CN117986327A - Antibacterial peptide RS12 and application thereof - Google Patents
Antibacterial peptide RS12 and application thereof Download PDFInfo
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- CN117986327A CN117986327A CN202410397116.7A CN202410397116A CN117986327A CN 117986327 A CN117986327 A CN 117986327A CN 202410397116 A CN202410397116 A CN 202410397116A CN 117986327 A CN117986327 A CN 117986327A
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an antibacterial peptide RS12, the amino acid sequence of which is PEMRPSMGKVAK, and the antibacterial amount of the antibacterial peptide is 1330 daltons. The antibacterial peptide has strong effect of inhibiting the growth of staphylococcus aureus and aeromonas hydrophila, can be used for preparing medicines for preventing or inhibiting the infection of staphylococcus aureus and aeromonas hydrophila, and can be used as food preservative and feed additive for preventing fish diseases.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antibacterial peptide RS12 and application thereof.
Background
Staphylococcus aureus, belonging to the genus staphylococcus, is a representative of gram-positive bacteria. As one of the most widely affecting pathogens for human health, the bacterium can cause food poisoning, toxic shock, etc. Aeromonas hydrophila is a human-livestock-fish co-pathogen widely distributed in fresh water environments, and has become an important food-borne pathogen. The strain not only causes animal illness, but also can cause human infection septicemia, intestinal tract infection, diarrhea and other diseases by polluting water and diseased fish. Although a series of physicochemical measures are adopted for control and prevention, the environment is not friendly, and the long-term use of antibiotics also leads to the increase of the drug resistance of pathogenic bacteria. Therefore, there is an urgent need for an antibacterial substance which is environmentally friendly and does not easily develop drug resistance.
Disclosure of Invention
The invention aims to provide an antibacterial peptide and application thereof, wherein the antibacterial peptide is an antibacterial peptide chain obtained from fermented mulberry leaves and is named as peptide RS12. The antibacterial activity and the heat stability of staphylococcus aureus and aeromonas hydrophila are researched, and the antibacterial activity and the heat stability can be used for food preservatives and aquatic feed additives for preventing fish diseases.
To achieve the technical object of the present invention, a first aspect of the present invention provides an antimicrobial peptide RS12, which has an amino acid sequence PEMRPSMGKVAK as shown in SEQ ID NO: 1.
Wherein, the molecular weight of the antibacterial peptide RS12 is 1330 daltons, the charged electric energy is +2, and the total hydrophobicity ratio is 33%.
The antibacterial peptide RS12 is a bacterial disruption based on the following principle: in one aspect, the antibacterial peptide RS12 has a positive charge that can interact with the bacterial cell membrane to increase the pore-forming activity of the bacterial cell membrane. On the other hand, the cell membrane is destroyed while the permeability of the bacterial cell membrane is changed, so that the intracellular substances are extravasated to cause bacterial death.
In particular, the antibacterial peptide provided by the invention is obtained from the mulberry leaves after fermentation. Comprising the following steps:
Inoculating mixed bacterial liquid of lactobacillus, bacillus subtilis and bacillus licheniformis into mulberry leaf powder, and fermenting to obtain the fermented mulberry leaf.
The lactic acid bacteria, the bacillus subtilis and the bacillus licheniformis used in the application are all commercially available.
Extracting peptide segments in the fermented mulberry leaves, and screening to obtain the antibacterial peptide RS12.
Wherein, the peptide fragment in the extracted and fermented mulberry leaves is obtained by carrying out protein extraction and quantitative protein analysis screening on a fermented sample.
The antibacterial peptide RS12 is obtained by synthesizing the peptide fragment sequence after screening. Wherein the peptide fragment sequence after screening is the amino acid sequence PEMRPSMGKVAK. The synthesis method can be any one of the technical fields, and the invention is not limited.
Wherein the fermented sample is a component which is desalted and freeze-dried and then is taken to be more than 1000 Da.
In particular, the liquid phase conditions for protein extraction and protein quantification are timsTOF PRO systems (Bruker); the analytical Column was a C18 reverse phase chromatography Column (Thermo SCIENTIFIC EASY Column,75 μm. Times.10 cm); mobile phase a was 0.1% formic acid, B was 84% acetonitrile and 0.1% formic acid, flow rate was 300 nL/min;
The mass spectrometry conditions were peptide fragment analysis using timsTOF Pro mass spectrometer (Bruker) which performs full scan acquisition (m/z 100-1700). The library searching software is MaxQuant, and the mass spectrum results of the protein and peptide fragments are identified by using a Morus alba database in Uniprot.
In particular, the antibacterial peptide RS12 obtained after the screening was obtained by screening the highest scoring peptide fragments in server APD3 according to the number of amino acids (< 50), total positive charges (+2 to +9) and hydrophobic amino acid percentage (> 30%), and scoring on-line server CAMP.
In particular, the amino acid sequence of the antibacterial peptide obtained after screening is PEMRPSMGKVAK, which consists of 12 amino acids, the total charge is +2, the hydrophobicity is 33%, and the antibacterial mass is 1330: 1330 Da, and is named as RS12.
To achieve the technical object of the present invention, a second aspect of the present invention provides an antibacterial drug comprising the antibacterial peptide RS12 of the first aspect.
Wherein the antibacterial agent can inhibit and/or kill staphylococcus aureus and aeromonas hydrophila. For example, it may be a peptide antibiotic.
It should be noted that the antibacterial drug can be prepared by adopting the prior art in the field, the invention is not limited, and the antibacterial drug prepared by using the antibacterial peptide RS12 provided by the invention belongs to the protection scope of the invention.
To achieve the technical object of the present invention, a second aspect of the present invention provides an application of the antimicrobial peptide RS12, S1) or S2) or S3):
s1) application of preparing antibacterial drugs;
s2) application of preparing a feed additive;
s3) preparing an application of a food preservative;
wherein, the amino acid sequence of the antibacterial peptide RS12 is shown in SEQ ID NO: 1.
It should be noted that the peptide may be prepared into an antibacterial agent, a feed additive or a food preservative by any conventional method, for example, conventional addition, and the present invention is not limited thereto.
The amino acid sequence of the antibacterial peptide RS12 provided by the invention is synthesized or expressed by a person skilled in the art, and the creative labor is not required, so that antibacterial drugs or feed additives or food preservatives prepared by using the antibacterial peptide provided by the invention belong to the protection scope of the invention.
The implementation of the invention has the following beneficial effects:
According to the invention, fermented mulberry leaves are taken as a research object, and the peptide fragment is subjected to bioinformatics prediction through LCMS mass spectrum results, so that polypeptide RS12 with a brand new amino acid sequence is found. Studying the antibacterial activity of the polypeptide RS12 on staphylococcus aureus and aeromonas hydrophila; and the damage degree of RS12 is observed by using a transmission electron microscope by taking staphylococcus aureus and aeromonas hydrophila as examples. Experimental results show that the peptide has a strong inhibition effect on staphylococcus aureus and aeromonas hydrophila. The antibacterial mechanism is that the antibacterial agent is firstly adsorbed on the surface of bacteria, then the cell membrane of the bacteria is destroyed, and the generation of the membrane is inhibited to enable substances in the cells to be extravasated, so that the effect of inactivating the bacteria is achieved. The antibacterial peptide RS12 has certain potential in the application of food preservatives and feed additives.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following description will briefly explain the drawings that are required to be used in the embodiments.
FIG. 1 is a schematic structural diagram of an antimicrobial peptide RS 12;
FIG. 2 is a graph of a Minimum Inhibitory Concentration (MIC) assay of antimicrobial peptide RS12 against Staphylococcus aureus. Wherein A: antibacterial peptide concentration 0 μg/mL; b: antibacterial peptide concentration 15.625 μg/mL; c: the concentration of the antibacterial peptide is 31.25 mug/mL; d: the concentration of the antibacterial peptide is 62.5 mug/mL; e: the concentration of the antibacterial peptide is 125 mug/mL; f: the concentration of the antibacterial peptide is 250 mug/mL;
FIG. 3 is a graph showing a control of the determination of the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide RS12 against Aeromonas hydrophila. Wherein A: antibacterial peptide concentration 0 μg/mL; b: the concentration of the antibacterial peptide is 62.5 mug/mL; c: the concentration of the antibacterial peptide is 125 mug/mL; d: the concentration of the antibacterial peptide is 250 mug/mL; e: the concentration of the antibacterial peptide is 500 mug/mL; f: the concentration of the antibacterial peptide is 1000 mug/mL;
FIG. 4 is a graph showing the time-kill profile of the antimicrobial peptide RS12 against Staphylococcus aureus. Wherein, (. A) blank; (■) MIC; (ζ) 2MIC;
FIG. 5 is a graph showing the time-dependent killing of the antibacterial peptide RS12 against Aeromonas hydrophila. Wherein, (. A) blank; (■) MIC; (ζ) 2MIC;
FIG. 6 is a transmission electron micrograph of Staphylococcus aureus. Wherein, (a) a blank group; (B) antimicrobial peptide RS 12-treated staphylococcus aureus;
FIG. 7 is a transmission electron microscope image of Aeromonas hydrophila. Wherein, (a) a blank group; (B) antibacterial peptide RS 12-treated Aeromonas hydrophila.
Detailed Description
It will be appreciated by those skilled in the art that the following examples are not intended to limit the scope of the invention and that any changes and modifications made on the basis of the present invention are within the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 antimicrobial peptide RS12
1. Fermentation of mulberry leaves
Pulverizing folium Mori, sieving with 40 mesh sieve, inoculating 2% bacterial solution into the pulverized folium Mori powder, diluting, adding pulverized folium Mori powder (water content of 50%), and stirring; sealing the fermentation bag by using a heat sealing machine, and fermenting at normal temperature for 5-7 d deg.C until the fermented material has sweet and sour fermentation smell.
Wherein, the mulberry leaves can be sun-dried mulberry leaves, and the moisture content and the source of the mulberry leaves are not particularly limited.
Wherein the bacterial liquid is a mixed bacterial liquid of lactobacillus, bacillus subtilis and bacillus licheniformis, the mixing proportion is not limited, and 1 can be adopted in one embodiment of the invention: 1:1, and mixing the components in a proportion of 1.
2. Synthesis of peptide fragments
The peptide fragments in the fermented mulberry leaves are detected by utilizing a liquid chromatography/mass spectrometry (LCMS) technology, and the specific method is as follows:
Accurately weighing the fermented mulberry leaf 1g fermented in the previous step, extracting protein by adopting an SDT (sodium dodecyl sulfate) cracking method, and quantifying the protein by adopting a BCA (broadcast and multicast) method. And desalting and freeze-drying, and taking the components with the size of more than 1000Da for on-machine testing.
Liquid phase conditions: timsTOF PRO systems (Bruker); the analytical Column was a C18 reverse phase chromatography Column (Thermo SCIENTIFIC EASY Column, 75 μm. Times.10 cm); mobile phase a was 0.1% formic acid, B was 84% acetonitrile and 0.1% formic acid, flow rate was 300 nL/min;
Mass spectrometry conditions: peptide fragment analysis was performed using timsTOF Pro mass spectrometers (Bruker), which perform full scan acquisitions (m/z 100-1700). And the library searching software is MaxQuant, and the mass spectrum results of the protein and peptide fragments are identified by using a Morus alba database in Uniprot to obtain 83 amino acid sequences.
3. Screening for antimicrobial peptides
The obtained 83 amino acid sequences are predicted by using antibacterial peptide prediction on-line servers APD3 and CAMP, according to the characteristics of the antibacterial peptide with typical antibacterial effect, namely the number of amino acids (< 50), total positive charges (+2 to +9) and the percentage of hydrophobic amino acids (> 30%), the highest-scoring peptide segment is screened according to the on-line server CAMP scoring, the amino acid sequence PEMRPSMGKVAK is finally screened out, chemical synthesis (synthesized by Beijing-middle-family sub-optical biotechnology Co., ltd.) is carried out, the synthesized peptide sequence consists of 12 amino acids, the total charge is +2, the hydrophobicity is 33%, and the antibacterial quality is 1330 Da, and is named as RS12.
Example 2 3D Structure prediction of antibacterial peptide RS12
The antibacterial peptide structure was predicted using the online server Swiss-model, while the resulting structure was edited and modified using pymol to obtain the secondary structure of antibacterial peptide RS12, as shown in FIG. 1.
Example 3 antibacterial test of antibacterial peptide RS12
1. Minimum Inhibitory Concentration (MIC) determination
Staphylococcus aureus and aeromonas hydrophila were incubated at 37 ℃ for 12h to log phase and diluted to 10 6-7 CFU/mL in 0.01M pH 7.2 phosphate buffer. Antibacterial peptide RS12 was dissolved in phosphate buffer and mixed with bacteria in an equal volume at 37 ℃ of 2h. As shown in FIGS. 2, 3 and 4, the Minimum Inhibitory Concentration (MIC) of RS12 for Staphylococcus aureus was 31.25 μg/mL and the Minimum Inhibitory Concentration (MIC) for Aeromonas hydrophila was 0.125 μg/mL.
2. Time kill Curve TIMEKILL determination
Staphylococcus aureus and aeromonas hydrophila were incubated at 37 ℃ for 12h to log phase and diluted to 10 3-4 CFU/mL in 0.01m phosphate buffer ph 7.2. MIC and 2 XMIC concentration peptides were mixed with bacteria at 37℃in equal volumes and incubated separately, plated plates were sampled every 30 minutes, and the total number of colonies was recorded after incubation at 37℃overnight. The result shows that the antibacterial peptide has obvious effect on staphylococcus aureus at 1.5 hours and on aeromonas hydrophila at 1 hour; and then continue to decrease in trend. Under the action of the antibacterial peptide, the bacterial count is reduced more rapidly. The antibacterial peptide RS12 has obvious inhibition effect on staphylococcus aureus and aeromonas hydrophila with the increase of the action time (figures 4 and 5).
3. Transmission electron microscope analysis
Bacteria at 10 6-7 CFU/mL were treated with 2×mic of RS12 at 37 ℃ for 2h and then centrifuged at 2700 g for 10min, washed twice with phosphate buffer (pH 7.2). After fixation with 1% osmium acid, dehydration was performed with 95% ethanol and then acetone treatment was performed on 20 min. The samples were baked 24 h at 70 ℃, sheets of 70-90 nm were prepared on copper grids and then stained with lead citrate and uranium acetate. Ultrastructural observation and capture were performed using an H-7650 transmission electron microscope.
As shown in fig. 6A, 7A, the intracellular tissue and structural integrity of bacterial cells was good for untreated bacteria. However, as shown in FIGS. 6B and 7B, after the peptide RS12 treatment, it can be seen that the bacterial cell membrane structure starts to blur with cavitation of the cells, the shape of the cells becomes irregular, the cell membranes completely collapse, and the cytoplasmic contents flow out. The result of a transmission electron microscope shows that the RS12 peptide has destructive effect on cell membranes and internal structures of staphylococcus aureus and aeromonas hydrophila.
In summary, the invention provides a brand new antimicrobial peptide RS12, wherein the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide RS12 on staphylococcus aureus is 31.25 mug/mL, and the Minimum Inhibitory Concentration (MIC) on aeromonas hydrophila is 0.125 mug/mL. Has strong inhibiting effect on staphylococcus aureus and aeromonas hydrophila, and has better thermal stability. The antibacterial peptide RS12 achieves the effect of inactivating bacteria by adsorbing on the surface of the bacteria, destroying the cell membrane of the bacteria and inhibiting the generation of the membrane to enable substances in the cells to extravasate.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Claims (6)
1. The amino acid sequence of the antibacterial peptide RS12 is shown as SEQ ID NO: 1.
2. The antimicrobial peptide RS12 of claim 1, wherein the molecular weight of the antimicrobial peptide is 1330 daltons.
3. Use of the antimicrobial peptide RS12 according to any one of claims 1 to 2 for inhibiting and/or killing staphylococcus aureus and aeromonas hydrophila.
4. The antimicrobial peptide RS12 of claim 1, wherein the antimicrobial peptide is derived from fermented mulberry leaves.
5. The antibacterial peptide RS12 of claim 4, obtained from fermented mulberry leaves comprising:
Inoculating mixed bacterial liquid of lactobacillus, bacillus subtilis and bacillus licheniformis into mulberry leaf powder, and fermenting to obtain fermented mulberry leaves;
analyzing peptide fragments in the fermented mulberry leaves, and screening to obtain the antibacterial peptide RS12.
6. The application of the antibacterial peptide RS12 is S1) or S2) or S3):
s1) application of preparing antibacterial drugs;
s2) preparing an aquatic feed additive;
s3) preparing an application of a food preservative;
wherein, the amino acid sequence of the antibacterial peptide RS12 is shown in SEQ ID NO: 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450590A (en) * | 2014-12-26 | 2015-03-25 | 山东宝来利来生物工程股份有限公司 | Bacillus subtilis with antibacterial activity and application of bacillus subtilis |
| CN104886117A (en) * | 2015-05-27 | 2015-09-09 | 华南师范大学 | Application of lepidoptera antibiotic peptide Lebocin to pest control |
| CN107446032A (en) * | 2017-07-03 | 2017-12-08 | 汕头大学 | A kind of antibacterial peptide and its application |
| CN111333700A (en) * | 2020-03-23 | 2020-06-26 | 集美大学 | Pseudosciaena crocea whey acidic protein antibacterial peptide and application thereof |
-
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- 2024-04-03 CN CN202410397116.7A patent/CN117986327B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450590A (en) * | 2014-12-26 | 2015-03-25 | 山东宝来利来生物工程股份有限公司 | Bacillus subtilis with antibacterial activity and application of bacillus subtilis |
| CN104886117A (en) * | 2015-05-27 | 2015-09-09 | 华南师范大学 | Application of lepidoptera antibiotic peptide Lebocin to pest control |
| CN107446032A (en) * | 2017-07-03 | 2017-12-08 | 汕头大学 | A kind of antibacterial peptide and its application |
| CN111333700A (en) * | 2020-03-23 | 2020-06-26 | 集美大学 | Pseudosciaena crocea whey acidic protein antibacterial peptide and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| DZIEWIT等: "Two novel temperate bacteriophages co-existing in Aeromonas sp. ARM81 - characterization of their genomes, proteomes, and DNA methyltransferases", 《 JOURNAL OF GENERAL VIROLOGY 》, vol. 97, no. 8, 31 December 2016 (2016-12-31), pages 2008 - 2022 * |
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