A European yellow-bellied toad modified peptide Feleucin-K3r, preparation method thereof and applications thereof

Abstract

The present invention provides a modified peptide of European yellow-bellied toad Feleucin-K3r, a preparation method thereof and an application thereof. Among them, the modified peptide contains 18 amino acid residues, has a structural feature of an N-terminal Phe (F) residue and a C-terminal Leu (L) amide residue, shows a typical α-helix structure, and has a molecular weight of 2212.00 daltons. The modified peptide Feleucin-K3r provided by the present invention has broad-spectrum and high-efficiency antibacterial and anti-inflammatory activities. In addition, it has beneficial characteristics such as wide source, easy synthesis and modification, low hemolytic activity, and simple preparation method, and is expected to become a new type of high-efficiency antibacterial drug, with broad development and application prospects.

Description

Technical Field

[0001] The present invention relates to a modified peptide Feleucin-K3r of European yellow-bellied toad, a preparation method thereof and an application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Bacterial infections constantly threaten human health. The inflammatory response is a defense mechanism of the body against the invasion of pathogenic factors. Moderate inflammation can eliminate pathogenic microorganisms and play a host defense function that is beneficial to life and health; while uncontrolled inflammatory cascade reactions are harmful to human life and health. Currently, drugs such as antibiotics are widely used clinically. However, the limitation of chemical drug treatment is that they cannot directly remove the endotoxins and exotoxins of pathogens, which can lead to organ failure of the body. Therefore, there is an urgent need to search for and develop new antibacterial and anti-inflammatory drugs.

[0003] Antimicrobial peptides are considered to be the first line of defense for the body to resist the invasion of external pathogenic factors. They are widely present in various organisms such as mammals, amphibians, plants, birds, and microorganisms. They are conservative components of the innate immune system, can selectively stimulate the body's innate immune response, and have good prevention and treatment effects on various infectious diseases. In addition to having broad-spectrum antibacterial effects, antimicrobial peptides have also been found to have anti-biofilm, anti-cancer and anti-inflammatory activities. In addition, they can also regulate cell functions and reduce the damage of inflammatory reactions to tissues and organs. Compared with traditional antibiotics, the main advantages of antimicrobial peptides include increasing the phagocytic ability of macrophages, stimulating the proliferation of lymphocytes and immunomodulatory activities, directly or indirectly chemotaxing monocytes, immature dendritic cells, etc., and enhancing the body's immune function. At the same time, they have the characteristics of wide source, easy synthesis and modification, and diverse administration methods. Based on the above, the emergence of antimicrobial peptides provides new ideas for the research and development of antibacterial and anti-inflammatory drugs. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem of the increase in bacterial drug resistance caused by the abuse of antibiotics, and to propose a modified peptide Feleucin-K3r of European yellow-bellied toad, a preparation method thereof and an application thereof.

[0005] The preparation method of the modified peptide Feleucin-K3r described in the present invention: By analyzing the amino acid sequence of the antimicrobial peptide Feleucin-BO1 of European yellow-bellied toad, the amino acid sequence of the modified peptide Feleucin-K3r is designed by using molecular modification methods such as amino acid substitution and sequence repetition, and is synthesized by a polypeptide automatic synthesizer, desalted and purified by HPLC reverse C18 column chromatography, and then its purity is identified by high performance liquid chromatography, its molecular weight is determined by mass spectrometry, and its amino acid sequence is determined by an automatic amino acid sequencer.

[0006] The amino acid sequence of the modified peptide Feleucin-K3r is as follows: Phe Leu Lys Leu Leu Lys Lys Leu Leu Phe Leu Lys Leu Leu Lys Lys Leu Leu

[0007] The obtained modified peptide Feleucin-K3r contains 18 amino acid residues, has the structural characteristics of an N-terminal Phe (F) residue and a C-terminal Leu (L) amide residue, shows a typical α-helical structure, and has a molecular weight of 2212.00 Daltons.

[0008] The modified peptide Feleucin-K3r obtained in the present invention has been determined through experimental research to be useful for preparing antibacterial drugs or anti-inflammatory drugs.

[0009] In summary, the beneficial effects of the present invention are as follows:

[0010] Compared with the template peptide, the modified peptide Feleucin-K3r prepared in the present invention has broad-spectrum and highly efficient antibacterial activity and extremely strong anti-inflammatory activity. In addition, it has beneficial characteristics such as a small molecular weight, wide sources, easy synthesis and modification, low hemolytic activity, and simple preparation method, and is expected to become a new type of highly efficient antibacterial drug with broad development and application prospects. Brief Description of the Drawings

[0011] Figure 1 It is a schematic diagram of the experimental results of the modified peptide Feleucin-K3r inhibiting the release of the pro-inflammatory factor IL-6 induced by LPS in the RAW264.7 macrophage cell line;

[0012] Figure 2 It is a schematic diagram of the experimental results of the modified peptide Feleucin-K3r inhibiting the release of the pro-inflammatory factor TNF-α induced by LPS in the RAW264.7 macrophage cell line.

[0013] Figure 3 It is a schematic diagram of the experimental results of the modified peptide Feleucin-K3r inhibiting the activation of the NF-κB signaling pathway downstream of TLR4 induced by LPS.

[0014] Figure 4 It is a schematic diagram of the experimental results of the modified peptide Feleucin-K3r inhibiting the activation of the MAPK signaling pathway downstream of TLR4 induced by LPS. Detailed Description of the Embodiments

[0015] The following combines the drawings and examples to further describe in detail the specific embodiments of the present invention. The following examples are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0016] Example 1:

[0017] I. Preparation of the Modified Peptide Feleucin-K3r from the European Yellow-Bellied Toad (Bombina variegata)

[0018] 1. Obtaining the Skin Secretions of the European Yellow-Bellied Toad

[0019] The skin secretions were obtained from adult specimens of the European yellow-bellied toad using mild electrocutaneous stimulation technology. Then, the secretions on the toad's skin surface were rinsed into a frozen beaker with distilled water, quickly frozen in liquid nitrogen, and freeze-dried. The freeze-dried cells were stored in a -20°C refrigerator.

[0020] 2. cDNA Molecular Cloning and Sequencing of the Skin Secretions from the European Yellow-Bellied Toad

[0021] a. Dissolve 5 mg of the freeze-dried skin secretions in 1 mL of mRNA stabilization buffer, clarify by centrifugation (5000 xg for 10 min), and then isolate polyadenylated mRNA from the supernatant using magnetic oligonucleotide beads and reverse transcribe it. Use the SMART-RACE kit to perform a 3'-RACE procedure on the cDNA to obtain the full-length nucleic acid sequence data. The PCR cycling program is as follows:

[0022] Initial denaturation step: 60 s at 94°C; 35 cycles; denaturation at 94°C for 30 s; primer annealing at 58°C for 30 s; extension at 72°C for 180 s.

[0023] b. Gel purify the PCR products, clone them using the pGEM-T vector system, and sequence them using an ABI 3100 automated sequencer.

[0024] 3. LC / MS Fractionation and Identification of Polypeptides

[0025] a. Perform LC / MS fractionation on the freeze-dried skin secretion samples using a gradient HPLC system interfaced with an LCQ FleetTM ion trap electrospray mass spectrometer. The high-performance liquid chromatography system is equipped with a Phenomenex C5 column, which is eluted with a gradient formed by trifluoroacetic acid / water - trifluoroacetic acid / water / acetonitrile at a flow rate of 1 mL / min within 240 min. Fractions are collected at 1-min intervals (10% to the MS, 90% to the fraction collector), and the column effluent is continuously monitored at 214 nm.

[0026] b. Locate the peptides with molecular masses consistent with those predicted by the cloned cDNA in the fractions, and then inject the samples into a spray mass spectrometer to capture appropriate ions for MS / MS fragmentation.

[0027] 4. Synthesis of Polypeptides

[0028] a. The primary structure of the newly predicted peptide was determined from the translated cDNA and synthesized separately by solid-phase Fmoc chemistry using a PS3 automatic solid-phase synthesizer.

[0029] b. After cleavage and deprotection from the resin, each synthetic peptide was analyzed by reverse HPLC and MALDI-TOF mass spectrometry to determine its purity and structure.

[0030] c. Feleucin-BO1 contains nine amino acid residues and has a random coil structure. The full sequence is Phe Leu Gly Leu Leu Gly Ser Leu Leu.

[0031] 5. Synthesis of the modified peptide Feleucin-K3r

[0032] The Gly at position 3, Gly at position 6, and Ser at position 7 in the sequence of the European yellow-bellied toad antimicrobial peptide Feleucin-BO1 were mutated to Leu. Then, a repeat of the new sequence was performed to obtain Feleucin-K3r, which was synthesized using a polypeptide automatic synthesizer. It was desalted and purified by reverse C18 column chromatography on HPLC, and then its purity was identified by high-performance liquid chromatography, its molecular weight was determined by mass spectrometry, and its amino acid sequence was determined by an automatic amino acid sequencer.

[0033] Measurement results:

[0034] The modified peptide Feleucin-K3r contains 18 amino acid residues, has a structural feature with an N-terminal Phe (F) residue and a C-terminal Leu (L) amide residue, shows a typical α-helix structure, and has a molecular weight of 2212.00 daltons. Its amino acid sequence is: Phe Leu Lys Leu Leu Lys Lys Leu Leu Phe Leu Lys Leu Leu Lys Lys Leu Leu.

[0035] Example 2:

[0036] II. Determination of the antibacterial efficacy of the modified peptide Feleucin-K3r

[0037] 1. Determination of the minimum inhibitory concentration (MIC)

[0038] Using Mueller-Hinton Broth (MHB) liquid medium as the culture medium, antibacterial detection was carried out by the two-fold dilution method.

[0039] The specific method is as follows: Place the bacteria to be tested on a shaker for cultivation. When the number of bacteria reaches about 1×108 CFU / mL (obtained from the growth curve), take 50 μL of the bacterial solution and inoculate it into 10 mL of sterilized MHB broth medium, diluting the number of bacteria 200 times to about 5×105 CFU / mL. Use sterile PBS solution to prepare the polypeptide concentration to a final peptide concentration of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 μM, with 3 replicates for each concentration. Use sterile PBS solution as the solvent control and sterilized MHB broth medium as the blank control. Place it on a shaker at 37°C and 200 rpm for 16 h, then measure the absorbance at 550 nm to determine the MIC value of the polypeptide. The experiment is repeated three times and the average value is taken. The results are shown in Table 1.

[0040] 2. Determination of the minimum bactericidal concentration (MBC)

[0041] Take 10 μL of the bacterial solution from each well in the 96-well plate with a concentration greater than MIC in the above experiment and inoculate it onto sterilized MHB agar medium. Let it air dry naturally in a clean bench and then invert it and place it in an incubator at 37°C for 16 h. Take it out and observe that the minimum concentration group without growing colonies is the minimum bactericidal concentration (MBC).

[0042] Table 1 Determination of the antibacterial efficacy of the modified peptide Feleucin-K3r

[0043]

[0044] As can be seen above, the modified peptide Feleucin-K3r exhibits high antibacterial activity against Gram-positive bacteria and Gram-negative bacteria.

[0045] Example 3:

[0046] III. Determination of the anti-inflammatory efficacy of the modified peptide Feleucin-K3r

[0047] 1. The modified peptide Feleucin-K3r inhibits the release of pro-inflammatory factors

[0048] A large number of pro-inflammatory cytokines are released during inflammation, and the concentrations of IL-6 and TNF-α are often used as indicators to evaluate the efficacy of anti-inflammatory drugs. To quantify the concentrations of IL-6 and TNF-α in the supernatant of primary mouse peritoneal macrophages, the present invention uses the enzyme-linked immunosorbent assay (ELISA), which is widely used because of its high sensitivity and specificity based on many mature quantitative methods.

[0049] C57s at 6 - 8 weeks old (MPMs) were extracted, cultured in serum-free 1640 medium for 2 hours, and then changed to 1640 medium containing 2% serum for culture. They were cultured overnight at 5×105 cells / well. The next day, the MPMs were washed with sterile PBS to remove non-adherent cells. The experiment was divided into four groups: the blank control group was without treatment of MPMs cells; the LPS group was added with LPS at a final concentration of 0.5 μg / mL; the drug addition group was 0.5 μg / mL LPS + polypeptides at different concentrations; the positive control group was 0.5 μg / mL LPS + 20 μM polymyxin B (PMB). Then the cells were cultured in an incubator for 24 hours. After that, the supernatant was collected and the contents of IL-6 and TNF-α were detected using an ELISA kit. The average value was taken from three repeated experiments. The experimental results are as Figure 1 and Figure 2 shown.

[0050] The modified peptide Feleucin-K3r can significantly inhibit the expression of LPS-induced pro-inflammatory factors IL-6 and TNF-α in primary mouse peritoneal macrophages. Its inhibitory effect is significantly concentration-dependent and is comparable to that of the positive control group, indicating that the modified peptide Feleucin-K3r has excellent anti-inflammatory activity.

[0051] 2. Exploration of the anti-inflammatory mechanism of the modified peptide Feleucin-K3r

[0052] To explore and evaluate the effect of the modified peptide Feleucin-K3r on the activation of downstream signals NF-κB / MAPK of LPS / TLR4, the expression of related proteins in primary mouse peritoneal macrophages was determined by Western blotting. The experimental results are as Figure 3 and Figure 4 shown.

[0053] In summary, it is proved that the modified peptide Feleucin-K3r regulates LPS-induced inflammatory responses by inhibiting the NF-κB and MAPK signaling pathways downstream of TLR4, and has certain development and application prospects in the preparation of antibacterial or anti-inflammatory drugs.

[0054] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the design concept of the present invention shall be included within the protection scope of the present invention.

Claims

1. A modified peptide of European yellow-bellied toad Feleucin-K3r, characterized in that: The modified peptide Feleucin-K3r contains 18 amino acid residues, has the structural characteristics of an N-terminal Phe (F) residue and a C-terminal Leu (L) amide residue, shows a typical α-helix structure, and has a molecular weight of 2212.00 Daltons. The amino acid sequence of the modified peptide is as shown in SEQ ID NO: 1 in the sequence listing.

2. The preparation method of a modified peptide Feleucin-K3r of European yellow-bellied toad according to claim 1, characterized in that: The 3rd Gly, 6th Gly, and 7th Ser on the sequence of the European yellow-bellied toad antimicrobial peptide Feleucin-BO1 were mutated to Leu, and then a repeat was performed on the new sequence in a repetitive sequence manner to obtain the amino acid sequence of the modified peptide Feleucin-K3r, and it was chemically synthesized using the method of solid-phase peptide synthesis.

3. Use of a modified peptide Feleucin-K3r of European yellow-bellied toad according to claim 2, characterized in that: The modified peptide Feleucin-K3r is used for preparing anti-inflammatory drugs or antibacterial drugs against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli.

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