CN116535481A - Hirudinaria manillensis antibacterial peptide RK22, precursor protein thereof and application - Google Patents
Hirudinaria manillensis antibacterial peptide RK22, precursor protein thereof and application Download PDFInfo
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- CN116535481A CN116535481A CN202310664955.6A CN202310664955A CN116535481A CN 116535481 A CN116535481 A CN 116535481A CN 202310664955 A CN202310664955 A CN 202310664955A CN 116535481 A CN116535481 A CN 116535481A
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- antibacterial peptide
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- 241000500851 Poecilobdella manillensis Species 0.000 title claims abstract description 28
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a Hirudo manillensis antibacterial peptide RK22, a precursor protein and application thereof, and belongs to the technical field of functional peptides. The invention provides a Hirudinaria manillensis antibacterial peptide RK22, the amino acid sequence of which is shown as SEQ ID NO. 1. The antibacterial peptide RK22 is obtained by screening saliva of Hirudinaria manillensis, is an antibacterial peptide which has no procoagulant activity, no cytotoxicity, low hemolytic activity and strong plasma stability, has excellent bactericidal effect on staphylococcus aureus including methicillin-resistant staphylococcus aureus, escherichia coli, acinetobacter baumannii and the like, is an ideal candidate medicament for resisting infection of staphylococcus aureus, acinetobacter baumannii, escherichia coli and the like, and has good safety.
Description
Technical Field
The invention belongs to the technical field of functional peptides, and particularly relates to a Hirudinaria manillensis antibacterial peptide RK22, a precursor protein thereof and application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus, s.aureus) is a gram-positive bacterium with a carrier rate of between 20-30% in healthy people, which can infect almost all host tissues, leading to serious morbidity and mortality. Staphylococcus aureus activates coagulation, and bacterial surface proteins and exotoxins promote thrombosis through the action on coagulation pathways and anticoagulation factors, which are important causes of exacerbation and even death of patients. Antibiotics have been the main therapeutic agents of staphylococcus aureus. However, due to the phenomenon of antibiotic abuse, more and more clinical strains of antibiotic resistance are present. Among them, methicillin-resistant staphylococcus aureus (MRSA) is called "superbacteria", and studies by the world health organization indicate that MRSA infected persons have a mortality rate that is 64% higher than other bacteria. The antibacterial peptide has high-efficiency and rapid sterilization capability (the time for killing bacteria of most antibacterial peptides is less than 1 minute) which is far less than that of the traditional antibiotics (the time for killing bacteria of most antibiotics is more than 3 hours), so that the rapid sterilization characteristic makes the antibacterial peptide difficult to cause the drug resistance of microorganisms, and therefore, the antibacterial peptide becomes an ideal substitute for the antibiotics.
Animal toxins are an important source of antimicrobial peptide molecules, and the environment in which leeches live and the blood sucking process of the leeches are inevitably infected by microorganisms. With advances in bioinformatics, molecular biology, etc., a variety of novel antimicrobial peptide molecules have been identified successively from salivary glands of theromoyzonatosum, hirudo medicinalis, whereas antimicrobial peptide molecules have not been identified in saliva of Poecilobdella manillensis.
Disclosure of Invention
In view of the above, the present invention aims to provide a Hirudinaria manillensis antibacterial peptide RK22 having antibacterial properties.
The invention provides a Hirudinaria manillensis antibacterial peptide RK22, the amino acid sequence of which is shown as SEQ ID NO. 1.
The invention provides a precursor protein of a Hirudinaria manillensis antibacterial peptide RK22, and the amino acid sequence of the precursor protein is shown as SEQ ID NO. 2.
The invention provides a gene for encoding a precursor protein of a Hirudinaria manillensis antibacterial peptide RK22, and the nucleotide sequence of the gene is shown as SEQ ID NO. 3.
The invention provides a broad-spectrum antibacterial bactericide which comprises the Hirudinaria manillensis antibacterial peptide RK22 and auxiliary materials.
The invention provides application of the Hirudinaria manillensis antibacterial peptide RK22 in preparation of antibacterial drugs.
Preferably, the bacteria to which the antibacterial agent is directed include at least one of: staphylococcus (Staphylococcus), acinetobacter (Acinetobacter) and Escherichia (Escherichia).
Preferably, the staphylococcus genus comprises staphylococcus aureus;
the Acinetobacter genus comprises Acinetobacter baumannii;
the genus Escherichia includes Escherichia coli.
Preferably, the bacteria against which the antimicrobial agent is directed further include clinically resistant bacteria.
Preferably, the clinically resistant bacteria comprise a methicillin-resistant staphylococcus aureus clinically resistant strain.
Preferably, the methicillin-resistant staphylococcus aureus clinical resistant strain comprises at least one of the following: MRSA-Z, MRSA11, MRSA22, SA220823 and SA15772.
The amino acid sequence of the Hirudinaria manillensis antibacterial peptide RK22 provided by the invention is shown as SEQ ID NO. 1. According to the invention, the antibacterial peptide RK22 which does not have procoagulant activity, has no cytotoxicity, low hemolytic activity and strong plasma stability is screened from saliva of Poecilobdella manillensis, has excellent bactericidal effect on staphylococcus aureus including methicillin-resistant staphylococcus aureus, escherichia coli, acinetobacter baumannii and the like, is an ideal candidate medicament for resisting infection of staphylococcus aureus, acinetobacter baumannii, escherichia coli and the like, and has good safety.
Drawings
FIG. 1 shows the bactericidal kinetics of the antibacterial peptide RK22 against Staphylococcus aureus (left) and MRSA-Z (right);
FIG. 2 shows the cytotoxicity results of the antibacterial peptide RK 22;
FIG. 3 shows the results of the hemolytic activity of the antibacterial peptide RK 22;
FIG. 4 shows the effect of the antibacterial peptide RK22 on thrombin, FXa activity;
FIG. 5 is a graph showing the effect of the antibacterial peptide RK22 on platelet activation, wherein A is a flow chart of results and B is a statistical analysis of the results of the A chart;
FIG. 6 is a graph showing the effect of the antibacterial peptide RK22 on the survival rate of mice;
FIG. 7 shows the effect of the antibacterial peptide RK22 on tissue damage.
Detailed Description
The invention provides a Hirudinaria manillensis antibacterial peptide RK22, the amino acid sequence of which is shown as SEQ ID NO. 1 (RKYKEKKDKSQNKKKKRKCMIL). The antibacterial peptide RK22 has a length of 22 amino acid residues and a net charge: +10, hydrophobicity: -0.316, polar amino acid residue (n/%): 17/77.27, non-polar amino acid residue (n/%): 5/22.73.
The invention provides a precursor protein of the Hirudinaria manillensis antibacterial peptide RK22, and the amino acid sequence of the precursor protein is shown as SEQ ID NO. 2 (MTEYKLVVVGAGGVGKSALTIQLIQNHFVEEYDPTIEDSY RKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCIFAVNNAKSF EDINQYREQIKRVKDADEVPMVLVGNKVDLPSRNVDTKLARSKAESFTIP YVETSAKTRQGVDDAFFTLVREIRKYKEKKDKSQNKKKKRKCMIL). The precursor protein is subjected to enzyme cleavage to obtain the antibacterial peptide RK22.
The invention provides a gene for encoding a precursor protein of the Hirudinaria manillensis antibacterial peptide RK22, and the nucleotide sequence of the gene is shown as SEQ ID NO. 3 (ATGACAGAGTATAAGCTAGTTGTTGTTGGAG CTGGTGGTGTTGGAAAAAGTGCACTGACAATTCAACTTATTCAAAACCACTTCGTTGAAGAGTATGATCCAACCATTGAGGATTCATATAGAAAGCAAGTTGTCATTGATGGAGAAACCTGCCTTTTGGACATTCTCGATACTGCAGGTCAAGAAGAATACAGTGCAATGAGGGATCAATACATGAGGACAGGAGAGGGATTTCTGTGCATATTTGCTGTCAACAATGCAAAGTCATTTGAAGATATTAACCAGTATAGAGAACAAATTAAGAGAGTAAAAGATGCTGATGAAGTTCCTATGGTTTTGGTTGGCAACAAGGTTGATCTACCATCAAGAAATGTTGACACCAAATTAGCAAGGTCTAAAGCAGAGTCCTTTACGATTCCTTATGTCGAGACTTCTGCAAAGACCCGACAGGGAGTCGATGATGCCTTCTTCACTTTAGTCAGGGAAATTCGAAAATATAAGGAGAAAAAAGATAAAAGCCAAAACAAGAAAAAGAAGAGGAAATGCATGATTCTGTGA).
The invention provides a broad-spectrum antibacterial bactericide which comprises the Hirudinaria manillensis antibacterial peptide RK22 and auxiliary materials.
In the invention, the Hirudinaria manillensis antibacterial peptide RK22 is used as an active substance to play an antibacterial and bactericidal role. The content of the Hirudinaria manillensis antibacterial peptide RK22 is preferably 0.005-0.15% by mass, and more preferably 0.00625% by mass. The antibacterial and bactericidal auxiliary materials are not particularly limited, and auxiliary materials of antibacterial products known in the art can be adopted. The preparation method of the broad-spectrum antibacterial bactericide is not particularly limited, and the preparation method of antibacterial products known in the art can be adopted.
The invention provides application of the Hirudinaria manillensis antibacterial peptide RK22 in preparation of antibacterial drugs.
In the present invention, the bacteria to which the antibacterial agent is directed preferably include at least one of the following: staphylococcus (Staphylococcus), acinetobacter (Acinetobacter) and Escherichia (Escherichia). The staphylococcus genus preferably comprises staphylococcus aureus; the acinetobacter genus preferably comprises acinetobacter baumannii. The genus Escherichia preferably includes Escherichia coli. The bacteria against which the antimicrobial agent is directed preferably also include clinically resistant bacteria. The clinically resistant bacteria preferably comprise a methicillin-resistant staphylococcus aureus clinically resistant strain. The methicillin-resistant staphylococcus aureus clinical resistant strain comprises at least one of the following components: MRSA-Z, MRSA11, MRSA22, SA220823 and SA15772.
In the embodiment of the invention, experiments show that the minimum inhibitory concentrations (minimal inhibit concentration, MIC) of RK22 on staphylococcus aureus, escherichia coli and acinetobacter baumannii are respectively as follows: 6.25 mug/ml, 25 mug/ml and 100 mug/ml, and in addition, RK22 has excellent bactericidal effect on clinical drug-resistant bacteria such as methicillin-resistant staphylococcus aureus and the like, and the minimum inhibitory concentration MIC is 6.25 mug/ml to 12.5 mug/ml; RK22 has rapid sterilization, no hemolytic activity, no cytotoxicity, no acute toxicity and no procoagulant activity, RK22 is a novel safe and effective antibacterial peptide molecule, has the characteristics of simple structure, convenient artificial synthesis, strong antibacterial activity and the like, can be used as a means for resisting infection of staphylococcus aureus (including clinical resistant bacteria), escherichia coli, acinetobacter baumannii and the like, and is applied to treatment of infection of staphylococcus aureus (including clinical resistant bacteria), escherichia coli, acinetobacter baumannii and the like of humans and other animals.
The following examples are provided to illustrate the invention in detail with respect to Hirudinaria manillensis antibacterial peptide RK22 and its precursor proteins and applications, but they should not be construed as limiting the scope of the invention.
Example 1
Screening method of Hirudinaria manillensis antibacterial peptide RK22
Saliva from Poecilobdella manillensis was collected and sent to Northhouse for transcriptome sequencing, ORF (open reading frame) with complete sequence was predicted based on assembled high quality transcriptome data, proteins with a potential antibacterial effect were simultaneously sequenced after cleavage of the signal peptide by SignalP, sequences of <80aa (amino acids) were screened and uploaded as Fasta files to Antimicrobial Peptide Scanner vr.2 (https:// www.dveltri.com/ascan/v 2/index.html),
and initially obtaining a predicted antibacterial peptide sequence. Comparing the predicted antibacterial peptide sequence with the existing antibacterial peptide library, eliminating sequences identical to or highly homologous to the library, further manually screening the antibacterial peptide sequence, and finally obtaining the polypeptide sequence with potential antibacterial activity in the transcriptome. The activity of RK22 against E.coli was predicted to be 0.52 and the activity against Staphylococcus aureus was predicted to be 0.48 using Antimicrobial Peptide Scannervr.2 (https:// www.dveltri.com/ascan/v 2/index.html).
Example 2
Antibacterial activity of Hirudinaria manillensis (Poecilobdella manillensis) -derived antibacterial peptide RK22 on standard strains
1. RK22 polypeptide was synthesized chemically, RK22 polypeptide (SEQ ID NO: 1) was synthesized artificially by solid phase Fmoc chemistry, and verified by reverse phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. Specifically, GL biochemical company (Shanghai China Co.) was commissioned for synthesis.
2. In vitro antibacterial experiments
The antibacterial effect of RK22 polypeptide on staphylococcus aureus, acinetobacter baumannii, escherichia coli and pseudomonas aeruginosa is studied by adopting a minimum inhibitory concentration (minimal inhibit concentration, MIC) measuring method, and the specific method for measuring the MIC is as follows: first 20 is toMu.l of 200. Mu.g/ml peptide solution was added to the first column of wells, 100. Mu.l of physiological saline was added to the subsequent wells, 100. Mu.l was aspirated from the first column and added to the second column of wells, 100. Mu.l was aspirated again after mixing and added to the third column of wells, and so on, and then 100. Mu.l of 2X 10 were added to each well, respectively 5 CFU/ml bacteria liquid, the final concentration of peptide was 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.125 μg/ml, 1.563 μg/ml, 0.781 μg/ml, 0 μg/ml, 3 replicates per group, and blank control group (1640 medium was added without bacteria liquid) was set. And (3) placing the 96-well plate into a constant temperature incubator at 37 ℃ for overnight culture, and detecting the absorbance at 600nm by using an enzyme-labeled instrument, wherein the MIC value is the peptide concentration of the well in which the bacterial growth cannot be detected.
As a result, as shown in Table 1, RK22 showed no significant antimicrobial activity against Staphylococcus aureus, escherichia coli, acinetobacter baumannii at 100. Mu.g/ml, with MIC of 6.25. Mu.g/ml, 25. Mu.g/ml, 100. Mu.g/ml, respectively.
TABLE 1 MIC determination of the antibacterial peptide RK22 for four Standard strains
Example 3
Antibacterial Activity of antibacterial peptide RK22 against clinical drug-resistant Staphylococcus aureus
The antibacterial effect of RK22 on clinically resistant strains of Staphylococcus aureus was studied using MIC assay, experimental procedure see example 1, vancomycin as positive control.
TABLE 2 antibacterial effect of RK22 on methicillin-resistant Staphylococcus aureus
As can be seen from Table 2, the MIC of RK22 polypeptide was 6.25. Mu.g/ml for both MRSA-Z, MRSA11 and MRSA22, and 12.5. Mu.g/ml for RK22 polypeptide versus SA220823 and SA15772.
Example 4
Sterilization kinetics study of antibacterial peptide RK22 on staphylococcus aureus and MRSA-Z
The bactericidal kinetics of RK22 were determined as follows: regulating Staphylococcus aureus and MRSA-Z bacterial liquid to 2×10 5 CFU/ml, then dividing the bacterial liquid into several parts by volume in 96-well plate according to time, 100 μl each, adding 100 μl of RK22 solution each, respectively, to make the final concentration be 1×mic, 5×mic, 10×mic, respectively, the positive control drug being vancomycin, the negative control group being physiological saline and bacterial liquid, mixing, 3 replicates each. Placing the plate into a constant temperature incubator at 37 ℃, taking out bacterial solutions at 0, 15min, 30min, 60min, 180min and 360min respectively, mixing bacterial solutions uniformly, discharging 20 mu l of bacterial solutions into 180 mu l of physiological saline, mixing uniformly, taking 20 mu l of bacterial solutions, coating the plate, placing the plate into the constant temperature incubator at 37 ℃ for inverted culture overnight, counting single bacterial colonies on the plate the next day, and calculating the bacterial numbers under different drug treatments along with the time.
The results are shown in FIG. 1, which shows a faster bactericidal effect than vancomycin against both Staphylococcus aureus and MRSA-Z.
Example 5
Cytotoxicity of the antibacterial peptide RK22
In order to determine the safety of the antibacterial peptide RK22, its toxicity to HEK293T cells was first determined by the following specific experimental methods: the cell suspension concentration was adjusted to 1X 10 6 After adding 100. Mu.l of cell suspension per well to a sterile 96-well plate, the outermost wells of the sterile 96-well plate are easily evaporated, so 100. Mu.l of PBS solution was added to maintain the humidity in the plate, 5% CO 2 Culturing overnight in a constant temperature cell incubator at 37deg.C to adhere cells, sucking the culture medium with a waste liquid suction pump after the cell adhesion is completed, adding 200 μl of RK22 polypeptide solution diluted with culture medium at different concentrations of 400, 200, 100, 50, 12.5, 6.25 and 0 μg/ml, respectively, taking 10% DMSO as positive control, and setting blank control hole (containing culture medium and CCK8, no cells and R)K22 A) is provided; placing 5% CO 2 Culturing in a constant temperature incubator at 37 ℃ for 24 hours, taking out the 96-well plate, adding 10 μl of CCK8 solution into each well, and mixing well; and (5) continuously placing the cells into an incubator for incubation for 1-4 hours, and detecting the absorbance value at 450nm of the 96-well plate by using an enzyme-labeled instrument. Cell viability was calculated according to formula I.
Cell viability (%) = (experimental group absorbance-blank group absorbance)/(positive control group absorbance-blank group absorbance) ×100% formula I
The experimental results are shown in FIG. 2, where RK22 is still not cytotoxic at concentrations up to 400. Mu.g/ml.
Example 6
Hemolytic Activity of antibacterial peptide RK22
The haemolytic activity of the antibacterial peptide RK22 was determined by the following method: the eyeballs of the mice were bled, fresh blood of the mice was collected with EDTA anticoagulation tube, then 1000g was centrifuged for 5min, the supernatant was discarded, and erythrocytes were collected. Then washing the red blood cells with physiological saline for 3 times, and no longer showing red color on the supernatant; the erythrocytes were resuspended in physiological saline, and a 10% suspension of erythrocytes was prepared. Thereafter, 96-well plates were prepared, each well containing 100. Mu.l of RK22 solution of varying concentration and 100. Mu.l of red blood cell suspension, with RK22 final concentrations of 400, 200, 100, 50, 25, 12.5, 6.25 and 0. Mu.g/ml, respectively. Taking 1% TritonX-100 solution as positive control, and then placing the EP tube in a 37 ℃ incubator for co-incubation for 1h; the 96-well plate was removed, centrifuged at 1000g for 10min, and 150. Mu.l of the supernatant was transferred to a new 96-well plate, and absorbance at 540nm was measured by an enzyme-labeled instrument. Sterile saline and 1% Triton X-100 represent 0% and 100% hemolysis, respectively. The haemolysis rate was calculated according to formula II.
Hemolysis ratio (%) = (experimental group absorbance-negative control group absorbance)/(positive control group absorbance-negative control group absorbance) ×100% formula II
As shown in FIG. 3, the RK22 hemolytic activity is lower than 1% within 400 μg/ml, which indicates that RK22 has no toxicity to erythrocytes and can be used for sterilizing mammals.
Example 7
Plasma stability of the antibacterial peptide RK22
The plasma stability of the antibacterial peptide RK22 was determined as follows: adding RK22 polypeptide of 5mg/ml into blood plasma, incubating in a constant temperature incubator at 37 ℃, taking out a part of blood plasma/RK 22 mixed solution respectively at 0, 2, 4, 6, 8 and 10 hours, dissolving RK22 into 200 mug/ml by using physiological saline, and measuring the MIC of RK22 to staphylococcus aureus and drug-resistant bacteria by adopting a double gradient dilution method to determine whether the MIC value of a sample to be tested is improved after the RK22 and the blood plasma are incubated together, so as to influence the antibacterial activity of the sample.
As shown in Table 3, the MIC values of RK22 against Staphylococcus aureus and drug-resistant bacteria were not affected by 10h incubation in plasma, indicating that RK22 exhibited relatively stable properties in plasma.
TABLE 3 plasma stability of polypeptide RK22
Example 8
Effect of the antibacterial peptide RK22 on the coagulation System
The coagulation system consists of a coagulation cascade and platelets, and the excessive activation of the coagulation system induced by staphylococcus aureus infection and thus the generation of thrombus are main causes for death of patients, so that the influence of the antibacterial peptide RK22 on the coagulation cascade and the platelet activation is detected, and the specific experimental method is as follows: (1) Effect of RK22 on thrombin and FXa Activity: in a 96-well plate, 5 μl of thrombin (0.1056 μg/ml) or FXa (3 μg/ml) solution was added to each well, then 5 μl of RK22 solution or LL-37 solution was added respectively, the final concentrations were 1000nM, 200nM and 40nM respectively, incubation was performed at room temperature for 10min, physiological saline was used as a negative control, LL-37 (delegated to be synthesized by GL Biochemical Co (Shanghai China Co., sequence LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was used as a positive control; each well was added with 90. Mu.l of a substrate solution of thrombin (S-223845. Mu.g/ml) or FXa (S-222290. Mu.g/ml) dissolved in an enzyme kinetic buffer, and the 96-well plate was immediately placed in a microplate reader, and absorbance was read every 1min at 405nm for 90min.
(2) Effect of RK22 on platelet activation: taking blood from eyeballs of mice, mixing whole blood of each mouse with 800 mu l of desk type A liquid, and centrifuging 150g of the mixture for 10min by a horizontal rotor centrifuge at room temperature; taking the supernatant, and centrifuging for 5min at room temperature by a 400g angle rotor centrifuge. Discarding the supernatant, and re-suspending the sediment by using table A solution; discarding the supernatant, and re-suspending and diluting the sediment to a required volume by using table type B liquid for later use; to the isolated platelets, 1. Mu.g/ml of anti-CD 62P-PE was added, and after incubation for 10min, RK22 and LL-37 were added at different concentrations (20, 10, 5, 2.5. Mu.g/ml), respectively, and the blank was added with an equal amount of physiological saline, and the proportion of positive platelets was detected by flow cytometry after 1h, each of 50000 cells was detected.
As shown in FIGS. 4 and 5, RK22 does not have significantly enhanced thrombin and FXa activity or induce platelet activation compared to the endogenous antimicrobial peptide LL-37, and therefore RK22 has better safety than the endogenous antimicrobial peptide LL-37.
Example 9
Acute toxicity of antibacterial peptide RK22
Acute toxicity of RK22 was tested by the following experiment: mice were divided into 3 groups, RK22 low dose group: tail vein injection of 200 μl RK22 (20 mg/kg), RK22 high dose group: tail vein injection of 200 μl RK22 (40 mg/kg), blank control: the tail vein was injected with an equal amount of physiological saline, 8 per group. The living state and the survival state of the mice are continuously observed within 96 hours after administration, the mice are deeply anesthetized by isoflurane after 96 hours, the mice are dissected and liver, lung, spleen and kidney tissues are collected, paraffin sections and Hematoxylin-Eosin staining (HE) are manufactured after fixation, and the damage condition of the tissues of the mice is observed.
Results As shown in FIGS. 6 and 7, the RK22 tail vein injection mice did not suffer from death, and the tissue sections of the mice were HE stained without obvious tissue damage. Thus RK22 is not acutely toxic.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The Hirudinaria manillensis antibacterial peptide RK22 is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A precursor protein of the hirudin antibacterial peptide RK22 of claim 1, wherein the amino acid sequence is shown in SEQ ID No. 2.
3. A gene encoding a precursor protein of the hirudin antibacterial peptide RK22 of claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 3.
4. A broad-spectrum antibacterial bactericide, which is characterized by comprising the Hirudinaria manillensis antibacterial peptide RK22 as claimed in claim 1 and auxiliary materials.
5. The use of the hirudin antibacterial peptide RK22 of claim 1 in the preparation of an antibacterial medicament.
6. The use according to claim 5, wherein the bacteria to which the antibacterial agent is directed comprise at least one of: staphylococcus (Staphylococcus), acinetobacter (Acinetobacter) and Escherichia (Escherichia).
7. The use according to claim 6, wherein the staphylococcus genus comprises staphylococcus aureus;
the Acinetobacter genus comprises Acinetobacter baumannii;
the genus Escherichia includes Escherichia coli.
8. The use of claim 6, wherein the bacteria against which the antimicrobial agent is directed further comprise clinically resistant bacteria.
9. The use according to claim 8, wherein the clinically resistant bacteria comprise a methicillin-resistant staphylococcus aureus clinically resistant strain.
10. The use according to claim 8, wherein the methicillin-resistant staphylococcus aureus clinical resistant strain comprises at least one of the following: MRSA-Z, MRSA11, MRSA22, SA220823 and SA15772.
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