Antibacterial derived peptide, composition thereof and use thereof

Abstract

The present invention belongs to the field of application of peptide antibiotics. Provided are antibacterial derived peptide LL-37 I20M / V21R, a composition thereof and the use thereof. The prepared antibacterial derived peptide has higher stability and stronger bactericidal effects. The sustained-release microcapsule of the antibacterial derived peptide has the characteristics that the preparation method is simple in operation, the encapsulation rate is up to 87.31%, and the stability is high. The prepared sustained-release microcapsule product of antibacterial peptide LL-37 I20M / V21R can slowly release the antibacterial peptide to exert an antibacterial effect, and has a significant in-vivo reduction effect on Campylobacter jejuni, with a maximum reduction of 4.69 lg (CFU / g).

Description

An antibacterial derivative peptide and its composition and application Technical Field

[0001] The present invention belongs to the application field of peptide antibiotics, and particularly relates to an antibacterial derivative peptide and a composition and application thereof. Background Art

[0002] Campylobacter jejuni is a spiral-shaped, microaerophilic Gram-negative bacterium and a significant foodborne pathogen worldwide. Infection can cause enteritis, fever, arthritis, and even Guillain-Barré syndrome (GBS). The bacterium typically colonizes the intestinal tract of poultry as a commensal. Despite high intestinal colonization rates in animals, it does not cause clinical symptoms. Humans are commonly infected with Campylobacter jejuni through ingestion of contaminated surface water, unpasteurized milk, and undercooked poultry and other meat products. It has been reported that a dose of only 500-800 CFU of Campylobacter jejuni is sufficient to cause human infection. Campylobacteriosis caused by Campylobacter jejuni infection each year imposes a significant medical and socioeconomic burden worldwide.

[0003] It's reported that reducing Campylobacter colonization in poultry by just 2-3 log can reduce the risk of campylobacteriosis in humans by 95%. Clinically, antibiotics are commonly used to treat infections caused by Campylobacter jejuni. However, the overuse of antibiotics has led to a growing problem of bacterial resistance. Therefore, there is an urgent need to develop new prevention, control, and treatment options.

[0004] As the first line of defense of the host immune defense system, antimicrobial peptides have broad-spectrum antibacterial properties and are not prone to drug resistance. They are considered to be ideal candidates for antibiotic alternatives. Among them, LL-37 is a cationic antimicrobial peptide composed of 37 amino acids. It is produced by cleavage of the C-terminus of the 18kDa human hCAP18 protein. It has an α-helical structure and carries 6 positive charges. LL-37 has antibacterial activity against many Gram-positive and Gram-negative bacteria and fungi, and has immunomodulatory effects. It is considered to be a potential alternative to conventional antibiotics. Therefore, the present invention is based on the point mutation modification of LL-37, aiming to further improve its anti-Campylobacter jejuni activity and other properties, and to find a new and effective method for the prevention and control of Campylobacter jejuni.

[0005] Summary of the Invention

[0006] Purpose of the invention: To solve the problems existing in the prior art, the present invention provides a novel antibacterial derivative peptide, the antibacterial derivative peptide LL-37 of the present invention. I20M / V21R It has stronger stability and antibacterial activity in vivo, and is expected to become a new and effective means of preventing and controlling Campylobacter jejuni. It can be used to prepare antibacterial drugs and has potential application value in the prevention and control of foodborne pathogens.

[0007] The present invention also provides an antibacterial derivative peptide LL-37I20M / V21R Preparation method, composition and application thereof.

[0008] Technical solution: In order to achieve the above purpose, the antibacterial derivative peptide LL-37 of the present invention I20M / V21R , its amino acid sequence is: LLGDFFRKSKEKIGKEFKRMRQRIKDFLRNLVPRTES.

[0009] Wherein, the derivative peptide LL-37 I20M / V21R The peptide LL-37 was used as a template for site-directed mutagenesis, and the 20th amino acid in the amino acid sequence was changed to methionine (M) and the 21st amino acid was changed to arginine (R).

[0010] The antibacterial derivative peptide LL-37 of the present invention I20M / V21R The preparation method is to use the amino acid sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES of the polypeptide LL-37 as a template to perform amino acid substitution, replace the 20th amino acid in the amino acid sequence with methionine (M) and the 21st amino acid with arginine (R), and synthesize the antibacterial derivative peptide LL-37 using a solid phase peptide synthesis method. I20M / V21R .

[0011] The antibacterial derivative peptide LL-37 of the present invention I20M / V21R Application in the preparation of drugs or reagents for resisting foodborne pathogens.

[0012] Wherein, the foodborne pathogens are Campylobacter jejuni and its resistant strains.

[0013] Preferably, the foodborne pathogens are Campylobacter jejuni and LL-37-resistant Campylobacter jejuni isolates.

[0014] Wherein, the bacterial derived peptide LL-37 I20M / V21R Application in the preparation of drugs or reagents for resisting foodborne pathogens in mice and chickens.

[0015] The anti-foodborne pathogen pharmaceutical composition of the present invention comprises the antimicrobial derivative peptide LL-37 I20M / V21R and pharmaceutically acceptable carriers thereof.

[0016] Wherein, the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

[0017] The pharmaceutical composition antimicrobial peptide sustained-release microcapsules are prepared by:

[0018] Chitosan and acetic acid were mixed; while stirring, antimicrobial peptide LL-37 I20M / V21RAdd dropwise to the chitosan solution and continue stirring; add trisodium phosphate aqueous solution to the above solution and continue stirring; wash the precipitate after centrifugation and freeze-dry; grind and sieve the dried microcapsules.

[0019] Preferably, the pharmaceutical composition antimicrobial peptide sustained-release microcapsules are prepared by:

[0020] (1) Chitosan (30 mg / mL) and acetic acid (1%, v / v, pH = 5) were dissolved in a magnetic stirrer.

[0021] (2) While stirring, add the antimicrobial peptide LL-37 I20M / V21R It was added dropwise to the chitosan solution at a concentration of 25 μM and stirred at 4 °C for 24 h;

[0022] (3) Trisodium phosphate (TPP) aqueous solution (1 mg / mL, pH = 5) was added to the above solution at a ratio of 1:3 and stirred at room temperature for 2 hours;

[0023] (4) Centrifugation at 14,000 g for 40 min at 4°C, the precipitate was washed several times with ultrapure water, and then freeze-dried for 12 h;

[0024] (5) Grind and sieve the dried microcapsules.

[0025] The invention relates to the use of the anti-foodborne pathogen pharmaceutical composition in the preparation of drugs or reagents for resisting foodborne pathogens.

[0026] The antibacterial derivative peptide LL-37 of the present invention I20M / V21R Application of sustained-release microcapsules in the preparation of drugs or reagents for the in vivo prevention and control of Campylobacter jejuni.

[0027] Antibacterial derivative peptide LL-37 of the present invention I20M / V21R It has the characteristics of high charge number (+7), high stability (increased by 3.18kcal / mol), high antibacterial ability (MIC value is at least 32 times higher than that of natural LL-37), non-toxicity and α-helical structure.

[0028] Antibacterial derivative peptide LL-37 prepared by the preparation method of the present invention I20M / V21R The appearance is spherical and evenly distributed, belonging to micron-sized microcapsules. Antibacterial derivative peptide LL-37 I20M / V21R The microcapsules have the characteristics of high encapsulation rate (87.31%), strong stability and high antibacterial ability.

[0029] Furthermore, the antibacterial derivative peptide LL-37 I20M / V21RThe sustained-release microcapsules showed significant Campylobacter jejuni reduction effects in vivo: in the mouse cecum, the Campylobacter jejuni colonization could be reduced by 2.37 lg (CFU / g); in the chicken intestine, the maximum reduction was 4.69 lg (CFU / g).

[0030] The present invention designs a specific antibacterial derivative peptide LL-37 I20M / V21R , the amino acid sequence of the template peptide polypeptide LL-37 was replaced, showing excellent antibacterial activity against Campylobacter jejuni and good stability.

[0031] LL-37 proposed by the present invention I20M / V21R The derivative peptide is based on the mutation sites of the 20th isoleucine Ile and the 21st valine Val of the LL-37 antimicrobial peptide. I20M / V21R Compared with other mutation combinations such as LL-37 I20Q&V21S (The 20th amino acid in the amino acid sequence of LL-37 was mutated to glutamine Q, and the 21st amino acid was mutated to serine S), the antibacterial effect was significantly reduced, and its MIC value was lower than that of LL-37. I20M / V21R The derivatized peptide was increased 16-fold.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0033] 1. Antibacterial derivative peptide LL-37 prepared by the present invention I20M / V21R It has higher stability and stronger bactericidal effect; the preparation method of antimicrobial peptide sustained-release microcapsules is simple to operate, the encapsulation rate is as high as 87.31%, and the stability is strong; the prepared antimicrobial peptide LL-37 I20M / V21R The sustained-release microcapsule product can slowly release antimicrobial peptides to exert an antibacterial effect, and has a significant effect on the reduction of Campylobacter jejuni in the body, with a maximum reduction of 4.69lg (CFU / g), and is expected to become a new means of prevention and control of Campylobacter jejuni.

[0034] 2. Antimicrobial derivative peptide LL-37 of the present invention I20M / V21R It has antibacterial activity and has little cytotoxic side effects.

[0035] 3. Antibacterial derivative peptide LL-37 of the present invention I20M / V21R The designed preparation method is simple and convenient, the design is novel, the raw materials are easily available, and it can be applied in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1: LL-37 I20M / V21R Structural diagram of derived peptides and LL-37 natural peptide; (A) LL-37 I20M / V21R ; (B) LL-37;

[0037] Figure 2 shows the LL-37 I20M / V21RHPLC and MS detection results of derivatized peptides; (A) HPLC results; (B) MS results;

[0038] Figure 3 shows the LL-37 I20M / V21R Toxicity assay of the derived peptides on Caco-2 cells;

[0039] Figure 4 shows the morphology of microcapsules observed by scanning electron microscopy; (A) LL-37 I20M / V21R ; (B) LL-37;

[0040] Figure 5 shows the pH stability of sustained-release microcapsules;

[0041] Figure 6 is the sustained-release curve of sustained-release microcapsules;

[0042] FIG7 shows the effect of microcapsules prepared with different wall materials on the growth of Campylobacter jejuni P116B;

[0043] Figure 8 shows the sustained-release microcapsules and LL-37 I20M / V21R Comparison of the antibacterial abilities of the derived peptides;

[0044] Figure 9 shows the LL-37 I20M / V21R Toxicity determination of sustained-release microcapsules on Caco-2 cells;

[0045] Figure 10: LL-37 I20M / V21R The reduction of Campylobacter jejuni in the cecal tissue of mice by sustained-release microcapsules;

[0046] Figure 11 shows the average load of Campylobacter jejuni in chicken anal swabs;

[0047] Figure 12 shows the reduction of Campylobacter jejuni in chicken anal swabs by sustained-release microcapsules; Group B is the empty carrier microcapsule group; Group C is the microcapsule group containing 2.75 μg / mL LL-37 I20M / V21R Protein solution group; Group D contained 2.75 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group E contained 5.5 μg / mL of LL-37 I20M / V21R sustained-release microcapsule group; Group F was a sustained-release microcapsule group containing 11 μg / mL LL-37;

[0048] Figure 13 shows continuous feeding of LL-37 I20M / V21R The effect of sustained-release microcapsules on the elimination of Campylobacter jejuni in cecal tissue; Group B is the empty carrier microcapsule group; Group C is the microcapsule group containing 2.75 μg / mL LL-37 I20M / V21R Protein solution group; Group D contained 2.75 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group E contained 5.5 μg / mL of LL-37 I20M / V21R sustained-release microcapsule group; Group F was a sustained-release microcapsule group containing 11 μg / mL LL-37;

[0049] Figure 14 shows continuous feeding of LL-37 I20M / V21R The effect of sustained-release microcapsules on the elimination of Campylobacter jejuni in colon tissue; Group B is the empty carrier microcapsule group; Group C is the microcapsule group containing 2.75 μg / mL LL-37 I20M / V21R Protein solution group; Group D contained 2.75 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group E contained 5.5 μg / mL of LL-37 I20M / V21R sustained-release microcapsule group; Group F was a sustained-release microcapsule group containing 11 μg / mL LL-37;

[0050] Figure 15 shows continuous feeding of LL-37 I20M / V21R The effect of sustained-release microcapsules on the reduction of Campylobacter jejuni in cecal contents; Group B is the empty carrier microcapsule group; Group C is the microcapsule group containing 2.75 μg / mL LL-37 I20M / V21R Protein solution group; Group D contained 2.75 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group E contained 5.5 μg / mL of LL-37 I20M / V21R sustained-release microcapsule group; Group F was a sustained-release microcapsule group containing 11 μg / mL LL-37. DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0052] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0053] The antibacterial derivative peptide LL-37 designed in the present invention I20M / V21R Synthesized directly by a biotechnology company.

[0054] 1. Strains and cells

[0055] C. jejuni 81-176 is a standard strain, and Caco-2 cells and C. jejuni P116B were provided by Yangzhou University. The in vitro and in vivo antibacterial experiments in this invention employed standard strains and C. jejuni P116B. C. jejuni P116B is an LL-37-resistant strain with a MIC > 88 μg / mL. Other LL-37-resistant strains can also be used in this invention, generally with a MIC > 22 μg / mL.

[0056] 2. Main reagents

[0057] Campylobacter Columbia (CCDA) agar medium was purchased from Oxoid, UK; LL-37 I20M / V21RThe derived peptides (purity ≥95%) and human antimicrobial peptide LL-37 (purity ≥95%) were purchased from Hefei Xingpeptide Biotechnology Co., Ltd.; chitosan was purchased from Shanghai Aladdin Company (molecular weight 161.16, product number C105799); tripolyphosphate (product number A501283-0500) was purchased from Shanghai Shenggong Company; micro BCA kit was purchased from Thermo Fisher Scientific, USA; Mueller-Hinton (MH) liquid medium was purchased from BD Biosciences, USA; MTT cytotoxicity detection kit was purchased from Beijing Solarbio Technology Co., Ltd.; trimethoprim, cycloheximide and cefoperazone were purchased from LKT Company, Japan; amphotericin B, polymyxin B and rifampicin were purchased from Wako Company, Japan; other reagents were all domestically produced analytical grade.

[0058] 3. Main instruments and equipment

[0059] Millipore water purifier Milli-Q low-heat prototype was purchased from Millipore, France; microplate reader was purchased from BioTek, USA; desktop small centrifuge MiniSpin, desktop refrigerated centrifuge Centrifuge 5810R, desktop refrigerated centrifuge Centrifuge 5424R and spectrophotometer were purchased from Eppendorf, Germany; anaerobic jar was purchased from MGC, Japan; vacuum freeze dryer was purchased from Shanghai Fu Ruijie; cryo-grinder was purchased from Shanghai Jingxin; CO2 incubator was purchased from Thermo, USA; shaker SHAKE SCS-24 was purchased from Shanghai Centrifugal Machinery Research Institute Co., Ltd.; insulated thermostatic incubator was purchased from Shanghai Yuejin Medical Equipment Factory; constant temperature metal bath CHB-100 was purchased from Hangzhou Biori Technology Co., Ltd.

[0060] 4. Experimental Animals

[0061] Six-week-old C57 / BL6 mice were purchased from the Comparative Medicine Center of Yangzhou University; one-day-old SPF chicks were purchased from Zhejiang Lihua Agricultural Technology Co., Ltd.; and both had animal quarantine certificates.

[0062] Example 1

[0063] Using LL-37 (SEQ ID NO. 2: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) as a template, the 20th amino acid in the amino acid sequence was changed to methionine (M) and the 21st amino acid was changed to arginine (R). The antibacterial derivative peptide LL-37 was synthesized by solid phase peptide synthesis. I20M / V21R .

[0064] Peptide purity determination (HPLC) and mass spectrometry analysis results. Liquid chromatography analysis conditions: C18 column (4.6×250 mm, 5 μm); mobile phase A: acetonitrile containing 0.1% trifluoroacetic acid; mobile phase B: purified water containing 0.1% TFA. Detection wavelength: 220 nm; flow rate: 1.0 mL / min; injection volume: 20 μl; gradient elution.

[0065] Antimicrobial derivative peptide LL-37 I20M / V21R The sequence is: LLGDFFRKSKEKIGKEFKRMRQRIKDFLRNLVPRTES (SEQ ID NO. 1), with a purity greater than 95%. Antibacterial derivative peptide LL-37 I20M / V21R The theoretical molecular weight is: 4.57kDa; antibacterial derivative peptide LL-37 I20M / V21R The AlphaFold2 predicted secondary structure is shown in Figure 1A; its HPLC and MS are shown in Figures 2A and 2B, respectively, which are consistent with the theoretical values.

[0066] Example 2

[0067] Antibacterial derivative peptide LL-37 of the present invention I20M / V21R Structural and biological phenotype analysis and in vitro antibacterial activity determination

[0068] 1. LL-37 I20M / V21R Bioinformatics analysis of derived peptides

[0069] Based on the amino acid sequence of LL-37, the 20th isoleucine (I) was replaced by methionine (M), and the 21st valine (V) was replaced by arginine (A). The sequence was LLGDFFRKSKEKIGKEFKRMRQRIKDFLRNLVPRTES, and it was named LL-37. I20M / V21R The thermal stability changes caused by amino acid mutations were calculated and it was found that LL-37 I20M / V21R The stability of the derived peptide was improved by 3.18 kcal / mol compared to the template LL-37. The net charge, molecular weight, isoelectric point, overall average hydrophobicity (GRAVY), toxicity and half-life were further analyzed. The results are shown in Table 1. Compared with the natural peptide LL-37, LL-37 I20M / V21R The net charge, molecular weight and isoelectric point of the derived peptides increased. The larger the negative value of the average hydrophilicity coefficient (GRAVY), the stronger the hydrophilicity, and the larger the positive value, the stronger the hydrophobicity. LL-37 I20M / V21R The hydrophilicity is slightly enhanced. Through toxicity analysis, it was found that LL-37 I20M / V21R It has no toxicity and the half-life is the same as LL-37. I20M / V21RThe structure of LL-37 was analyzed, as shown in Figure 1. Compared with the structure of LL-37 (Figure 1B), LL-37 I20M / V21R There is an additional coil structure at the N-terminus, but the overall structure still exhibits an α-helical structure (see Figure 1A).

[0070] Table 1 LL-37 I20M / V21R Prediction of biological activity, water solubility and toxicity

[0071] 2. LL-37 I20M / V21R Determination of the antibacterial effect of derived peptides in vitro

[0072] To determine LL-37 I20M / V21R The in vitro antibacterial activity of the derived peptides was determined by the broth microdilution method using Campylobacter jejuni standard strains 81-176 and P116B human isolates according to CLSI standards. I20M / V21R The minimum inhibitory concentration (MIC) of the derived peptide and the natural peptide LL-37 is as follows:

[0073] (1) Resuscitation and subculture of Campylobacter jejuni standard strain 81-176 and human isolate P116B;

[0074] (2) Harvest the cells using MH liquid medium and adjust the OD 600 =0.05, 100 μL was taken and added to 96-well plates; then LL-37 was added I20M / V21R Derivatized peptides and native peptide LL-37 were added to a final concentration of 5.5 μg / mL to 88 μg / mL;

[0075] (3) The 96-well plate was incubated at 37°C under microaerobic conditions (5% O2, 85% N2, 10% CO2) for 24 hours, and the A value of each well was read by a microplate reader. 600 Each experiment was repeated three times, with no LL-37 or bacteria added as a positive control or a negative control, respectively.

[0076] The results showed that the standard strain 81-176 was extremely sensitive to the natural peptide LL-37, while the human isolate P116B was extremely resistant to it, with an MIC value of >88μg / mL. I20M / V21R The antibacterial activity was significantly enhanced, especially for the LL-37 resistant strain P116B, whose MIC value was significantly reduced from >88μg / mL to 2.75μg / mL. I20Q&V21S (LLGDFFRKSKEKIGKEFKRQSQRIKDFLRNLVPRTES) double-point mutation strategy, and LL-37 I20M / V21RIn comparison, its antibacterial effect is poor, especially for LL-37 sensitive strain 81-176, its antibacterial effect is even worse than that of natural peptide LL-37; once again confirming that LL-37 I20M / V21R Advantages of the derivatized peptides (see Table 2).

[0077] Table 2 LL-37 I20M / V21R MIC determination

[0078] 3. LL-37 I20M / V21R Cytotoxicity assay of derived peptides

[0079] Detection of LL-37 by MTT assay I20M / V21R Whether the derived peptide is cytotoxic to Caco-2 cells, the specific steps are as follows:

[0080] (1) Resuscitation and subculture of Caco-2 cells;

[0081] (2) After washing twice with PBS and digesting with trypsin, the cells were harvested with DMEM and counted. The cell concentration was adjusted to 1×10 5 cells / well, cultured overnight at 37°C;

[0082] (3) Antimicrobial peptide LL-37 I20M / V21R Mix with DMEM containing blood and no antibodies; add 100 μL of LL-37 to each well I20M / V21R The final concentrations were 5.5 μg / mL, 11 μg / mL, and 22 μg / mL, respectively. DMEM containing 1% DMSO was used as a control. Four replicate wells were placed in a CO2 incubator at 37°C for each group.

[0083] (4) After 2 h, follow the instructions of the MTT cytotoxicity assay kit, add MTT, and incubate at 37°C in the dark;

[0084] (5) Wash once with PBS, add formazan solvent, dilute it 2 times after the formazan is dissolved, and measure the OD using a microplate reader. 570 , record the data.

[0085] The results showed that 5.5μg / mL, 11μg / mL, and 22μg / mL of LL-37 were selected. I20M / V21R There was no significant change in cell viability when co-incubated with Caco-2 cells. This indicates that LL-37 I20M / V21R There is no obvious cytotoxicity and it can be used for subsequent animal experiments (see Figure 3).

[0086] Example 3

[0087] LL-37I20M / V21R Preparation and characterization of sustained-release microcapsules of derivatized peptides

[0088] 1. Preparation of sustained-release microcapsules and determination of encapsulation rate

[0089] Chitosan and trisodium phosphate (TPP) are used as wall materials, antimicrobial peptides are used as core materials, and sustained-release microcapsules are prepared by ion crosslinking gel method. The main steps include:

[0090] Preparation of empty carrier microcapsules:

[0091] (1) Chitosan (final concentration 30 mg / mL) was dissolved in acetic acid (1% v / v, pH = 5), and trisodium phosphate (TPP) was dissolved in deionized water (final concentration 1 mg / mL, pH = 5);

[0092] (2) The TPP aqueous solution was added dropwise to the chitosan solution at a volume ratio of 1:3, and the mixture was stirred at room temperature for 2 hours;

[0093] (3) centrifugation at 14,000 rpm for 40 min to separate the nanoparticles;

[0094] (4) Washed repeatedly with ultrapure water three times to remove excess chitosan and TPP, and then dried using freeze-drying technology for 12 hours;

[0095] (5) Grind and sieve the dried microcapsules.

[0096] Preparation of antimicrobial peptide sustained-release microcapsules:

[0097] (1) Chitosan (final concentration 30 mg / mL) and acetic acid (1%, v / v, pH = 5) were dissolved in a magnetic stirrer;

[0098] (2) While stirring, add the antimicrobial peptide LL-37 I20M / V21R Alternatively, LL-37 was added dropwise to the chitosan solution at a concentration of 25 μM and stirred at 4 °C for 24 h;

[0099] (3) Trisodium phosphate (TPP) aqueous solution (1 mg / mL, pH = 5) was added to the above solution at a volume ratio of 1:3 and stirred at room temperature for 2 hours;

[0100] (4) Centrifugation at 14,000 g for 40 min at 4°C, the precipitate was washed several times with ultrapure water, and then freeze-dried for 12 h;

[0101] (5) Grind and sieve the dried microcapsules.

[0102] After centrifugation of the prepared antimicrobial peptide sustained-release microcapsules, the micro BCA kit was operated according to the instructions and the amount of free antimicrobial peptides in the supernatant was measured at a wavelength of 565 nm. The encapsulation efficiency (EE) was calculated by the following formula: Encapsulation efficiency (%) = (Total peptides - free peptides) / Total peptides * 100. Wherein, Total peptides represents the total amount of antimicrobial peptides, and Free peptides represents the amount of free antimicrobial peptides in the supernatant. The results are shown in Table 3. LL-37 I20M / V21R The encapsulation rate of microcapsules was 87.31%, and the encapsulation rate of LL-37 microcapsules was 89.38%.

[0103] Table 3 Encapsulation rate of microcapsules

[0104] Scanning electron microscopy observation of the morphology of sustained-release microcapsules

[0105] The prepared LL-37 I20M / V21R The results are shown in Figure 4. The cationic polymer chitosan and the negatively charged sodium tripolyphosphate (TPP) are cross-linked through electrostatic adsorption to wrap the antibacterial body into the wall. I20M / V21R ( Figure 4A ) and LL-37 ( Figure 4B ) microcapsules are spherical and evenly distributed, belonging to micron-sized microcapsules.

[0106] Example 4

[0107] LL-37 I20M / V21R Stability determination of sustained-release microcapsules of derived peptides

[0108] To determine the antimicrobial peptide LL-37 I20M / V21R The pH stability of LL-37 sustained-release microcapsules was studied. According to the pH range in the host body, 3 mg of sustained-release microcapsules were incubated with 2 mL of phosphate buffer solution with pH values ​​of 2, 4, 6, 8, and 10 at room temperature for 30 minutes. The solution was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected. The antimicrobial peptide content in the supernatant was determined according to the instructions of the micro BCA kit, and the retention rate of the antimicrobial peptide in the sustained-release microcapsules was analyzed. The results are shown in Figure 5. When the pH value is within the range of 2 to 10, LL-37 I20M / V21R The peptide contents in the and LL-37 sustained-release microcapsules were relatively stable, with only a slightly lower retention rate at pH = 10 (P>0.05), which were 87.67% and 86.67%, respectively; this showed that the microcapsules with chitosan and TPP cross-linked as wall materials had pH stability, which to a certain extent protected the antimicrobial peptides from premature exposure to the external adverse environment.

[0109] Example 5

[0110] Determination of antibacterial ability of sustained-release microcapsules

[0111] 1. Determination of sustained-release curve of microcapsules

[0112] LL-37 in sustained-release microcapsules I20M / V21R The release kinetics of LL-37 antimicrobial peptide were monitored for 10 days. The specific steps are as follows:

[0113] (1) The prepared sustained-release microcapsules were dispersed in 1 mL of PBS (pH = 7.4) and stirred at 37°C and 120 rpm.

[0114] (2) At specific time points, the samples were centrifuged at 4°C, 14,000 rpm for 30 minutes. The supernatant was collected and the antimicrobial peptide content was determined using a micro BCA kit. Fresh PBS (1 mL) was added and resuspended.

[0115] (3) Statistical analysis of the release percentage of antimicrobial peptides from sustained-release microcapsules.

[0116] The results showed that after 1 day, the sustained-release microcapsules released about 33.39% of LL-37 I20M / V21R Peptide; over time, the outer polymer material begins to degrade, LL-37 I20M / V21R The LL-37 diffused from the core of the sustained-release microcapsule to the external environment, showing a gradual linear release, and was almost completely released after about 6 days; during this period, about 75.87% of LL-37 I20M / V21R The release kinetics of LL-37 sustained-release microcapsules were similar, with approximately 72.80% of the LL-37 antimicrobial peptide released after 6 days (see Figure 6).

[0117] 2. Determination of the antibacterial curve of sustained-release microcapsules

[0118] In order to evaluate the antibacterial effect of the prepared antimicrobial peptide microcapsules, chitosan and TPP were first prepared to encapsulate LL-37 I20M / V21R The specific steps of forming microcapsules are as follows:

[0119] (1) Chitosan single embedding LL-37 I20M / V21R Chitosan (30 mg / mL) and acetic acid (1%, v / v, pH=5) were dissolved in a magnetic stirrer. I20M / V21RThe chitosan solution was added dropwise at a concentration of 25 μM and stirred at 4°C for 24 hours. The solution was centrifuged at 14,000 g for 40 minutes at 4°C. The precipitate was washed with ultrapure water several times and dried by freeze drying for 12 hours. The dried microcapsules were ground and sieved.

[0120] (2) TPP single encapsulation LL-37 I20M / V21R : Dissolve trisodium phosphate (TPP) in aqueous solution (1 mg / mL, pH=5) in a magnetic stirrer; I20M / V21R Alternatively, LL-37 was added dropwise to the TPP solution at a concentration of 25 μM and stirred at 4°C for 24 hours; the solution was centrifuged at 14,000 g for 40 minutes at 4°C, the precipitate was washed multiple times with ultrapure water, and dried for 12 hours using freeze-drying technology; the dried microcapsules were ground and sieved.

[0121] Empty carrier microcapsules, chitosan, and TPP were separately encapsulated with LL-37 I20M / V21R The microcapsules formed, and the chitosan and TPP cross-linked co-encapsulation of LL-37 in Example 2 I20M / V21R The antibacterial ability of the formed microcapsules was tested, and except for the empty microcapsules, the peptide content of each microcapsule was consistent.

[0122] The specific steps are as follows:

[0123] (1) Resuscitation and subculture of Campylobacter jejuni P116B;

[0124] (2) MH liquid harvest and dilute the bacterial solution to OD 600 = 0.1, LL-37 was added to a final concentration of 2.75 μg / mL (calculated as polypeptide) I20M / V21R The sustained-release microcapsules and other microcapsules of equal mass were co-cultured with Campylobacter jejuni P116B at 37°C and 120 rpm in a microaerophilic (5% O2, 85% N2, 10% CO2) environment;

[0125] (3) The plate counts were performed every 2 h; the data were collected and the growth curve was drawn. The experiment was repeated three times.

[0126] The results showed that compared with the control group, empty carrier microcapsules and TPP single-encapsulated LL-37 I20M / V21R The microcapsules formed by the derivative peptides had no significant effect on the growth of Campylobacter jejuni (P>0.05). I20M / V21R The number of bacteria surviving in the microcapsules formed by the derivative peptides decreased by only 0.58 lg (CFU / mL) after 12 h of action; however, the LL-37 was encapsulated by cross-linking chitosan and TPP. I20M / V21RThe resulting sustained-release microcapsules exhibited significantly enhanced antibacterial activity (P<0.05), reducing the number of surviving bacteria by 4.87 lg (CFU / mL) after 12 hours compared to the control group. These experimental results demonstrate that the antibacterial activity of the antimicrobial peptides co-encapsulated with chitosan and TPP is the most significant, and this encapsulation method was selected for subsequent preparation (see Figure 7).

[0127] 3. For the prepared LL-37 I20M / V21R The sustained-release microcapsules were further evaluated for their antibacterial ability. The specific steps are as follows:

[0128] (1) Resuscitation and subculture of Campylobacter jejuni P116B;

[0129] (2) MH liquid harvest and dilute the bacterial solution to OD 600 =0.1, and LL-37 was added to a final concentration of 5.5 μg / mL I20M / V21R Peptide protein (free peptide) and LL-37 with a final concentration of 5.5 μg / mL (peptide) I20M / V21R The sustained-release microcapsules were co-cultured with Campylobacter jejuni P116B at 37°C and 120 rpm in a microaerophilic (5% O2, 85% N2, 10% CO2) environment. At the same time, sustained-release microcapsules containing 11 μg / mL (in terms of polypeptide) of LL-37 were used as a control.

[0130] (3) The plate counts were performed every 2 h; the data were collected and the growth curve was drawn. The experiment was repeated three times.

[0131] The results showed that LL-37 I20M / V21R Compared with protein, LL-37 I20M / V21R The antibacterial ability of the sustained-release microcapsules was significantly enhanced (P < 0.05), suggesting that the sustained-release microcapsules release LL-37 through linear I20M / V21R Antimicrobial peptides, to a certain extent, avoid premature exposure to external adverse environments. At the same time, the antibacterial ability of LL-37 sustained-release microcapsules as the control group was weak, indicating that LL-37 I20M / V21R Sustained-release microcapsules have great application potential (see Figure 8).

[0132] Example 5

[0133] LL-37 I20M / V21R Evaluation of sustained-release microcapsules on the elimination of Campylobacter jejuni in mice

[0134] 1. Determination of cytotoxicity of sustained-release microcapsules

[0135] Targeting LL-37 I20M / V21R The Caco-2 cell toxicity test of the sustained-release microcapsules includes the following steps:

[0136] (1) Resuscitation and subculture of Caco-2 cells;

[0137] (2) After washing twice with PBS and digesting with trypsin, the cells were harvested with DMEM and counted. The cell concentration was adjusted to 1×10 5 cells / well, cultured overnight at 37°C;

[0138] (3) Take LL-37 at a final concentration of 5 μg / mL (calculated as polypeptide) and 11 μg / mL (calculated as polypeptide) respectively I20M / V21R The microcapsules were co-incubated with Caco-2 cells; DMEM containing 1% DMSO was used as a control, with 4 replicate wells in each group, and the cells were cultured in a CO2 incubator at 37°C.

[0139] (4) After 2 h, follow the instructions of the MTT cytotoxicity assay kit, add MTT, and incubate at 37°C in the dark;

[0140] (5) Wash once with PBS, add formazan solvent, dilute it 2 times after the formazan is dissolved, and measure the OD using a microplate reader. 570 , record the data.

[0141] The results showed that, as shown in Figure 9, within the concentration range of 11 μg / mL LL-37 I20M / V21R The sustained-release microcapsules have no obvious cytotoxicity to cells and can be used in subsequent animal experiments.

[0142] 2. LL-37 I20M / V21R Effect of sustained-release microcapsules on the elimination of Campylobacter jejuni in the intestines of mice

[0143] To explore LL-37 I20M / V21R The sustained-release microcapsules can reduce the number of Campylobacter jejuni in the intestine of mice. Six-week-old C57 / BL6 mice were divided into two groups: a PBS (pH 7.4) blank group, an empty carrier microcapsule solution group (PBS, pH 7.4), and a 2.75 μg / mL LL-37 group. I20M / V21R Protein solution group (PBS, pH 7.4), containing 2.75 μg / mL LL-37 peptide I20M / V21R The sustained-release microcapsule solution group (PBS, pH 7.4) and the sustained-release microcapsule solution group containing LL-37 peptide (PBS, pH 7.4) were dissolved in PBS. 30 minutes before the challenge, each mouse was gavaged with sodium bicarbonate solution to neutralize gastric acid. Campylobacter jejuni P116B was inoculated with 1×10 8 According to the colonization status of Campylobacter jejuni in mice, mice in each group were gavaged with 200 μL of PBS, LL-37 containing 2.75 μg / mL peptide, and 4 days after the infection, the mice in each group were gavaged with 200 μL of PBS, LL-37 containing 2.75 μg / mL peptide, and LL-37 containing 2.75 μg / mL peptide.I20M / V21R Protein solution (LL-37 I20M / V21R ), LL-37 containing 2.75 μg / mL peptide I20M / V21R Sustained-release microcapsules (CS-TPP / LL-37 I20M / V21R ), LL-37 sustained-release microcapsules containing 11 μg / mL peptide (CS-TPP / LL-37), and blank carrier microcapsules (CS-TPP). The mice were then killed on the second day, and the bacterial load of Campylobacter jejuni in the cecum was statistically analyzed. The results are shown in Figure 10. In the cecal tissue, the average colonization of the PBS control group was 5.72 lg (CFU / g); in comparison, the LL-37 control group was fed I20M / V21R The protein solution, LL-37 sustained-release microcapsules and blank carrier microcapsules groups reduced the number of Campylobacter jejuni colonization in the mouse cecum by 0.53lg, 0.13lg and 0.33lg respectively. It is worth noting that LL-37 I20M / V21R The sustained-release microcapsule test group significantly decreased by 2.37lg (CFU / g), indicating that LL-37 I20M / V21R Sustained-release microcapsules can significantly reduce the colonization of Campylobacter jejuni in mice.

[0144] Example 6

[0145] LL-37 I20M / V21R Evaluation of sustained-release microcapsules on the elimination of Campylobacter jejuni in chickens

[0146] 1. LL-37 I20M / V21R Effect of sustained-release microcapsules on the elimination of Campylobacter jejuni in chicken feces

[0147] The experiment selected one-day-old chicks and started to challenge Campylobacter jejuni P116B at 5 days old. Each chicken was orally administered 200 μL 1×10 9 CFU / mL bacteria. To determine the bacterial load of the chickens after the challenge, anal swabs were collected regularly to detect the amount of Campylobacter jejuni carried. The average load of Campylobacter jejuni in the anal swabs of the chickens was measured continuously for one week from 5 days of age to 11 days of age. The results are shown in Figure 11. The bacterial load in the anal swabs of the chickens gradually increased after the challenge and stabilized at 9 days of age, reaching 10 8 ~10 9 CFU / g.

[0148] As the bacterial load in chickens tended to be stable, the chickens were divided into groups A and B, which were the control group for Campylobacter jejuni, and the group B was the empty carrier microcapsule solution group (PBS, pH 7.4). The group C was the group containing 2.75 μg / mL LL-37 I20M / V21R Protein solution group, group D is LL-37 containing 2.75μg / mL peptide I20M / V21RSustained-release microcapsule solution group (PBS, pH 7.4), group E containing LL-37 containing 5.5 μg / mL polypeptide I20M / V21R The sustained-release microcapsule solution group (PBS, pH 7.4) and the F group were LL-37 sustained-release microcapsule solution group (PBS, pH 7.4) containing 11 μg / mL peptide. The microcapsules in each group were dissolved in PBS. At 14, 21 and 28 days of age, the rats were fed 200 μL of PBS (Group A), blank carrier microcapsule solution group (Group B), 2.75 μg / mL LL-37 I20M / V21R Protein solution (Group C), LL-37 containing 2.75 μg / mL peptide I20M / V21R Sustained-release microcapsule solution group (Group D), LL-37 containing 5.5 μg / mL peptide I20M / V21R The sustained-release microcapsule solution group (Group E) and the sustained-release microcapsule solution group containing LL-37 (Group F) containing 11 μg / mL peptide were used. After the start of feeding with protein solution and microcapsules, anal swabs were collected regularly at 17, 19, and 24 days of age, and the bacterial load of Campylobacter jejuni was statistically analyzed. The results are shown in Figure 12. I20M / V21R After 3 days of sustained-release microcapsules, the bacterial load of Campylobacter jejuni in chickens showed a significant downward trend in a dose-dependent manner. Compared with the control group, group D (containing 2.75 μg / mL of LL-37 I20M / V21R sustained-release microcapsule group) and group E (containing 5.5 μg / mL LL-37 I20M / V21R The load of Campylobacter jejuni in the anal swab samples of the sustained-release microcapsule group) decreased by 2.06lg and 3.22lg (CFU / g), respectively. At 19 days of age, compared with the control group, the load of Campylobacter jejuni in the anal swab samples of the D and E groups decreased by 3.17lg and 4.17lg (CFU / g), respectively; at 24 days of age, the D and E groups still had a significant reduction effect, with a decrease of 3.17lg and 3.67lg (CFU / g), respectively. At the same time, feeding 2.75μg / mL LL-37 I20M / V21R However, the protein solution in group C had no significant reduction effect (P>0.05), indicating that chitosan microcapsules protected the antimicrobial peptide LL-37 in vivo. I20M / V21R In addition, no significant in vivo reduction effect was observed in group B (empty carrier microcapsule group) and group F (LL-37 sustained-release microcapsule group) as controls (P>0.05).

[0149] 2. LL-37 I20M / V21R Evaluation of the effect of microcapsules on the elimination of Campylobacter jejuni in the intestinal tract of chickens

[0150] To explore LL-37 I20M / V21RThe sustained-release microcapsules had an in vivo reduction effect on Campylobacter jejuni in the intestinal tract of chickens. At 14, 21 and 28 days of age, the chickens were divided into groups according to the method in step 1 and fed with 200 μL of empty carrier microcapsule solution (group B) containing 2.75 μg / mL of LL-37. I20M / V21R Protein solution (Group C), containing 2.75 μg / mL LL-37 peptide I20M / V21R Sustained-release microcapsule solution group (Group D), containing 5.5 μg / mL LL-37 polypeptide I20M / V21R The sustained-release microcapsule solution group (Group E) and the LL-37 (11 μg / mL) polypeptide sustained-release microcapsule solution group (Group F) were used as the control group. On the second day, some chickens were slaughtered and the bacterial load of Campylobacter jejuni in their cecum, colon and intestinal contents was quantitatively determined and calculated. The results showed that the bacterial load in the cecum of the control group was approximately 10 at 14, 21 and 28 days of age, respectively. 9 CFU / g, 10 10 CFU / g and 10 9 CFU / g. At 14 days of age, compared with the control group, the D and E groups decreased by 3.57lg and 3.99lg, respectively (P < 0.001, P < 0.001); at 21 days of age, the D and E groups decreased by 3.68lg and 4.62lg, respectively (P < 0.001, P < 0.001); at 28 days of age, the D and E groups still had a reducing effect, decreasing by 2.83lg and 3.66lg, respectively (P < 0.05, P < 0.001); while the control group fed with 2.75μg / mL LL-37 I20M / V21R The protein solution and LL-37 sustained-release microcapsules did not show significant in vivo depletion effects (see Figure 13).

[0151] In the colon tissue, at 14, 21 and 28 days of age, the colonization amount of the control group was approximately 10 7 CFU / g, 10 8 CFU / g and 10 7 CFU / g. At 14 days of age, compared with the control group, the D and E groups decreased by 2.12lg and 2.96lg, respectively (P < 0.001, P < 0.001); at 21 days of age, the D and E groups decreased by 2.50lg and 3.50lg, respectively (P < 0.01, P < 0.001); at 28 days of age, the D and E groups still had a reducing effect, decreasing by 2.17lg and 3.00lg, respectively (P < 0.001, P < 0.001); while the control group fed with 2.75μg / mL LL-37 I20M / V21R The protein solution and LL-37 sustained-release microcapsules did not show significant in vivo depletion effects (see Figure 14).

[0152] In the intestinal contents, at 14, 21 and 28 days of age, the colonization amount of the control group was approximately 10 10 CFU / g, 10 9 CFU / g and 10 9 CFU / g. At 14 days of age, compared with the control group, the D and E groups decreased by 3.90lg and 4.69lg, respectively (P < 0.001, P < 0.001); at 21 days of age, the D and E groups decreased by 2.62lg and 3.72lg, respectively (P < 0.001, P < 0.001); at 28 days of age, the D and E groups decreased by 3.53lg and 4.30lg, respectively (P < 0.001, P < 0.001); while the control group fed with 2.75μg / mL LL-37 I20M / V21R The protein solution and LL-37 microcapsules still did not show obvious in vivo reduction effect (see Figure 15).

[0153] In summary, the antimicrobial peptide LL-37 provided by the present invention I20M / V21R The antibacterial effect in vitro is significant; the sustained-release microcapsules also have a significant reduction effect in vivo. In the mouse cecum, the colonization of Campylobacter jejuni can be reduced by 2.37 lg (CFU / g); in the chicken intestine, the maximum reduction can be 4.69 lg (CFU / g). The antimicrobial peptide LL-37 provided by the present invention I20M / V21R Sustained-release microcapsules are expected to become a new means of prevention and control of Campylobacter jejuni, with a huge early stage of application, and are of great significance for the prevention and control of Campylobacter jejuni.

Claims

1. An antimicrobial derivative peptide LL-37 I20M / V21R , characterized in that, Its amino acid sequence is: LLGDFFRKSKEKIGKEFKRMRQRIKDFLRNLVPRTES.

2. The derivative peptide LL-37 according to claim 1 I20M / V21R , characterized in that, The derivative peptide LL-37 I20M / V21R The polypeptide LL-37 was used as a template for site-directed mutagenesis, and the 20th amino acid in the amino acid sequence was changed to methionine (M) and the 21st amino acid to arginine (R).

3. An antimicrobial derivative peptide LL-37 according to claim 1 I20M / V21R The preparation method is characterized in that The amino acid sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES of the peptide LL-37 was used as a template for amino acid substitution, the 20th amino acid in the amino acid sequence was replaced with methionine (M), and the 21st amino acid was replaced with arginine (R), and the antibacterial derivative peptide LL-37 was synthesized by solid phase peptide synthesis. I20M / V21R .

4. An antimicrobial derivative peptide LL-37 according to claim 1 I20M / V21R Application in the preparation of drugs or reagents for resisting foodborne pathogens.

5. The use according to claim 4, characterized in that: The foodborne pathogens are Campylobacter jejuni and its resistant strains.

6. The use according to claim 5, characterized in that: The bacterial derived peptide LL-37 I20M / V21R Application in the preparation of drugs or reagents for resisting foodborne pathogens in mice and chickens.

7. A pharmaceutical composition for resisting foodborne pathogens, characterized in that: Comprising the antibacterial derivative peptide LL-37 according to claim 1 I20M / V21R and a pharmaceutically acceptable carrier thereof.

8. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is preferably a capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

9. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition antimicrobial peptide sustained-release microcapsules are prepared by: Chitosan and acetic acid were mixed; while stirring, the antimicrobial peptide LL-37 I20M / V21R Add dropwise to the chitosan solution and continue stirring; add trisodium phosphate aqueous solution to the above solution and continue stirring; wash the precipitate after centrifugation and freeze-dry; grind and sieve the dried microcapsules.

10. Use of the anti-foodborne pathogens pharmaceutical composition according to claim 7 in the preparation of drugs or reagents for resisting foodborne pathogens.

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