Anti-enzymolysis antibacterial peptide derived from porcine PR-39 as well as preparation method and application of anti-enzymolysis antibacterial peptide

Abstract

The invention discloses a porcine PR-39-derived anti-enzymolysis antibacterial peptide and a preparation method and application thereof, and belongs to the technical field of bioengineering, the sequence of the antibacterial peptide is shown as SEQ ID No.1, an Arg-Pro amino acid structure is used for ensuring the positive charge number and providing the trypsin enzymolysis resisting capacity at the same time, phenylalanine is added for improving the hydrophobicity, and the antibacterial peptide can be used for preparing an anti-enzymolysis antibacterial peptide. The sequence is obtained by taking an RPPF amino acid sequence as a basic structural unit and repeating for six times. The invention further discloses application of the antibacterial peptide in preparation of drugs for treating infectious diseases caused by gram-negative bacteria or / and gram-positive bacteria. The antibacterial peptide has extremely high antibacterial activity and extremely low cytotoxicity to various bacteria, the cell survival rate can still reach 80% or above under the high concentration, and in addition, the antibacterial peptide has high stability under the conditions of high-concentration trypsin and artificial intestinal juice; therefore, the antibacterial peptide has relatively strong potential of replacing antibiotics.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to an anti-enzymatic antimicrobial peptide derived from porcine PR-39 and a preparation method and application thereof. Technical Background

[0002] Antimicrobial peptides (AMPs) have encountered many obstacles in clinical application, such as their poor stability to various inhibitory factors (physiological salts, serum and proteases) in the body, which leads to the reduction or even loss of their antimicrobial activity. The most critical limitation is the effect of proteases (mainly chymotrypsin and trypsin) on the stability of AMPs, because it greatly reduces the availability in the body.

[0003] The host defense peptide PR-39, derived from the pig intestine, contains a repeating sequence of "XPPX" and is a linear peptide with only 7 amino acids, mainly rich in Arg and Pro. According to the progress of protease cleavage sites, peptides rich in Pro can effectively resist the degradation of serine proteases, trypsin or aminopeptidase proteins, and to a certain extent improve their stability in the enzyme-containing microenvironment in the body. Related studies have also found that AMPs with Arg-Pro structure usually have good protease stability and can show antibacterial and hemolytic activities that are almost the same as those of the parent peptide. The antimicrobial peptide PR-39 has strong antibacterial activity and multiple biological functions, but its resistance to enzymatic hydrolysis is not strong. The presence of positively charged amino acids helps AMPs to produce electrostatic effects with negatively charged bacterial cell membranes, while hydrophobic amino acids are the key structural parameters that determine the bacterial killing rate of AMPs. Therefore, how to reasonably use the Arg-Pro structure and positive charge and hydrophobicity to modify the antimicrobial peptide PR-39 is a problem that needs to be solved at present, so that it has the ability to resist enzymatic hydrolysis while ensuring its original biological activity and biosafety. Summary of the invention

[0004] Based on the above shortcomings, the present invention provides a porcine PR-39-derived antimicrobial peptide (RPPF) 6 to solve the problem of poor stability to trypsin.

[0005] The technical scheme adopted by the present invention is as follows: a porcine PR-39-derived antimicrobial peptide (RPPF) 6, whose amino acid sequence is shown in SEQ ID No. 1, and whose C-terminus is amidated with -NH2 amino group.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned porcine PR-39-derived antimicrobial peptide (RPPF) 6, which is as follows: an Arg-Pro amino acid structure is used to provide the ability to resist trypsin hydrolysis while ensuring the positive charge number, and phenylalanine is added to improve the hydrophobicity, and the RPPF amino acid sequence is repeated six times as the basic structural unit. The amino acid sequence of the obtained polypeptide is shown in SEQ ID No. 1, and the -NH2 amino group is amidated at its C-terminus; the polypeptide is synthesized by solid phase chemical synthesis, and the preparation of the polypeptide is completed after mass spectrometry identification; the polypeptide is subjected to antibacterial activity detection and cytotoxicity detection, and then the effect of trypsin and artificial intestinal fluid on the antibacterial activity is evaluated, and finally named as antimicrobial peptide (RPPF) 6.

[0007] Another object of the present invention is to provide a porcine PR-39-derived antimicrobial peptide (RPPF) 6 for use in the preparation of drugs for treating and / or preventing infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

[0008] Furthermore, the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii or Salmonella typhimurium.

[0009] Furthermore, the Gram-negative bacteria are Gram-positive bacteria such as Bacillus subtilis or Streptococcus suis.

[0010] Another object of the present invention is to provide a drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, wherein the drug contains the porcine PR-39-derived antimicrobial peptide (RPPF) 6 as described above.

[0011] Beneficial effects and advantages of the present invention: The sequence length of the antimicrobial peptide (RPPF) 6 of the present invention is shorter than that of PR-39, and the structure is stable. After the antimicrobial peptide (RPPF) 6 was tested for antibacterial, cytotoxicity, and enzyme stability, it was found that the antimicrobial peptide (RPPF) 6 showed excellent inhibitory effects on various Gram-negative strains such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium. The geometric mean of the minimum inhibitory concentration of four Gram-negative bacteria reached 4.59 μM, and it had very low cytotoxicity. The survival rate of mouse mononuclear macrophages reached more than 80% at all tested concentrations. In addition, the antimicrobial peptide (RPPF) 6 can still maintain excellent stability under high concentration trypsin conditions. In summary, the antimicrobial peptide (RPPF) 6 is an antimicrobial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the HPLC chromatogram of antimicrobial peptide (RPPF) 6;

[0013] Figure 2 This is the mass spectrum of antimicrobial peptide (RPPF) 6. DETAILED DESCRIPTION

[0014] 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.

[0015] Example 1

[0016] Design of antimicrobial (RPPF)6

[0017] Based on the PR-39 repeat sequence "XPPX" and the trypsin cleavage site, and according to the effects of positive charge and hydrophobicity on AMPs, the sequence was modified and repeated to obtain the polypeptide template: (YPPX)n-NH2, where X is a hydrophobic amino acid, Y is a positively charged amino acid, and n is the number of repetitions. Studies have shown that the Arg-Pro (RP) structure can provide resistance to trypsin hydrolysis while ensuring the number of positive charges. After measuring AMPs with the (RPPX)n-NH2 sequence pattern, it was found that when X = Phe and n = 6, the AMPs had the strongest antibacterial activity, named antimicrobial peptide (RPPF) 6, and its amino acid sequence is shown in Table 1.

[0018] Table 1 Antimicrobial peptide (RPPF) 6 amino acid sequence

[0019]

[0020] The antimicrobial peptide (RPPF) 6 sequence length is 24 amino acids, and the basic structural unit is RPPF. The C-terminus of the antimicrobial peptide (RPPF) 6 is amidated with -NH2 to increase a positive charge, and the charge number is +7. The antimicrobial peptide designed in this way has high antibacterial activity and excellent trypsin stability.

[0021] Example 2

[0022] Solid phase chemical synthesis of antimicrobial peptide (RPPF) 6

[0023] 1. The preparation of AMPs 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 the Wang resin, and then the Fmoc group is removed to obtain the 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 the side chain protection resin with the Fmoc group removed is obtained;

[0024] 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;

[0025] 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;

[0026] 4. Identification of antimicrobial peptides: The AMPs obtained above were analyzed by electrospray mass spectrometry. The molecular weights (e.g. Figure 2 The molecular weight of the antimicrobial peptide (RPPF) 6 is basically consistent with the theoretical molecular weight in Table 1, and the purity of the antimicrobial peptide (RPPF) 6 is greater than 95% (as shown in Figure 1 shown).

[0027] Example 3

[0028] Bioactivity assay of antimicrobial peptides

[0029] 1. Determination of antibacterial activity

[0030] The bacteria were grown in cation-adjusted MHB broth at 37°C and 220 g in a shaking incubator until the logarithmic growth phase and diluted to an OD 600nm =0.4(3×10 8 -9×10 8 CFU mL -1 ). The bacterial solution was diluted 1000 times before use. Equal volumes (50 μL) of bacterial suspension and solutions containing different concentrations of amphiphiles (0.25×10 -6 -128×10 -6Bovine serum albumin solution (BSA, 0.2%; acetic acid, 0.01%) was added to a round-bottom transparent polypropylene 96-well plate. MHB medium containing bacteria was used as a positive control, and MHB without bacteria was used as a negative control. The 96-well plate was incubated in a 37°C constant temperature incubator for 16-18h. The minimum inhibitory concentration was measured by naked eye and microplate reader OD 492nm The minimum concentration at which no bacterial growth was observed was 1.38 Å. Each test was performed in three independent parallels, with two replicates for each parallel. The test results are shown in Table 2.

[0031] Table 2 Minimum inhibitory concentration (μM) of antimicrobial peptide (RPPF) 6

[0032]

[0033]

[0034] It can be seen from Table 2 that antimicrobial peptide (RPPF) 6 maintains the antibacterial activity of PR-39 and has good antibacterial activity against Gram-negative bacteria and some Gram-positive bacteria.

[0035] 2. Cytotoxicity Assay:

[0036] (1) Preparation of cell suspension: Mouse mononuclear macrophage RAW264.7 cells frozen in liquid nitrogen were revived and then transferred to 5 mL of RPMI-1640 medium containing 10% fetal bovine serum and cultured at 37°C. 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 1 mL of 0.25% trypsin to digest the cells. At the same time, observe the cell morphology during digestion. When the intercellular gaps increase and most of them become round, aspirate the digestion solution, add 5 mL of RPMI-1640 culture medium (containing 10% fetal bovine serum), and gently blow with a pipette to mix to form a single cell suspension. Adjust the cell concentration, and then add 50 μL of cell suspension to wells 1 to 11 in each row of the 96-well plate. The final concentration is about 3×10 5 cells / well, 50 μL of culture medium was added to well 12;

[0037] (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 in a carbon dioxide incubator for 3-4 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;

[0038] (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 in a carbon dioxide incubator for 3-4 h. Then, 150 μL of DMSO was added and shaken for 10 min to dissolve the crystals. The OD was measured using an ELISA reader. 492nm Absorbance.

[0039] (4) Each experiment was repeated more than 3 times, and the cell survival rate was calculated according to the following formula:

[0040] Cell viability (100%) = (OD 测定值 / OD 阳性对照 )×100%

[0041] The survival rate of mouse mononuclear macrophages was above 80% at all tested concentrations. The results are shown in Table 3.

[0042] Table 3 Determination of cytotoxicity of antimicrobial peptide (RPPF) 6

[0043]

[0044]

[0045] It can be seen from Table 3 that when the concentration is 32, the survival rate of PR-39 cells is only 71.26%, while the cell survival rate of (RPPF)6 in the detection range is more than 80%. The toxicity of antimicrobial peptide (RPPF)6 to mouse macrophages is far less than that of PR-39.

[0046] 3. To evaluate the effect of protease and simulated intestinal fluid on antibacterial activity, the antimicrobial peptides were incubated with trypsin solution (8 mg / mL) and artificial intestinal fluid for 4 hours and then the antibacterial activity was determined. The test results are shown in Table 4.

[0047] Table 4 Antibacterial activity of antimicrobial peptide (RPPF) 6 and PR-39 against Escherichia coli 25922 under trypsin and simulated intestinal fluid conditions (μM)

[0048]

[0049] According to the results in Table 4, PR-39 lost its activity under the conditions of 8 mg / mL trypsin and artificial intestinal fluid, while (RPPF)6 still maintained good antibacterial activity.

[0050] Based on all the above results, the repetitive sequence antimicrobial peptide (RPPF) 6 designed for the trypsin cleavage site has lower cytotoxicity and extremely strong stability while almost maintaining the original antibacterial activity of PR-39, indicating that it is superior to PR-39 and has a strong potential to replace antibiotics.

Claims

1. A porcine PR-39-derived antimicrobial peptide (RPPF) 6, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, and its C-terminus is amidated with -NH2 amino group.

2. The method for preparing a porcine PR-39-derived antimicrobial peptide (RPPF) 6 according to claim 1, characterized in that: The method is as follows: an Arg-Pro amino acid structure is used to provide the ability of resisting trypsin hydrolysis while ensuring the positive charge number, and phenylalanine is added to improve the hydrophobicity, and the RPPF amino acid sequence is repeated six times as the basic structural unit. The amino acid sequence of the obtained polypeptide is shown in SEQ ID No. 1, and the -NH2 amino group is amidated at its C-terminus; the polypeptide is synthesized by solid phase chemical synthesis, and the preparation of the polypeptide is completed after mass spectrometry identification; the polypeptide is subjected to antibacterial activity detection and cytotoxicity detection, and then the effect of trypsin and artificial intestinal fluid on the antibacterial activity is evaluated, and finally it is named antimicrobial peptide (RPPF) 6.

3. The use of a porcine PR-39-derived antimicrobial peptide (RPPF) 6 according to claim 1 in the preparation of a drug for treating and / or preventing infectious diseases caused by Gram-negative bacteria and / or Gram-positive bacteria.

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

5. The use according to claim 3, wherein the Gram-positive bacteria is Bacillus subtilis or Streptococcus suis.

6. A drug suitable for treating and / or preventing Gram-positive and / or Gram-negative bacterial infections, characterized in that: The drug contains a porcine PR-39-derived antimicrobial peptide (RPPF) 6 resistant to enzymatic degradation as described in claim 1.

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