CN116731150B - An HD6 biomimetic nanocapture peptide and its preparation method and application - Google Patents

Abstract

The invention provides an HD6 bionic nano capture peptide, a preparation method and application thereof, wherein the amino acid sequence of the HD6 bionic nano capture peptide is shown as SEQ ID No. 1; the preparation method comprises the following steps: alternately arranging arginine with positive charges and glutamine with uncharged polarity, continuously arranging nonpolar residual phenylalanine to obtain a sequence structural unit of RFQF, and repeating the structural unit for 4 times to be used as a self-assembly to form the cationic peptide fiber skeleton. GSGS is used as a flexible joint to be connected with the active recognition region RKVRGPP of lactoferrin 28-34, so as to construct a bionic nano capture peptide sequence structure. The bionic nano-capture peptide can form a compact nano-fiber net in a physiological environment, has strong capture capability on escherichia coli, and promotes phagocytic capability of macrophages on bacterial clusters. In conclusion, the HD6 bionic nano-capture peptide has great development potential and higher application value.

Description

An HD6 biomimetic nanocapture peptide and its preparation method and application

Technical field

The invention belongs to the field of biotechnology, and specifically relates to an HD6 bionic nanocapture peptide and its preparation method and application.

Background technique

Antimicrobial Peptides (AMPs), also called host defense peptides, are a type of small molecule polypeptides synthesized by ribosomes that are highly conserved during the evolution of the body's innate immune system. When the host fights an external infection, antimicrobial peptides are precisely released at the infection site and kill pathogenic bacteria by interacting with low-affinity targets such as cell membranes. The unique mechanism of action of antimicrobial peptides and their multiple biological functions make antimicrobial peptides the most potential contenders to replace traditional antibiotics.

Human α-defensin 6 (HD6) is a small molecule short peptide composed of 32 amino acid residues. Its structure contains three pairs of conserved disulfide bonds (Cys1-Cys6, Cys2-Cys4, Cys3-Cys5), forming a stable Three-stranded antiparallel β-sheet domain. HD6 itself lacks broad-spectrum antibacterial activity similar to other human α-defensins. It mainly forms a nanofiber network to trap pathogenic bacteria, thereby preventing pathogenic bacteria from invading the host epithelium and subsequently spreading to other organs. This kind of "siege but not attack" "The natural supramolecular strategy can minimize the selective pressure exerted on pathogenic bacteria and reduce the possibility of pathogenic bacteria developing resistance. This provides a new strategy for the development of a new generation of antibacterial agents." However, due to the particularity of the source of HD6 and the complexity of its sequence structure, HD6 cannot be prepared and applied on a large scale through natural extraction or chemical synthesis.

Contents of the invention

Based on the above background technical problems, the purpose of the present invention is to provide a HD6 bionic nano-capture peptide RFQF ₄ -hLF _28-34 to effectively solve the application problems of human α-defensin 6 (HD6). The peptide can form a nano-network structure, identify and capture Escherichia coli, and promote the phagocytosis of cell clusters by macrophages, effectively reducing the possibility of Escherichia coli developing drug resistance.

The technical solution adopted by the present invention is as follows: an HD6 bionic nanocapture peptide RFQF ₄ -hLF _28-34 , the amino acid sequence of which is shown in SEQ ID No. 1.

Further, the nano self-assembly conditions of the HD6 biomimetic nanocapture peptide RFQF ₄ -hLF _28-34 as described above are as follows: the concentration is 8-256 μM, and incubated at 37°C for 24 hours.

Furthermore, E. coli can be captured after self-assembly into nanostructures.

Another object of the present invention is to provide a preparation method of HD6 bionic nanocapture peptide RFQF ₄ -hLF _28-34 , as follows:

(1) Alternately arrange the positively charged arginine R and the polar uncharged glutamine Q, and select phenylalanine F as the hydrophobic amino acid in the middle to obtain a structural unit with the sequence RFQF, and repeat the structural unit 4 times as a self-assembled cationic peptide fiber skeleton, using GSGS as a flexible linker to connect to the 28-34 active recognition region RKVRGPP of lactoferrin to construct the sequence structure of the polypeptide, and its amino acid sequence is shown in SEQ ID No. 1;

(2) Use solid-phase chemical synthesis to obtain peptide resin through a peptide synthesizer, and then cleave the obtained peptide resin with TFA to obtain the peptide;

(3) After reversed-phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed, and then the nanomorphological characterization, biocompatibility, capture antibacterial ability and ability to promote macrophage phagocytosis of the polypeptide are carried out. After determination, the polypeptide was finally named HD6 biomimetic nanocapture peptide RFQF4-hLF _28-34 .

Another object of the present invention is to provide the application of the above-mentioned nanocapture peptide RFQF ₄ -hLF _28-34 in the preparation of drugs for treating Escherichia coli infectious diseases.

Beneficial effects and advantages of the present invention: The HD6 bionic nanocapture peptide RFQF ₄ - _{hLF 28-34} of the present invention can form a dense nanofiber network in a physiological environment, has high biocompatibility, and is resistant to the tested Salmonella typhimurium, gold The average bactericidal activity of Staphylococcus aureus and Staphylococcus epidermidis is low (64μM), it has antibacterial ability against Escherichia coli, and has strong capture ability. It can effectively capture Escherichia coli at 16μM and promote macrophages to attack bacterial groups. With the phagocytic ability, this antibacterial substitution mode with the help of the innate immune system can effectively reduce the selective pressure on bacteria and reduce the development of drug resistance. In summary, RFQF ₄ -hLF _28-34 is a HD6 biomimetic nanocapture peptide with great development potential and high application value.

Description of drawings

Figure 1 shows the mass spectrum of RFQF ₄ -hLF _28-34 ;

Fig. 2 is a chromatogram of RFQF ₄ -hLF _28-34 ;

Figure 3 is a critical aggregation concentration diagram of RFQF ₄ -hLF _28-34 ;

Figure 4 shows the nanostructure characterization diagram of RFQF ₄ -hLF _28-34 , where (a) the concentration is 16 μm, (b) the concentration is 64 μm;

Figure 5 is a measurement chart of the hemolytic activity of RFQF ₄ -hLF _28-34 ;

Figure 6 is a measurement chart of RFQF ₄ -hLF _28-34 cytotoxicity;

Figure 7 shows the killing activity of RFQF ₄ -hLF _28-34 against common pathogenic bacteria, including (a) E.coli 25922, (b) E.coli K88, (c) S.typhimurium 7731, (d) S.typhimurium 14028, (e) S. aureus 29213, (f) S. epidermidis 12228;

Figure 8 is a graph showing the ability of RFQF ₄ -hLF _28-34 to capture bacteria by sedimentation, wherein (a) E. coli 25922, (b) E. coli 25922 data statistics graph, (c) E. coli K88, (d) E. coli K88 data statistics graph, (e) S. aureus 29213, (f) S. aureus 29213 data statistics graph;

Figure 9 is a graph showing that RFQF ₄ -hLF _28-34 promotes macrophage phagocytosis of E.coli ATCC 25922, including (a) colony number observation graph, (b) colony number determination graph;

Figure 10 is a super-resolution fluorescence microscope observation of RFQF ₄ -hLF _28-34 promoting macrophage phagocytosis of E.coli, in which (a) DAPI staining in the control group, (b) EGFP observation in the control group, (c) combined control groups, ( d) DAPI staining of RFQF ₄ -hLF _28-34 treatment group, (e) EGFP observation of RFQF ₄ -hLF _28-34 treatment group, (f) RFQF ₄ -hLF _28-34 treatment group combined;

Detailed ways

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.

Example 1

Design of RFQF ₄ -hLF _28-34 : Arg and Gln were selected as the charged core and polar uncharged residues, the two amino acids were alternately arranged, and Phe was selected as the hydrophobic amino acid in the middle to obtain the amino acid sequence template to form the nanofiber scaffold (ArgPhe Gln Phe) _n , when n=4, it is named RFQF ₄ . The 28-34 active recognition region RKVGPP of lactoferrin was then connected through the linker GSGS. The amino acid sequence of this peptide is shown in Table 1.

Table 1 Amino acid sequence of HD6 biomimetic nanocapture peptide RFQF ₄ -hLF _28-34

Example 2

Synthesis of HD6 biomimetic nanocapture peptide RFQF ₄ -hLF _28-34 using solid-phase chemical synthesis method

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 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); synthesize from the C-terminus to the N-terminus in sequence as above until the synthesis is completed, and obtain a resin with the side chain protection of the Fmoc group removed;

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 with anhydrous ether, and vacuum dry to obtain a polypeptide. The cleavage agent is a mixture of TFA, water and TIS (triisopropylchlorosilane) in a mass ratio of 95:2.5:2.5;

3. Fmoc-S5-OH (1mmol), HATU (1mmol), HOAT (1mmol), DIPEA (1mmol) DMF (6mL) were mixed for 15min and then added to the resin at room temperature. After 2 hours, the resin was washed with DMF (3 times), DCM (3X, 5mL) and DMF (3X, 5mL). The ring-closing metathesis reaction was carried out in 1,2-dichloroethane (DCE) at 35°C using Grubbs' first generation catalyst. The resin was washed with DCM (3X, 5mL) and DCE (3X, 5mL) and then treated with a 10mM solution of Grubbs' first generation catalyst in DCE;

4. Use 0.2moL/L sodium sulfate (phosphoric acid adjusted to pH=7.5) for column equilibration for 30 minutes, dissolve the peptide with 90% acetonitrile aqueous solution, filter, use C18 reversed-phase atmospheric pressure column, and use gradient elution (eluent is methanol and The sodium sulfate aqueous solution is mixed according to the volume ratio of 30:70 to 70:30), the flow rate is 1mL/min, and the detection wave is 220nm. Collect the main peak and freeze-dry it; then use a reversed-phase C18 column to further purify, and the eluent A is 0.1% TFA/water solution; eluent B is 0.1% TFA/acetonitrile solution, the elution concentration is 25% B ~ 40% B, the elution time is 12min, the flow rate is 1mL/min, and the main peak is collected as above and lyophilized;

5. Identification of polypeptide: The polypeptide obtained above was analyzed by electrospray mass spectrometry. The molecular weight shown in the mass spectrum (see attached figure) is basically consistent with the theoretical molecular weight in Table 1. The purity of the polypeptide is greater than 95%.

Example 3

Nano-characterization assay of RFQF ₄ -hLF _28-34 :

1. Determination of critical aggregation concentration: In order to detect the ability of RFQF ₄ -hLF _28-34 to form nanostructures, the critical aggregation concentration (CAC) was measured using 1-aniline-8-naphthalenesulfonic acid (ANS) fluorescent probe. Add 1 μL ANS (final concentration: 1 mM, dissolved in 100% DMF) to different concentrations of polypeptides (dissolved in deionized water), and incubate at 37°C for 15 min. Transfer the mixed sample to a 96-well plate, and use a fluorescent microplate reader to scan the fluorescence spectrum. The excitation wavelength is 369nm and the emission wavelength is 440nm-550nm. Origin software was then used to calculate the CAC value of the peptide. The test results are shown in Figure 3.

As can be seen from Figure 3, as the concentration increases, the fluorescence intensity of RFQF ₄ -hLF _28-34 gradually increases within 440nm-550nm, indicating the presence of nanomacromolecules in the solution. The formation of nanostructures was initially determined. Then Origin software was used The CAC value of RFQF ₄ -hLF _28-34 from the fitting analysis is 8.84 μM.

2. Nanomorphology analysis: In order to further analyze the nanomorphology of RFQF ₄ -hLF _28-34 , the peptide (1.28mM) was diluted in deionized water to 16, 64 and 256μM concentrations and incubated in a 37°C incubator for 24 hours. The sample was deposited on a carbon-coated mesh and observed using a Hitachi H-7800TEM (Hitachi, Japan) with 2% uranyl acetate negative staining at 100KV for 15 seconds. The test results are shown in Figure 4.

As can be seen from Figure 4(a), RFQF ₄ -hLF _28-34 formed narrow fibers with a diameter of 7.78 nm at 16 μM; as can be seen from Figure 4(b), when the concentration reached 64 μM, the diameter and length of the nanofibers increased, forming a dense network structure with a diameter of 9.50 nm.

Example 4

RFQF ₄ -hLF _28-34 In vitro hemolytic activity, cytotoxicity and bactericidal activity determination:

1. Hemolytic activity assay: Collect 1 mL of fresh human blood in a sodium heparin anticoagulant tube, centrifuge at 1000 g for 5 min to collect red blood cells, rinse with PBS 3 times and resuspend with 10 mL PBS, add different concentrations of peptides to a 96-well plate containing 50 μL PBS, and then add an equal volume of red blood cell suspension. The hRBC suspension treated with 0.1% Triton X-100 was used as a positive control, and the untreated hRBC suspension was used as a negative control. After incubation at 37°C in an incubator for 1 hour, take out and centrifuge at 4°C and 1000 g for 5 minutes; take out the supernatant and use an enzyme reader to measure the light absorption value at 570 nm, and the concentration that causes 5% hemolysis is the minimum hemolytic concentration. The hemolysis rate is calculated using the following formula:

Hemolysis rate (%) = [(sample OD _{570 nm} - negative control OD _{570 nm} )/(positive control OD _{570 nm} - negative control OD _{570 nm} )] × 100%

The hemolysis results (Figure 5) showed that RFQF ₄ -hLF _28-34 had no damaging effect on blood cells, and the minimum hemolysis concentration was >128 μM.

2. Cytotoxicity measurement: Resuscitate the cells frozen in liquid nitrogen and inoculate them into a culture medium containing 10% fetal calf serum and 1% double antibody, and subculture at 37°C and 5% _CO2 . The cultured cells were digested with 0.25% trypsin, and the culture medium was adjusted to 2 to 4×10 ⁵ cells/mL. Mix 50 μL of cell suspension and 50 μL of polypeptides of different concentrations in a 96-well plate, incubate for 24 h at 37°C and 5% _CO2 , then add 25 μL of MTT (5 mg/mL) to each well and continue incubation for 4 h. After the incubation, discard the supernatant, use 100 μL DMSO to dissolve the crystals at the bottom of the well, and measure the absorbance value of each well at 570 nm with a microplate reader. The medium well served as a blank control. The test results are shown in Figure 6.

As can be seen from Figure 6, RFQF ₄ -hLF _28-34 has no significant toxicity to RAW 264.7 and is less toxic to HEK 293T, indicating that RFQF ₄ -hLF _28-34 has good biocompatibility and has the potential to become an antibiotic substitute. potential.

3. Determination of bactericidal activity: Use microdilution method to determine the minimum inhibitory concentration of antibacterial peptides. Different concentrations of peptides were added to 0.2% BSA dilution (containing 0.01% acetic acid) in a 96-well plate, and then an equal volume of bacterial suspension with a final concentration of 1×10 ⁵ CFUmL ^-1 was added. The final peptide in the 96-well plate Concentration range is 0.25 to 128 μM. After incubation at 37°C for 3 hours, aspirate 50 μL after gradient dilution in PBS, and use the plate counting method to determine the peptide concentration that kills 99.99% of bacteria as the minimum bactericidal concentration. The test results are shown in Figure 7 and Table 2.

Table 2 Bactericidal activity, MHC and TI values of RFQF ₄ - _{hLF 28-34}

^1When the minimum hemolytic concentration is >128μM, use 256μM to calculate the selectivity index

^2TI ＝MHC/ _GMMBC

As can be seen from Figure 7(af) and Table 2, RFQF ₄ - _{hLF 28-34} shows strong bactericidal activity against E. coli, but has low bactericidal activity against other test bacteria.

Example 5

RFQF ₄ - hLF _28-34 determination of bacterial capture ability:

In order to evaluate the ability of RFQF ₄ -hLF _28-34 to capture pathogenic bacteria, 3 mL of E.coli 25922 bacterial solution with OD _{600 nm} = 0.4 was placed in an ethanol-sterilized cuvette, and 8-256 μM of RFQF ₄ -hLF _28-34 was added. Leave the peptide solution at room temperature for 6 hours, observe the bacterial agglutination state and take photos, and use the untreated group as the control group. Take 50 μL of bacterial supernatant at 0, 0.5, 1, 2, 4, and 6 h respectively, add 450 μL of high-temperature sterilized PBS for gradient dilution, and take 100 μL of the dilution and inoculate it evenly on the MHA plate culture medium. Cultivate overnight in a 37°C incubator, count the number of colonies of each sample, and calculate the CFU value of each sample. The test is repeated three times. The test results are shown in Figure 8.

As can be seen from Figure 8(af), RFQF ₄ -hLF _28-34 has an obvious capture and sedimentation effect on both E.coli 25922 and E.coli K88, and is concentration- and time-dependent. 256 μM RFQF ₄ -hLF _28-34 treatment for 2 hours can completely settle the E. coli. RFQF ₄ -hLF _28-34 has a weak effect on the sedimentation of Staphylococcus aureus S. aureus29213. Treatment with 256 μM RFQF ₄ -hLF _28-34 for 6 hours failed to completely sedimentation of Staphylococcus aureus.

Example 6

Analysis of phagocytosis of RFQF ₄ - _{hLF 28-34} captured bacterial pellets by macrophages:

1. Gentamicin protection test: In order to evaluate whether RFQF ₄ - _{hLF 28-34} can promote the phagocytosis of bacterial clumps by macrophages, the bacterial content in macrophages was detected by gentamicin protection test. Take the E.coli 25922 bacterial liquid in the logarithmic growth phase, centrifuge at 3000 rpm for 5 minutes to collect the cells, wash 3 times with PBS buffer, resuspend in PBS buffer, and adjust the bacterial concentration to OD _600nm = 0.4. Add 16 μM RFQF ₄ -hLF _28-34 and co-culture with bacterial suspension at 37°C for 2 hours. Add the peptide-pretreated bacterial particles to the cell culture medium of RAW 264.7 and culture it at 37°C for 2 hours. Aspirate the cell culture medium and wash it 3 times. Add a gentamicin solution containing 50 μg/mL to remove extracellular bacteria. Lyse with 0.1% Triton Cultivate overnight in a 37°C incubator and calculate the number of colonies (CFU) of each sample. The test is repeated three times. The test results are shown in Figure 9.

As can be seen from Figure 9(ab), compared with the control group, RFQF ₄ -hLF _28-34 significantly promoted the phagocytosis of bacterial clumps by RAW264.7 macrophages after pretreating bacteria.

2. Super-resolution fluorescence microscopy observation: In order to further explore the phagocytosis of bacterial clumps mediated by RFQF ₄ -hLF _28-34 by macrophages, super-resolution fluorescence microscopy was used to observe the phagocytosis of E. coli by RAW 264.7. Prepare E. coli particles containing green fluorescent protein, centrifuge at 3000 rpm for 5 minutes to collect E. coli cells, and resuspend them in PBS buffer. Add the peptide solution with a final concentration of 16 μM and co-culture with the bacterial suspension at 37°C for 2 hours. Add the peptide-pretreated bacterial particles to the cell culture medium of RAW 264.7, and incubate at 37°C for 2 hours. Aspirate the cell culture medium and wash it 3 times. Add Triton X-100 to fix it for 10 minutes. Aspirate the Triton X-100 and wash it 3 times. Once again, add an antifade reagent containing DAPI. Observe with a Deltavision OMX SR fluorescence microscope. The test results are shown in Figure 10.

The results are shown in Figure 10(af). Compared with the control, E. coli particles treated with RFQF ₄ -hLF _28-34 aggregated, and the aggregated E. coli particles increased the internalization of RAW 264.7. This shows that RFQF ₄ -hLF _28-34 can capture E. coli and promote its uptake by phagocytes.

Claims (4)

1. A HD6 biomimetic nano-capture peptide RFQF ₄ -hLF _28-34 , characterized in that its amino acid sequence is shown in SEQ ID No.1. 2. The self-assembly method of a kind of HD6 bionic nanocapture peptide RFQF ₄ -hLF _28-34 according to claim 1, characterized in that: its nanoself-assembly conditions are as follows: concentration is 8-256 μM, incubated at 37°C for 24 hours , capable of capturing E. coli after self-assembling into nanostructures. 3. The preparation method of HD6 bionic nanocapture peptide RFQF4-hLF _28-34 according to claim 1, characterized in that the method steps are as follows: (1) Arrange positively charged arginine R and polar uncharged glutamine Q alternately, select phenylalanine F as a hydrophobic amino acid in the middle, obtain a structural unit with a sequence of RFQF, repeat the structural unit 4 times as a self-assembled cationic peptide fiber skeleton, use GSGS as a flexible linker to connect with the 28-34 active recognition region RKVRGPP of lactoferrin, and construct a polypeptide sequence structure, the amino acid sequence of which is shown in SEQ ID No. 1; (2) Use solid-phase chemical synthesis to obtain peptide resin through a peptide synthesizer, and then cleave the obtained peptide resin with TFA to obtain the peptide; (3) After reversed-phase high performance liquid chromatography purification and mass spectrometry identification, the preparation of the polypeptide is completed, and then the nanomorphological characterization, biocompatibility, capture antibacterial ability and ability to promote macrophage phagocytosis of the polypeptide are carried out. After determination, the polypeptide was finally named HD6 biomimetic nanocapture peptide RFQF4-hLF _28-34 . 4. Application of the HD6 bionic nanocapture peptide RFQF4-hLF _28-34 according to claim 1 in the preparation of drugs for treating Escherichia coli infectious diseases.