Anti-enzymolysis nano capture peptide as well as preparation method and application thereof

Abstract

The invention provides an anti-enzymolysis nano capture peptide as well as a preparation method and application thereof, and belongs to the field of bioengineering, and the sequence of the anti-enzymolysis nano capture peptide is shown as SEQ ID No.1. The invention also discloses a preparation method of the antibacterial peptide. The capture peptide disclosed by the invention has a relatively strong capture effect instead of a direct sterilization effect on escherichia coli and staphylococcus aureus, and can effectively reduce the selective pressure of bacteria; the protein is almost free of toxicity to mammalian cells, has relatively high resistance under a protease condition test, and has the application potential of becoming an antibiotic substitute.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an enzyme-resistant nano-capture peptide, a preparation method thereof, and an application thereof. Background Art

[0002] The natural supramolecular strategy of human α-defensin 6 (HD6) to capture pathogenic bacteria by forming a nanofiber network can reduce the trend of acquired drug resistance of bacteria, which provides a new strategy for the research and development of a new generation of antibacterial agents. Such peptides are called capture peptides. However, the sensitivity of capture peptides to protease digestion is still an important factor restricting their oral application. Digestive proteases such as pepsin and trypsin may hydrolyze capture peptides into amino acids or short peptides such as dipeptides, thus losing their original biological activity. Therefore, the key point of capture peptide molecule modification is to enhance its ability to resist protease hydrolysis and retain its characteristics of chemical stability and low cytotoxicity to increase its clinical application potential. Summary of the Invention

[0003] Based on the above deficiencies, the purpose of the present invention is to provide an enzyme-resistant nano-capture peptide with a more stable structure, which can avoid the digestion and hydrolysis of digestive proteases in the physiological environment and further improve the application potential of capture peptides.

[0004] The technical solution adopted by the present invention is as follows: An enzyme-resistant nano-capture peptide RF-29, whose amino acid sequence is shown in SEQ ID No.1.

[0005] Another object of the present invention is to provide a preparation method of an enzyme-resistant nano-capture peptide RF-29, and the steps are as follows:

[0006] Step S1: Select phenylalanine F to provide hydrophobic interaction; select arginine R to provide positive charge to meet the basic conditions of antibacterial peptides; the polypeptide structure adopts a surfactant-like mode to promote the self-assembly of the whole molecule; at the same time, select proline P to avoid protease hydrolysis;

[0007] Step S2: Use GSGS as a flexible Linker to connect with the 123-131 active region: LPEHAIVQF of the natural peptide Rv2626c of Mycobacterium tuberculosis to construct a polypeptide whose amino acid sequence is shown in SEQ ID No.1;

[0008] Step S3: Use solid-phase chemical synthesis method and mass spectrometry identification to prepare the polypeptide, and then measure the nano-morphology, in vitro cytotoxicity, protease stability and the ability to capture bacteria of the polypeptide, and finally name it enzyme-resistant nano-capture peptide RF-29.

[0009] Furthermore, for the self-assembly method of the anti-proteolytic nano-capture peptide RF-29 as described above, the self-assembly conditions are as follows: the concentration is 2 - 512 μM, and it is incubated at 37 °C for 24 hours.

[0010] Another object of the present invention is to provide the use of the anti-proteolytic nano-capture peptide RF-29 as described above in the preparation of drugs for treating infectious diseases caused by Escherichia coli or / and Staphylococcus aureus.

[0011] Furthermore, the anti-proteolytic nano-capture peptide RF-29 as described above can capture Escherichia coli and Staphylococcus aureus.

[0012] The present invention has the following advantages and beneficial effects: The anti-proteolytic nano-capture peptide RF-29 of the present invention has strong biological activity and significant effect in resisting proteases; by measuring the bacterial agglutination ability, cytotoxicity, and protease stability of the prepared nano-antibacterial peptide, it is found that the anti-proteolytic nano-capture peptide RF-29 has a strong capturing effect on Escherichia coli and Staphylococcus aureus rather than a direct bactericidal effect, has almost no toxicity to mammalian cells, and has strong resistance under protease condition tests, and has the application potential to become an alternative to antibiotics. Description of the Drawings

[0013] Figure 1 It is the high-performance liquid chromatography diagram of the anti-proteolytic nano-capture peptide RF-29;

[0014] Figure 2 It is the mass spectrometry diagram of the anti-proteolytic nano-capture peptide RF-29;

[0015] Figure 3 It is the scanning electron microscope nano-characterization diagram of the anti-proteolytic nano-capture peptide RF-29; (a) 2 μM; (b) 512 μM;

[0016] Figure 4 It is the cytotoxicity determination diagram of the anti-proteolytic nano-capture peptide RF-29;

[0017] Figure 5 It is the diagram for determining the ability of the anti-proteolytic nano-capture peptide RF-29 to capture Escherichia coli; (a) SYTO9 staining of the control group; (b) SYTO9 staining of the RF-29 group; (c) SYTO9 staining of the melittin group; (d) PI staining of the control group; (e) PI staining of the RF-29 group; (f) PI staining of the melittin group; (g) merged picture of the control group; (h) merged picture of the RF-29 group; (i) merged picture of the melittin group.

[0018] Figure 6Graph for measuring the ability of the anti-proteolytic nano-capture peptide RF-29 to capture Staphylococcus aureus; (a) SYTO9 staining of the control group; (b) SYTO9 staining of the RF-29 group; (c) SYTO9 staining of the melittin group; (d) PI staining of the control group; (e) PI staining of the RF-29 group; (f) PI staining of the melittin group; (g) merged picture of the control group; (h) merged picture of RF-29; (i) merged picture of the melittin group.

[0019] Figure 7 Graph for the protease stability of the anti-proteolytic nano-capture peptide RF-29. Specific implementation manners

[0020] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0021] Embodiment 1

[0022] Design of the anti-proteolytic nano-capture peptide

[0023] Design of the anti-proteolytic nano-capture peptide RF-29: Phenylalanine (F) was selected to provide hydrophobic interaction; Arginine (R) was selected to provide positive charge to meet the basic conditions of the antibacterial peptide; the polypeptide structure adopted a surfactant-like mode to promote the self-assembly of the whole molecule; at the same time, proline (P) was used to protect F and R to avoid their hydrolysis by proteases; GSGS was used as a flexible Linker to connect with the 123-131 active region of the natural peptide Rv2626c of Mycobacterium tuberculosis: LPEHAIVQF, and the amino acid sequence of this peptide is shown in Table 1.

[0024] Table 1 Amino acid sequence of the anti-proteolytic nano-capture peptide RF-29

[0025]

[0026] Embodiment 2

[0027] Synthesis of the capture peptide by solid-phase chemical synthesis method

[0028] a. Resin swelling: The polypeptide synthesis is carried out sequentially from the C-terminus to the N-terminus. Weigh Fmoc-Dab-OH-Wang resin and pour it into the reaction column, soak it for 30 minutes, and then drain it.

[0029] b. Deprotection: After the resin is soaked for 30 minutes, drain the solution, and then deprotect it. Carry out the deprotection reaction with piperidine for 30 minutes.

[0030] c. Weighing: Within 30 minutes of deprotection, calculate the amount of amino acid, condensing agent, and NMM required for each step according to the amount to be made, and then weigh the next amino acid.

[0031] d. Deprotection and washing: After 30 minutes of deprotection, pipidine was removed by suction, and then washed 6 times with DMF. After 6 washes, the deprotection color was detected and recorded.

[0032] e. Feeding: After the deprotection washing and detection, in sequence, the weighed materials were added one by one, then a little reaction solution was added, followed by the addition of base and NMM. The gas was adjusted evenly, and the resin adhered to the inner wall of the reaction column was rinsed off with DCM, and then the reaction time was recorded. The reaction was carried out for 30 minutes.

[0033] f. Washing after reaction: The solution in the reaction column was drained by suction, an appropriate amount of DMF was added for washing, and nitrogen was bubbled for 2 minutes, then drained by suction. This operation was repeated 3 times.

[0034] g. Detection: After 30 minutes of reaction, the reaction solution was removed by suction, washed 3 times with DMF and then detected to check whether the reaction was complete.

[0035] h. Washing and drying after synthesis: For the above polypeptide, it was drained by suction. An appropriate amount of methanol was added to the reaction column, and nitrogen was bubbled for 2 minutes, then drained by suction. Then an appropriate amount of DCM was added, and nitrogen was bubbled for 2 minutes, and drained by suction. This operation was repeated 3 times. Finally, an appropriate amount of methanol was added to the reaction kettle, nitrogen was bubbled for 2 minutes, and drained by suction. This operation was repeated 2 times. The resin was loaded into a suitable container and placed in a vacuum dryer for vacuum drying for 12 hours pending cleavage.

[0036] Cleavage: The dried resin was loaded into a suitable round-bottom flask, an appropriate amount of the prepared cleavage solution (1 g / 10 mL) was added, and it was placed in a constant-temperature shaker and shaken at 25 °C for 2 hours.

[0037] Filtration: The resin particles were filtered off with a 50 mL sintered glass funnel, and then the filtrate was poured into a 100 mL centrifuge tube. 6 - 8 times the volume of anhydrous ether was added while stirring. The precipitated white solid was the crude polypeptide product required.

[0038] Washing: The centrifuge tube was sealed and placed in a centrifuge and centrifuged at a speed of 4000 revolutions per minute for 3 minutes. It was taken out, the supernatant was poured off, and ether was added again. It was stirred evenly with a glass rod and centrifuged again; this operation was repeated 5 times for washing.

[0039] Drying: The polypeptide that had been washed 5 times was placed in a vacuum dryer for vacuum drying for 24 hours. The finally obtained white powder was the crude polypeptide product required. It was weighed and awaited purification.

[0040] Purification: The column was equilibrated with 0.2 mol / L sodium sulfate (phosphate pH = 7.4) for 30 min. The polypeptide was dissolved in 90% aqueous acetonitrile solution, filtered, and added to a normal pressure reverse-phase column. Gradient elution was used (the eluent was a mixture of methanol and aqueous sodium sulfate solution in a volume ratio of 30:70 to 70:30), the flow rate was 1 mL / min, the detection wavelength was 220 nm, the main peak was collected, and freeze-dried; then further purified using a reverse-phase C18 column. Eluent A was 0.1% TFA / aqueous solution; eluent B was 0.1% TFA / acetonitrile solution, the flow rate was 1 mL / min, and the main peak was collected as above and freeze-dried;

[0041] Identification: The captured peptide obtained above was analyzed by electrospray mass spectrometry (as Figure 1 shown), and the purity of the captured peptide was greater than 95% (as Figure 2 shown).

[0042] Example 3

[0043] Scanning electron microscopy nano-characterization map of the anti-proteolytic nano-captured peptide RF-29:

[0044] Nano-morphology analysis: To further analyze the nano-morphology of the anti-proteolytic nano-captured peptide RF-29, the peptide (1.28 mM) was diluted to 512 μM and 2 μM concentrations in deionized water and incubated in a 37 °C incubator for 24 hours. The samples were smeared on glass slides and coated with metal using a Polaron SC7640 sputter coater after natural drying. The samples were observed by a Hitachi S-4800 SEM (Hitachi, Japan) at 5 kV, and the detection results are shown in Figure 3 .

[0045] It can be seen from Figure 3 (a) that the 2 μM anti-proteolytic nano-captured peptide RF-29 formed nano-particles with larger sizes and loose structures; it can be seen from Figure 3 (b) that the 512 μM anti-proteolytic nano-captured peptide RF-29 formed nano-particles with smaller sizes and compact structures.

[0046] Example 4

[0047] In vitro cytotoxicity assay of the anti-proteolytic nano-captured peptide RF-29: After the cells cryopreserved in liquid nitrogen were revived, they were inoculated into a medium containing 10% fetal bovine serum and 1% double antibody and subcultured under the conditions of 37 °C and 5% CO 2 . The cultured cells were digested with 0.25% trypsin and adjusted to 2 - 4×10 5 cells / mL with the medium. 50 μL of the cell suspension was mixed with 50 μL of polypeptides at different concentrations in a 96-well plate and incubated at 37 °C and 5% CO 2Incubate for 24 h under the conditions, then add 25 μL of MTT (5 mg / mL) to each well and continue to incubate for 4 h. After the incubation, discard the supernatant, dissolve the crystals at the bottom of the wells with 100 μL of DMSO, and measure the absorbance value of each well at 570 nm using an enzyme-linked immunosorbent assay reader. The medium well serves as a blank control. The test results are shown in Figure 4 .

[0048] From Figure 4 It can be seen that even after treatment with the high-concentration anti-proteolytic nanocapture peptide RF-29, the cell viability of porcine jejunal epithelial cells (IPEC-J2) and mouse peritoneal macrophages (RAW 264.7) still remains at 80%, indicating that the anti-proteolytic nanocapture peptide RF-29 has good biocompatibility and the potential to become an alternative to antibiotics.

[0049] Example 5

[0050] Determination of the ability of the anti-proteolytic nanocapture peptide RF-29 to capture bacteria: To evaluate the ability of the anti-proteolytic nanocapture peptide RF-29 to capture pathogenic bacteria, the aggregation effect of the anti-proteolytic nanocapture peptide RF-29-mediated bacterial aggregates was observed using a super-high-resolution fluorescence microscope. Prepare Escherichia coli particles containing green fluorescent protein, centrifuge the Escherichia coli cells at 3000 rpm for 5 min, and resuspend them in PBS buffer. Add the peptide solution and melittin at a final concentration of 32 μM, co-culture with the bacterial suspension at 37 °C for 2 h, then add the PI dye at a final concentration of 10 μg / mL, incubate at 4 °C for 15 min, and centrifuge to wash away the free PI. After resuspending the sample, coat it on a glass slide, air-dry, cover it with a coverslip, and seal it with nail polish. Observe the sample using a super-high-resolution fluorescence microscope under excitation wavelengths of 488 nm and 535 nm. Untreated cells serve as a negative control.

[0051] From Figure 5 and Figure 6 (a-i), it can be seen that different from the direct bactericidal effect of melittin, the 32 μM anti-proteolytic nanocapture peptide RF-29 has an obvious capture effect on Escherichia coli E. coli 25922 and Staphylococcus aureus S. aureus 29213, and has no direct killing effect on bacteria. This antibacterial mechanism of action can minimize the selective pressure exerted on pathogenic bacteria and reduce the possibility of pathogenic bacteria developing drug resistance.

[0052] Example 6

[0053] Determination of the protease stability of the anti-proteolytic nanocapture peptide RF-29: To detect the protease resistance ability of the peptide, artificial simulated gastric / intestinal fluid, 10 mg / mL of pepsin, trypsin, and chymotrypsin were mixed with the peptide (2.56 mM) in equal volumes and incubated at 37 °C for different times. The peptide untreated with protease was used as a control, and then the Tricine-SDS-PAGE protein gel was used to reflect the banding situation.

[0054] It can be seen that Figure 7 after treatment with proteases, the anti-proteolytic nanocapture peptide RF-29 showed protein bands similar to those in the control group, further demonstrating that the anti-proteolytic nanocapture peptide RF-29 has strong resistance to multiple proteases.

Claims

1. An enzymatically resistant nano-capture peptide RF-29, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The method for preparing the enzymatically resistant nano-capture peptide RF-29 according to claim 1, characterized in that: The steps are as follows: Step S1: selecting phenylalanine F to provide hydrophobic interaction, selecting arginine R to provide positive charge, and selecting proline P to avoid protease hydrolysis; Step S2: using GSGS as a flexible linker to connect with the 123-131 active region of the natural peptide Rv2626c of Mycobacterium tuberculosis: LPEHAIVQF, to construct a polypeptide with an amino acid sequence as shown in SEQ ID No. 1; Step S3: The peptide was prepared by solid phase chemical synthesis and mass spectrometry, and then the nano-morphology of the peptide, in vitro cytotoxicity, protease stability and bacterial capture ability were measured. Finally, it was named as the enzymatically resistant nano-capture peptide RF-29.

3. The self-assembly method of the enzymatically resistant nano-capture peptide RF-29 according to claim 1, characterized in that: The self-assembly conditions are as follows: the concentration is 2-512 μM, and incubation at 37° C. for 24 hours enables the self-assembly into a nanostructure.

4. Use of the enzymatically resistant nano-capture peptide RF-29 according to claim 1 in the preparation of drugs for treating infectious diseases caused by Escherichia coli and / or Staphylococcus aureus.

5. The use according to claim 4, characterized in that: The enzymatically resistant nano-capture peptide RF-29 can capture Escherichia coli and Staphylococcus aureus.

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