Aggregate ineales antibacterial peptide and application thereof

Abstract

The invention relates to the technical field of antibacterial peptides, and discloses an Aggregate ineales antibacterial peptide and application thereof, and the amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO: 1. The antibacterial peptide obtained by the invention has a relatively good antibacterial effect on staphylococcus aureus, klebsiella pneumoniae and acinetobacter baumannii.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and in particular to an Aggregatilineales antimicrobial peptide and application thereof. Background Art

[0002] Pathogenic microorganisms are pathogenic microorganisms that can cause diseases in humans, animals and plants. Microorganisms refer to biological groups with individual volume diameters generally less than 1 mm. They have simple structures, most of them are single cells, and some do not even have cell structures. Common pathogenic microorganisms in the prior art include Staphylococcus aureus ( Staphylococcus aureus, S. aureus ), Klebsiella pneumoniae and Acinetobacter baumannii. Among them, Staphylococcus aureus often parasitizes on the skin, nasal cavity, throat, stomach, carbuncle, suppurative sores of humans and animals, and is also ubiquitous in air, sewage and other environments. Under appropriate conditions, Staphylococcus aureus can produce enterotoxins, thus causing food poisoning and affecting consumer safety; Klebsiella pneumoniae can cause pneumonia; Acinetobacter baumannii usually causes bacteremia, pneumonia, meningitis, peritonitis, endocarditis, as well as urinary tract and skin infections.

[0003] Since food is rich in nutrients, improper handling during food production, storage or transportation may lead to the rapid reproduction of pathogenic bacteria, which in turn may cause food poisoning, gastroenteritis or even serious systemic infection after ingestion by consumers. Antimicrobial peptides (AMPs) are important components of prokaryotes and eukaryotes. These small peptides are usually composed of 12-50 amino acids and have broad-spectrum antibacterial, antifungal and antiviral abilities, and are of great value in pharmacological treatment and preventive applications. Therefore, there is an urgent need to obtain an antimicrobial peptide that can be used in the production, storage or transportation of food to reduce the harm of pathogenic bacteria to consumers. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an Aggregatilineales antimicrobial peptide and its application. The antimicrobial peptide obtained by the present invention has good antibacterial effect on Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0005] The present invention provides an Aggregatilineales antimicrobial peptide, the amino acid sequence of the antimicrobial peptide is shown as SEQ ID NO:1.

[0006] The present invention also provides an antimicrobial composition, which comprises an antimicrobial peptide with an amino acid sequence such as SEQ ID NO: 1.

[0007] The present invention also provides a gene encoding the antimicrobial peptide.

[0008] The present invention also provides a recombinant vector, an expression box, a transgenic cell line, a transgenic plant or a recombinant microorganism containing the encoding gene.

[0009] The present invention also provides the use of the antimicrobial peptide in preparing medicines for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0010] The present invention also provides the use of the antibacterial composition in preparing medicines for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0011] The present invention also provides the use of the antimicrobial peptide in preparing an additive, wherein the additive is any one of a cosmetic, feed or food additive.

[0012] The present invention also provides the use of the antimicrobial peptide in inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii for non-disease treatment and / or non-disease diagnosis.

[0013] Furthermore, the minimum inhibitory concentration of the antimicrobial peptide for inhibiting Staphylococcus aureus is 12.5 μM, the minimum inhibitory concentration for inhibiting Klebsiella pneumoniae is 200 μM, and the minimum inhibitory concentration for inhibiting Acinetobacter baumannii is 100 μM.

[0014] The embodiments of the present invention have the following technical effects: The invention obtains a new antimicrobial peptide, which has good antibacterial effect on Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is the genome spectrum of the antimicrobial peptide provided in the embodiment of the present invention.

[0017] Figure 2 It is a three-dimensional structure diagram of the antimicrobial peptide provided in the embodiment of the present invention.

[0018] Figure 3 is a diagram showing the antibacterial effect of the antimicrobial peptide provided in an embodiment of the present invention, wherein Figure 3 (a) is the inhibition zone of the antimicrobial peptide of the present invention against Staphylococcus aureus, Figure 3 (b) is the inhibition zone of the antimicrobial peptide of the present invention against Klebsiella pneumoniae, Figure 3 (c) is the inhibition zone of the antimicrobial peptide of the present invention against Acinetobacter baumannii.

[0019] Figure 4 This is a data graph of the minimum inhibitory concentration of antimicrobial peptides provided in an embodiment of the present invention.

[0020] Figure 5 The antimicrobial peptide provided in the embodiment of the present invention is applied to chicken.

[0021] Figure 6 The antimicrobial peptide provided by the embodiment of the present invention is applied to milk. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0023] In a first aspect, some embodiments of the present invention provide an Aggregatilineales antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0024] In a second aspect, some embodiments of the present invention further provide an antimicrobial composition, wherein the antimicrobial composition comprises an antimicrobial peptide having an amino acid sequence such as SEQ ID NO: 1.

[0025] In a third aspect, some embodiments of the present invention also provide a gene encoding the antimicrobial peptide.

[0026] In a fourth aspect, some embodiments of the present invention also provide a recombinant vector, an expression cassette, a transgenic cell line, a transgenic plant or a recombinant microorganism containing the encoding gene.

[0027] In a fifth aspect, some embodiments of the present invention also provide use of the antimicrobial peptide in the preparation of drugs for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0028] In a sixth aspect, some embodiments of the present invention also provide use of the antibacterial composition in the preparation of drugs for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0029] In a seventh aspect, some embodiments of the present invention further provide the use of the antimicrobial peptide in the preparation of an additive, wherein the additive is any one of a cosmetic, feed or food additive.

[0030] In an eighth aspect, some embodiments of the present invention also provide use of the antimicrobial peptide in inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii for non-disease treatment and / or non-disease diagnosis.

[0031] In some embodiments, the minimum inhibitory concentration of the antimicrobial peptide for inhibiting Staphylococcus aureus is 12.5 μM, the minimum inhibitory concentration for inhibiting Klebsiella pneumoniae is 200 μM, and the minimum inhibitory concentration for inhibiting Acinetobacter baumannii is 100 μM.

[0032] The following is described in conjunction with the embodiments and comparative examples: Example 1: Obtaining microbial genomes based on metagenomic component-related technology: CRR807596 was downloaded from the NGDC public database of the National Center for Bioinformation, which was derived from a hot spring environment. Trim Galore (v. 0.5.0) was used for quality control to remove adapter sequences and low-quality reads (quality score < 20 points) to obtain clean reads. MEGAHIT was used to assemble clean reads. Seqtk was used to filter the contigs of the assembly results according to the sequence length (retaining the length of 1500bp). Bowtie2 was used to map the clean reads to the corresponding contigs. Samtools was used to convert the mapping results to BAM format. SAMtools was used to sort and index the BAM files, and then CONCOCT was used to generate the genome. The genome 133.CRR807596_concoct_sub was obtained. CheckM2 was used to analyze the integrity and contamination of the genome, which were 59.99% and 3.63%, respectively, meeting the requirements of MIMAG for the genome. GTDB-Tk was used for species annotation, and the results showed that the genome was CP2-2F sp938005675 of the order Aggregatilineales of bacteria (such as Figure 1 The detailed annotation information is as follows: d__Bacteria;p__Chloroflexota;c__Anaerolineae;o__Aggregatilineales;f__UBA2796;g__CP2-2F;s__CP2-2F sp938005675.

[0033] The amino acid sequence of the antimicrobial peptide (antimicrobial peptide 744) formed by the genome is shown in SEQ ID NO: 1, and SEQ ID NO: 1 is: NSKLAAKIAKLIGVKLKKLKVG.

[0034] According to the amino acid sequence of the obtained antimicrobial peptide, the antimicrobial peptide is synthesized: (1) According to the first amino acid at the C-terminus of the peptide sequence, 0.5 mmol of the corresponding Fmoc-protected amino acid-WangResin was selected and added to the solid phase reactor. After adding DCM to swell the resin for 30 minutes, the resin was drained and washed three times with DMF. A 20% volume ratio of hexahydropyridine DMF solution was added and reacted for 5 minutes. A 20% volume ratio of hexahydropyridine DMF solution was added again and reacted for 10 minutes. The resin was washed once with DMF in the middle. After the reaction was completed, the resin was drained and washed three times with DMF.

[0035] (2) According to the sequence of the peptide from C-terminus to N-terminus, the condensation reaction and Fmoc removal reaction were carried out alternately according to the reaction amount of 1.5 mmol of amino acids. According to the amino acid sequence of the antimicrobial peptide, all the amino acids were condensed on the resin. After the last amino acid was condensed, a 20% by volume solution of hexahydropyridine in DMF was added and reacted for 5 minutes. A 20% by volume solution of hexahydropyridine in DMF was added again and reacted for 10 minutes. The mixture was washed with DMF once in the middle. After the reaction was completed, the mixture was drained and washed with DMF three times.

[0036] (3) Add 1 mmol of FITC and N-methylmorpholine to the reactor and react for 5-10 minutes. Use ninhydrin to detect whether the reaction is complete. After the reaction is complete, wash it alternately with DMF and DMC three times, and then wash and shrink it with methanol to obtain a dry polypeptide-resin.

[0037] (4) Place the peptide-resin in a round-bottom flask, slowly add the prepared lysis solution (the volume ratio of the reagent formula of the lysis solution is TFA: thioanisole: phenol: triisopropylsilane: water = 82.5:7.5:5:3:2) at 0°C, stir slowly, react at low temperature for 0.5 hour, react at room temperature for 2 hours, filter to obtain the lysis solution, slowly add the lysis solution into anhydrous ice ether and stir, filter and separate the crude peptide, wash with ice ether 3 times to obtain the crude peptide.

[0038] (5) Use mass spectrometry to detect whether the molecular weight of the crude product is correct. If correct, purify and separate it using high performance liquid chromatography, freeze-dry it, and then obtain the pure polypeptide.

[0039] The antimicrobial peptide obtained in Example 1 was tested: (1) Use AlphaFold2 to predict its three-dimensional structure. The results are as follows Figure 2 shown.

[0040] (2) Determination of the antibacterial spectrum of antimicrobial peptides: Take out the pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Salmonella enteritidis, Salmonella typhimurium, Listeria monocytogenes) stored in the previous period from the -80℃ refrigerator, take a small amount of bacterial liquid with an inoculation loop, make three-zone stripes on LB solid culture medium (commercially available), and culture in a constant temperature incubator at 37℃ for 16-24h.

[0041] After a single colony grows on the plate, use a sterilized inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid culture medium and place it in a 37°C constant temperature shaker for activation for 16-24 hours. 1 mL of the activated pathogenic bacteria culture solution was taken and added to 100 mL of LB liquid medium (commercially available), and cultured at 37°C and 200 rpm for 16-24 h. The biomass was measured using an ELISA instrument, and the final biomass OD 600 Reach between 1.0-1.2.

[0042] Take 40 μL of the cultured bacterial solution and spread it evenly on the LB solid culture medium with a spreading rod. Place 32 sterilized filter papers (the diameter of the filter paper is 6 mm) at the same intervals on the plate covered with pathogenic bacteria. Take 20 μL of the 400 μM antimicrobial peptide solution (the antimicrobial peptide is dispersed in sterilized water) and spot it on the filter paper. After spotting, culture the LB solid culture medium at 37°C for 16-24 hours.

[0043] The LB solid medium after culture was taken, and the inhibition zone formed was observed and measured. The test was repeated three times for each strain to be tested, and three parallels were made each time. Each inhibition zone was measured three times and the average value was taken. Finally, the average value of all the measured inhibition zone diameters represented the final inhibition zone diameter of the antimicrobial peptide for the strain. It was found that the antimicrobial peptide of the present invention formed an inhibition zone against Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii. Figure 3 shown.

[0044] (3) Determination of minimum inhibitory concentration (MIC) of antimicrobial peptides: An LB medium solution containing antimicrobial peptides was prepared, and the concentration of the antimicrobial peptides was 400 μM.

[0045] Take 500 μL of the above LB culture medium solution containing antimicrobial peptides and add it to 500 μL of LB culture medium to prepare a culture medium solution containing antimicrobial peptides with a concentration of 200 μM.

[0046] Similarly, the culture medium solution containing the antimicrobial peptide was diluted to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM in sequence, and 6 mL of the culture medium solution containing the antimicrobial peptide at each concentration was prepared.

[0047] Similarly, an LB medium solution containing the natural antimicrobial peptide Nisin was prepared, and the concentration of the natural antimicrobial peptide Nisin was 400 μM. According to the above steps, the natural antimicrobial peptide Nisin was diluted in turn to 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM antimicrobial peptide medium solutions.

[0048] The pathogenic bacteria Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii stored previously were taken out from the -80°C refrigerator and activated and cultured on LB solid culture medium at 37°C for 16-24h.

[0049] After a single colony grows on the plate, use a sterilized inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid medium. Place it in a 37°C constant temperature shaker for activation for 16-24 hours, and use an ELISA reader to measure the biomass. The final biomass OD 600 When the concentration reaches between 1.0-1.2, dilute the activated bacterial solution 100 times and put it into a 2mL sterile centrifuge tube for later use.

[0050] 200 μL of the culture medium solution containing different concentrations of antimicrobial peptides were taken and placed in a sterile 96-well culture plate, and three parallel experiments were performed for each concentration. Then, 10 μL of diluted Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii solutions were added to the culture medium solutions containing different concentrations of antimicrobial peptides, and the culture medium solution containing antimicrobial peptides without the addition of pathogenic bacteria was used as a blank control group.

[0051] According to the above method, Nisin control was performed in the same way.

[0052] The 96-well plate was placed in a shaker at 37°C and 200 rpm for 16-24 h, and the biomass was measured using an ELISA reader to determine the minimum inhibitory concentration (MIC).

[0053] (4) Application of antimicrobial peptides in chicken preservation: Cut the raw chicken into 1cm pieces 3 The square blocks (about 0.27g) were first sterilized by soaking in 0.5% sodium hypochlorite for 1 minute, then washed three times with PBS (phosphate buffered saline), and then sterilized by soaking in 75% ethanol for 1 minute. After washing three times with PBS, the raw chicken was evenly irradiated with ultraviolet light for 1.2 hours to make it sterile.

[0054] Taking Staphylococcus aureus as an example, the previously stored Staphylococcus aureus was taken out from the -80°C refrigerator and activated and cultured on LB solid culture medium at 37°C for 16-24h.

[0055] After a single colony grows on the plate, use a sterilized inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid medium. Place it in a 37°C constant temperature shaker for activation for 16-24 hours, and use an ELISA reader to measure the biomass. The final biomass OD 600 Reach between 1.0-1.2.

[0056] The activated bacterial solution was diluted to a concentration of 5×10 3 CFU / mL, apply the diluted bacterial solution evenly on the surface of the chicken cubes with a sterile cotton swab and place it in a 25℃ incubator for 2 hours to infect the chicken.

[0057] The antimicrobial peptide with a concentration of 12.5 μM was evenly spread on the surface of the chicken cubes with a sterile cotton swab and cultured in a 25°C incubator for 32 hours. PBS buffer solution was used as a PBS control.

[0058] The chicken pieces were homogenized and centrifuged, and the supernatant was diluted and spread on the corresponding selective medium to enumerate the remaining viable bacteria.

[0059] Formula of PBS buffer solution (pH 7.2): Take 100 mL of 0.2 mol / L potassium dihydrogen phosphate solution and 70 mL of 0.2 mol / L sodium hydroxide solution, add freshly boiled cold water to dilute to 400 mL, and adjust the pH to between 7.2 and 7.4.

[0060] (5) Application of antimicrobial peptides in milk preservation: Take 30 mL of commercially available fresh milk and dispense it into a 50 mL conical bottle that is resistant to high temperatures and seal the bottle mouth with a sterile film. Set the autoclave to 115°C for 30 minutes to sterilize the fresh milk. After sterilization, slowly cool it down and immediately store it in a 4°C refrigerator.

[0061] Taking Staphylococcus aureus as an example, the previously stored Staphylococcus aureus was taken out from the -80°C refrigerator and activated and cultured on LB solid culture medium at 37°C for 16-24h.

[0062] After a single colony grows on the plate, use a sterilized inoculation loop to pick up a single colony and inoculate it into 5 mL of LB liquid medium. Place it in a 37°C constant temperature shaker for activation for 16-24 hours, and use an ELISA reader to measure the biomass. The final biomass OD 600 Reach between 1.0-1.2.

[0063] The activated bacterial solution was diluted to a concentration of 5×10 3 CFU / mL, add the diluted bacterial solution into the above conical flask containing sterilized milk, place it in a 25℃ incubator and culture it for 2h to infect the milk.

[0064] The antimicrobial peptide with a concentration of 12.5 μM was added to the conical flask containing the bacterial solution and milk, and cultured in a 25° C. incubator for 32 h, and sterilized sterile water was used as a blank control.

[0065] The above milks were diluted and spread on the corresponding selective medium to count the remaining live bacteria.

[0066] Results and analysis: exist Figure 2 It can be found that the antimicrobial peptide of the present invention has a typical α-helical structure. Antimicrobial peptide 744 (NSKLAAKIAKLIGVKLKKLKVG) is a peptide with potential antibacterial activity. The results show that the peptide has a high structural credibility (pLDDT=90.5) and a good match with the experimental structure (pTM=0.204). Its electrostatic charge is 7, indicating that the peptide has a positive charge under physiological conditions, which is conducive to interacting with negatively charged bacterial membranes. The standardized hydrophobic moment is 0.87 and the amphipathic index is 1.17, indicating that the peptide has certain hydrophobicity and amphipathicity and can interact with the lipid bilayer of the bacterial membrane. The isoelectric point penetration depth is 15, which further indicates that it has a strong penetration ability near the isoelectric point. The peptide has a typical α-helical structure, a helix number of 5, and a triple helix tendency of 0.88. These structural features help it to bind and insert into the bacterial membrane, thereby exerting an antibacterial effect.

[0067] Figure 3 It can be found that the antimicrobial peptides obtained by the present invention can achieve the inhibition of Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii. Figure 3 (a) It can be found that the diameter of the inhibition zone of Staphylococcus aureus is 7 mm, the diameter of the inhibition zone of Klebsiella pneumoniae is 7 mm, and the diameter of the inhibition zone of Acinetobacter baumannii is 12 mm.

[0068] Figure 4 It can be found that the antimicrobial peptide obtained by the present invention has a mic of 12.5 μM for Staphylococcus aureus, and its inhibitory effect on Staphylococcus aureus is significantly better than Nisin in the prior art; in addition, the antimicrobial peptide of the present invention has a mic of 200 μM for Klebsiella pneumoniae and a mic of 100 μM for Acinetobacter baumannii, while Nisin in the prior art has no inhibitory effect on Klebsiella pneumoniae and Acinetobacter baumannii. It can be seen that the antimicrobial peptide of the present invention has significant advantages in inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

[0069] Figure 5 It can be found that the antimicrobial peptide obtained by the present invention can be used to inhibit Staphylococcus aureus in chicken, and its inhibitory effect on Staphylococcus aureus within 32 hours is significant.

[0070] Figure 6 It can be found that the antimicrobial peptide obtained by the present invention can be used to inhibit Staphylococcus aureus in milk, and its inhibitory effect on Staphylococcus aureus within 32 hours is significant.

[0071] In summary, the antimicrobial peptides obtained in the present invention can inhibit Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii, and the antibacterial effect is significantly better than that of the prior art.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. An Aggregatilineales antimicrobial peptide, characterized in that The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. An antibacterial composition, characterized in that The antimicrobial composition comprises an antimicrobial peptide with an amino acid sequence such as SEQ ID NO:

1.

3. A coding gene, characterized in that The coding gene is used to encode the antimicrobial peptide according to claim 1.

4. A recombinant vector, expression cassette, transgenic cell line, transgenic plant or recombinant microorganism containing the encoding gene according to claim 3.

5. Use of the antimicrobial peptide according to claim 1 in inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii for non-disease treatment and / or non-disease diagnosis.

6. Use of the antimicrobial peptide according to claim 1 in the preparation of a drug for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

7. Use of the antibacterial composition according to claim 2 in the preparation of a medicament for inhibiting Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii.

8. Use of the antimicrobial peptide according to claim 1 in the preparation of an additive, characterized in that: The additive is any one of a cosmetic, feed or food additive.

9. The use according to claim 5, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide for inhibiting Staphylococcus aureus is 12.5 μM, the minimum inhibitory concentration for inhibiting Klebsiella pneumoniae is 200 μM, and the minimum inhibitory concentration for inhibiting Acinetobacter baumannii is 100 μM.

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