CN113943348A - Antibacterial peptide for bacterial tracing and application thereof - Google Patents
Antibacterial peptide for bacterial tracing and application thereof Download PDFInfo
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- CN113943348A CN113943348A CN202111201705.6A CN202111201705A CN113943348A CN 113943348 A CN113943348 A CN 113943348A CN 202111201705 A CN202111201705 A CN 202111201705A CN 113943348 A CN113943348 A CN 113943348A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
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- 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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Abstract
The invention relates to an antibacterial peptide for bacteria tracing and application thereof, belonging to the technical field of biological medicines. The amino acid sequence of the antibacterial peptide for bacteria tracing provided by the invention is shown in SEQ ID NO. 1. Can be used for bacteria tracing, the antimicrobial peptide has good biocompatibility, no obvious cytotoxicity and hemolysis even under the concentration of 1mg/mL, no obvious hepatorenal toxicity in vivo application, and no clinical UBI (UBI) currently applied29‑41Compared with the prior art, the biological compatibility and the bacteria tracing capability are better.
Description
Technical Field
The invention relates to an antibacterial peptide for bacteria tracing and application thereof, belonging to the technical field of biological medicines.
Background
Bacterial infections are the major diseases frequently occurring in clinic and complications after surgical operations, and particularly, due to the current abuse of antibiotics, antibiotic resistance is more and more common, so that the clinical treatment of bacterial infections is particularly troublesome. Early diagnosis of bacterial infection is of great significance to guide treatment. Traditional diagnosis of infection involves measuring the number of leukocytes or C-reactive protein (CRP) biomarkers, Erythrocyte Sedimentation Rate (ESR), procalcitonin, etc. Although these biomarkers have been widely used in the clinic and show good infection diagnosis efficiency. However, biomarkers seem to be less promising for biomaterial-related or periprosthetic infections, and there is currently no gold standard for diagnosing periprosthetic infections, as these biomarkers do not track bacteria directly (Yue B, Tang T., Nucl Med Commun.2015Apr; 36(4): 305-11). The in vivo bacterial tracing can carry out positioning and qualitative diagnosis on bacterial infection, the diagnosis is more accurate, and the positioning diagnosis of bacteria is beneficial to surgeons to make clinical decisions, so that the treatment efficiency of the bacterial infection is improved. The bacterial tracer comprises antibiotic, antibacterial peptide, and ubiquitin protein (UBI) 29-41) Is a cationic synthetic antibacterial peptide fragment containing 6 n-amino groups and preferentially binding to the anionic microbial cell membrane at the site of infection. Based on UBI29-41The diagnosis of infections or the targeting of bacterial tracking has been extensively studied (Aryana, K.; et al., Nuklermedizin. Nuclear medicine 2012,51, (4), 133-9). Recently, 99 mTc-ubiquitin (UBI 29-41) has been investigated as an aid to bone scanning for periprosthetic infection and aseptic loosening (Shinto, A.S, et al, Nuclear medicine communications 2017,38, (4), 285-290). Currently, antimicrobial peptides are extensively studied in bacterial tracking, on the one hand they can accumulate at the site of infection, rather than in sterile inflammatory lesions, because they bind bacteria and fungi preferentially to mammalian cells. On the other hand, the antimicrobial peptides maintain a broad antimicrobial spectrum, thereby eliminating bacteria. The currently widely used antibacterial peptide is UBI29-41. However UBI29-41Lack of specificity. Therefore, there is a need in the art to find more antibacterial peptides to compensate for UBI29-41Deficiency or function ofA substitute for bacterial tracers.
Disclosure of Invention
The invention aims to solve the technical problems of how to obtain a better antibacterial peptide and the application of the antibacterial peptide.
In order to solve the above problems, the technical scheme adopted by the present invention is to provide an antibacterial peptide for bacterial tracing, wherein the amino acid sequence of the antibacterial peptide is shown as SEQ ID No.1, and the amino acid sequence is: KRIVQRIKDFLR are provided.
The invention provides an application of antibacterial peptide for bacterial tracing in preparing a medicament for bacterial tracing.
The invention provides an application of antibacterial peptide for bacterial tracing in preparing a diagnostic kit for diagnosis and prognosis of bacterial infection.
The invention provides an application of antibacterial peptide for bacterial tracing in preparing a diagnostic kit for diagnosis and prognosis of aseptic inflammation.
The invention provides an application of antibacterial peptide for bacteria tracing in preparing antibacterial drugs.
Compared with the prior art, the invention has the following beneficial effects:
the invention synthesizes a novel antibacterial peptide and is used for bacteria tracing, the biocompatibility of the antibacterial peptide is good, no obvious cytotoxicity and hemolysis are caused even under the concentration of 1mg/mL, no obvious hepatorenal toxicity is caused in vivo application, and the antibacterial peptide has no obvious UBI applied to clinic at present29-41Compared with the prior art, the biological compatibility and the bacteria tracing capability are better.
Drawings
FIG. 1 shows KR-12 and UBI 29-41And KR-12 and UBI29-41A graph of the results of the cytotoxicity test;
wherein, A is the chemical structure diagram of KR-12; b is diagram of UBI29-41The chemical structure of (1); panel C is KR-12 cytotoxicity evaluation diagram; d is diagram of UBI29-41Cytotoxicity evaluation chart.
FIG. 2 is an in vitro assay for KR-12 and UBI29-41Affinity for staphylococcus aureus and escherichia coli: wherein A is quantitativeKR-12 and UBI29-41Efficiency of adhesion to the surface of s. Panel B shows quantitative KR-12 and UBI29-41Efficiency of adhesion to E.coli cell surface.
FIG. 3 is a flow cytometry quantification of FITC-KR-12 and FITC-UBI29-41Efficiency profile of adhesion to bacterial cell surface.
Wherein, A is FITC-KR-12 and FITC-UBI29-41Adherence to bacterial cell surface maps after incubation at 37 ℃ for 15 min, 30 min, 45 min, 60 min, 90 min, 120 min; panel B shows quantification of FITC-KR-12 and FITC-UBI29-41A graph of adhesion efficiency to the surface of staphylococcus aureus cells; FIG. C is a diagram for quantifying FITC-KR-12 and FITC-UBI29-41Coli bacterial cell surface adhesion efficiency map.
FIG. 4 shows KR-12 and UBI29-41In vitro haemolysis profile.
FIG. 5 shows in vivo FITC-UBI29-41And FITC-KR-12 bacteria in vivo tracing and identifying aseptic inflammation performance diagram;
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:
the invention provides an antibacterial peptide for bacteria tracing, and the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO. 1.
The invention provides an application of antibacterial peptide for bacterial tracing in preparing a medicament for bacterial tracing.
The invention provides an application of antibacterial peptide for bacterial tracing in preparing a diagnostic kit for diagnosis and prognosis of bacterial infection.
The invention provides an application of antibacterial peptide for bacterial tracing in preparing a diagnostic kit for diagnosis and prognosis of aseptic inflammation.
The invention provides an application of antibacterial peptide for bacteria tracing in preparing antibacterial drugs.
The invention provides an antibacterial peptide KR-12 for in vivo bacterial tracing, and researches the feasibility of the KR-12 peptide as a bacterial tracer in vitro and in vivo, thereby providing a novel tracer for bacterial diagnosis. And to evaluate the diagnostic value of KR-12 as a bacterial tracer in distinguishing bacterial infections from sterile inflammation.
The preparation method of the novel antibacterial peptide is a solid-phase polypeptide synthesis method, wherein an insoluble high polymer is used as a carrier, and one of reactants is fixed on the high polymer carrier through an active group of the carrier, so that organic synthesis is carried out on a solid phase. The method has the advantages of mature technology, simple process, high efficiency and low cost, and can synthesize the polypeptide with any monomer sequence.
Examples
The present invention is further described below in conjunction with the following detailed description and the accompanying drawings, it being understood that the following detailed description and/or the drawings are only illustrative of the invention and are not limiting.
1. Preparation of peptide: the antibacterial peptide KR-12 is synthesized by a solid-phase synthesis method, and has the amino acid sequence as follows: KRIVQRIKDFLR, each peptide was over 95% pure by HPLC analysis.
2. The amino acid arrangement of the peptide is shown in FIG. 1.
3. KR-12 cell activity and apoptosis studies are shown in FIG. 1:
MC3T3 was cultured in 10% FBS alpha-MEM medium at 37 ℃ in the presence of 5% CO2(ii) a Cells harvested in logarithmic growth phase at 5X 103Cell density of individual cells/well was seeded into 96-well plates. After 24 hours, UBI with different concentrations (1-1000 mu g/mL)29-41And KR-12 were added to the cells and incubated for 48 hours. Apoptosis was studied by flow cytometry.
4. FIG. 1 shows the result of cytotoxicity assay of antimicrobial peptide KR-12, UBI29-41Is a positive control. On day three, KR-12 concentrations up to 1000. mu.g/mL did not show any significant growth inhibition compared to the control without peptide. KR-12 showed cytotoxicity to mammalian cells at concentrations over 1000. mu.g/mL at day 5, and the experimental results showed that the antimicrobial peptide KR-12 has good biocompatibility in vitro.
5. The affinity research of the antimicrobial peptide KR-12 on staphylococcus aureus and escherichia coli comprises the following steps: for the study ofBacterial targeting ability of KR-12 peptide to Staphylococcus aureus and Escherichia coli at 1X 106CFU/mL was suspended in PBS and reacted with KR-12, UBI29-41(32. mu.g/ml) and PBS. After incubation at 37 ℃ for 15 minutes, 30 minutes, 1 hour, 2 hours. The bacteria were centrifuged and the peptide concentration of the supernatant was simply calculated and the amount of non-affine bacterial membrane peptide was determined using the biuret reagent. Briefly, different concentrations of Gly-Gly-Tyr-Arg peptide standard solutions (0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8mg/ml) were prepared, mixed with the biuret reagent for 10min, and the mixed solution was centrifuged at 2000r/min for 10 min. The absorbance of the supernatant at 540nm was measured using a microplate reader. And then obtaining a standard curve of the peptide concentration and the absorbance, and determining the non-covalent grafting peptide according to the standard curve. FIG. 2 shows that the bacterial affinity rate of KR-12 antibacterial peptide reaches 58.6 +/-2.8%, UBI29-41The bacterial affinity reaches 52.4 +/-3.1% at 90min, while the UBI29-41The highest affinity was reached at 60 min and KR-12 peptide reached the highest affinity at 90 min. The results show both KR-12 and UBI29-41Has the same affinity for Staphylococcus aureus and Escherichia coli in vitro, while UBI 29-41Showing a faster maximum bacterial binding concentration compared to KR-12, KR-12 shows a faster maximum bacterial binding concentration than UBI29-41Compared with higher affinity.
6. Detecting the affinity of the antimicrobial peptide KR-12 to staphylococcus aureus and escherichia coli by using a flow cytometry: staphylococcus aureus and Escherichia coli at 1X 106CFU/mL was suspended in PBS and mixed with FITC-KR-12, FITC-UBI29-41(32. mu.g/mL) and PBS were incubated in different suspensions. After incubation at 37 ℃ for 15 min, 30min, 45min, 60 min, 90 min, 120 min. The bacteria were centrifuged and resuspended in PBS. FITC-KR-12 and FITC-UBI29-41The efficiency of adhesion to the bacterial surface was quantified by flow cytometry. FIG. 3 shows that the antimicrobial peptide KR-12(KRIVQRIKDFLR-NH2) contains 5 positively charged amino acids (3 Rs: arginine and 2 Ks: lysine) as UBI29-41(TGRAKRRMQTNRR) contains 6 positively charged amino acids (5Rs arginine, 1K: lysine) as positive controls. FIG. 3 flow-through experimental results show KR-12-FITC and UBI29-41FITC has a better affinity for Staphylococcus aureus inKR-12-FITC and UBI within 10-30min29-41FITC has no obvious difference on the affinity of staphylococcus aureus, and the affinity is 45min-120minUBI29-41FITC is superior to KR-12-FITC. KR-12-FITC and UBI 29-41FITC has good affinity for G + and G-bacteria, KR-12 binds to E.coli in more amounts than UBI at each time point29-41-FITC. Therefore, the fluorescent tracer is a good tracer for targeting bacteria.
KR-12 hemolytic study: heparin-supplemented blood from 5 healthy individuals (after informed consent and approval by the institutional ethics committee) was collected and centrifuged at 2000rpm for 15 minutes. The erythrocytes were washed 3 times with PBS, centrifuged at 1000 Xg for 10 minutes, and suspended in PBS to 2% (v/v). Addition of KR-12 and UBI29-41To 5 red blood cell suspensions (final concentration 6.25-200 g/mL), incubated at 37 ℃ for 1 hour, and then centrifuged at 5 ℃ at 1000 Xg for 5 minutes. 200uL of the supernatant was transferred to a flat bottom 96 well plate and the release of hemoglobin was determined by absorbance at 450 nm. FIG. 4 shows the results of measurement of hemolytic activity of peptide. The results in the figure show that KR-12 did not show any significant hemolytic activity even at concentrations as high as 1000. mu.g/ml, indicating that KR-12, an antimicrobial peptide of the present invention, did not show hemolytic activity.
8. The antibacterial peptide KR-12 can distinguish aseptic inflammation and infection in vivo
A local infectious focus is made in the mouse axilla by using staphylococcus aureus and escherichia coli, and LPS is used as a focus of local inflammatory reaction. FITC-KR-12, FITC-UBI 29-41And normal saline is used as a control group, and local tissues are taken out at each time point to be frozen and sliced to observe the fluorescence intensity and the gram staining. FIG. 5 shows the results obtained in FITC-KR-12, FITC-UBI29-41FITC fluorescence can be seen after 2h and 4h of injection into the animal. Demonstration of FITC-KR-12, FITC-UBI29-41It is proved that the in vivo has the bacterial tracing performance and can distinguish aseptic inflammation.
9. In vivo hepatotoxicity test in rats
The acute toxicity of the antimicrobial peptides was studied in normal mice. Briefly, 30 mice were randomly divided into saline group, UBI29-41Three groups of groups and KR-12 groups, through the tail veinThe antimicrobial peptides were injected into mice. Blood samples were drawn from the orbit on day 7 post-injection to prepare serum samples and whole blood. And (3) detecting serum biochemical indexes of Blood Urea Nitrogen (BUN), creatinine (Cr), Uric Acid (UA) and other indexes of the blood sample. White blood cells and neutrophils were also measured from the whole blood samples. As shown in Table 1, the groups did not show any significant difference at day 7, indicating that the bacterial tracers KR-12 and UBI29-41Is safe.
TABLE 1 mice injected with NaCl, KR-12, UBI29-41Renal toxicity after 7 days, data are presented as mean ± standard deviation.
The invention synthesizes the antimicrobial peptide KR-12 which has good biocompatibility, no obvious cytotoxicity and hemolysis even under the concentration of 1mg/mL, no obvious hepatorenal toxicity in vivo application and no obvious UBI applied to clinic at present and is used for bacteria tracing 29-41Compared with the prior art, the compound has better biocompatibility and bacteria tracing capability.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Sequence listing
<120> antibacterial peptide for bacterial tracing and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg
1 5 10
Claims (5)
1. An antibacterial peptide for bacteria tracing is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. Use of an antimicrobial peptide according to claim 1 for bacterial tracking in the manufacture of a medicament for bacterial tracking.
3. Use of an antimicrobial peptide for bacterial tracking according to claim 1 for the preparation of a diagnostic kit for the diagnosis and prognosis of bacterial infections.
4. Use of an antimicrobial peptide for bacterial tracking according to claim 1 for the preparation of a diagnostic kit for the diagnosis and prognosis of sterile inflammation.
5. The use of an antimicrobial peptide according to claim 1 for bacterial tracking in the manufacture of an antimicrobial medicament.
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