PH-responsive antibacterial peptide LIH as well as preparation method and application thereof

Abstract

The invention provides a pH response antibacterial peptide LIH and a preparation method and application thereof, the pH response antibacterial peptide LIH comprises a main chain and a side chain, the amino acid sequence of the main chain is shown as SEQ ID No.1, the amino acid sequence of the side chain is shown as SEQ ID No.2, and the side chain is connected with epsilon-amino of lysine residues at the tail end of the main chain. The pH response antibacterial peptide LIH has no bactericidal activity on all detected pathogenic microorganisms under the condition that the pH value is equal to 7.0, has efficient bactericidal activity on escherichia coli, staphylococcus aureus and enterococcus faecalis under the condition that the pH value is equal to 6.0, and has no toxicity on human red blood cells, so that the pH response antibacterial peptide LIH has the potential of becoming a feed antibiotic substitute.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly 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 natural active polypeptides, usually composed of 10 to 30 amino acid residues, and exhibit broad-spectrum antibacterial activity. Different from antibiotics that interfere with the key metabolic pathways of microorganisms, antimicrobial peptides usually destroy bacterial cell membranes physically to eliminate pathogenic microorganisms. Therefore, compared with antibiotics, antimicrobial peptides are less likely to produce drug resistance and show great potential as alternatives to feed antibiotics.

[0003] In recent years, the creation technology of antimicrobial peptides has become increasingly mature. However, their non-discriminatory killing effect of "difficult to distinguish between friend and foe" has hindered the application of antimicrobial peptides in actual production. There is an urgent need to develop an antimicrobial peptide that exerts specific bactericidal activity according to the microenvironment response in organisms. The existing antimicrobial peptide LI has broad-spectrum antibacterial activity, but its utilization rate in vivo is poor, and effective release at specific infection sites cannot be achieved. Therefore, a pH-responsive antimicrobial peptide is needed to provide a theoretical basis and technical support for the research and development of feed-type antimicrobial peptides. Summary of the Invention

[0004] Based on the above deficiencies, 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, making it have the potential to become a substitute for feed antibiotics.

[0005] The purpose of the present invention is achieved by adopting the following technical scheme: A pH-responsive antimicrobial peptide LIH, comprising a main chain and a side chain, wherein the amino acid sequence of the main chain is as shown in SEQ ID No.1, the amino acid sequence of the side chain is as 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 as shown in formula (I):

[0007]

[0008] Another object of the present invention is to provide a method for preparing a pH-responsive antibacterial peptide LIH, as follows: Using antibacterial peptide LI as a template, lysine K is connected to the N-terminus of antibacterial peptide LI, and two short peptides are respectively connected to the N-terminus of lysine K and the ε-amino group of its residue. The amino acid sequence of the short peptide is: HHHH. Therefore, the amino acid sequence of the obtained polypeptide is GKEFKRIVKWPWWPRRKHHHH(HHHH). The polypeptide is synthesized by solid-phase chemical synthesis method and purified by reverse-phase high performance liquid chromatography and identified by mass spectrometry, and then the bactericidal activity and hemolytic activity are measured, and finally it is named antibacterial peptide LIH.

[0009] Another object of the present invention is to provide an application of a pH-responsive antibacterial peptide LIH in the preparation of a drug for treating Gram-positive bacteria or / and Gram-negative bacteria infectious diseases.

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

[0011] Further, in the above application, the Gram-positive bacteria is Staphylococcus aureus or Enterococcus faecalis.

[0012] Further, in the above application, antibacterial peptide LIH has bactericidal activity against Escherichia coli, Staphylococcus aureus or Enterococcus faecalis under the condition of pH = 6.0.

[0013] Another object of the present invention is to provide a drug applicable to the treatment and / or prevention of Escherichia coli, Staphylococcus aureus or Enterococcus faecalis infection under the condition of pH = 6.0, and the drug contains a pH-responsive antibacterial peptide LIH as described above.

[0014] The beneficial effects and advantages of the present invention are as follows: The antibacterial peptide LIH of the present invention can convert the level of bactericidal activity according to environmental changes. By detecting the biological activity of antibacterial peptide LIH, it is found that it has no bactericidal activity against all detected pathogenic microorganisms under the condition of pH 7.0, while it has high bactericidal activity against Escherichia coli, Staphylococcus aureus and Enterococcus faecalis under the condition of pH 6.0. This antibacterial peptide causes only 4.71% hemolysis of red blood cells at a concentration of 128 μM, and it already has the potential to become a substitute for feed antibiotics. Description of the Drawings

[0015] Figure 1 It is the high performance liquid chromatography diagram of the pH-responsive antibacterial peptide of the present invention;

[0016] Figure 2 It is the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry diagram of the pH-responsive antibacterial peptide of the present invention;

[0017] Figure 3This is the hemolytic activity diagram of the pH-responsive antimicrobial peptide and melittin ME of the present invention. Detailed implementation mode

[0018] The present invention will be further described in detail below with reference to the accompanying drawings of the specification. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0019] Example 1

[0020] Design of pH-responsive antimicrobial peptide LIH

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

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

[0023]

[0024] The net charge number 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 method

[0027] 1. The preparation of the pH-responsive antimicrobial peptide is carried out one by one from the C-terminus to the N-terminus and completed by 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-fluorenylmethoxycarbonyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); this procedure is carried out sequentially from the C-terminus to the N-terminus until the synthesis is completed, and the resin with side-chain protection after removing the Fmoc group is obtained;

[0028] 2. Add a cleavage reagent to the obtained peptide resin and react for 2 h at 20 °C in the dark, then filter; wash the precipitate with TFA (trifluoroacetic acid), mix the washing solution with the above filtrate, concentrate it using a rotary evaporator, add about 10 volumes of pre-cooled anhydrous ether, precipitate at -20 °C for 3 h to precipitate a white powdery substance, centrifuge at 2500 g for 10 min, collect the precipitate, wash the precipitate with anhydrous ether again, and dry it under vacuum to obtain a polypeptide. The cleavage reagent is composed of TFA, water, and TIS (triisopropylchlorosilane) mixed in a mass ratio of 95:2.5:2.5;

[0029] 3. Perform column equilibration for 30 min using 0.2 M sodium sulfate (adjusted to pH 7.5 with phosphoric acid), dissolve the polypeptide in a 90% aqueous acetonitrile solution, filter, use a C18 reversed-phase normal pressure column, and perform gradient elution (the eluent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), with a flow rate of 1 mL / min, a detection wavelength of 220 nm, collect the main peak, and freeze-dry; further purify using a reversed-phase C18 column. Eluent A is a 0.1% TFA / aqueous solution; eluent B is a 0.1% TFA / acetonitrile solution, the elution concentration is 25% B to 40% B, the elution time is 12 min, the flow rate is 1 mL / min, collect the main peak as above, and freeze-dry;

[0030] 4. Identification of antibacterial peptide LIH: Analyze the obtained antibacterial peptide LIH by electrospray mass spectrometry. The molecular weight shown in the mass spectrum (as Figure 1 、 2 shown) is basically consistent with the theoretical molecular weight in Table 1, and the purity of antibacterial peptide LIH is greater than 95%.

[0031] Example 3

[0032] 1. Determination of bactericidal activity: Use the serial dilution method to treat pH-responsive antibacterial peptide LIH and then determine the minimum bactericidal concentration on LB solid agar. Use 2 mg / ml BSA (containing 0.01% acetic acid) as the diluent, and then use PBS solution as the buffer solution to adjust the pH value of the BSA diluent to 6.0. Use the two-fold dilution method to prepare a series of gradient antibacterial peptide solutions in sequence. Take 100 μL of the above solution and place it in a 96-well cell culture plate, and then add an equal volume of the test bacterial solution (~10 5 CFU / mL) to each well. Set positive controls (containing bacterial solution but no antibacterial peptide) and negative controls (neither containing bacterial solution nor antibacterial peptide) respectively. Incubate at 37 °C for 2 h, take 10 μl of the culture solution, dilute it to an appropriate concentration, and evenly spread it on LB solid agar. After incubating at 37 °C for 14 - 18 h, calculate the number of colonies on the agar. The antibacterial peptide concentration with a bacterial killing rate greater than 99.9% is 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 showed 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 heparin anticoagulation, centrifuge at 3000 rpm for 10 min, and collect red blood cells; wash them 3 times with PBS solution and then resuspend them with 10 mL of PBS solution; take 50 μL of red blood cell suspension and mix it evenly with 50 μL of antimicrobial peptide solutions with different concentrations, incubate at a constant temperature in a 37 °C incubator for 1 h; then centrifuge at 4 °C and 3000 rpm for 10 min; take out the supernatant and measure the optical absorption value at 570 nm with an enzyme-linked immunosorbent assay instrument. Among them, 50 μL of red blood cells plus 50 μL of PBS solution was used as a negative control, and 50 μL of red blood cells plus 50 μL of 0.1% Tritonx-100 was used as a positive control. The minimum hemolytic concentration is the antimicrobial peptide concentration when the antimicrobial peptide causes a 10% hemolysis rate. The test results are shown in Figure 3 . Through Figure 3 it can be seen that the antimicrobial peptide LIH did not show hemolytic activity within the detection range, caused 4.71% red blood cell hemolysis at a concentration of 128 μM, and failed to cause 10% red blood cell hemolysis, showing a significant difference from the control group melittin, indicating 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 in that: 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.

2. A pH-responsive antimicrobial peptide LIH according to claim 1, characterized in that: Its molecular formula is shown in formula (I):

3. 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, connecting lysine K to the N-terminus of antimicrobial peptide LI, and connecting two short peptides to the N-terminus of lysine K and the ε-amino group of its residue, respectively, the amino acid sequence of the short peptides is: HHHH, so the amino acid sequence of the obtained polypeptide is GKEFKRIVKWPWWPRRKHHHH (HHHH), synthesizing the polypeptide by solid phase chemical synthesis, and subjecting to reverse phase high performance liquid chromatography purification and mass spectrometry identification, and then subjecting to bactericidal activity determination and hemolytic activity determination, the polypeptide is finally named antimicrobial peptide LIH.

4. Use of a pH-responsive antimicrobial peptide LIH according to claim 1 in the preparation of a drug for treating Gram-positive bacteria and / or Gram-negative bacteria infectious diseases.

5. The use according to claim 4, characterized in that: The Gram-negative bacteria is Escherichia coli.

6. The use according to claim 4, characterized in that: The Gram-positive bacteria are Staphylococcus aureus or Enterococcus faecalis.

7. The use according to claim 4, characterized in that: At pH = 6.0, the antimicrobial peptide LIH has bactericidal activity against Escherichia coli, Staphylococcus aureus or Enterococcus faecalis.

8. 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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