CN116375805B - Polypeptide with antibacterial and osteogenic activities, preparation and application thereof - Google Patents
Polypeptide with antibacterial and osteogenic activities, preparation and application thereof Download PDFInfo
- Publication number
- CN116375805B CN116375805B CN202310306245.6A CN202310306245A CN116375805B CN 116375805 B CN116375805 B CN 116375805B CN 202310306245 A CN202310306245 A CN 202310306245A CN 116375805 B CN116375805 B CN 116375805B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- formulation
- antibacterial
- good
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 79
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 79
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 79
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 31
- 230000002188 osteogenic effect Effects 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 28
- 230000007547 defect Effects 0.000 claims abstract description 24
- 208000015181 infectious disease Diseases 0.000 claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 15
- 230000002458 infectious effect Effects 0.000 claims abstract description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims description 25
- 238000009472 formulation Methods 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 4
- 239000012669 liquid formulation Substances 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- -1 amine salt Chemical class 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 229940049920 malate Drugs 0.000 claims description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 2
- 239000002674 ointment Substances 0.000 claims description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 claims description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 19
- 229940079593 drug Drugs 0.000 abstract description 9
- 238000013461 design Methods 0.000 abstract description 7
- 241000894006 Bacteria Species 0.000 abstract description 6
- 230000008929 regeneration Effects 0.000 abstract description 6
- 238000011069 regeneration method Methods 0.000 abstract description 6
- 230000001737 promoting effect Effects 0.000 abstract description 5
- 239000004480 active ingredient Substances 0.000 abstract description 4
- 150000003384 small molecules Chemical class 0.000 abstract 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 13
- 241000194032 Enterococcus faecalis Species 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 9
- 241000222122 Candida albicans Species 0.000 description 8
- 206010041925 Staphylococcal infections Diseases 0.000 description 8
- 230000002949 hemolytic effect Effects 0.000 description 8
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 230000000845 anti-microbial effect Effects 0.000 description 6
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 206010059866 Drug resistance Diseases 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000011164 ossification Effects 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 2
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 101100328884 Caenorhabditis elegans sqt-3 gene Proteins 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000002924 anti-infective effect Effects 0.000 description 2
- 239000012867 bioactive agent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 229960003085 meticillin Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000010267 two-fold dilution method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- RSWGJHLUYNHPMX-UHFFFAOYSA-N 1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,6,10,10a-octahydrophenanthrene-1-carboxylic acid Chemical compound C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C(O)=O RSWGJHLUYNHPMX-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241001116468 Cunninghamia Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007621 bhi medium Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000010478 bone regeneration Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000002173 cutting fluid Substances 0.000 description 1
- 238000001804 debridement Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000797 effect on infection Effects 0.000 description 1
- 229940032049 enterococcus faecalis Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 208000004480 periapical periodontitis Diseases 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Rheumatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biological medicines, and discloses a polypeptide with antibacterial and osteogenic activities, a preparation and application thereof. The invention designs a polypeptide with antibacterial and osteogenic activities, and the amino acid sequence of the polypeptide is shown as any one sequence of SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3. The invention provides a preparation containing the polypeptide or the derivative of the polypeptide, and the application of the polypeptide and the preparation in preparing medicines for treating jawbone infection or maxillofacial infectious bone defects. The polypeptide has small molecules and good effect of resisting maxillofacial infectious bone defect related bacteria; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. The invention solves the antibacterial problem and regeneration promoting problem of maxillofacial infectious bone defect at the same time, and has new design concept and good clinical application potential.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a polypeptide with antibacterial and osteogenic activities, a preparation and application thereof.
Background
The maxillofacial infectious bone defect is one of the common diseases of the stomatology, is caused by various bacterial infections, such as the bone defect of the jawbone, the defect of the alveolar bone, the defect of periapical periodontitis, and the like, and is different from the common bone defect, the range of the bone defect can be continuously expanded due to the existence and the diffusion of the infection, and the local debridement combined with the implantation of bioactive materials is required to promote the healing of the bone defect and the application of antibiotics to control the infection in serious cases. Bone regeneration materials generally studied and used are focused on the induction regeneration of bone tissue, but lack the effect on infection control; the use of conventional antibiotics is also at risk of drug-resistant bacteria. Thus, there is a need for bioactive agents that have both antimicrobial and osteogenic properties. However, a large number of bioactive agents associated with bone defect repair have focused on promoting regeneration or anti-infection, and the usual agents are simply simple functional stacks of osteogenic material and antimicrobial components, and the usual bone defect repair materials do not have anti-infection effect, and antibiotics have the risk of causing drug resistance.
The antibacterial peptide is a polypeptide with antibacterial activity and derivatives thereof, is not easy to generate drug resistance, and can improve the antibacterial capability of the antibacterial peptide and enable the antibacterial peptide to have additional biological activity by modifying the sequence of the antibacterial peptide. Thus, engineering a polypeptide to have both antimicrobial and osteogenic activity is a potential means of achieving an "antimicrobial-osteogenic" dual-purpose formulation. However, the design of multifunctional polypeptides is difficult because the antimicrobial functional polypeptide fragment and the osteogenic functional polypeptide fragment may interfere with each other due to charge, hydrophilicity and hydrophobicity, structure, resulting in a decrease in antimicrobial and osteogenic capabilities. Few polypeptides claim to have antibacterial-osteogenic ability, but the effective concentration is high, the high concentration application is economical and the action efficiency is still further improved. Therefore, there is a need to develop a safe and effective polypeptide with the dual effects of antibacterial and osteogenesis so as to overcome the technical difficulty of the active preparation for infectious bone defect and apply the polypeptide to clinic.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a polypeptide with antibacterial and osteogenic activities, a preparation and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention designs a polypeptide with antibacterial and osteogenic activities, wherein the amino acid sequence of the polypeptide is shown as any one sequence of SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.
The polypeptide molecule with antibacterial and osteogenic activities is small, and the effect of resisting the bacteria related to maxillofacial infectious bone defects is good; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. The polypeptide provided by the invention simultaneously solves the antibacterial problem and regeneration promoting problem faced by maxillofacial infectious bone defect by a single molecule, and has new design concept and good clinical application potential.
In a second aspect, the invention provides a formulation having both antibacterial and osteogenic activity, said formulation comprising said polypeptide or a derivative of said polypeptide.
As a preferred embodiment of the formulation according to the invention, the derivative is an ester derivative.
As a preferred embodiment of the formulation according to the invention, the formulation further comprises a pharmaceutically acceptable salt.
Further, the pharmaceutically acceptable salt comprises at least one of hydrochloride, sulfate, acetate, mesylate, succinate, fumarate, citrate, malate, and organic amine salt.
As a preferred embodiment of the formulation according to the invention, the formulation further comprises a pharmaceutically acceptable carrier and/or adjuvant.
As a preferred embodiment of the formulation according to the invention, the formulation is a liquid formulation, a solid formulation or a semisolid formulation.
Further, the liquid preparation is a solution or an injection; the solid preparation is a tablet or a capsule; the semisolid preparation is ointment or gel.
In a third aspect, the invention applies the polypeptide and the preparation in preparing medicines for treating jawbone infection.
In a fourth aspect, the polypeptide and the preparation are applied to the preparation of drugs for maxillofacial infectious bone defects.
Compared with the prior art, the invention has the beneficial effects that:
the polypeptide molecule with antibacterial and osteogenic activities is small, and the effect of resisting the bacteria related to maxillofacial infectious bone defects is good; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. After the polypeptide is prepared into the antibacterial peptide medicine, compared with the traditional antibiotics, the antibacterial peptide medicine is difficult to cause drug resistance. The polypeptide provided by the invention simultaneously solves the antibacterial problem and regeneration promoting problem faced by maxillofacial infectious bone defect by a single molecule, and has new design concept and good clinical application potential.
Drawings
FIG. 1 is a helix pattern of polypeptides WL14, GK16, GL 14;
FIG. 2 is a mass spectrum of the prepared polypeptide WL 14;
FIG. 3 is a mass spectrum of the prepared polypeptide GK 16;
FIG. 4 is a mass spectrum of the prepared polypeptide GL 14;
FIG. 5 is a high performance liquid chromatogram of the prepared polypeptide WL 14;
FIG. 6 is a high performance liquid chromatogram of the prepared polypeptide GK 16;
FIG. 7 is a high performance liquid chromatogram of the prepared polypeptide GL 14;
FIG. 8 is a graph of the detection of hemolytic toxicity of polypeptide WL 14;
FIG. 9 is a graph showing the detection of hemolytic toxicity of the polypeptide GK 16;
FIG. 10 is a graph showing the detection of hemolytic toxicity of polypeptide GL 14;
FIG. 11 is the effect of polypeptide WL14 on BMSCs osteogenesis-related gene expression;
FIG. 12 is a graph showing the effect of polypeptide GK16 on BMSCs osteogenic related gene expression;
FIG. 13 shows the effect of polypeptide GL14 on BMSCs osteogenic related gene expression.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1: polypeptide with antibacterial and osteogenic activities
Designing a group of polypeptides with antibacterial and osteogenic activities, namely 'antibacterial osteogenic' polypeptides which are respectively marked as WL14, and the amino acid sequence of the polypeptides is WLKRWLRKLYKFGG (SEQ ID No. 1); GK16 with the amino acid sequence of GKLIWKLLRKAYKFGG-NH 2 (SEQ ID No. 2); GL14, amino acid sequence GLKRWLRKLYKFGG-NH 2 (SEQ ID No.3)。
A spiral wheel diagram of the polypeptides WL14, GK16, GL14 is shown in FIG. 1.
The preparation method of the polypeptide comprises the following steps:
(1) Fmoc-His (Trt) -Wang Resin is selected as Resin (carrier);
(2) The resin was fully swollen with Dichloromethane (DCM);
(3) The Fmoc-protecting group was removed with the appropriate concentration of DBLK (hexahydropyridine+DMF);
(4) Washing with N, N-Dimethylformamide (DMF) for several times to wash off DBLK;
(5) Weighing a proper condensing agent and an activator (HBTU, NMM) for coupling, wherein the second Fmoc-protected amino acid at the C terminal (Fomc-Leu-OH);
(6) The ninhydrin detection method ensures that the connection is complete;
(7) Washing with DMF for several times to remove residual residues and activator condensing agent;
(8) Coupling according to the amino acid sequences of the polypeptides WL14, GK16 and GL14, wherein the method refers to the steps (3) to (7);
(9) Removing the final Fmoc-protecting group by adopting the methods of the steps (3) and (4) after all amino acid connection is finished;
(10) Cracking with TFA cutting fluid, removing resin and amino acid protecting group to obtain crude product;
(11) Sending mass spectrum to confirm the correctness of the product;
(12) The crude product is sent to purification and separation, and the purity is improved.
Mass spectra of the prepared polypeptides WL14, GK16 and GL14 are shown in figures 2-4. The high performance liquid chromatograms of the prepared polypeptides WL14, GK16 and GL14 are shown in figures 5-7.
Example 2: determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of polypeptide
The minimum inhibitory concentration MIC and the minimum bactericidal concentration MBC are indicators of the antimicrobial activity of the drug, indicating the ability of the drug to inhibit and kill pathogenic microorganisms. The antibacterial ability of the polypeptides WL14, GK16 and GL14 prepared in example 1 on the common bacteria of maxillofacial infectious bone defects is detected by using a maxillofacial infectious bone defect common bacteria staphylococcus aureus (Staphylococcus aureus), methicillin-resistant staphylococcus aureus (Methicillin-resistant Staphylococcus aureus), periapical infection common bacteria enterococcus faecalis (Enterococcus faecalis) and candida Albicans (Canidia Albicans) through a microplate dilution method drug sensitivity test.
The method comprises the following specific steps:
(1) Picking the preserved single colony S.aureus and MRSA in 10mL LB liquid medium, and culturing E.faecalis in BHI medium at 37 ℃ by shaking table overnight; albicans were grown overnight in YPD medium at 35℃on a constant temperature shaker.
(2) Absorbing 20 μL of bacterial liquid in 10mL of corresponding culture medium, subculturing for 2 generations at 35 ℃ or 37 ℃ by a constant temperature shaking table, taking bacterial liquid in mid-log growth, diluting S.aureus and MRSA with MH culture medium, E.faecalis and C.albicans with RPMI 1640 culture medium containing 10% fetal bovine serum to 1×10 6 CFU/mL was ready for use.
(3) The polypeptides WL14, GK16, GL14 were added to 96-well plates using a 2-fold dilution method, respectively, at 10. Mu.L per well. 90 mu L of bacterial liquid is added into each hole to ensure that the final concentration of the antibacterial peptide is 2 mu g/mL-128 mu g/mL. Sterile water served as a negative control.
(4) The 96-well plate is placed in a constant temperature culture tank at 35 ℃ or 37 ℃ for culture for 24 hours.
(5) MIC is the lowest antimicrobial peptide concentration of the plate-inside clearing wells.
(6) 50 mu L of bacterial liquid in the clarification hole is sucked, S.aureus and MRSA are coated on an LB agar plate, E.faecalis is coated on a BHI agar plate, C.albicans is coated on a YPD agar plate, and the culture is carried out for 24 hours at a constant temperature of 35 ℃ or 37 ℃. MBC is the lowest polypeptide concentration for sterile colony growth on plates.
As shown in Table 1, the polypeptides WL14, GK16 and GL14 have low MIC and MBC for S.aureus, MRSA, E.faecalis and C.albicans, and have good antibacterial capability between 8 mug/mL and 32 mug/mL.
TABLE 1 MIC and MBC of polypeptides WL14, GK16, GL14 vs S.aureus, MRSA, E.faecalis, C.albicans
WL14 | MIC(μg/mL) | MBC(μg/mL) |
S.aureus | 16.00±0.00 | 24.00±8.00 |
MRSA | 16.00±0.00 | 24.00±8.00 |
E.faecalis | 32.00±0.00 | 32.00±0.00 |
C.albicans | 16.00±0.00 | 16.00±0.00 |
GK16 | MIC(μg/mL) | MBC(μg/mL) |
S.aureus | 16.00±0.00 | 32.00±0.00 |
MRSA | 16.00±0.00 | 32.00±0.00 |
E.faecalis | 32.00±0.00 | 32.00±0.00 |
C.albicans | 10.67±3.77 | 16.00±0.00 |
GL14 | MIC(μg/mL) | MBC(μg/mL) |
S.aureus | 32.00±0.00 | 32.00±0.00 |
MRSA | 32.00±0.00 | 32.00±0.00 |
E.faecalis | 32.00±0.00 | 32.00±0.00 |
C.albicans | 8.00±0.00 | 12.00±4.00 |
Note that: MIC and MBC are expressed as mean ± standard deviation of six independent replicates
Example 3: haemolytic toxicity of Polypeptides
The hemolytic test is an important index for commonly testing the safety of the antibacterial peptide, and the polypeptides WL14, GK16 and GL14 prepared in example 1 are taken for performing a hemolytic toxicity test to evaluate the biosafety.
The method comprises the following specific steps:
(1) Defibrinated sheep blood was centrifuged at 1000rpm for 10min, the supernatant was discarded, washed with PBS and centrifuged again to obtain red blood cells. Erythrocytes were resuspended with ten volumes of PBS to obtain a suspension of erythrocytes.
(2) The polypeptides WL14, GK16, GL14 were each added to the EP tube by a 2-fold dilution method, 100. Mu.L per tube. 900. Mu.L of the red blood cell suspension was added to each well to give a final concentration of the antimicrobial peptide of 8. Mu.g/mL to 128. Mu.g/mL. Another 100. Mu.L of 1% Triton X-100 was used as positive control and 100. Mu.L of PBS was used as negative control. Incubate at 37℃for 1h. After centrifugation at 1000rpm for 10min, the supernatant was taken and the OD was measured 540 。
Percent hemolysis = (OD 540 processing group -OD 540 negative control group )/(OD 540Triton X-100 -OD 540 negative control group )×100%。
As shown in FIGS. 8-10, different lower case letters and Roman numerals indicate that there is a statistical difference, P<0.05。OD 540 Can characterize the lytic action of the polypeptides on the mammal red blood cells, and the hemolytic toxicity of the three polypeptides for treating the mammal red blood cells for 1h is extremely weak at the concentration of 64 mug/mL or below, and is only below 4%; the three polypeptides only showed a significant hemolytic activity at a high concentration of 128. Mu.g/mL, with a hemolysis rate of WL14 of 20% and GL14 and GK16 of about 10%. The three polypeptides are proved to have good biological safety under the antibacterial concentration.
Example 4: effect of polypeptide on bone marrow mesenchymal Stem cell (BMSCs) osteogenic Gene expression
The effect of the polypeptides WL14, GK16 and GL14 prepared in example 1 on the expression of BMSCs bone formation related genes ALP, col-1, OCN, OPN, runx2 and BSP was examined, and the potential of the polypeptides to promote bone was evaluated.
The method comprises the following specific steps:
(1) BMSCs were inoculated in 12-well plates using DMEM/F-12 medium containing diabody, 10% fetal calf serum in CO 2 In incubator (5% CO) 2 Culturing at constant temperature of 95% air, 100% humidity and 37 ℃).
(2) Cultured adherent cells were changed daily for fresh medium containing the polypeptide. The polypeptides WL14, GK16 and GL14 are subjected to gradient dilution by using a culture medium, wherein the concentration range is 8-32 mug/mL of antibacterial concentration; the negative control group used a medium without antibacterial peptide. Cells were cultured for 14 days.
(3) Cells were harvested after 14 days and subjected to real-time quantitative PCR to measure the expression of osteogenic related genes. RNA rapid extraction kit (Yi Cunninghamia, shanghai, china) is used for separating and purifying RNA. RNA concentration and purity were measured using a NanoDrop 2000 (Thermo Scientific, USA). cDNA was synthesized using PrimeScript reverse transcription kit (TaKaRa, japan) containing gDNA Eraser. Using cDNA as templateqPCR/>Green Master Mix (next holy, shanghai, china) in +.>480-II (Roche, switzerland) was subjected to real-time quantitative PCR. GADPH is used as an internal reference, 2 -ΔΔCt And calculating the gene expression fold difference by a method.
As shown in fig. 11-13, different lowercase letters indicate that there was a statistical difference in gene expression for this group, P <0.05. Under the antibacterial concentration, the polypeptides WL14, GK16 and GL14 have obvious up-regulation on the expression of BMSCs osteogenesis related genes ALP, col-1, OCN, OPN, runx and BSP, which shows that the polypeptide has the antibacterial effect and simultaneously has the bone-promoting capability and the antibacterial-osteogenesis double effect.
In conclusion, the polypeptide molecule with antibacterial and osteogenic activities is small, and the effect of resisting maxillofacial infectious bone defect related bacteria is good; the biological safety is good, and the bone potential is good; good activity, low concentration of active ingredients, good effect and low cost required by clinical application. After the polypeptide is prepared into the antibacterial peptide medicine, compared with the traditional antibiotics, the antibacterial peptide medicine is difficult to cause drug resistance. The polypeptide provided by the invention simultaneously solves the antibacterial problem and regeneration promoting problem faced by maxillofacial infectious bone defect by a single molecule, and has new design concept and good clinical application potential.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (9)
1. A polypeptide with antibacterial and osteogenic activities, which has the amino acid sequence of WLKRWLRKLYKFGG, GKLIWKLLRKAYKFGG-NH 2 、GLKRWLRKLYKFGG-NH 2 Any sequence shown in the specification.
2. A formulation having both antibacterial and osteogenic activity, wherein the formulation comprises the polypeptide of claim 1.
3. The formulation of claim 2, further comprising a pharmaceutically acceptable salt.
4. The formulation of claim 3, wherein the pharmaceutically acceptable salt comprises at least one of a hydrochloride, sulfate, acetate, mesylate, succinate, fumarate, citrate, malate, organic amine salt.
5. The formulation of claim 2, further comprising a pharmaceutically acceptable carrier and/or adjuvant.
6. The formulation of claim 2, wherein the formulation is a liquid formulation, a solid formulation, or a semi-solid formulation.
7. The formulation of claim 6, wherein the liquid formulation is a solution or an injection; the solid preparation is a tablet or a capsule; the semisolid preparation is ointment or gel.
8. Use of the polypeptide of claim 1, the formulation of any one of claims 2 to 7 in the manufacture of a medicament for treating a jawbone infection.
9. Use of the polypeptide of claim 1, the formulation of any one of claims 2 to 7, in the manufacture of a medicament for treating maxillofacial infectious bone defects.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310306245.6A CN116375805B (en) | 2023-03-27 | 2023-03-27 | Polypeptide with antibacterial and osteogenic activities, preparation and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310306245.6A CN116375805B (en) | 2023-03-27 | 2023-03-27 | Polypeptide with antibacterial and osteogenic activities, preparation and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116375805A CN116375805A (en) | 2023-07-04 |
CN116375805B true CN116375805B (en) | 2023-09-08 |
Family
ID=86962757
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310306245.6A Active CN116375805B (en) | 2023-03-27 | 2023-03-27 | Polypeptide with antibacterial and osteogenic activities, preparation and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116375805B (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3526235A1 (en) * | 2016-10-12 | 2019-08-21 | Feldan Bio Inc. | Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same |
CN112646026A (en) * | 2020-12-24 | 2021-04-13 | 四川大学 | Lactoferrin-based bionic antibacterial functional polypeptide and preparation method and application thereof |
CN113717254A (en) * | 2021-09-23 | 2021-11-30 | 中山大学附属口腔医院 | Antibacterial peptide and application thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103360464A (en) * | 2013-07-17 | 2013-10-23 | 武汉摩尔生物科技有限公司 | Polypeptide, DNA molecule encoding polypeptide, vector, preparation method and application thereof |
-
2023
- 2023-03-27 CN CN202310306245.6A patent/CN116375805B/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3526235A1 (en) * | 2016-10-12 | 2019-08-21 | Feldan Bio Inc. | Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same |
CN112646026A (en) * | 2020-12-24 | 2021-04-13 | 四川大学 | Lactoferrin-based bionic antibacterial functional polypeptide and preparation method and application thereof |
CN113717254A (en) * | 2021-09-23 | 2021-11-30 | 中山大学附属口腔医院 | Antibacterial peptide and application thereof |
Non-Patent Citations (1)
Title |
---|
Targeting cariogenic pathogens and promoting competitiveness of commensal bacteria with a novel pH-responsive antimicrobial peptide;Wentao Jiang等;Journal of Oral Microbiology;第15卷;1-17 * |
Also Published As
Publication number | Publication date |
---|---|
CN116375805A (en) | 2023-07-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2018086516A1 (en) | Antibacterial peptide of inhibiting drug-resistant bacteria and application thereof | |
CN109369792B (en) | Antibacterial peptide and application thereof | |
CN110950947B (en) | Rana temporaria chensinensis host defense peptide DMS-PS2, and gene and application thereof | |
US9090655B2 (en) | Low hemolytic antimicrobial peptide, pharmaceutical composition and use thereof | |
CN110156875B (en) | Antibacterial peptide H5-p5, and preparation method and application thereof | |
CN104031135B (en) | Rhacophorus defense peptides PopuDef and gene thereof and application | |
CN113321708B (en) | Preparation of artificially designed antibacterial peptide and application of artificially designed antibacterial peptide in aquatic products | |
CN113583103B (en) | Antibacterial peptide HeHamp I (67-92) and application thereof | |
CN116375805B (en) | Polypeptide with antibacterial and osteogenic activities, preparation and application thereof | |
WO2011120359A1 (en) | Low hemolytic antimicrobial peptide, pharmaceutical composition and use thereof | |
JP2013521330A (en) | Peptide compounds useful as antibacterial agents | |
CN113045628B (en) | Application of antibacterial peptide or variant thereof in preparation of antibacterial product | |
CN112646026B (en) | Lactoferrin-based bionic antibacterial functional polypeptide and preparation method and application thereof | |
CN111253474B (en) | Antibacterial peptide RG-27 and application thereof | |
CN116804046B (en) | Cyclic cation antibacterial peptide and application thereof | |
CN113527458B (en) | Antibacterial peptide HeHamp II-2 (63-86) and application thereof | |
CN113880933B (en) | Antibacterial peptide SsNKL27 and application thereof | |
CN115819544A (en) | Antibacterial peptide and preparation method and application thereof | |
CN113698464B (en) | Antibacterial peptide HeHamp II-4 (63-88) and application thereof | |
CN105777875B (en) | Antibacterial peptide CSTC24 and application thereof | |
CZ285281B6 (en) | Minor components of the group a of streptogramines | |
CN102250216A (en) | Rana nigromaculata antimicrobial peptide as well as gene and application thereof | |
CN106432459B (en) | Antibacterial peptide of hypsizigus marmoreus, gene thereof and application thereof in pharmacy | |
CN106496317B (en) | Rana chensinensis secretory peptide, gene thereof and application thereof in pharmacy | |
KR20200071416A (en) | Poecilocorisin-1 peptide isolated from Poecilocoris lewisi or antimicrobial, antimycotic and antiinflammatory composition comprising it |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |