A porcine-derived PR-39-derived anti-proteolytic antibacterial peptide, its preparation method and application

Abstract

The present invention discloses a porcine-derived PR-39-derived anti-proteolytic antibacterial peptide, its preparation method and application, belonging to the field of bioengineering technology. The sequence of this antibacterial peptide is shown as SEQ ID No.1. The Arg-Pro amino acid structure is used to provide the ability to resist trypsin digestion while ensuring the number of positive charges, and phenylalanine is added to increase hydrophobicity. This sequence is obtained by repeating the RPPF amino acid sequence six times as the basic structural unit. It also discloses the application of this antibacterial peptide in the preparation of drugs for treating infectious diseases caused by Gram-negative bacteria or / and Gram-positive bacteria. This antibacterial peptide has extremely strong antibacterial activity against a variety of bacteria and extremely low cytotoxicity. The cell survival rate can still reach more than 80% at its high concentration. In addition, it has high stability under the conditions of high concentration of trypsin and artificial intestinal juice. Therefore, this antibacterial peptide has great potential to replace antibiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a porcine-derived PR-39-derived anti-proteolytic antibacterial peptide, its preparation method and application. Technical Background

[0002] Antimicrobial peptides (AMPs) have encountered many obstacles in clinical applications. For example, their stability against various inhibitory factors (physiological salts, serum, and proteases) present in the body is poor, which in turn leads to a decrease or even loss of their antimicrobial activity. The most critical limitation is the impact of proteases (mainly chymotrypsin and trypsin) on the stability of AMPs, as it significantly reduces their bioavailability in the body.

[0003] The host defense peptide PR-39 derived from the porcine intestine, containing the repeated sequence "XPPX", 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 the Arg-Pro structure usually have good protease stability and can exhibit antibacterial and hemolytic activities similar to those of the parent peptide. The antibacterial peptide PR-39 has strong antibacterial activity and multiple biological functions, but its anti-proteolytic ability is not strong. The presence of positively charged amino acids helps AMPs to have an electrostatic interaction with the negatively charged bacterial cell membrane, while hydrophobic amino acids are the key structural parameters determining the bacterial killing rate of AMPs. Therefore, how to reasonably use the Arg-Pro structure and positive and hydrophobic properties to modify the antibacterial peptide PR-39 so that it has anti-proteolytic ability while ensuring its original biological activity and biosafety is the problem that needs to be solved currently. Summary of the Invention

[0004] Based on the above deficiencies, the present invention provides a porcine-derived PR-39-derived anti-proteolytic antibacterial peptide (RPPF)6 to solve the problem of its poor stability against trypsin.

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

[0006] Another object of the present invention is to provide a method for preparing a porcine-derived PR-39-derived anti-proteolytic antibacterial peptide (RPPF)6 as described above, as follows: The Arg-Pro amino acid structure is used to provide the ability to resist trypsin digestion while ensuring the number of positive charges, and phenylalanine is added to increase hydrophobicity. The RPPF amino acid sequence is used as the basic structural unit for six repetitions, and the amino acid sequence of the resulting polypeptide is shown in SEQ ID No.1, and -NH2 amide is used at its C-terminus; The polypeptide is synthesized by solid-phase chemical synthesis method, and after being identified by mass spectrometry, the preparation of the polypeptide is completed; The antibacterial activity and cytotoxicity of the polypeptide are detected, and then the effects of trypsin and artificial intestinal juice on antibacterial activity are evaluated through experiments, and finally it is named antibacterial peptide (RPPF)6.

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

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

[0009] Further, the Gram-positive bacteria are 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 bacteria and / or Gram-negative bacteria infections, and the drug contains a porcine-derived PR-39-derived anti-proteolytic antibacterial peptide (RPPF)6 as described above.

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

[0012] Figure 1 It is the high-performance liquid chromatography diagram of the antibacterial peptide (RPPF)6;

[0013] Figure 2 Mass spectrum of antimicrobial peptide (RPPF)6 Detailed implementation mode

[0014] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation modes of the present invention are not limited thereto.

[0015] Embodiment 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 influence of positive charge and hydrophobicity on AMPs, the polypeptide template obtained by modifying and repeating the sequence is: (YPPX)n-NH2, where X is a hydrophobic amino acid, Y is a positively charged amino acid, and n is the number of repetitions. Existing studies have shown that the Arg-Pro (RP) structure can provide the ability to resist trypsin digestion while ensuring the number of positive charges. After measuring the AMPs with the (RPPX)n-NH2 sequence pattern, it was found that the AMPs with the strongest antibacterial activity had X = Phe and n = 6, which was named antimicrobial peptide (RPPF)6, and its amino acid sequence is shown in Table 1.

[0018] Table 1 Amino acid sequence of antimicrobial peptide (RPPF)6

[0019]

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

[0021] Embodiment 2

[0022] Synthesis of antimicrobial peptide (RPPF)6 by solid-phase chemical synthesis method

[0023] 1. The preparation of AMPs is carried out one by one from the C-terminus to the N-terminus and completed by a polypeptide 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); according to this procedure, the synthesis is carried out from the C-terminus to the N-terminus in turn until the synthesis is completed, and the resin with side-chain protection with the Fmoc group removed is obtained;

[0024] 2. In the obtained peptide resin, a cleavage reagent is added, and the reaction is carried out at 20 °C in the dark for 2 h, followed by filtration; the precipitate is washed with TFA (trifluoroacetic acid), the washing solution is mixed with the above filtrate, concentrated by a rotary evaporator, and then about 10 times the volume of pre-cooled anhydrous ether is added. Precipitation is carried out at -20 °C for 3 h to precipitate a white powder, which is centrifuged at 2500 g for 10 min, the precipitate is collected, washed with anhydrous ether again, and dried in vacuo 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;

[0025] 3. Column equilibration is carried out for 30 min using 0.2 mol / L sodium sulfate (adjusted to pH 7.4 with phosphoric acid). The polypeptide is dissolved in a 90% aqueous acetonitrile solution, filtered, and passed through a C18 reversed-phase normal pressure column. Gradient elution is used (the eluent is a mixture of methanol and sodium sulfate aqueous solution in a volume ratio of 30:70 to 70:30), the flow rate is 1 mL / min, the detection wavelength is 220 nm, the main peak is collected and freeze-dried; further purification is carried out using a reversed-phase C18 column. Eluent A is 0.1% TFA / aqueous solution; eluent B is 0.1% TFA / acetonitrile solution. The elution concentration is 25% B - 40% B, the elution time is 12 min, the flow rate is 1 mL / min, and the main peak is collected and freeze-dried as above;

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

[0027] Example 3

[0028] Bioactivity determination of antibacterial peptides

[0029] 1. Antibacterial activity determination

[0030] Under the conditions of a shaker at 37 °C and 220 g, the bacteria are cultured in cation-adjusted MHB broth medium until the logarithmic growth phase and diluted to OD 600nm = 0.4 (3×10 8 -9×10 8 CFU mL -1 ). The bacterial solution is diluted 1000 times before use. Equal volumes (50 μL) of the bacterial suspension and amphiphiles with different concentrations (0.25×10 -6 -128×10 -6A bovine serum albumin solution (BSA, 0.2%; acetic acid, 0.01%) of (M) was added to a round-bottom transparent polypropylene 96-well plate. The MHB medium containing bacteria was used as a positive control, and the MHB without bacteria was used as a negative control. The 96-well plate was incubated in a 37 °C constant temperature incubator for 16 - 18 h. The minimum inhibitory concentration is the minimum concentration at which no bacterial growth can be observed under the optical density of 492nm and the minimum concentration at which no bacterial growth can be observed under the optical density of

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

[0032]

[0033]

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

[0035] 2. Cytotoxicity assay:

[0036] (1) Preparation of cell suspension: Mouse mononuclear macrophages RAW264.7 cryopreserved in liquid nitrogen were resuscitated and then transferred to 5 mL of RPMI-1640 medium containing 10% fetal bovine serum and incubated at a constant temperature of 37 °C. When the cells covered about 70% of the bottom of the 25 cm 2 culture flask, it indicated that the cells entered the rapid growth phase and passage was carried out. The cells were rinsed twice with sterile PBS, and 1 mL of 0.25% trypsin was added to digest the cells. At the same time, the cell morphology was observed during digestion. When the cell gaps increased and most of the cells became round, the digestion solution was aspirated, 5 mL of RPMI-1640 medium (containing 10% fetal bovine serum) was added, and the cells were gently pipetted and mixed to form a single-cell suspension. The cell concentration was adjusted, and then 50 μL of the cell suspension was added to wells No. 1 to 11 in each row of the 96-well plate, with a final concentration of about 3×10 5 cells / well, and 50 μL of medium was added to well No. 12;

[0037] (2) Antimicrobial peptide treatment: 50 μL of the antimicrobial peptide serially diluted with the medium was respectively added to wells No. 1 to 10 of the 96-well plate and incubated in a carbon dioxide incubator for 3 - 4 h. Well No. 12 containing only the medium was used as a negative control, well No. 11 containing cells but no antimicrobial peptide was used as a positive control, and wells No. 1 to 10 were the assay wells;

[0038] (3) Result judgment: After the culture is completed, 50 μL of 5 mg / mL MTT is aspirated and added to each well of the 96-well plate, and cultured in a carbon dioxide incubator for 3 - 4 h. Then, 150 μL of DMSO is added, and shaken for 10 min to dissolve the crystals. The OD 492nm Absorbance is measured with an enzyme-linked immunosorbent assay (ELISA) reader.

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

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

[0041] The survival rate of mouse mononuclear macrophages reaches over 80% at all detected 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 cell survival rate of PR-39 is only 71.26%, while the cell survival rate of (RPPF)6 reaches over 80% within the detection range. The cytotoxicity of antimicrobial peptide (RPPF)6 to mouse macrophages is much less than that of PR-39.

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

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

[0048]

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

[0050] Based on all the above results, the repetitive sequence antimicrobial peptide (RPPF)6 designed against the trypsin cleavage site almost maintains the original antibacterial activity of PR-39, has lower cytotoxicity and extremely strong stability, indicating that it is superior to PR-39 and has great 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. 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; the Gram-negative bacteria are Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium or Acinetobacter baumannii; the Gram-positive bacteria are Bacillus subtilis or Streptococcus suis.

4. 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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