CN116731150B - HD6 bionic nano capture peptide and preparation method and application thereof - 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

HD6 bionic nano capture peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an HD6 bionic nano capture peptide, a preparation method and application thereof.

Background

Antibacterial peptides (Antimicrobial Peptides, AMPs), also known as host defensin peptides, are a class of small molecule polypeptides synthesized by ribosomes that are highly conserved during the evolution of the body's innate immune system. When the host is against external infection, the antibacterial peptide is precisely released at the infection site, and pathogenic bacteria are killed through interaction with low-affinity targets such as cell membranes. The unique mechanism of action and multiple biological functions of the antibacterial peptide make the antibacterial peptide the most potential competitor to replace traditional antibiotics.

Human alpha-defensin 6 (HD 6) is a small short peptide consisting of 32 amino acid residues with three pairs of conserved disulfide bonds (Cys 1-Cys6, cys2-Cys4, cys3-Cys 5) in its structure forming a stable three-ply antiparallel beta-sheet domain. HD6 itself lacks broad-spectrum antimicrobial activity similar to other human α -defensins, and captures the pathogenic bacteria mainly by forming a nanofiber web, thereby preventing the pathogenic bacteria from invading host epithelium and then spreading to other organs, and the "surrounding but not attacking" natural supermolecular strategy can reduce the selective pressure applied to the pathogenic bacteria to the greatest extent, and reduce the possibility of drug resistance of the pathogenic bacteria, which provides a brand new strategy for the development of new generation antimicrobial agents. However, the specificity of HD6 source and the complexity of its sequence structure cannot be applied to large-scale preparation of HD6 by natural extraction or chemical synthesis.

Disclosure of Invention

Based on the background technology, the invention aims to provide the HD6 bionic nano capture peptide RFQF ₄ -hLF _28-34 The peptide can form a nano-network structure, identify and capture escherichia coli, promote phagocytosis of macrophages on cell clusters, and effectively reduce the possibility of drug resistance of the escherichia coli.

The technical scheme adopted by the invention is as follows: HD6 bionic nano capture peptide RFQF ₄ -hLF _28-34 The amino acid sequence is shown as SEQ ID No. 1.

Further, the HD6 bionic nano capture peptide RFQF is as described above ₄ -hLF _28-34 Nano self-organization thereofThe mounting conditions are as follows: the concentration was 8-256. Mu.M and incubated at 37℃for 24 hours.

Furthermore, it is capable of capturing E.coli after self-assembly into nanostructures.

Another object of the present invention is to provide an HD6 bionic nano-capture peptide RFQF ₄ -hLF _28-34 The preparation method of (2) is as follows:

(1) Alternately arranging arginine R with positive charges and glutamine Q with uncharged polarity, selecting phenylalanine F as hydrophobic amino acid to obtain a structural unit with a sequence of RFQF, repeating the structural unit for 4 times to be used as a self-assembly to form a cationic peptide fiber skeleton, connecting GSGS as a flexible joint with a 28-34 active recognition region RKVRGPP of lactoferrin to construct a polypeptide sequence structure, wherein the amino acid sequence is shown as SEQ ID No. 1;

(2) Obtaining peptide resin by a solid-phase chemical synthesis method through a polypeptide synthesizer, and cutting the obtained peptide resin by TFA to obtain polypeptide;

(3) After reversed-phase high performance liquid chromatography purification and mass spectrum identification, the preparation of the polypeptide is completed, the nano morphological characterization, biocompatibility, capture antibacterial capacity and macrophage phagocytic capacity of the polypeptide are measured, and finally the polypeptide is named as HD6 bionic nano capture peptide RFQF4-hLF _28-34 。

It is another object of the present invention to provide the nano-capture peptide RFQF as described above ₄ -hLF _28-34 The application of the composition in preparing medicines for treating escherichia coli infectious diseases.

The invention has the beneficial effects and advantages that: the HD6 bionic nano capture peptide RFQF of the invention ₄ -hLF _28-34 Can form a compact nanofiber web in a physiological environment, has higher biocompatibility, has lower average bactericidal activity (64 mu M) on tested salmonella typhimurium, staphylococcus aureus and staphylococcus epidermidis, has antibacterial capability on escherichia coli, has stronger capturing capability on escherichia coli, can effectively capture escherichia coli at 16 mu M, and promotes phagocytic capability of macrophages on bacterial clusters, and can effectively reduce bacteria by virtue of an antibacterial agent mode of an innate immune systemSelective pressure reduces the occurrence of drug resistance. To sum up, RFQF ₄ -hLF _28-34 Is a HD6 bionic nano capture peptide with great development potential and higher application value.

Drawings

FIG. 1 shows RFQF ₄ -hLF _28-34 Mass spectrum of (3);

FIG. 2 is a RFQF ₄ -hLF _28-34 Is a chromatogram of (2);

FIG. 3 is an RFQF ₄ -hLF _28-34 Is a critical aggregation concentration plot of (2);

FIG. 4 is a RFQF ₄ -hLF _28-34 Wherein (a) the concentration is 16 μm and (b) the concentration is 64 μm;

FIG. 5 is an RFQF ₄ -hLF _28-34 A graph of hemolytic activity;

FIG. 6 is an RFQF ₄ -hLF _28-34 A cytotoxicity assay;

FIG. 7 is an RFQF ₄ -hLF _28-34 A graph of killing activity against common pathogens, wherein (a) e.coll 25922, (b) e.coll K88, (c) s.tyrphinium 7731, (d) s.tyrphinium 14028, (e) s.aureus29213, (f) s.epididis 12228;

FIG. 8 is an RFQF ₄ -hLF _28-34 A sedimentation capture bacterial capacity profile, wherein (a) e.coll 25922, (b) e.coll 25922 data statistics profile, (c) e.coll K88, (d) e.coll K88 data statistics profile, (e) s.aureus29213, (f) s.aureus29213 data statistics profile;

FIG. 9 is an RFQF ₄ -hLF _28-34 A plot of promotion of phagocytosis by macrophages, coll ATCC 25922, wherein (a) a plot of colony count is observed and (b) a plot of colony count is determined;

FIG. 10 shows the observation of RFQF by ultra-high resolution fluorescence microscope ₄ -hLF _28-34 Promoting phagocytosis of E.coli by macrophages, wherein (a) control DAPI staining, (b) control EGFP observation, (c) control combination and, (d) RFQF ₄ -hLF _28-34 DAPI staining of treatment group, (e) RFQF ₄ -hLF _28-34 Treatment group EGFP observations, (f) RFQF ₄ -hLF _28-34 Processing combination and combining;

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1

RFQF ₄ -hLF _28-34 Is designed according to the following steps: arg and Gln are selected as charged core and polar uncharged residue, two amino acids are alternately arranged, phe is selected as hydrophobic amino acid in the middle, and an amino acid sequence template (Arg Phe Gln Phe) for forming a nanofiber scaffold is obtained _n Named RFQF when n=4 ₄ . The 28-34 active recognition region RKVGPP of lactoferrin was then ligated via linker GSGS, the amino acid sequence of this peptide is shown in Table 1.

TABLE 1HD6 bionic nano-capture peptide RFQF ₄ -hLF _28-34 Amino acid sequence of (2)

Example 2

Synthesis of HD6 bionic nano capture peptide RFQF by solid phase chemical synthesis method ₄ -hLF _28-34

1. The preparation of the antibacterial peptide is carried out from the C end to the N end one by one and is completed by a polypeptide synthesizer. Firstly, fmoc-X (X is the first amino acid at the C end of each antibacterial peptide) is inoculated into Wang resin, and Fmoc groups are removed to obtain X-Wang resin; fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y being the second amino acid at the C-terminus of each antimicrobial peptide); sequentially synthesizing from the C end to the N end until the synthesis is completed, and obtaining the side chain protected resin from which Fmoc groups are removed;

2. adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; washing with precipitated TFA (trifluoroacetic acid), mixing the washing solution with the filtrate, concentrating by a rotary evaporator, adding pre-cooled anhydrous ether with volume of about 10 times, precipitating at-20 ℃ for 3 hours, separating out white powder, centrifuging for 10 minutes at 2500g, collecting the precipitate, washing with anhydrous ether, drying in vacuum to obtain polypeptide, and mixing TFA, water and TIS (triisopropylchlorosilane) according to a mass ratio of 95:2.5:2.5 to obtain a cutting reagent;

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

4. performing column equilibrium for 30min by using 0.2moL/L sodium sulfate (the pH value is regulated to be 7.5), dissolving polypeptide by using 90% acetonitrile water solution, filtering, performing reverse phase normal pressure column C18, performing gradient elution (the eluent is methanol and sodium sulfate water solution are mixed according to the volume ratio of 30:70-70:30), performing flow rate of 1mL/min, detecting wave of 220nm, collecting main peak, and performing freeze drying; further purification using a reverse phase C18 column, eluent a was 0.1% TFA/water; eluting with 0.1% TFA/acetonitrile solution, eluting with 25% -40% B, eluting for 12min at flow rate of 1mL/min, collecting main peak, and lyophilizing;

5. identification of the polypeptide: the obtained polypeptide is analyzed by electrospray mass spectrometry, the molecular weight (shown in the attached drawing) in the mass spectrum is basically consistent with the theoretical molecular weight in the table 1, and the purity of the polypeptide is more than 95%.

Example 3

RFQF ₄ -hLF _28-34 Nano-characterization assay of (c):

1. critical aggregation concentration determination: to detect RFQF ₄ -hLF _28-34 The ability to form nanostructures, critical Aggregation Concentration (CAC) was determined using a 1-aniline-8-naphthalene sulfonic Acid (ANS) fluorescent probe. mu.L of ANS (final concentration 1mM, dissolved in 100% DMF) was added to various concentrations of polypeptide (dissolved in deionized water) and incubated at 37℃for 15min. Transferring the mixed sample to a 96-well plate, and performing fluorescence spectrum scanning by using a fluorescence enzyme-labeled instrument, wherein the excitation wavelength is 369nm, and the emission wavelength is 440-550 nm. The CAC value of the polypeptide was then calculated using Origin software. The detection result is shown in FIG. 3.

As can be seen from FIG. 3, RFQF increases with increasing concentration ₄ -hLF _28-34 Fluorescence intensity within 440nm-550nmThe degree gradually rises, indicating that nano macromolecules exist in the solution, preliminarily judging the formation of the nano structure, and then using Origin software to fit and analyze RFQF ₄ -hLF _28-34 The CAC value of (C) was 8.84. Mu.M.

2. Nanotopography analysis: to further analyze RFQF ₄ -hLF _28-34 The peptides (1.28 mM) were diluted to 16, 64 and 256. Mu.M concentrations in deionized water and incubated for 24 hours in a 37℃incubator. Samples were deposited on carbon coated mesh and observed by negative staining with 2% uranyl acetate at 100KV for 15 seconds using Hitachi H-7800TEM (Hitachi, japan). The detection results are shown in FIG. 4.

As can be seen from FIG. 4 (a), RFQF ₄ -hLF _28-34 Forming a fine and narrow fiber sample at 16 μm with a diameter of 7.78nm; as can be seen from FIG. 4 (b), the diameter and length of the nanofibers increased to a concentration of 64. Mu.M, forming a dense network structure with a diameter of 9.50nm.

Example 4

RFQF ₄ -hLF _28-34 In vitro hemolytic, cytotoxic and bactericidal activity assays:

1. hemolytic activity assay: human fresh blood 1mL was collected in heparin sodium anticoagulation tube, red blood cells were collected by centrifugation at 1000g for 5min, washed 3 times with PBS, resuspended in 10mL PBS, peptides of different concentrations were added to 96-well plates containing 50 μl PBS, followed by addition of equal volumes of red blood cell suspension. hRBC suspension treated with 0.1% Triton X-100 was used as positive control and untreated hRBC suspension was used as negative control. Incubating for 1h at constant temperature in a 37 ℃ incubator, taking out, and centrifuging at 4 ℃ for 5min at 1000 g; the supernatant was taken out and the absorbance was measured at 570nm with a microplate reader to give a concentration causing 5% hemolysis as the minimum hemolysis concentration. The haemolysis rate was 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 (FIG. 5) show RFQF ₄ -hLF _28-34 Has no damage to blood cells, and has minimum hemolysis concentration of>128μM。

2. Cytotoxicity assay: resuscitating cells frozen in liquid nitrogen, inoculating into culture medium containing 10% foetal calf serum and 1% double antibody, and inoculating into culture medium containing 5% CO at 37deg.C ₂ Subculturing under the condition. The cultured cells were digested with 0.25% pancreatin and adjusted to 2 to 4X 10 with medium ⁵ cells/mL. 50. Mu.L of the cell suspension was mixed with 50. Mu.L of the polypeptide at different concentrations in 96-well plates at 37℃with 5% CO ₂ Incubation was carried out for 24h, followed by addition of 25. Mu.L MTT (5 mg/mL) per well and incubation was continued for 4h. After the incubation, the supernatant was discarded, the bottom crystals were dissolved in 100. Mu.L of DMSO, and the absorbance value per well was measured at 570nm using an ELISA reader. Medium wells served as blank. The detection results are shown in FIG. 6.

As can be seen from fig. 6, RFQF ₄ -hLF _28-34 No obvious toxicity to RAW 264.7 and less toxicity to HEK 293T, which indicates RFQF ₄ -hLF _28-34 Has good biocompatibility and potential of becoming an antibiotic substitute.

3. Determination of bactericidal activity: the minimum inhibitory concentration of the antimicrobial peptide was determined by microdilution. Peptides of different concentrations were added to a 0.2% BSA dilution (containing 0.01% acetic acid) in 96-well plates, followed by an equal volume of final concentration of 1X 10 ⁵ CFUmL ^-1 The final peptide concentration in 96-well plates ranged from 0.25 to 128 μm. After incubation at 37℃for 3 hours, 50. Mu.L of the peptide was aspirated and diluted in PBS, and the concentration of the peptide which killed 99.99% of the bacteria was determined as the minimum bactericidal concentration by plate counting. The results of the detection are shown in FIG. 7 and Table 2.

TABLE 2RFQF ₄ -hLF _28-34 Bactericidal activity, MHC and TI values of (C)

¹ Minimum hemolysis concentration>At 128. Mu.M, the selectivity index was calculated with 256. Mu.M

² TI＝MHC/GM _MBC

From FIGS. 7 (a-f) and Table 2 and as can be seen, RFQF ₄ -hLF _28-34 The bactericidal composition has stronger bactericidal activity on escherichia coli, but has lower bactericidal activity on other test bacteria.

Example 5

RFQF ₄ -hLF _28-34 Measurement of Capture bacteria:

to evaluate RFQF ₄ -hLF _28-34 Ability to capture pathogenic bacteria, OD _{600 nm} 3mL of E.coli 25922 bacterial liquid with the concentration of 0.4 is placed in a cuvette sterilized by ethanol, and 8-256 mu M RFQF is added ₄ -hLF _28-34 The peptide solution was left to stand at room temperature for 6 hours, the bacterial aggregation state was observed and photographed, and the group without peptide treatment was used as a control group. 50. Mu.L of bacterial supernatant was aspirated at 0, 0.5, 1,2, 4, 6h, respectively, and then added to 450. Mu.L of autoclaved PBS for gradient dilution, 100. Mu.L of the dilution was inoculated on MHA plate medium. The colonies of each sample were counted after overnight incubation in an incubator at 37℃and CFU value was calculated for each sample, and the test was repeated three times, and the detection results are shown in FIG. 8.

As can be seen from FIGS. 8 (a-f), RFQF ₄ -hLF _28-34 Has obvious capturing sedimentation effect on E.coli 25922 and E.coli K88 and has concentration and time dependence, 256 mu M RFQF ₄ -hLF _28-34 E.coli was allowed to settle completely after 2 hours of treatment. RFQF (radio frequency quad Flat No-lead) ₄ -hLF _28-34 Has weaker sedimentation effect on staphylococcus aureus S.aureus29213 and 256 mu M RFQF ₄ -hLF _28-34 The staphylococcus aureus could not be completely settled after 6 hours of treatment.

Example 6

Macrophage pair RFQF ₄ -hLF _28-34 Phagocytosis analysis of captured bacterial pellet:

1. gentamicin protection assay: to evaluate RFQF ₄ -hLF _28-34 Whether the phagocytosis of the bacterial groups by the macrophages is promoted or not, and the bacterial content in the macrophages is detected through a gentamicin protection test. Collecting E.coli 25922 bacterial liquid in logarithmic phase, centrifuging at 3000rpm for 5min, collecting bacterial body, washing with PBS buffer for 3 times, suspending in PBS buffer, and regulating bacterial concentration to OD _600nm =0.4. Add 16. Mu. MRFQF ₄ -hLF _28-34 Co-culture with bacterial suspension at 37 ℃ for 2 hours. The peptide-pretreated bacterial particles were added to the cell culture broth of RAW 264.7, incubated at 37℃for 2 hours, the cell culture broth was aspirated and washed 3 times, extracellular bacteria were removed by adding a gentamicin solution containing 50. Mu.g/mL, lysis was performed for 10 minutes using 0.1% Triton X-100, 50. Mu.L of the lysate was aspirated, and 450. Mu.L of high-temperature sterilized PBS was added for gradient dilution, and 100. Mu.L of the dilution was uniformly inoculated on MHA plate medium. The colonies were Counted (CFU) for each sample by overnight incubation in an incubator at 37℃and the test was repeated three times, and the detection results are shown in FIG. 9.

As can be seen from FIGS. 9 (a-b), RFQF was compared with the control group ₄ -hLF _28-34 After pretreatment of bacteria, phagocytosis of bacterial clusters by RAW 264.7 macrophages is significantly promoted.

2. Ultra-high resolution fluorescent microscope observation: to further explore macrophage versus RFQF ₄ -hLF _28-34 Mediate phagocytosis of bacterial groups and observe phagocytosis of E.coli by RAW 264.7 using ultra-high resolution fluorescence microscopy. E.coli particles containing green fluorescent protein were prepared, and E.coli cells were collected by centrifugation at 3000rpm for 5min and resuspended in PBS buffer. A final concentration of 16. Mu.M of peptide was added and incubated with the bacterial suspension at 37℃for 2h. The peptide-pretreated bacterial pellet was added to the cell culture broth of RAW 264.7, incubated at 37℃for 2h, the cell culture broth was aspirated and washed 3 times, triton X-100 was added for 10min, triton X-100 was aspirated and washed 3 times, and the anti-quencher containing DAPI was added. Observed with a Deltavision OMX SR fluorescence microscope. The detection results are shown in FIG. 10.

The results are shown in FIGS. 10 (a-f), compared with the control, via RFQF ₄ -hLF _28-34 The treated E.coli particles aggregate and the aggregated E.coli particles increase the internalization of RAW 264.7. This illustrates RFQF ₄ -hLF _28-34 Can capture Escherichia coli and promote uptake thereof by phagocytes.

Claims (4)

1. HD6 bionic nano capture peptide RFQF ₄ -hLF _28-34 The method is characterized in thatThe amino acid sequence is shown as SEQ ID No. 1.

2. The HD6 bionic nano-capture peptide RFQF according to claim 1 ₄ -hLF _28-34 The self-assembly method of (2) is characterized in that: the nano self-assembly condition is as follows: the concentration is 8-256 mu M, and the escherichia coli can be captured after the escherichia coli is self-assembled into a nano-structure after incubation for 24 hours at 37 ℃.

3. The HD6 bionic nano-capture peptide RFQF4-hLF according to claim 1 _28-34 The preparation method is characterized by comprising the following steps:

(1) Alternately arranging arginine R with positive charges and glutamine Q with uncharged polarity, selecting phenylalanine F as hydrophobic amino acid to obtain a structural unit with a sequence of RFQF, repeating the structural unit for 4 times to be used as a self-assembly to form a cationic peptide fiber skeleton, connecting GSGS as a flexible joint with a 28-34 active recognition region RKVRGPP of lactoferrin to construct a polypeptide sequence structure, wherein the amino acid sequence is shown as SEQ ID No. 1;

(2) Obtaining peptide resin by a solid-phase chemical synthesis method through a polypeptide synthesizer, and cutting the obtained peptide resin by TFA to obtain polypeptide;

(3) After reversed-phase high performance liquid chromatography purification and mass spectrum identification, the preparation of the polypeptide is completed, the nano morphological characterization, biocompatibility, capture antibacterial capacity and macrophage phagocytic capacity of the polypeptide are measured, and finally the polypeptide is named as HD6 bionic nano capture peptide RFQF4-hLF _28-34 。

4. The HD6 bionic nano-capture peptide RFQF4-hLF according to claim 1 _28-34 The application of the composition in preparing medicines for treating escherichia coli infectious diseases.