An antibacterial derivative peptide and its composition and application

Abstract

The present invention discloses an antibacterial derivative peptide LL-37 I20M / V21R And its composition and application, belonging to the application field of peptide antibiotics, antibacterial derivative peptide LL-37 I20M / V21R , and its amino acid sequence is: LLGDFFRKSKEKIGKEFKRMRQRIKDFLRNLVPRTES. The antimicrobial peptide sustained-release microcapsules prepared by the present invention have higher stability and stronger bactericidal effect. The preparation method of the 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 elimination of Campylobacter jejuni in the body, with a maximum reduction of 4.69lg (CFU / g). It is expected to become a new product and means of prevention and control of Campylobacter jejuni.

Description

Technical Field

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

[0002] Campylobacter jejuni is a spiral, microaerophilic Gram-negative bacterium and an important foodborne pathogen worldwide. Infection can cause enteritis, fever, arthritis, and even Guillain-Barré syndrome (GBS). The bacterium usually colonizes the intestines of poultry as a commensal bacterium. Although the colonization rate of the bacterium in the animal intestine is high, it does not cause clinical symptoms. Humans are usually infected with Campylobacter jejuni by ingesting contaminated surface water, unpasteurized milk, and undercooked poultry and other meat products. It is reported that only a dose of 500-800 CFU of Campylobacter jejuni can cause human infection. Campylobacteriosis caused by Campylobacter jejuni infection every year brings serious medical and socioeconomic burdens to the world.

[0003] It is reported that reducing the colonization of Campylobacter in poultry by 2-3 lg can reduce the risk of campylobacteriosis in humans by 95%. Clinically, medical staff often use antibiotics to treat infections caused by Campylobacter jejuni. However, with the abuse of antibiotics, the problem of bacterial resistance has become more serious. Therefore, it is urgent to develop a new prevention, control and treatment method.

[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, and are considered to be ideal candidates for antibiotic substitutes. Among them, LL-37 is a cationic antimicrobial peptide composed of 37 amino acids, produced by C-terminal cleavage of the 18kDa human hCAP18 protein, with an α-helical structure and 6 positive charges. LL-37 has antibacterial activity against many Gram-positive, Gram-negative bacteria and fungi, and has an immunomodulatory effect, and is considered to be a potential substitute for 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 Campylobacter jejuni prevention and control method. Summary of the invention

[0005] Purpose of the invention: In view of 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 Campylobacter jejuni prevention and control. It can be used to prepare antibacterial drugs and has potential application value in the prevention and control of foodborne pathogens.

[0006] The present invention also provides an antibacterial derivative peptide LL-37 I20M / V21R Preparation method, composition and application thereof.

[0007] Technical solution: In order to achieve the above purpose, the antibacterial derivative peptide LL-37 described in the present invention I20M / V21R , and its amino acid sequence is: LLGDFFRKSKEKIGKEFKRMRQRIKDFLRNLVPRTES.

[0008] Wherein, 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).

[0009] 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 .

[0010] The antibacterial derivative peptide LL-37 of the present invention I20M / V21R Application in the preparation of drugs or reagents for resisting foodborne pathogens.

[0011] Wherein, the foodborne pathogens are Campylobacter jejuni and its resistant strains.

[0012] Preferably, the foodborne pathogens are Campylobacter jejuni and LL-37-resistant Campylobacter jejuni isolates.

[0013] Among them, the bacterial derived peptide LL-37 I20M / V21R Application in the preparation of drugs or reagents for resisting foodborne pathogens in mice and chickens.

[0014] The anti-foodborne pathogenic bacteria pharmaceutical composition of the present invention comprises the antibacterial derivative peptide LL-37 I20M / V21R and a pharmaceutically acceptable carrier thereof.

[0015] Wherein, the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

[0016] Wherein, the pharmaceutical composition antimicrobial peptide sustained-release microcapsules are prepared by:

[0017] Chitosan and acetic acid were mixed; while stirring, the 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.

[0018] Preferably, the pharmaceutical composition antimicrobial peptide sustained-release microcapsules are prepared by:

[0019] (1) Dissolve chitosan (30 mg / mL) and acetic acid (1%, v / v, pH = 5) in a magnetic stirrer;

[0020] (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;

[0021] (3) adding trisodium phosphate (TPP) aqueous solution (1 mg / mL, pH = 5) to the above solution at a ratio of 1:3 and stirring at room temperature for 2 hours;

[0022] (4) centrifugation at 14,000 g for 40 min at 4°C, the precipitate was washed several times with ultrapure water, and then dried by freeze drying for 12 h;

[0023] (5) Grind and sieve the dried microcapsules.

[0024] The invention discloses an application of the foodborne pathogen resistance drug composition in the preparation of drugs or reagents for resisting foodborne pathogens.

[0025] 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.

[0026] Antibacterial derivative peptide LL-37 of the present invention I20M / V21R It has the characteristics of high charge number (+7), high stability (increased by 3.18 kcal / mol), high antibacterial ability (MIC value is at least 32 times higher than that of natural LL-37), non-toxicity and α-helical structure.

[0027] 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.

[0028] Furthermore, the antibacterial derivative peptide LL-37 I20M / V21RThe sustained-release microcapsules showed significant Campylobacter jejuni reduction effects in vivo: in the cecum of mice, the Campylobacter jejuni colonization could be reduced by 2.37 lg (CFU / g); in the intestine of chickens, the maximum reduction was 4.69 lg (CFU / g).

[0029] 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.

[0030] 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 In comparison, 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.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0032] 1. Antimicrobial 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 elimination of Campylobacter jejuni in the body, with a maximum reduction of 4.69lg (CFU / g), and is expected to become a new prevention and control method for Campylobacter jejuni.

[0033] 2. Antimicrobial derivative peptide LL-37 of the present invention I20M / V21R It has antibacterial activity and little cytotoxicity.

[0034] 3. Antibacterial derivative peptide LL-37 of the present invention I20M / V21R The 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

[0035] Figure 1 For LL-37 I20M / V21R Structural diagram of derived peptides and LL-37 natural peptide; (A) LL-37 I20M / V21R ; (B) LL-37;

[0036] Figure 2 For LL-37I20M / V21R HPLC and MS test results of the derivatized peptides; (A) HPLC results; (B) MS results;

[0037] Figure 3 For LL-37 I20M / V21R Determination of toxicity of derived peptides on Caco-2 cells;

[0038] Figure 4 The morphology of microcapsules was observed by scanning electron microscopy; (A) LL-37 I20M / V21R ; (B) LL-37;

[0039] Figure 5 pH stability for sustained-release microcapsules;

[0040] Figure 6 This is the sustained-release curve of the sustained-release microcapsules;

[0041] Figure 7 Effects of microcapsules prepared with different wall materials on the growth of Campylobacter jejuni P116B;

[0042] Figure 8 Sustained release microcapsules with LL-37 I20M / V21R Comparison of the antibacterial abilities of the derived peptides;

[0043] Fig. 9 For LL-37 I20M / V21R Toxicity determination of sustained-release microcapsules on Caco-2 cells;

[0044] Fig.10 For LL-37 I20M / V21R The reduction of Campylobacter jejuni in the cecal tissue of mice by sustained-release microcapsules;

[0045] Fig.11 is the average load of Campylobacter jejuni in chicken anal swabs;

[0046] Fig.12 The results of sustained-release microcapsules on the reduction of Campylobacter jejuni in chicken anal swabs; 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 contains 5.5 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group F was a group containing 11 μg / mL LL-37 sustained-release microcapsules;

[0047] Fig.13 For continuous feeding of LL-37 I20M / V21R The reduction of Campylobacter jejuni in cecal tissue by sustained-release microcapsules; Group B is the empty carrier microcapsule group; Group C is the microcapsule group containing 2.75 μg / mL LL-37I20M / V21R Protein solution group; Group D contained 2.75 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group E contains 5.5 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group F was a group containing 11 μg / mL LL-37 sustained-release microcapsules;

[0048] Fig.14 For continuous feeding of LL-37 I20M / V21R The reduction of Campylobacter jejuni in colon tissue 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 contains 5.5 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group F was a group containing 11 μg / mL LL-37 sustained-release microcapsules;

[0049] Fig.15 For continuous feeding of LL-37 I20M / V21R The reduction of Campylobacter jejuni in cecal contents 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 contains 5.5 μg / mL LL-37 I20M / V21R Sustained-release microcapsule group; Group F was a sustained-release microcapsule group containing 11 μg / mL LL-37. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with embodiments and drawings.

[0051] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0052] The antibacterial derivative peptide LL-37 designed in the present invention I20M / V21R Synthesized directly by a biotechnology company.

[0053] 1. Strains and cells

[0054] Among them, Campylobacter jejuni 81-176 is a standard strain, and Caco-2 cells and Campylobacter jejuni P116B are provided by Yangzhou University. The in vitro and in vivo antibacterial experiments of the present invention use standard strains and Campylobacter jejuni P116B, wherein Campylobacter jejuni P116B is a LL-37 resistant strain with MIC>88μg / mL. The present invention can also use other LL-37 resistant strains, generally with MIC>22μg / mL.

[0055] 2. Main reagents

[0056] Campylobacter Columbia (CCDA) agar medium was purchased from Oxoid Company, UK; LL-37 I20M / V21R The 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 Company, USA; Mueller-Hinton (MH) liquid culture medium was purchased from BD Company, USA; MTT cytotoxicity detection kit was purchased from Beijing Solarbio Technology Co., Ltd.; trimethoprim, cycloheximide and cefoperazone were purchased from Japan LKT Company; amphotericin B, polymyxin B and rifampicin were purchased from Japan Wako Company; other reagents were all domestic analytical grade.

[0057] 3. Main instruments and equipment

[0058] 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.

[0059] 4. Experimental Animals

[0060] 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 they had animal quarantine certificates.

[0061] Example 1

[0062] 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), and the antibacterial derivative peptide LL-37 was synthesized by solid phase peptide synthesis. I20M / V21R .

[0063] Purity determination of peptides (HPLC method) and mass spectrometry analysis results, liquid chromatography analysis conditions: C18 column (4.6×250mm, 5μm); mobile phase A is acetonitrile solution containing 0.1% trifluoroacetic acid, mobile phase B is pure water containing 0.1% TFA. Detection wavelength is 220nm; flow rate is 1.0mL / min; injection volume is 20μl, and gradient elution is performed.

[0064] Antimicrobial derivative peptide LL-37 I20M / V21R , whose sequence is: LLGDFFRKSKEKIGKEFKRMRQRIKDFLRNLVPRTES (SEQ ID NO.1), and whose purity is 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 as follows Figure 1 A; its HPLC and MS are as follows Figure 2 A and Figure 2 B shows that it is consistent with the theoretical value.

[0065] Example 2

[0066] Antibacterial derivative peptide LL-37 of the present invention I20M / V21R Structural and biological phenotype analysis and in vitro antibacterial activity determination

[0067] 1. LL-37 I20M / V21R Bioinformatic analysis of derived peptides

[0068] 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 / V21RThe stability of the derived peptide was increased by 3.18 kcal / mol compared with 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 number, molecular weight and isoelectric point of the derivative peptide 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 The half-life of LL-37 is the same as that of LL-37. I20M / V21R Analyze the structure of Figure 1 As shown, the LL-37 structure ( Figure 1 B) Compared with LL-37 I20M / V21R There is a coiled structure at the N-terminus, but the overall structure is still an α-helical structure (see Figure 1 A).

[0069] Table 1LL-37 I20M / V21R Prediction of biological activity, water solubility and toxicity

[0070]

[0071] 2. LL-37 I20M / V21R Determination of 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 the Campylobacter jejuni standard strain 81-176 and the P116B human isolate according to the CLSI standard. 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 P116B human isolate;

[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 The derived peptides and the 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 of each well was read using an ELISA reader. 600 Each experiment was repeated three times, with no addition of LL-37 or bacteria as a positive control or negative control, respectively.

[0076] The results showed that for the natural peptide LL-37, the standard strain 81-176 was extremely sensitive to it, while the human isolate P116B was extremely tolerant to it, with an MIC value of up to >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 / V21R In 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 derived peptides (see Table 2).

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

[0078]

[0079] 3. LL-37 I20M / V21R Cytotoxicity assay of derived peptides

[0080] 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:

[0081] (1) Resuscitation and subculture of Caco-2 cells;

[0082] (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;

[0083] (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 / V21RThe 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 culture.

[0084] (4) After 2 h, follow the instructions of the MTT cytotoxicity assay kit, add MTT, and incubate at 37°C in the dark;

[0085] (5) Wash once with PBS, add formazan solvent, dilute it 2-fold after the formazan is dissolved, and measure the OD using an enzyme reader 570 , record the data.

[0086] The results showed that 5.5 μg / mL, 11 μg / mL, and 22 μg / 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 No obvious cytotoxicity, can be used for subsequent animal experiments (see Figure 3 ).

[0087] Example 3

[0088] LL-37 I20M / V21R Preparation and characterization of sustained-release microcapsules of derived peptides

[0089] 1. Preparation of sustained-release microcapsules and determination of encapsulation rate

[0090] 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 cross-linking gel method. The main steps include:

[0091] Preparation of empty carrier microcapsules:

[0092] (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);

[0093] (2) adding the TPP aqueous solution to the chitosan solution at a volume ratio of 1:3, and stirring the mixture at room temperature for 2 hours;

[0094] (3) centrifugation to separate nanoparticles at 14,000 rpm for 40 min;

[0095] (4) washing with ultrapure water for three times to remove excess chitosan and TPP, and then drying by freeze drying for 12 h;

[0096] (5) Grind and sieve the dried microcapsules.

[0097] Preparation of antimicrobial peptide sustained-release microcapsules:

[0098] (1) Dissolve chitosan (final concentration 30 mg / mL) and acetic acid (1%, v / v, pH = 5) in a magnetic stirrer;

[0099] (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;

[0100] (3) adding trisodium phosphate (TPP) aqueous solution (1 mg / mL, pH = 5) into the above solution at a volume ratio of 1:3 and stirring at room temperature for 2 hours;

[0101] (4) centrifugation at 14,000 g for 40 min at 4°C, the precipitate was washed several times with ultrapure water, and then dried by freeze drying for 12 h;

[0102] (5) Grind and sieve the dried microcapsules.

[0103] After centrifugation of the prepared antimicrobial peptide sustained-release microcapsules, the micro BCA kit instructions were followed to determine the amount of free antimicrobial peptides in the supernatant at a wavelength of 565 nm. The encapsulation efficiency (EE) was calculated using the following formula: Encapsulation efficiency (%) = (Total peptides-free peptides) / Total peptides*100. 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 the microcapsules was 87.31%, and the encapsulation rate of the LL-37 microcapsules was 89.38%.

[0104] Table 3 Encapsulation rate of microcapsules

[0105]

[0106] Scanning electron microscopy observation of the morphology of sustained-release microcapsules

[0107] The prepared LL-37 I20M / V21R The results are shown in Table 1. Figure 4 As shown in the figure, 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; the prepared LL-37 I20M / V21R ( Figure 4 A) and LL-37( Figure 4 B) The microcapsules are all spherical in shape and evenly distributed, belonging to the micron-sized microcapsules.

[0108] Example 4

[0109] LL-37 I20M / V21R Stability determination of sustained-release microcapsules of derived peptides

[0110] 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 min. The supernatant was collected and the antimicrobial peptide content in the supernatant was determined according to the instructions of the micro BCA kit, and then the retention rate of antimicrobial peptides in the sustained-release microcapsules was analyzed. The results are shown in Figure 5 As shown, when the pH value is in the range of 2 to 10, LL-37 I20M / V21R The polypeptide contents in the and LL-37 sustained-release microcapsules were relatively stable, with the retention rates being slightly lower only at pH = 10 (P > 0.05), which were 87.67% and 86.67%, respectively; this indicates that the microcapsules with chitosan and TPP cross-linked as wall materials have pH stability, which to a certain extent protects the antimicrobial peptides from premature exposure to the external adverse environment.

[0111] Example 5

[0112] Determination of antibacterial ability of sustained-release microcapsules

[0113] 1. Determination of sustained-release curve of microcapsules

[0114] LL-37 in sustained-release microcapsules I20M / V21R The release kinetics of LL-37 antimicrobial peptide was monitored for 10 days. The specific steps are as follows:

[0115] (1) The prepared sustained-release microcapsules were dispersed in 1 mL of PBS (pH = 7.4) and stirred at 37° C. and 120 rpm.

[0116] (2) At specific time points, the samples were centrifuged at 4°C, 14,000 rpm for 30 min. The supernatant was taken and the antimicrobial peptide content was determined using a microBCA kit, and fresh PBS (1 mL) was added for resuspending.

[0117] (3) Statistical analysis of the release percentage of antimicrobial peptides from sustained-release microcapsules.

[0118] The results showed that after 1 day, the sustained-release microcapsules released about 33.39% of LL-37 I20M / V21RPeptide; 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, and about 72.80% of LL-37 antimicrobial peptides were released after 6 days (see Figure 6 ).

[0119] 2. Determination of the antibacterial curve of sustained-release microcapsules

[0120] 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 the microcapsules are as follows:

[0121] (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; while stirring, the antimicrobial peptide LL-37 I20M / V21R Add it dropwise into the chitosan solution at a concentration of 25 μM, stir it at 4°C for 24 hours, centrifuge it at 4°C, 14000g for 40 minutes, wash the precipitate with ultrapure water for several times, dry it by freeze-drying technology for 12 hours, grind and sieve the dried microcapsules.

[0122] (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; centrifuged at 4°C, 14000 g for 40 minutes, the precipitate was washed with ultrapure water for multiple times, and dried for 12 hours using freeze-drying technology; the dried microcapsules were ground and sieved.

[0123] Empty carrier microcapsules, chitosan, and TPP were separately encapsulated with LL-37 I20M / V21R The formed microcapsules 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 measured, and except for the empty microcapsules, the peptide content of each microcapsule was consistent.

[0124] The specific steps are as follows:

[0125] (1) Resuscitation and subculture of Campylobacter jejuni P116B;

[0126] (2) MH liquid harvest and dilute the bacterial solution to OD 600 = 0.1, LL-37 at a final concentration of 2.75 μg / mL (in terms of 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 microaerobic (5% O2, 85% N2, 10% CO2) environment;

[0127] (3) The plate counts were performed every 2 h. The statistics were calculated and the growth curve was drawn. The experiment was repeated three times.

[0128] The results showed that compared with the control group, empty carrier microcapsules and TPP-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 encapsulation of LL-37 by cross-linking chitosan and TPP I20M / V21R The antibacterial ability of the formed sustained-release microcapsules was significantly enhanced (P<0.05). Compared with the control group, the number of bacteria surviving in 12 hours was reduced by 4.87lg (CFU / mL). The above experimental results show that the antibacterial ability of chitosan and TPP co-crosslinked encapsulated antimicrobial peptides is the most significant, so this encapsulation method was selected as the subsequent preparation scheme (see Figure 7 ).

[0129] 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:

[0130] (1) Resuscitation and subculture of Campylobacter jejuni P116B;

[0131] (2) MH liquid harvest and dilute the bacterial solution to OD 600 = 0.1, and LL-37 at 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 (based on peptide) I20M / V21R The sustained-release microcapsules were co-cultured with Campylobacter jejuni P116B at 37°C and 120 rpm in a microaerobic (5% O2, 85% N2, 10% CO2) environment, and the sustained-release microcapsules containing 11 μg / mL (in terms of polypeptide) of LL-37 were used as controls;

[0132] (3) The plate counts were performed every 2 h. The statistics were calculated and the growth curve was drawn. The experiment was repeated three times.

[0133] 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, prevented it from being exposed to the external adverse environment early. 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 good application potential (see Figure 8 ).

[0134] Example 5

[0135] LL-37 I20M / V21R Evaluation of sustained-release microcapsules on the elimination of Campylobacter jejuni in mice

[0136] 1. Determination of cytotoxicity of sustained-release microcapsules

[0137] Targeting LL-37 I20M / V21R The sustained-release microcapsules were used to test the cytotoxicity of Caco-2 cells, including the following steps:

[0138] (1) Resuscitation and subculture of Caco-2 cells;

[0139] (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;

[0140] (3) Take LL-37 at a final concentration of 5 μg / mL (based on polypeptide) and 11 μg / mL (based on polypeptide) respectively. I20M / V21R The microcapsules were co-incubated with Caco-2 cells; DMEM containing 1% DMSO was used as a control, and 4 replicate wells in each group were placed in a CO2 incubator at 37°C for culture;

[0141] (4) After 2 h, follow the instructions of the MTT cytotoxicity assay kit, add MTT, and incubate at 37°C in the dark;

[0142] (5) Wash once with PBS, add formazan solvent, dilute it 2-fold after the formazan is dissolved, and measure the OD using an enzyme reader 570 , record the data.

[0143] The results showed that Fig. 9 As shown, in 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.

[0144] 2. LL-37 I20M / V21R Effect of sustained-release microcapsules on the elimination of Campylobacter jejuni in the intestines of mice

[0145] 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 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 LL-37 sustained-release microcapsule solution group containing 11 μg / mL polypeptide (PBS, pH 7.4) were dissolved in PBS. Each mouse was gavaged with sodium bicarbonate solution 30 minutes before the challenge to neutralize gastric acid. Campylobacter jejuni P116B was inoculated with 1×10 8 According to the colonization of Campylobacter jejuni in mice, mice in each group were gavaged with 200 μL of PBS, 2.75 μg / mL of LL-37 peptide, and 100 μL of PBS. 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), and the mice were killed on the second day, and the bacterial load of Campylobacter jejuni in the cecum was statistically analyzed. Fig.10 As shown in the figure, in the cecal tissue, the average colonization amount of the PBS control group was 5.72lg (CFU / g); in comparison, the LL-37 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 was 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.

[0146] Example 6

[0147] LL-37 I20M / V21R Evaluation of sustained-release microcapsules on the elimination of Campylobacter jejuni in chickens

[0148] 1. LL-37I20M / V21R Effect of sustained-release microcapsules on the elimination of Campylobacter jejuni in chicken feces

[0149] The experiment selected one-day-old chicks and started to challenge Campylobacter jejuni P116B at 5 days of age. 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 as follows Fig.11 As shown in Figure 2, the bacterial load in the anal swab samples of the chickens gradually increased after the challenge, tended to stabilize at 9 days of age, and reached 10 8 ~10 9 CFU / g.

[0150] As the bacterial load in chickens tended to be stable, group A was divided into the Campylobacter jejuni control group, group B was divided into the empty carrier microcapsule solution group (PBS, pH 7.4), and group C was divided into the LL-37 solution group containing 2.75 μg / mL. I20M / V21R Protein solution group, group D is LL-37 containing 2.75 μg / mL peptide I20M / V21R Sustained-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 polypeptide. The microcapsules in each group were dissolved in PBS. At 14, 21, and 28 days of age, the mice were continuously fed with 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 LL-37 sustained-release microcapsule solution group containing 11 μg / mL peptide (Group F). After the start of feeding 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. Fig.12 As shown, feeding LL-37 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 / V21RThe load of Campylobacter jejuni in the anal swab samples of the sustained-release microcapsule group and 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, decreasing by 3.17lg and 3.67lg (CFU / g), respectively. At the same time, feeding 2.75μg / mL LL-37 I20M / V21R However, the protein solution 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 the control groups B (empty carrier microcapsule group) and F (LL-37 sustained-release microcapsule group) (P>0.05).

[0151] 2. LL-37 I20M / V21R Evaluation of the effect of microcapsules on the elimination of Campylobacter jejuni in the intestinal tract of chickens

[0152] To explore LL-37 I20M / V21R The sustained-release microcapsules have 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 of LL-37 peptide I20M / V21R The sustained-release microcapsule solution group (Group E) and the LL-37 (11 μg / mL) polypeptide sustained-release microcapsule solution group control group (Group F). 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 at 14, 21 and 28 days of age, the bacterial load in the cecum of the control group was approximately 10 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 reduction effect, decreasing by 2.83lg and 3.66lg, respectively (P < 0.05, P < 0.001); while the feeding of 2.75μg / mL LL-37 I20M / V21RThe protein solution and LL-37 sustained-release microcapsules did not show significant in vivo reduction effects (see Fig.13 ).

[0153] 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 reduction effect, decreasing by 2.17lg and 3.00lg, respectively (P < 0.001, P < 0.001); while the feeding of 2.75μg / mL LL-37 I20M / V21R The protein solution and LL-37 sustained-release microcapsules did not show significant in vivo reduction effects (see Fig.14 ).

[0154] 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 Protein solution and LL-37 microcapsules still did not show obvious in vivo reduction effect (see Fig.15 ).

[0155] In summary, the antimicrobial peptide LL-37 provided by the present invention I20M / V21R The in vitro antibacterial effect is significant; the sustained-release microcapsules also have significant in vivo reduction effects. 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. 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 .

3. An antimicrobial derivative peptide LL-37 according to claim 1 I20M / V21R Application in the preparation of drugs or reagents for resisting foodborne pathogens, wherein the foodborne pathogens are Campylobacter jejuni.

4. The use according to claim 3, characterized in that: The bacterial derived peptide LL-37 I20M / V21R Application of the invention in the preparation of medicines or reagents for resisting foodborne pathogens in mice and chickens, wherein the foodborne pathogens are Campylobacter jejuni.

5. 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.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

7. The pharmaceutical composition according to claim 5, 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.

8. Use of the anti-foodborne pathogen pharmaceutical composition according to claim 5 in the preparation of drugs or reagents for resisting foodborne pathogens, wherein the foodborne pathogen is Campylobacter jejuni.

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