A new antimicrobial peptide GG25 and its application

Abstract

The present invention is applicable to the field of polypeptide technology, and provides a novel antimicrobial peptide GG25 and its application, wherein the amino acid sequence of the antimicrobial peptide GG25 is shown in SEQ ID No. 1. The novel antimicrobial peptide GG25 of the present invention has a molecular weight of 2890.52 Da, and has antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella typhimurium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Bacillus subtilis; it has low hemolytic activity, low cytotoxicity, and is not easy to induce drug resistance in Escherichia coli and methicillin-resistant Staphylococcus aureus; in addition, the gelatin chitosan food coating containing GG25 also has a good beef preservation effect, and can be applied to the fields of food, animal husbandry, and medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and in particular relates to a novel antimicrobial peptide GG25 and application thereof. Background Art

[0002] Antibiotics are a milestone drug in the history of modern medicine. Since their clinical use, they have significantly reduced deaths caused by bacterial infections. However, antimicrobial resistance caused by overuse of antibiotics has become a long-term public health problem worldwide. When bacteria develop antimicrobial resistance, they can continue to reproduce at antibiotic treatment levels, which can cause the infected person or animal to become more ill and increase their risk of death. In order to solve diseases caused by pathogenic microorganisms and reduce the development of antimicrobial resistance, it is urgent to develop new antimicrobial drugs for the prevention and treatment of diseases caused by pathogenic microorganisms.

[0003] Antimicrobial peptides are a large class of small molecule active peptides that have resistance to harmful organisms (or pathogens) such as bacteria, fungi, parasites and viruses. They have broad-spectrum antimicrobial activity and low drug resistance development potential. Therefore, antimicrobial peptides are one of the potential alternatives to antibiotics and are expected to solve the problem of drug-resistant bacterial infections that plague humans; Jilin Paenibacillus ( Paenibacillus jilinensis sp. nov. ) The model strain is YPG26, which was deposited in the China Center for Type Culture Collection on December 14, 2020, with the number CCTCC M2020899 and the GenBank database accession number OK324374. It is a new strain of the genus Paenibacillus that can produce a variety of active metabolites. This strain has great potential for mining; with the development of bioinformatics, mining of active substances based on bacterial genomes has become a new trend; statistical and computer science methods have the advantages of high efficiency and low cost in developing antimicrobial peptide lead compounds with better activity and less toxicity. Therefore, it is of great significance and broad application prospects to develop an antimicrobial peptide with strong antibacterial effect, low toxicity, low hemolysis, and low potential for drug resistance development based on the genome of Jilin Paenibacillus. Summary of the invention

[0004] The purpose of the embodiments of the present invention is to provide a novel antimicrobial peptide GG25, aiming to solve the problems raised in the above background technology.

[0005] The embodiment of the present invention is implemented as follows: a novel antimicrobial peptide GG25, whose amino acid sequence is shown in SEQ ID No.1.

[0006] Another object of the embodiment of the present invention is to provide an application of a novel antimicrobial peptide GG25 in the preparation of antimicrobial drugs.

[0007] Preferably, the pathogen targeted by the antibacterial drug is at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella typhimurium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Bacillus subtilis.

[0008] Preferably, the minimum inhibitory concentration of the antimicrobial peptide against Escherichia coli is 3.13 μM, the minimum inhibitory concentration against Klebsiella pneumoniae is 25 μM, the minimum inhibitory concentration against Pseudomonas aeruginosa is 6.25 μM, the minimum inhibitory concentration against Enterococcus faecalis is 12.5 μM, the minimum inhibitory concentration against Salmonella typhimurium is 25 μM, the minimum inhibitory concentration against methicillin-resistant Staphylococcus aureus is 12.5 μM, the minimum inhibitory concentration against Staphylococcus aureus is 25 μM, the minimum inhibitory concentration against Staphylococcus epidermidis is 3.13 μM, the minimum inhibitory concentration against Listeria monocytogenes is 3.13 μM, and the minimum inhibitory concentration against Bacillus subtilis is 1.56 μM.

[0009] Another object of the embodiment of the present invention is to provide an application of a novel antimicrobial peptide GG25 in the preparation of a food preservative.

[0010] Preferably, the food preservative is a gelatin chitosan food coating material containing antimicrobial peptide GG25, which is used for preserving beef.

[0011] Preferably, the beef is immersed in the food coating material, and then taken out and dried, so that the coating material forms a thin film on the surface of the beef.

[0012] A novel antimicrobial peptide GG25 provided in an embodiment of the present invention has an amino acid sequence as shown in SEQ ID No. 1, specifically GCKAFSLRWRGKARMRIGLRLKKTG, a molecular weight of 2890.52 Da, and can be chemically synthesized by solid phase peptide synthesis, with simple preparation and novel design; it has antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella typhimurium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Bacillus subtilis; and the antimicrobial peptide GG25 has low hemolytic activity and low cytotoxicity, and is not easy to induce drug resistance in Escherichia coli and methicillin-resistant Staphylococcus aureus; in addition, the gelatin chitosan food coating containing GG25 also has a good beef preservation effect and can delay beef spoilage; it can be used to prepare drugs, additives and preservatives for anti-bacterial infection, and is applied to the fields of food, animal husbandry and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a graph showing the HPLC test results of the antimicrobial peptide GG25 provided in Example 1 of the present invention;

[0014] Figure 2 This is a graph showing the ESI-MS detection results of the antimicrobial peptide GG25 provided in Example 1 of the present invention;

[0015] Figure 3 The hemolytic assay result of the antimicrobial peptide GG25 provided in Example 2 of the present invention;

[0016] Figure 4 The cytotoxicity test results of the antimicrobial peptide GG25 provided in Example 2 of the present invention;

[0017] Figure 5 The results of the determination of the inducing bacterial resistance of the antimicrobial peptide GG25 provided in Example 2 of the present invention;

[0018] Figure 6 The appearance change results of the beef sample treated with the gelatin chitosan food coating provided in Example 3 of the present invention during storage;

[0019] Figure 7 The weight change result of the beef sample treated with the gelatin chitosan food coating during storage provided in Example 3 of the present invention;

[0020] Figure 8 The result of the change of the total viable bacteria count of the beef sample treated with the gelatin chitosan food coating provided in Example 3 of the present invention during storage;

[0021] Fig. 9 The TBARS change results of the beef sample treated with the gelatin chitosan food coating during storage provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0024] Example 1: A novel antimicrobial peptide GG25, the design steps of which are as follows:

[0025] The embodiment of the present invention mines novel antimicrobial peptides based on the whole genome of Paenibacillus jilinense. First, the EditSeq module of DNASTAR software is used to find ORFs less than 100 bp in the whole genome of Paenibacillus jilinense and translate them into amino acid sequences.

[0026] Then through CAMP R4and HydrAMP online software to predict the antibacterial activity of peptides. R4 Select Synthetic in the data setting, check Random Forest (RF), Support Vector Machine (SVM), Artificial Neural Network (ANN) in the algorithm, keep the default settings in HydrAMP, submit the peptide sequence, collect CAMP R4 The prediction probability values ​​of antimicrobial peptides predicted by the three algorithms were all >0.9 and the peptides whose prediction values ​​of antimicrobial peptides and antimicrobial activity in HydrAMP were both 1;

[0027] The obtained peptides were analyzed for physicochemical properties by APD3 and compared with the known antimicrobial peptide database to remove the peptide sequences consistent with the known database. ToxinPred online software was used to predict toxicity, DRAMP online software was used to predict hemolysis probability, and ExPasy online software was used to predict stability.

[0028] The predicted antimicrobial peptides were chemically synthesized using solid phase peptide synthesis, and the purity and accurate molecular weight of the antimicrobial peptides were detected using high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS), respectively.

[0029] The amino acid sequence of the antimicrobial peptide GG25 in the embodiment of the present invention is: GCKAFSLRWRGKARMRIGLRLKKTG;

[0030] The HPLC test results were as follows: Figure 1 As shown, according to Figure 1 It can be seen that the purity of the synthesized antimicrobial peptide GG25 is greater than 95%;

[0031] The ESI-MS test results are as follows: Figure 2 As shown, according to Figure 2 It can be seen that the exact molecular weight of the synthesized antimicrobial peptide GG25 is 2890.52 Da.

[0032] Example 2. Activity analysis of antimicrobial peptide GG25

[0033] (1) Determination of minimum inhibitory concentration. The specific steps are as follows:

[0034] Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella typhimurium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis were inoculated into CAMHB medium at a 1% inoculum, and Listeria monocytogenes was inoculated into BHI medium at a 1% inoculum. After culturing overnight at 37°C and 120 rpm, the bacterial solution was diluted to a concentration of 10 6CFU / mL; the test was performed in a 96-well plate, 50 μL of diluted bacterial solution and 50 μL of serially diluted antimicrobial peptide solution were added to each well of the sample well, 50 μL of culture medium and 50 μL of diluted bacterial solution were added to each well of the negative control well, and 50 μL of culture medium and 50 μL of deionized water were added to each well of the blank control well; the 96-well plate was incubated in a 37°C incubator for 16 to 20 h, and the lowest peptide concentration with no visible bacterial growth was the minimum inhibitory concentration. The results are shown in Table 1:

[0035]

[0036] It can be seen from Table 1 that the antimicrobial peptide GG25 provided in the embodiment of the present invention has an inhibitory effect on Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella typhimurium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Bacillus subtilis;

[0037] Among them, Enterococcus faecalis ( Enterococcus faecalis 4P-SA ), Staphylococcus epidermidis ( Staphylococcus epidermidis YYF-771 ) was isolated by establishing a bacterial library from cattle and sheep, and confirmed by 16S rRNA sequence comparison. The cattle samples were collected from Guangze Ranch in Changchun City, Jilin Province, and the sheep samples were collected from Hongwei Live Sheep Trading Market in Da'an City, Jilin Province (with quarantine certificates);

[0038] Escherichia coli ( Escherichia coli O157:H7 ), Klebsiella pneumoniae ( Klebsiella pneumoniae K7 ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ATCC27853 ), Salmonella typhimurium ( Salmonella Typhimurium SL1344 ), methicillin-resistant Staphylococcus aureus ( Methicillin-Resistant Staphylococcus aureus USA30 0)、Staphylococcus aureus( Staphylococcus aureus ATCC25904 ), Listeria monocytogenes ( Listeria monocytogenes 10403S ) and Bacillus subtilis ( Bacillus subtilis BL-4 ) were donated by the Veterinary Pharmacology and Toxicology Laboratory, College of Veterinary Medicine, Jilin University.

[0039] (2) Hemolysis assay. The specific steps are as follows:

[0040] Fresh mouse erythrocytes (the mice used were C57BL / 6, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were collected and stored in polycarbonate tubes containing anticoagulants at 4°C. When used, the anticoagulated blood was slowly injected into a centrifuge tube, centrifuged at 1500rpm for 5 min, and the upper serum was removed. 0.9% sodium chloride solution was added to the precipitated erythrocytes, shaken well, and centrifuged at 2000 rpm for 10 min to remove the supernatant. This was repeated 2 to 3 times until the supernatant was no longer red, and then the supernatant was aspirated. The obtained erythrocytes were made into a 2% (v / v) suspension with 0.9% sodium chloride solution and stored in a 4°C refrigerator for later use. The antimicrobial peptides were graded diluted with 0.9% sodium chloride solution. 50 μL of erythrocyte suspension and an equal volume of antimicrobial peptide solution were added to a 96-well plate and mixed evenly, and incubated at 37°C for 1 h. The positive control was 50 μL of 1% Triton X-100 solution and 50 μL of 2% erythrocyte suspension, and the negative control was 50 50 μL0.9% sodium chloride solution and 50 μL 2% red blood cell suspension; after incubation, centrifuge the mixture at 1000 rpm for 5 min, transfer the supernatant (50 μL) to a clean 96-well plate, measure the absorbance at 570 nm using an ELISA reader and calculate the hemolysis rate;

[0041] The result is as follows Figure 3 As shown, according to Figure 3 It can be seen that there is almost no hemolysis when the concentration of antimicrobial peptide GG25 reaches 100 μM.

[0042] (3) Cytotoxicity assay. The specific steps are as follows:

[0043] The density is 5×10 4 L929 cells with a concentration of cells / mL were inoculated into a 96-well plate at 100 μL per well and cultured until the cells adhered to the wall. An equal volume of antimicrobial peptide solution was added to each well to make the final concentrations of 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. The plates were incubated at 37°C and 5% CO2 for 24 hours. After removing the original culture medium, 100 μL of culture medium containing 10% CCK8 solution was added to each well. After incubation at 37°C in the dark for 2 hours, the absorbance at 450 nm was measured using a microplate reader and the cell viability was calculated.

[0044] The result is as follows Figure 4 As shown, according to Figure 4 It can be seen that the antimicrobial peptide GG25 had no significant effect on L929 cells when the concentration reached 200 μM.

[0045] (4) Determination of induced bacterial resistance. The specific steps are as follows:

[0046] Escherichia coli and methicillin-resistant Staphylococcus aureus were inoculated into LB and TSB liquid culture media at a 1% inoculation rate, respectively, and cultured at 37°C 120 rpm overnight, and the bacterial solution concentration was adjusted to 10 6 CFU / mL; MIC detection was performed in a 96-well plate, 50 μL of diluted bacterial solution and 50 μL of continuously diluted antimicrobial peptide solution were added to each sample well, 50 μL of diluted bacterial solution and 50 μL of continuously diluted antibiotic solution were added to each positive control well, 50 μL of culture medium and 50 μL of diluted bacterial solution were added to each negative control well, and 50 μL of culture medium and 50 μL of deionized water solution were added to each blank control well; the 96-well plate was incubated in a 37°C incubator for 16 to 20 h, and the lowest peptide concentration with no visible bacterial growth was the minimum inhibitory concentration; the bacterial suspension with sub-inhibitory concentration (1 / 2×MIC) was re-inoculated into the liquid culture medium, and the MIC was determined according to the above steps. The above method was used for continuous subculture determination, and the growth of MIC value was used to evaluate the development of bacterial resistance to antimicrobial peptides and antibiotics;

[0047] The result is as follows Figure 5 As shown, according to Figure 5 It can be seen that the antimicrobial peptide GG25 is not easy to induce drug resistance in Escherichia coli and methicillin-resistant Staphylococcus aureus.

[0048] Example 3: Study on the effect of GG25-containing gelatin chitosan food coating on beef preservation

[0049] (1) Preparation of gelatin chitosan food coating containing GG25, the specific steps are as follows:

[0050] Chitosan was dissolved in 2% acetic acid solution, and magnetically stirred at 60°C for 1 h to prepare a 2% chitosan solution. Gelatin was dissolved in deionized water, and stirred in a water bath at 70°C for 1 h to prepare a 4% gelatin solution. Chitosan and gelatin solution were mixed in a ratio of 1:1 (w / w), stirred for 1 h, and then ultrasonically defoamed for 30 min to obtain a gelatin-chitosan food coating. Antimicrobial peptide GG25 solution was added to the mixed gelatin-chitosan solution to a final concentration of GG25 of 12.5 μM and 25 μM, stirred for 1 h, and ultrasonically defoamed for 30 min to obtain a gelatin-chitosan food coating containing GG25.

[0051] (2) The specific steps for beef processing and testing are as follows:

[0052] Fresh beef purchased from the market was evenly cut into small pieces with a side length of 2.5 cm×2.5 cm×2.5 cm in a clean bench and randomly divided into a blank control group (Control), a gelatin chitosan food coating treatment group (CG), a gelatin chitosan food coating treatment group containing 12.5 μM GG25 (CG-AMP1) and a gelatin chitosan food coating treatment group containing 25 μM GG25 (CG-AMP2); the blank control group did not undergo any preservation treatment, while the treated groups soaked the beef pieces in the food coating for 5 minutes, took them out and dried them, and stored them in trays at 4°C; samples were taken from each group at 0, 2, 4, 6, 9, 12, and 15 days of storage for beef appearance record, pH value determination, total viable bacteria determination, and lipid oxidation (TBARS) determination, and the results were as follows: Figures 6 to 9 As shown in Table 2:

[0053]

[0054] according to Figure 6-9 As can be seen from Table 2, after 15 days of storage, compared with the spoiled control group, the beef treated with the food coating containing GG25 had no obvious change in appearance and little weight loss. In addition, the food coating containing GG25 could significantly reduce the increase of total viable bacteria and TBARS in beef and maintain the pH value of beef.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel antimicrobial peptide GG25, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. Use of the novel antimicrobial peptide GG25 as claimed in claim 1 in the preparation of antimicrobial drugs, characterized in that: The pathogens targeted by the antibacterial drug are at least one of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Salmonella typhimurium, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, and Listeria monocytogenes.

3. The use according to claim 2, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide against Escherichia coli is 3.13 μM, the minimum inhibitory concentration against Klebsiella pneumoniae is 25 μM, the minimum inhibitory concentration against Pseudomonas aeruginosa is 6.25 μM, the minimum inhibitory concentration against Enterococcus faecalis is 12.5 μM, the minimum inhibitory concentration against Salmonella typhimurium is 25 μM, the minimum inhibitory concentration against methicillin-resistant Staphylococcus aureus is 12.5 μM, the minimum inhibitory concentration against Staphylococcus aureus is 25 μM, the minimum inhibitory concentration against Staphylococcus epidermidis is 3.13 μM, and the minimum inhibitory concentration against Listeria monocytogenes is 3.13 μM.

4. Use of the novel antimicrobial peptide GG25 as claimed in claim 1 in the preparation of a food preservative.

5. The use according to claim 4, characterized in that: The food preservative is a gelatin chitosan food coating material containing antimicrobial peptide GG25, and is used for preserving beef.

6. The use according to claim 5, characterized in that: The beef is immersed in the food coating material, and then taken out and air-dried, so that the coating material forms a thin film on the surface of the beef.

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