Trp-label cross-chain interaction beta-hairpin antibacterial peptide as well as preparation method and application of Trp-label cross-chain interaction beta-hairpin antibacterial peptide

Abstract

The invention provides a beta-hairpin antibacterial peptide based on Trp-label cross-chain interaction as well as a preparation method and application of the beta-hairpin antibacterial peptide based on Trp-label cross-chain interaction. A beta-hairpin antibacterial peptide WRFPG is used as a basis, the amino acid sequence of the beta-hairpin antibacterial peptide WRFPG is shown as SEQ ID No.2, five polypeptides are obtained and synthesized through a solid-phase chemical synthesis method, the antibacterial peptide with excellent antibacterial activity and low toxicity is screened out through determination of antibacterial activity and determination of hemolytic activity and cytotoxicity, the amino acid sequence of the antibacterial peptide is shown as SEQ ID No.1, the C end of the antibacterial peptide is amidated by-NH2, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No.1. The antibacterial peptide is finally named as the antibacterial peptide KKRRPG. According to the antibacterial peptide KKRRPG disclosed by the invention, the antibacterial activity is ensured, the hemolytic activity is reduced, and the selection index of human embryo kidney cells HEK293t is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Trp-cation cross-chain interactive beta-hairpin antimicrobial peptide and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides widely distributed in animals, insects and plants. As an important component of innate host defense, most AMPs can bind to negatively charged cell membranes, thereby destroying the integrity of bacterial cell membranes. This unique membrane-breaking mechanism, which is different from antibiotics, makes it difficult for bacteria to develop resistance to AMPs. β-folded antimicrobial peptides are mostly stabilized by disulfide bonds and present an antiparallel folding structure, and the chemical synthesis of disulfide bonds is difficult and costly. The tryptophan-cation effect of the π bond in the benzene ring of tryptophan and the positively charged amino acid to form a cross-chain intramolecular tryptophan molecule provides cross-chain interaction in the β-hairpin structure, avoiding the introduction of complex and lengthy polypeptide sequences and the poor antibacterial activity caused by disulfide bonds. The in vivo application of AMPs is hindered by the common problems of high toxicity and high hemolytic activity of AMPs. Summary of the invention

[0003] Based on the above shortcomings, the purpose of the present invention is to provide a Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG, which is highly efficient and low toxic, and solves the problems of high hemolytic activity and large toxic and side effects of existing antimicrobial peptides.

[0004] The technical scheme adopted by the present invention is as follows: a Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG, wherein the antimicrobial peptide uses PG as a turn unit and forms a β-hairpin structure through Tryptophan-cation cross-chain interactive interactions. Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

[0005] Another object of the present invention is to provide a method for preparing the β-hairpin antimicrobial peptide KKRRPG with Trp-cation cross-chain interaction as described above, as follows: based on the β-hairpin antimicrobial peptide WRFPG, whose amino acid sequence is shown in SEQ ID No.2, the four positively charged amino acids therein are exchanged to obtain five polypeptides, which are then synthesized by solid phase chemical synthesis, and the antimicrobial peptide with excellent antimicrobial activity and low toxicity is screened out by measuring the antimicrobial activity, hemolytic activity and cytotoxicity, whose amino acid sequence is shown in SEQ ID No.1, and its C-terminus is amidated with -NH2, and finally named as antimicrobial peptide KKRRPG.

[0006] Another object of the present invention is to provide the use of the Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG as described above in the preparation of drugs for treating and / or preventing infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

[0007] Furthermore, in the application described above, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.

[0008] Furthermore, in the application described above, the Gram-positive bacteria are Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

[0009] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive bacteria and / or Gram-negative bacteria infection, wherein the drug contains the antimicrobial peptide KKRRPG as described above.

[0010] Beneficial effects and advantages of the present invention: The antimicrobial peptide KKRRPG of the present invention forms a stable β-hairpin structure without a disulfide bond formed by a cysteine ​​pair, and the amino acid sequence length is only 12. It reduces the hemolytic activity and cytotoxicity while maintaining good antibacterial activity. In summary, the antimicrobial peptide KKRRPG is an antimicrobial peptide with high application value and has the advantages of high efficiency and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the HPLC chromatogram of the antimicrobial peptide KKRRPG;

[0012] Figure 2 This is the mass spectrum of the antimicrobial peptide KKRRPG. DETAILED DESCRIPTION

[0013] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1

[0015] Design of the antimicrobial peptide KKRRPG

[0016] Its amino acid sequence is:

[0017]

[0018] Based on the arrangement of β-hairpin amphiphilic peptides, the Trp-cation cross-chain interaction is used as the force to assist the PG turn unit to form the β-hairpin structure, which is named KKRRPG. The sequence is shown in Table 1.

[0019] 1 Amino acid sequence of the derived peptide

[0020]

[0021] By exchanging the position of positively charged amino acids in AMPs, an antimicrobial peptide KKRRPG with high antibacterial activity and low hemolytic activity was obtained. Its sequence length is 12, the turn unit is a PG unit, and the β-hairpin structure is formed through the Trp-cation cross-chain interaction.

[0022] Example 2

[0023] Antimicrobial peptide KKRRPG synthesized by solid phase chemical synthesis (synthesized by Shanghai Sangon Biotechnology Co., Ltd.)

[0024] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminus to the N-terminus, and is 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-fluorenylmethoxycarboxyl-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus in sequence until the synthesis is completed, and a resin with side chain protection removed from the Fmoc group is obtained;

[0025] 2. Add a cleavage agent to the peptide resin obtained above, react at 20°C in the dark for 2 hours, and filter; wash with precipitated TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate on a rotary evaporator, add about 10 times the volume of pre-cooled anhydrous ether, precipitate at -20°C for 3 hours, precipitate a white powder, centrifuge at 2500g for 10 minutes, collect the precipitate, wash the precipitate with anhydrous ether, and vacuum dry to obtain a polypeptide, wherein the cleavage agent is a mixture of TFA, water and TIS (triisopropylsilyl chloride) in a mass ratio of 95:2.5:2.5;

[0026] 3. Use 0.2 mol / L sodium sulfate (phosphoric acid adjusted to pH 7.4) to balance the column for 30 min, dissolve the polypeptide with 90% acetonitrile aqueous solution, filter, C18 reverse phase atmospheric pressure column, use gradient elution (eluent is methanol and sodium sulfate aqueous solution mixed in a volume ratio of 30:70 to 70:30), flow rate is 1 mL / min, detection wave is 220 nm, collect the main peak, and freeze-dry; further purify using reverse phase C18 column, eluent A is 0.1% TFA / water solution; eluent B is 0.1% TFA / acetonitrile solution, elution concentration is 25% B to 40% B, elution time is 12 min, flow rate is 1 mL / min, collect the main peak as above, and freeze-dry;

[0027] 4. Identification of antimicrobial peptides: The antimicrobial peptides obtained above were analyzed by electrospray ionization mass spectrometry. The molecular weights (e.g. Figure 1 , 2The molecular weight of the antimicrobial peptide was basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide was greater than 95%.

[0028] Example 3

[0029] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antimicrobial peptides was determined by the broth microdilution method. Bacteria were incubated at 37°C overnight with constant shaking at 220 rpm and then transferred to new MHB until the logarithmic phase of growth. 50 μL of bacterial culture (~10 6 CFU / mL) was added to 50 μL of BSA containing different concentrations of antimicrobial peptides and incubated at 37°C for 18 h. The absorbance at 492 nm was observed and measured at the end of the 18 h incubation. The antimicrobial peptide concentration at which no microbial growth was observed visually and spectrophotometrically was the minimum inhibitory concentration. Broth containing microbial cells was used as a negative control, and uninoculated broth was used as a positive control. The test was repeated 3 times, with two parallels for each repeat.

[0030] The test results are shown in Table 2.

[0031] Table 2 Antibacterial activity of antimicrobial peptides (μM)

[0032]

[0033]

[0034] It can be seen from Table 2 that the polypeptide KKRRPG exhibits high antibacterial activity against both Gram-negative and Gram-positive bacteria.

[0035] 2. Determination of hemolytic activity: 1 mL of fresh blood from a healthy person was centrifuged at 1000 g for 5 min, and red blood cells were collected; washed with PBS 3 times, and then resuspended with 10 mL PBS; 50 μL of red blood cell suspension was mixed evenly with 50 μL of antimicrobial peptide solution of different concentrations dissolved in PBS, and incubated in a 37°C incubator for 1 hour; taken out after 1 hour, centrifuged at 4°C and 1000 g for 5 minutes; the supernatant was taken out and the light absorption value was measured at 570 nm with an enzyme marker; the average value of each group was taken and compared and analyzed. Among them, 50 μL of red blood cells plus 50 μL of PBS was used as a negative control; 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 Table 3.

[0036] Table 3 Determination of hemolytic activity of antimicrobial peptides and therapeutic index

[0037]

[0038] It can be seen from Table 3 that, compared with the known antimicrobial peptide WKFPG, the antimicrobial peptide KKRRPG reduced the hemolytic activity and increased the therapeutic index, with the therapeutic index reaching 140.11.

[0039] 3. Cytotoxicity assay: (1) Preparation of cell suspension: Porcine intestinal epithelial cells IPEC-J2 frozen in liquid nitrogen were revived and then transferred to 5 mL of DMEM medium containing 10% fetal bovine serum and cultured at 37°C. When the cells were confluent over 25 cm 2 When the bottom of the culture flask is about 70%, it means that the cells have entered a rapid growth phase and are subcultured. Rinse the cells twice with sterile PBS and add 2mL of 0.25% trypsin to digest the cells. At the same time, observe the cell morphology during digestion. When the intercellular gap increases and most of them become round, aspirate the digestion solution, add 5mL of DMEM culture medium (containing 10% calf serum), gently blow and mix to form a single cell suspension. Adjust the cell concentration, and then add 50μL of cell suspension to wells 1 to 11 in each row of the 96-well plate. The final concentration is about 2×10 4 cells / well, 50 μL of culture medium was added to well 12;

[0040] (2) Antimicrobial peptide treatment: 50 μL of antimicrobial peptide diluted in culture medium was added to wells 1 to 10 of a 96-well plate and cultured at 37°C for 16-18 h. Well 12 contained only culture medium as a negative control, well 11 contained cells but no antimicrobial peptide as a positive control, and wells 1 to 10 were assay wells;

[0041] (3) Result evaluation: After the incubation, 50 μL of 5 mg / mL MTT was added to each well of the 96-well plate and incubated at 37°C for 4 h. Then 150 μL of DMSO was added and shaken for 10 min to dissolve the crystals. The OD was measured using an enzyme-labeled instrument. 492 Absorbance.

[0042] (4) Each experiment was repeated more than 3 times, and the cell survival rate was calculated according to the following formula:

[0043] Cell viability (100%) = (OD 测定值 / OD 阳性对照 )×100%

[0044] Table 4 Determination of the selection index of antimicrobial peptides HEK293t cells

[0045]

[0046] The above results show that by changing the position of positively charged amino acids to enable Trp-cation cross-chain interactions, the antimicrobial peptide KKRRPG designed with PG as the turn unit has good antibacterial activity and lower hemolytic activity, and improves the selection index for human embryonic kidney cells HEK293t, indicating that it has a strong potential to replace antibiotics.

Claims

1. A β-hairpin antimicrobial peptide KKRRPG with Trp-cation cross-chain interaction, characterized in that: The antimicrobial peptide uses PG as a turn unit and forms a β-hairpin structure through tryptophan-cation cross-chain interaction. Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2.

2. The method for preparing a Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG according to claim 1, characterized in that: The method is as follows: based on β-hairpin antimicrobial peptide WRFPG, whose amino acid sequence is shown in SEQ ID No.2, four positively charged amino acids therein are exchanged to obtain five polypeptides, which are then synthesized by solid phase chemical synthesis. Antimicrobial activity, hemolytic activity and cytotoxicity are measured to screen out antimicrobial peptides with excellent antimicrobial activity and low toxicity, whose amino acid sequence is shown in SEQ ID No.1, and the C-terminus is amidated with -NH2, and finally named as antimicrobial peptide KKRRPG.

3. Use of the Trp-cation cross-chain interactive β-hairpin antimicrobial peptide KKRRPG according to claim 1 in the preparation of a drug for treating and / or preventing infectious diseases caused by Gram-positive bacteria and / or Gram-negative bacteria.

4. The use according to claim 3, wherein the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa or Salmonella typhimurium.

5. The use according to claim 3, wherein the Gram-positive bacteria is Staphylococcus aureus, Enterococcus faecalis or Staphylococcus epidermidis.

6. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The medicine contains the antimicrobial peptide KKRRPG as claimed in claim 1.

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