A pH-responsive nanoantibacterial peptide with immunomodulatory function, its preparation method and application

Abstract

The present invention provides a pH-responsive nanoantibacterial peptide with immunomodulatory function, its preparation method and application, belonging to the field of biotechnology. The antibacterial peptide sequence is as shown in SEQ ID No.1, and its N-terminus is acetylated with 2-naphthylbutyric acid. This antibacterial peptide can spontaneously assemble into a nano-network structure, and has a good killing effect on pathogenic bacteria, especially Gram-negative bacteria, in an acidic environment with a pH of 5.5, and has good biocompatibility. Although it does not have antibacterial activity at pH 7.4, it can capture pathogenic bacteria, but can promote the phagocytosis of cell aggregates by macrophages, effectively reducing the possibility of bacteria developing drug resistance. In summary, the present invention is a pH-responsive nanoantibacterial peptide with immunomodulatory function that has great development potential and high application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a pH-responsive nanoantimicrobial peptide with immunomodulatory function, its preparation method and application. Background Art

[0002] Antimicrobial peptides (AMPs), also known as host defense peptides, are important components of the body's innate immune system. Due to their membrane-breaking bactericidal mechanism that is not easily induced bacteria to produce drug resistance, they are considered the most promising alternatives to antibiotics. Although antimicrobial peptides show incomparable superiority, they still face challenges in clinical applications and livestock production. On the one hand, traditional antimicrobial peptides usually kill beneficial bacteria in the host due to their broad-spectrum antibacterial activity, and also have a killing effect on normal microbial flora in the intestine, thus disrupting the balance of the intestinal microecological environment and inducing serious secondary infections. On the other hand, although bacteria are not easily resistant to antimicrobial peptides, the drug resistance of antimicrobial peptides themselves cannot be ignored. Therefore, mobilizing the body's immune function to jointly resist bacterial infection is an effective strategy. Since bacteria metabolize to produce various acids, the microenvironment at the lesion site is weakly acidic, which easily causes changes in the structure of antimicrobial peptides and thus affects their antibacterial activity. Therefore, the new design or molecular modification of existing natural antimicrobial peptides to have pH-responsive targeted antimicrobial peptides is of great significance for solving the drug resistance problem and finding alternatives to antibiotics. Summary of the Invention

[0003] Based on the above problems in the background art, the purpose of the present invention is to provide a pH-responsive self-assembled nanoantimicrobial peptide with immunomodulatory function. This antimicrobial peptide can spontaneously assemble into a nano-network structure, has a good killing effect on pathogenic bacteria, especially Gram-negative bacteria, in an acidic environment with a pH of 5.5, and has good biocompatibility. Although it does not have antibacterial activity at a pH of 7.4, it can capture pathogenic bacteria and promote the phagocytosis of cell clusters by macrophages, effectively reducing the possibility of bacteria developing drug resistance.

[0004] The technical solution adopted by the present invention is as follows: A pH-responsive nanoantimicrobial peptide with immunomodulatory function, whose amino acid sequence is as shown in SEQ ID No.1, and its N-terminus is acetylated with 2-naphthylbutyric acid.

[0005] Further, for the pH-responsive nanoantimicrobial peptide with immunomodulatory function as described above, its molecular formula is as shown in formula (Ⅰ):

[0006]

[0007]

[0008] Another object of the present invention is to provide a method for preparing a pH-responsive nanoantibacterial peptide with immunomodulatory function as described above, the steps are as follows:

[0009] S1: Alternately arrange polar uncharged phenylalanine and histidine with imidazole group to obtain the sequence structural unit of FH and repeat it 6 times to construct a β-sheet secondary structure with an alternating hydrophilic and hydrophobic pattern. At physiological pH, the neutral imidazole group can act as both a hydrogen bond donor and a hydrogen bond acceptor, which can promote the self-assembly of peptide chain molecules into supramolecular nanostructures through hydrogen bond interactions with hydrophobic amino acid phenylalanine, forming a stable nanofiber structure; under weak acid pH conditions, the imidazole group undergoes protonation of H, causing an increase in positive charge. The positively charged polypeptide recognizes and accumulates on the negatively charged bacterial membrane surface, exerting antibacterial activity and forming a stable nanofiber structure; Use GGG as a flexible linker to connect with the 5-13 active recognition region VKTLTGKTI of ubiquitin to construct a polypeptide with the sequence shown in SEQ ID No.1, and then acetylate its N-terminus with 2-naphthylbutyric acid to provide hydrophobic force as a hydrophobic scaffold;

[0010] S2: Synthesize by solid-phase chemical synthesis method, and then complete the preparation of the polypeptide after chromatographic purification and mass spectrometry identification;

[0011] S3: Then perform nano-morphology characterization measurement, biocompatibility measurement, capture ability measurement, antibacterial ability measurement and macrophage phagocytosis promotion ability measurement on the polypeptide, and finally name it antibacterial peptide Nap-FH 6 -Ubi 5-13 。

[0012] Further, the self-assembly conditions of a pH-responsive nanoantibacterial peptide with immunomodulatory function as described above are as follows: pH = 5.5, concentration 1-256 μM, incubation at 37 °C for 24 hours or pH = 7.4, concentration 9-256 μM, incubation at 37 °C for 24 hours.

[0013] Further, a pH-responsive nanoantibacterial peptide with immunomodulatory function as described above can kill Escherichia coli or Salmonella typhimurium in an acidic environment with a pH of 5.5 and can capture Escherichia coli in an environment with a pH of 7.4.

[0014] Another object of the present invention is to provide the application of a pH-responsive nanoantibacterial peptide with immunomodulatory function as described above in the preparation of drugs for treating and / or preventing infectious diseases caused by Gram-negative bacteria.

[0015] Further, in the application as described above, the Gram-negative bacteria are Escherichia coli or Salmonella typhimurium.

[0016] The present invention also provides a drug suitable for treating and / or preventing Gram-positive bacterial infections. The Gram-negative bacteria are Escherichia coli or Salmonella typhimurium, and the drug contains a pH-responsive nanoantibacterial peptide with immunomodulatory function as described above.

[0017] Beneficial effects and advantages of the present invention: The antibacterial peptide Nap-FH 6 -Ubi 5-13 can spontaneously assemble into a nano-network structure, has a good killing effect on Gram-negative bacteria in an acidic environment with a pH of 5.5, and has good biocompatibility. Although it does not have antibacterial activity at pH 7.4, it can capture pathogenic bacteria and promote the phagocytosis of cell aggregates by macrophages. This can both capture bacteria under neutral conditions and trigger the innate immune system to achieve antibacterial purposes, and can also disrupt the cell membrane and kill bacteria in a weakly acidic environment, fully considering the pathological acidic environment and avoiding the overuse of antibacterial peptides, effectively reducing the generation of drug resistance. Detecting the antibacterial activity and biocompatibility of the antibacterial peptide Nap-FH 6 -Ubi 5-13 found that the antibacterial peptide Nap-FH 6 -Ubi 5-13 has an obvious inhibitory effect on Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium in a weakly acidic environment, and has no obvious effect on Gram-positive bacteria such as Staphylococcus aureus. The antibacterial peptide Nap-FH 6 -Ubi 5-13 did not show hemolysis when measured at the maximum test concentration under acidic and neutral conditions. In summary, the antibacterial peptide Nap-FH 6 -Ubi 5-13 is a pH-responsive self-assembling antibacterial peptide with high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the mass spectrum of the antibacterial peptide Nap-FH 6 -Ubi 5-13 ;

[0019] Figure 2 is the chromatogram of the antibacterial peptide Nap-FH 6 -Ubi 5-13 ;

[0020] Figure 3 is the critical aggregation concentration of the antibacterial peptide Nap-FH 6 -Ubi 5-13 and the linear fitting value graph of the critical aggregation concentration;

[0021] Figure 4 is the antibacterial peptide Nap-FH 6 -Ubi 5-13Determination diagram of hemolytic activity;

[0022] Figure 5 For the antimicrobial peptide Nap-FH 6 -Ubi 5-13 Determination diagram of cytotoxicity; (a) Cytotoxicity determination on IPEC J2 cells, (b) Cytotoxicity determination on RAW 264.7 cells;

[0023] Figure 6 For the antimicrobial peptide Nap-FH 6 -Ubi 5-13 Diagram of killing activity against common pathogenic bacteria; (a) E.coli 25922, (b) E.coli K88, (c) S.typhimurium 7731, (d) S.typhimurium 14028, (e) S.aureus 43300, (f) S.aureus 29213;

[0024] Figure 7 For the antimicrobial peptide Nap-FH 6 -Ubi 5-13 Determination of the ability to sediment and capture E.coli ATCC 25922; (a) Bacterial sedimentation diagram, (b) Colony count determination diagram;

[0025] Figure 8 For the antimicrobial peptide Nap-FH 6 -Ubi 5-13 Promote macrophage phagocytosis of E.coli 25922; Diagram

[0026] Figure 9 For the antimicrobial peptide Nap-FH 6 -Ubi 5-13 Diagram of promoting macrophage phagocytosis of Escherichia coli, where (a) Control group Bright, (b) Control group EGFP staining, (c) Control group DAPI observation merged, (d) Control group merged, (e) Nap-FH 6 -Ubi 5-13 Treatment group Bright, (f) Nap-FH 6 -Ubi 5-13 Treatment group EGFP staining, (g) Nap-FH 6 -Ubi 5-13 Treatment group DAPI observation merged, (h) Nap-FH 6 -Ubi 5-13 Treatment group merged. Specific implementation manners

[0027] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0028] Example 1

[0029] Antibacterial peptide Nap-FH 6 -Ubi 5-13 Design

[0030] Phenylalanine with polar neutral charge and histidine with imidazole group are arranged alternately to obtain the sequence structural unit of FH, which is repeated 6 times to construct a β-sheet secondary structure with an amphiphilic alternating pattern. At physiological pH, the neutral imidazole group can act as both a hydrogen bond donor and a hydrogen bond acceptor, promoting the self-assembly of peptide chain molecules into supramolecular nanostructures through hydrogen bond interactions with hydrophobic amino acid phenylalanine, forming a stable nanofiber structure; under weak acidic pH conditions, the imidazole group undergoes protonation of H, causing an increase in positive charge. The positively charged polypeptide recognizes and accumulates on the negatively charged bacterial membrane surface to exert antibacterial activity; GGG is selected as a flexible linker to connect with the 5-13 active recognition region VKTLTGKTI of ubiquitin, and the polypeptide sequence is constructed as shown in SEQ ID No.1. Then, the N-terminus is acetylated with 2-naphthylbutyric acid Nap as a hydrophobic scaffold to provide hydrophobic interaction, and finally, a nano-antibacterial peptide with immunomodulatory function is constructed.

[0031] The amino acid sequence of the antibacterial peptide is as follows:

[0032] Nap-FHFHFHFHFHFHGGGVKTLTGKTI

[0033] Table 1 Amino acid sequence

[0034]

[0035] The molecular structural formula is shown in formula (I):

[0036]

[0037] Example 2

[0038] Synthesize the nano-antibacterial peptide Nap-FH 6 -Ubi 5-13

[0039] 1. The preparation of the antibacterial peptide is carried out step by step from the C-terminus to the N-terminus and completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminus of each antibacterial peptide) is attached to Wang resin, and the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trityl-Y, Y is the second amino acid at the C-terminus of each antibacterial peptide); and so on, synthesizing from the C-terminus to the N-terminus in sequence until the synthesis is completed to obtain the resin with side-chain protection of the Fmoc group removed.

[0040] 2. In the obtained peptide resin, a cleavage reagent is added, and the reaction is carried out for 2 hours at 20 °C in the dark, 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, about 10 times the volume of precooled anhydrous ether is added, and precipitation is carried out at -20 °C for 3 h to precipitate a white powder, centrifuged at 2500 g for 10 min, the precipitate is collected, washed with anhydrous ether again, and dried in vacuo to obtain the polypeptide. The cleavage reagent is composed of TFA, water and TIS (triisopropylchlorosilane) mixed according to a mass ratio of 95:2.5:2.5;

[0041] 3. Fmoc-S5-OH (1 mmol), HATU (1 mmol), HOAT (1 mmol), DIPEA (1 mmol) are mixed in DMF (6 mL) for 15 min, and then added to the resin at room temperature. After 2 hours, the resin is washed successively with DMF (3 times), DCM (3X, 5 mL) and DMF (3X, 5 mL). The ring-closing metathesis reaction is carried out in 1,2-dichloroethane (DCE) at 35 °C using the first-generation Grubbs catalyst. The resin is washed with DCM (3X, 5 mL) and DCE (3X, 5 mL), and then treated with a solution of the first-generation Grubbs catalyst in DCE at 10 mM;

[0042] 4. Column equilibration is carried out for 30 min using 0.2 moL / L sodium sulfate (adjusted to pH = 7.5 with phosphoric acid), the polypeptide is dissolved in 90% aqueous acetonitrile solution, filtered, and passed through a C18 reversed-phase normal-pressure column. Gradient elution is adopted (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 to 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;

[0043] 5. Identification of the polypeptide: The obtained polypeptide is analyzed by electrospray mass spectrometry, and the molecular weight shown in the mass spectrum (see attachment Figure 1 ) is basically consistent with the theoretical molecular weight in Table 1, and the purity of the polypeptide is greater than 95%.

[0044] Example 3

[0045] Antibacterial peptide Nap-FH 6 -Ubi 5-13 Nanoscale characterization determination:

[0046] Critical aggregation concentration determination: In order to detect the antibacterial peptide Nap-FH 6 -Ubi 5-13The ability to form nanostructures was determined using the 1-anilino-8-naphthalenesulfonic acid (ANS) fluorescence probe. 1 μL of ANS (final concentration 1 mM, dissolved in 100% DMF) was added to different concentrations of the polypeptide (dissolved in deionized water at different pH values), and incubated at 37 °C for 15 min. The mixed samples were transferred to a 96-well plate, and fluorescence spectra were scanned using a fluorescence microplate reader with an excitation wavelength of 369 nm and an emission wavelength range of 440 nm - 550 nm. Subsequently, the CAC value of the polypeptide was calculated using Origin software. The detection results are shown in Figure 3 .

[0047] As can be seen from Figure 3 (a, c), as the concentration increased, the fluorescence intensity of the antimicrobial peptide Nap-FH 6 -Ubi 5-13 gradually increased within the range of 440 nm - 550 nm, indicating the presence of nanomacromolecules in the solution, and initially determining the formation of nanostructures. Subsequently, fitting analysis was performed using Origin software, as shown in Figure 3 (b, d), the CAC value of Nap-FH 6 -Ubi 5-13 was 0.94 μM at pH = 5.5 and 8.34 μM at pH = 7.4.

[0048] Example 4

[0049] Determination of in vitro hemolytic activity, cytotoxicity and bactericidal activity of the antimicrobial peptide Nap-FH 6 -Ubi 5-13 :

[0050] 1. Determination of hemolytic activity: 1 mL of fresh human blood was collected into a heparin sodium anticoagulant tube, centrifuged at 1000 g for 5 min to collect red blood cells, washed 3 times with PBS and then resuspended in 10 mL of PBS. Different concentrations of the peptide were added to a 96-well plate containing 50 μL of PBS, and then an equal volume of the red blood cell suspension was added. The hRBC suspension treated with 0.1% Triton X-100 was used as the positive control, and the untreated hRBC suspension was used as the negative control. After incubation at a constant temperature in a 37 °C incubator for 1 h, it was taken out, centrifuged at 4 °C and 1000 g for 5 min; the supernatant was taken out and the absorbance was measured at 570 nm using a microplate reader, and the concentration causing 5% hemolysis was taken as the minimum hemolytic concentration.

[0051] The hemolysis results ( Figure 4 ) showed that the antimicrobial peptide Nap-FH 6 -Ubi 5-13 had no hemolytic toxicity to human red blood cells, whether in an acidic environment or a neutral environment.

[0052] 2. Cytotoxicity assay: The cells cryopreserved in liquid nitrogen were thawed and seeded in a medium containing 10% fetal bovine serum and 1% double antibiotics, 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 for 24 h under the conditions of 37 °C and 5% CO 2 . Then, 25 μL of MTT (5 mg / mL) was added to each well and incubation continued for 4 h. After incubation, the supernatant was discarded, the crystals at the bottom of the wells were dissolved with 100 μL of DMSO, and the absorbance value of each well was measured at 570 nm with an enzyme-linked immunosorbent assay reader. The medium well was used as a blank control. The test results are shown in Figure 5 .

[0053] It can be seen from Figure 5 that whether in an acidic environment or a neutral environment, the antimicrobial peptide Nap-FH 6 -Ubi 5-13 showed no significant toxicity to IPEC J2 and RAW 264.7 cells, indicating that the antimicrobial peptide Nap-FH 6 -Ubi 5-13 has good biocompatibility and the potential to become an alternative to antibiotics.

[0054] 3. Antibacterial activity assay:

[0055] To evaluate the antibacterial effect of the antimicrobial peptide Nap-FH 6 -Ubi 5-13 under weak acid conditions, the minimum inhibitory concentration of the antimicrobial peptide was determined by the microdilution method. Different concentrations of the peptide were added to a 0.2% BSA dilution (adjusted to pH = 5.5 with acetic acid) in a 96-well plate, and then an equal volume of a bacterial suspension with a final concentration of 1×10 5 CFU / mL -1 was added. The final peptide concentration range in the 96-well plate was 0.25 to 128 μM. Incubate at 37 °C for 3 hours, aspirate 50 μL, dilute it in PBS gradient, and calculate the number of colonies after treatment with Nap-FH 6 -Ubi 5-13 by the viable cell counting method. The test results are shown in Figure 6 .

[0056] It can be seen from Figure 6 that under weak acidic conditions, the antimicrobial peptide Nap-FH 6 -Ubi 5-13 showed strong antibacterial activity against Gram-negative bacteria, but low antibacterial activity against Gram-positive bacteria.

[0057] Example 5

[0058] Antibacterial peptide Nap-FH 6 -Ubi 5-13 Determination of the ability to capture bacteria:

[0059] To evaluate the ability of antibacterial peptide Nap-FH 6 -Ubi 5-13 to capture pathogenic bacteria under physiological conditions, 3 mL of E. coli 25922 bacterial solution with an OD 600 nm = 0.4 was placed in a colorimetric cuvette sterilized with ethanol, and 16 - 256 μM of Nap-FH6-Ubi 5-13 peptide solution was added. It was left standing at room temperature for 6 h, the bacterial agglutination state was observed and photographed, and the group without peptide treatment was used as the control group. At 0, 0.5, 1, 2, 4, and 6 h respectively, 50 μL of the bacterial supernatant was aspirated, added to 450 μL of high-temperature sterilized PBS for gradient dilution, and 100 μL of the diluted solution was evenly inoculated on MHA plate medium. It was cultured overnight in a 37 °C incubator, the number of colonies of each sample was counted, the CFU value of each sample was calculated, and the test was repeated three times. The test results are shown in Figure 7 .

[0060] It can be seen from Figure 7 that under neutral conditions, the antibacterial peptide Nap-FH 6 -Ubi 5-13 has an obvious capture and sedimentation effect on Escherichia coli E. coli 25922, and has concentration and time dependence. 256 μM of the antibacterial peptide Nap-FH 6 -Ubi 5-13 can completely sediment Escherichia coli after 1 hour of treatment.

[0061] Example 6

[0062] Antibacterial peptide Nap-FH 6 -Ubi 5-13 Determination of immunomodulatory ability:

[0063] 1. Gentamicin protection test: To evaluate whether the antibacterial peptide Nap-FH 6 -Ubi 5-13 under physiological conditions will promote the phagocytosis of bacteria by macrophages, the bacterial content in macrophages was detected by the gentamicin protection test. Take the E. coli 25922 bacterial solution in the logarithmic growth phase, centrifuge at 3000 rpm for 5 minutes to collect the bacteria, wash them 3 times with PBS buffer, resuspend them in PBS buffer, and adjust the bacterial concentration to OD 600nm = 0.4. Add 16 μM and 32 μM of Nap-FH 6 -Ubi 5-13Co-cultured with the bacterial suspension at 37 °C for 2 hours. The peptide-pretreated bacterial particles were added to the cell culture medium of RAW 264.7, cultured at 37 °C for 2 hours, the cell culture medium was aspirated and washed 3 times, a gentamicin solution containing 50 μg / mL was added to remove extracellular bacteria, lysed with 0.1% Triton X-100 for 10 minutes, 50 μL of the lysate was aspirated, added to 450 μL of high-temperature sterilized PBS for gradient dilution, and 100 μL of the diluted solution was evenly inoculated on MHA plate medium. Incubated overnight in a 37 °C incubator, the colony count (CFU) of each sample was calculated, and the experiment was repeated three times. The test results are shown in Figure 8 。

[0064] From Figure 8 it can be seen that compared with the control group, the antibacterial peptides Nap-FH 6 -Ubi 5-13 significantly increased the internalization of Escherichia coli particles in RAW 264.7 after pretreatment, and showed a dose-dependent manner (the significance in the figure is that the larger value is a). This indicates that the antibacterial peptide Nap-FH 6 -Ubi 5-13 can capture bacteria and further enhance the phagocytic ability of phagocytes.

[0065] 2. Macrophage phagocytosis of bacteria assay: To visually observe the promotion of macrophage phagocytosis of bacterial aggregates by the antibacterial peptide Nap-FH 6 -Ubi 5-13 we observed the phagocytosis of Escherichia coli by RAW 264.7 using a fluorescence inverted microscope. Prepare Escherichia coli particles containing green fluorescent protein, centrifuge the Escherichia coli cells at 3000 rpm for 5 min, resuspend them in PBS buffer. Add a peptide solution with a final concentration of 16 μM, co-culture with the bacterial suspension at 37 °C for 2 h. The peptide-pretreated bacterial particles were added to the cell culture medium of RAW 264.7, cultured at 37 °C for 2 h, the cell culture medium was aspirated and washed 3 times, fixed with Triton X-100 for 10 min, the Triton X-100 was aspirated and washed 3 times, and an anti-quencher containing DAPI was added. Observed with a fluorescence inverted microscope. The test results are shown in Figure 9 。

[0066] The results are as Figure 9 shown. Compared with the control, the Escherichia coli particles treated with the antibacterial peptide Nap-FH 6 -Ubi 5-13 aggregated, and the phagocytosis of the aggregated Escherichia coli particles by RAW 264.7 macrophages was enhanced. This indicates that the nano-capture peptide can capture bacteria and further promote the uptake of phagocytes.

[0067] Therefore, the antimicrobial peptide Nap-FH of the present invention 6 -Ubi 5-13 can capture bacteria under neutral conditions and trigger the innate immune system to achieve the antibacterial purpose, and can also disrupt the cell membrane and kill bacteria in a weakly acidic environment. It fully takes into account the pathological acidic environment and at the same time avoids the overuse of antimicrobial peptides, effectively reducing the generation of drug resistance and mobilizing the body's immune function to jointly resist bacterial infections.

Claims

1. A pH-responsive nano antimicrobial peptide with immunomodulatory function, characterized in that: Its amino acid sequence is shown in SEQ ID No. 1, its N-terminus is acylated by 2-naphthoic acid, and its molecular formula is shown in formula (I):

2. The self-assembly method of a pH-responsive nano antimicrobial peptide with immunomodulatory function according to claim 1, characterized in that: The self-assembly conditions were as follows: pH = 5.5, concentration 1-256 μM, incubation at 37° C. for 24 hours or pH = 7.4, concentration 9-256 μM, incubation at 37° C. for 24 hours.

3. The method for preparing a pH-responsive nano antimicrobial peptide with immunomodulatory function according to claim 1, characterized in that: The steps are as follows: S1: Alternately arrange polar uncharged phenylalanine and histidine with imidazole groups to obtain the sequence structural unit of FH and repeat it 6 times to construct a β-folded secondary structure with a hydrophilic-hydrophobic alternating pattern, select GGG as a flexible linker to connect with the 5th to 13th active recognition region VKTLTGKTI of ubiquitin, and construct the sequence of the polypeptide as shown in SEQ ID No. 1, and then acylate its N-terminus with 2-naphthoic acid to provide hydrophobic interaction as a hydrophobic scaffold; S2: Synthesize by solid phase chemical synthesis, and then complete the preparation of the peptide after chromatography purification and mass spectrometry identification; S3: The peptide was then subjected to nanomorphological characterization, biocompatibility, capture ability, antibacterial ability, and macrophage phagocytosis ability tests, and was finally named the antimicrobial peptide Nap-FH6-Ubi 5-13 .

4. Use of a pH-responsive nano antimicrobial peptide with immunomodulatory function according to claim 1 in the preparation of a drug for treating and / or preventing diseases caused by Gram-negative bacteria, wherein the Gram-negative bacteria are Escherichia coli or Salmonella typhimurium.

5. A drug for treating and / or preventing Gram-negative bacteria infection, wherein the Gram-negative bacteria are Escherichia coli or Salmonella typhimurium, characterized in that: The medicine contains the pH-responsive nano antimicrobial peptide with immunomodulatory function as described in claim 1.

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