly with 50 μL of antimicrobial peptide solution of different concentrations, incubate it in a constant temperature incubator at 37°C for 1 hour; then centrifuge it at 4°C and 3000 rpm for 10 minutes; take out the supernatant and use an enzyme-labeled instrument to measure the light absorption value at 570 nm. 50 μL of red blood cells plus 50 μL of PBS solution is used as a negative control, and 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 is used as a positive control. The minimum hemolytic concentration is the concentration of antimicrobial peptide when the antimicrobial peptide causes a hemolysis rate of 10%. The test results are shown in Figure 3 .pass Figure 3 It can be seen that the antimicrobial peptide LIH did not show hemolytic activity within the detection range. At a concentration of 128 μM, it caused 4.71% hemolysis of erythrocytes and failed to cause 10% hemolysis of erythrocytes, which was significantly different from the control group of melittin. This shows that a pH-responsive antimicrobial peptide LIH of the present invention has the potential to be developed into a feed-type antimicrobial peptide.

Claims

1. A pH-responsive antimicrobial peptide LIH, characterized by: It comprises a main chain and a side chain, wherein the amino acid sequence of the main chain is shown in SEQ ID No. 1, the amino acid sequence of the side chain is shown in SEQ ID No. 2, and the side chain is connected to the ε-amino group of the lysine residue at the end of the main chain. Its molecular formula is shown in formula (I):

2. The method for preparing a pH-responsive antimicrobial peptide LIH according to claim 1, characterized in that: The method is as follows: using antimicrobial peptide LI as a template, lysine K is connected to the C-terminus of antimicrobial peptide LI, and two short peptides are respectively connected to the carboxyl terminus of lysine K and the ε-amino group of its residue, wherein the amino acid sequence of the short peptide is: HHHH. Thus, the amino acid sequence of the obtained polypeptide is GKEFKRIVKWPWWPRRKHHHH (HHHH). The polypeptide is synthesized by solid-phase chemical synthesis and purified by reverse-phase high-performance liquid chromatography and identified by mass spectrometry. Then, the bactericidal activity and hemolytic activity are determined, and finally, the polypeptide is named antimicrobial peptide LIH.

3. Use of the pH-responsive antimicrobial peptide LIH according to claim 1 in the preparation of a medicament for treating Gram-positive and / or Gram-negative bacterial infectious diseases, wherein the Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is Staphylococcus aureus or Enterococcus faecalis, and the use is under the condition of pH = 6.

0.

4. A drug suitable for treating and / or preventing Escherichia coli, Staphylococcus aureus or Enterococcus faecalis infection at pH 6.0, characterized in that: The drug contains the pH-responsive antimicrobial peptide LIH as claimed in claim 1.

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