CN109485701A - Antibacterial peptide, antibacterial drug and preparation method - Google Patents
Antibacterial peptide, antibacterial drug and preparation method Download PDFInfo
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- CN109485701A CN109485701A CN201811431760.2A CN201811431760A CN109485701A CN 109485701 A CN109485701 A CN 109485701A CN 201811431760 A CN201811431760 A CN 201811431760A CN 109485701 A CN109485701 A CN 109485701A
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 229940124350 antibacterial drug Drugs 0.000 title abstract 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 39
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 32
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 20
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000011347 resin Substances 0.000 claims description 30
- 229920005989 resin Polymers 0.000 claims description 30
- 229920001184 polypeptide Polymers 0.000 claims description 27
- 239000002994 raw material Substances 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 claims description 12
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 12
- 229940088710 antibiotic agent Drugs 0.000 claims description 11
- 150000003053 piperidines Chemical class 0.000 claims description 10
- 230000000903 blocking effect Effects 0.000 claims description 8
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 claims description 7
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 7
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 6
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- SWZCTMTWRHEBIN-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(O)C=C1 SWZCTMTWRHEBIN-QFIPXVFZSA-N 0.000 claims description 5
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 241000222122 Candida albicans Species 0.000 abstract description 18
- 241000589517 Pseudomonas aeruginosa Species 0.000 abstract description 18
- 229940095731 candida albicans Drugs 0.000 abstract description 18
- 241000588724 Escherichia coli Species 0.000 abstract description 17
- 230000002401 inhibitory effect Effects 0.000 abstract description 8
- 239000003814 drug Substances 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 23
- 239000000523 sample Substances 0.000 description 18
- 239000000243 solution Substances 0.000 description 16
- 239000000843 powder Substances 0.000 description 14
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 244000131844 Dendrobium aphyllum Species 0.000 description 9
- 150000001413 amino acids Chemical group 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 239000012530 fluid Substances 0.000 description 8
- 235000015097 nutrients Nutrition 0.000 description 8
- 230000001408 fungistatic effect Effects 0.000 description 7
- 235000014655 lactic acid Nutrition 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 6
- 102000007079 Peptide Fragments Human genes 0.000 description 5
- 108010033276 Peptide Fragments Proteins 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108700032080 Hyalophora cecropia cecropin D Proteins 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 125000003345 AMP group Chemical group 0.000 description 1
- VPSHHQXIWLGVDD-ZLUOBGJFSA-N Asp-Asp-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O VPSHHQXIWLGVDD-ZLUOBGJFSA-N 0.000 description 1
- USENATHVGFXRNO-SRVKXCTJSA-N Asp-Tyr-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 USENATHVGFXRNO-SRVKXCTJSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241001647783 Lactobacillus amylolyticus Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229920006227 ethylene-grafted-maleic anhydride Polymers 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1021—Tetrapeptides with the first amino acid being acidic
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- 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
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Animal Behavior & Ethology (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an antibacterial peptide, an antibacterial drug and a preparation method thereof, and relates to the technical field of antibacterial peptides. The amino acid sequence of the antibacterial peptide disclosed by the invention is shown in SEQ ID NO.1 or SEQ ID NO. 2. The antibacterial peptide has antibacterial effect on Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The compound can be used for preparing antibacterial drugs, such as drugs for inhibiting pseudomonas aeruginosa, escherichia coli or candida albicans, and has wide application prospects.
Description
Technical field
The present invention relates to antibacterial peptide technical fields, in particular to a kind of antibacterial peptide, antibacterials and preparation side
Method.
Background technique
Drug resistance is the imminent threat being effectively prevented and treated to bacterium infection, and there is an urgent need to take the antibiotic of substitution to fight
Slightly.Antibacterial peptide (AMPs) may be the promising substitute of current antibiotic or improve antibiotic effect as adjuvant.
Antibacterial peptide is basic polypeptide in vivo with antibacterial activity, is that most of organisms resist the natural of pathogen invasion
The important component of defense mechanism.Antibacterial peptide has extensive inhibiting effect, has good answer in agricultural, medicine and other fields
Use prospect.
Summary of the invention
The purpose of the present invention is to provide a kind of antibacterial peptides, and the antibacterial peptide is to pseudomonas aeruginosa, Escherichia coli and white
Candida albicans has fungistatic effect.
Another object of the present invention is to provide a kind of antibacterials, the antibacterials with above-mentioned antibacterial peptide be activity at
Point, there is fungistatic effect to pseudomonas aeruginosa, Escherichia coli and Candida albicans.
Another object of the present invention is to provide a kind of method for preparing above-mentioned antibacterial peptide, this method can be made above-mentioned anti-
Bacterium peptide, obtained antibacterial peptide have fungistatic effect to pseudomonas aeruginosa, Escherichia coli and Candida albicans.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of antibacterial peptide, amino acid sequence such as SEQ ID NO.1 or SEQ ID NO.2
It is shown.
The present invention is from using Dendrobium aphyllum (Roxb.) C. E. Fisch. as raw material, using lactic acid bacteria to separate, purify, through surveying in the product that strain ferments
Sequence identifies two kinds of peptides, and base acid sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.And by experimental verification, this two
Kind peptide all has good fungistatic effect to pseudomonas aeruginosa, Escherichia coli and Candida albicans.
Wherein, peptide shown in SEQ ID NO.1, to the minimum of pseudomonas aeruginosa, Escherichia coli and Candida albicans
Mlc (MIC) is respectively as follows: 18.08,2.26,1.13mg/mL.
Peptide shown in SEQ ID NO.2 is antibacterial dense to the minimum of pseudomonas aeruginosa, Escherichia coli and Candida albicans
Degree (MIC) is respectively as follows: 4.44,8.87,2.22mg/mL.
Two kinds of above-mentioned antibacterial peptides can be used for preparing antibacterials, such as is used to prepare and inhibits pseudomonas aeruginosa, big
The drug of enterobacteria or Candida albicans has wider application prospect.
On the other hand, the application the present invention provides above-mentioned antibacterial peptide in preparation antibacterials.
For example, above-mentioned is anti-based on antibacterial peptide to the fungistatic effect of pseudomonas aeruginosa, Escherichia coli and Candida albicans
Bacterium peptide can be used for preparing the drug for inhibiting pseudomonas aeruginosa, Escherichia coli or Candida albicans.It can be the preparation of antibacterials
Raw material provides broader selection.
On the other hand, the present invention provides a kind of antibacterials, contain above-mentioned antibacterial peptide and pharmaceutically acceptable
Carrier.
Based on antibacterial peptide to the fungistatic effect of pseudomonas aeruginosa, Escherichia coli and Candida albicans, above-mentioned antimicrobial
Also having the same fungistatic effect of the object to pseudomonas aeruginosa, Escherichia coli and Candida albicans.
On the other hand, the present invention provides a kind of preparation methods for preparing above-mentioned antibacterial peptide comprising: toward equipped with resin
Polypeptide shown in the following raw material synthesis SEQ ID NO.1: Fmoc-Tyr (otbu)-OH, Fmoc-Asp is sequentially added in reactor
(otbu)-OH, Fmoc-Asp (otbu)-OH and Fmoc-Asp (otbu)-OH；
Alternatively, sequentially adding polypeptide shown in the following raw material synthesis SEQ ID NO.2 into the reactor equipped with resin:
Fmoc-Asp (otbu)-OH, Fmoc-Asp (otbu)-OH, Fmoc-Tyr (otbu)-OH and Fmoc-Asp (otbu)-OH.
Further, in some embodiments of the present invention, it after raw material is added every time, proceeds as follows:
Step (a): reactor is placed in shaking table and is reacted；
Step (b): piperidine solution is added into reactor and is washed to slough Fmoc blocking group, then with DMF.
Further, in some embodiments of the present invention, in step (a), the temperature control of shaking table is 29-31
DEG C, reaction time control is 90-150min.
Further, in some embodiments of the present invention, in step (b), piperidine solution contains piperidines and DMF.
Further, in some embodiments of the present invention, the volume ratio of piperidines and DMF are 1:4.
Further, in some embodiments of the present invention, when raw material is added into reactor at the 1st time, by DCM and
DIEA is added together with this added raw material into reactor；
In the 2nd any primary addition raw material into reactor into the 4th, HOBT and DIC is added with this time
The raw material added is added into reactor together.
Further, in some embodiments of the present invention, it is added after raw material, carries out into reactor at the 1st time
Following operation: methanol solution is added into reactor and is closed.
The present invention provides preparation methods to prepare above-mentioned antibacterial peptide using the method for Peptide systhesis, has easy to operate, institute
The antibacterial peptide obtained has the characteristics that purity is high, the rate of recovery are high.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the HPLC testing result of the freeze-dried powder sample of the DDDY polypeptide synthesized in embodiment 2；
Fig. 2 is the MS testing result of the freeze-dried powder sample of the DDDY polypeptide synthesized in embodiment 2；
Fig. 3 is the HPLC testing result of the freeze-dried powder sample of the DYDD polypeptide synthesized in embodiment 3；
Fig. 4 is the MS testing result of the freeze-dried powder sample of the DYDD polypeptide synthesized in embodiment 3.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
Fermented by lactic acid bacteria prepares antibacterial peptide
1. sample preparation
The new stem of Dendrobium aphyllum (Roxb.) C. E. Fisch. is cut off into leaf and root, obtains Dendrobium aphyllum (Roxb.) C. E. Fisch. scapus to constant weight using drying machine drying afterwards.
It is pulverized into powder with pulverizer, and leads to and sieve with 100 mesh sieve, obtain Dendrobium aphyllum (Roxb.) C. E. Fisch. powder.
2. microorganism solid fermentation
By selecting lactic acid bacteria Lactobacillus amylolyticus 6 and being sent out as microbe in solid state to existing research
Yeast-like fungi kind.First by MRS broth bouillon 54g/L concentration configure, and with autoclave sterilize (121 DEG C, 15min).It will save
Recover in -40 DEG C of bacterial strains, after the frozen bacteria that has melted 50 μ l instill in 25mlMRS broth bouillon, sealed with sealing strip,
It avoids pollution, is placed in 37 DEG C of incubators and cultivates 48 hours.Lactic acid bacteria after being recovered with sterile water wash.By the lactic acid bacteria after culture
It is centrifuged (3000r/min, 10min, room temperature) 2 times, outwells supernatant every time, after 25ml sterile water wash is added, outwell supernatant
And 10ml sterile water is added, take its suspension stand-by.The dry Dendrobium aphyllum (Roxb.) C. E. Fisch. powder of 20g is respectively placed in 4 culture dishes, is used
Distilled water wetting to keep 50% water content, and sterilizes (121 DEG C, 15min) in autoclave.After be inoculated with sterile sampling
(10% inoculum concentration), lactic acid bacteria suspension is uniformly sprinkling upon and has been soaked in Dendrobium aphyllum (Roxb.) C. E. Fisch. sample powder, sealed with sealing strip in order to avoid
Pollution, is placed in 37 DEG C of incubator 48 hours.
3. the extraction of the thick polypeptide of Dendrobium aphyllum (Roxb.) C. E. Fisch.
After fermentation, it by sample in an oven with 60 DEG C of dryings, and is stored in -20 DEG C of refrigerators Celsius immediately.Afterwards with
The solid-liquid ratio of 1:10 is dissolved in distilled water, is centrifuged until completely dissolved using centrifuge using the magnetic stirring apparatus 30min that stirs
(3500r/min, 10min) 3 times takes supernatant every time and adds water centrifugation again, save under the conditions of -40 DEG C, rear to use freeze-drying
Machine freeze-drying, obtains Dendrobium aphyllum (Roxb.) C. E. Fisch. polypeptide study, and be stored in -20 DEG C of refrigerators Celsius immediately.
4.DEAE Sepharose-FF anion-exchange chromatography
Ion column can be separated according to the electrically charged difference of polypeptide institute during elution.The study that freeze-drying is obtained
Be dissolved in distilled water with 10mg/ml concentration, use pure water respectively, the sodium chloride solution of 0.05mol/L, 0.15mol/L to sample into
Row elution.First 15ml sample solution is slowly instilled in pillar along tube wall, after start to collect, keep pump revolution be 26.8, collect
60 pipes, and its absorbance is surveyed in 220nm using spectrophotometer.It is every to have eluted one group of sample, first rinsed with 4mol/L sodium chloride
Ion column 100 is managed, then is managed with distilled water flushing ion column 100.Obtained Dendrobium aphyllum (Roxb.) C. E. Fisch. polypeptide is named as DPP.
5.HPLC-MS/MS analysis
Use amino acid sequence in high-resolution ultrahigh pressure liquid phase chromatography characterization DPP component.Appropriate DPP component is taken, is used
Ultrapure water configures 1mg/ml solution, and crosses the miillpore filter of 0.22um.20ul solution is taken to inject chromatographic column (Agilent SB-
C18RRHD, 1.8um, 2.1 × 50mm).It is carried out with the formic acid water (Mobile phase B) of acetonitrile (mobile phase A) and 0.1% as mobile phase
Gradient elution.Gradient are as follows: 0-2-5-7-8-9-10min, 90-75-75-15-15-90-90%A.Detection wavelength 220nm,
Flow velocity 0.2ml/min.The peptide fragment of elution pass through electrospray interface spray to tandem mass spectrometer (Bruker Daltonik GmbH,
Germany).In terms of acquisition parameter, Source Type ESI is scanned using cation, and drying heater is set as 180 DEG C.Scanning
Range is 50-1000 (m/z).Peptide sequence is analyzed with Proteomic Toolkit software combination manual calculations.
6. analyzing result
2 kinds of peptides are identified, amino acid sequence is respectively: DDDY (SEQ ID NO.1) and DYDD (SEQ ID NO.2).
Embodiment 2
According to the sequence of aforementioned polypeptides, synthesis polypeptide DDDY.
1. calculating the weight of every kind of raw material according to the weight of target polypeptides.
2. 5g resin is put into 150ml reactor, and 50ml DCM is added and impregnates 2 hours
3. washing resin with DMF, then drain, be so repeated four times, resin is drained.
4. weighing 0.02molFmoc-Tyr (tBu)-OH (first amino acid of C-terminal)+10ml DCM+5ml DIEA addition
Into reactor, then reactor is placed in 30 DEG C of shaking table and is reacted 2 hours.
5. closing (methanol: DIEA:DCM=1:1:2) half an hour with 50ml methanol solution, then washed four times, is taken out with DMF
It is dry.
6. 20% piperidine solution of 50ml (piperidines/DMF=1:4) is added into reactor, Fmoc blocking group is sloughed.It is de-
It is washed four times after complete protection with DMF, is then drained.
7. a small amount of resin is taken to be detected with ninhydrin method, resin has color, illustrates to be deprotected successfully.
8. weighing 0.06mlFmoc-Asp (otbu)-OH (second amino acid of C-terminal)+20ml HOBT+10ml DIC addition
Into reactor, then reactor is placed in 30 DEG C of shaking table and is reacted 1 hour.
9. a small amount of resin is taken to detect, detected with ninhydrin method, if resin has color, illustrates condensation not exclusively, the reaction was continued.
If resin be it is colourless, illustrate fully reacting；After complete reaction, it is washed resin four times with DMF, is then drained.
10. a certain amount of 20% piperidines (piperidines/DMF=1:4) is added into reactor, it is placed on decolorization swinging table and rocks
20min sloughs the Fmoc blocking group on resin with this.It is washed four times after having taken off protection with DMF, then drains detection protection
Whether slough.
11. a small amount of resin is taken to be detected with ninhydrin method, resin has color, illustrates to be deprotected successfully.
12. sequentially adding Fmoc-Asp (otbu)-OH+HOBT+DIC and Fmoc-Asp (otbu)-OH according to step 8-11
+ HOBT+DIC raw material connects amino acid.
13. with cutting reagent by polypeptide blocking group complete resection, and cutting down from resin, purifying is sent.
14. target peptide fragment is separated with impurity by high performance liquid chromatograph device (HPLC), by 500mg target peptide fragment DDDY
It is lyophilized into powder, and send QC quality inspection.Testing result is shown in Fig. 1 and Fig. 2.Fig. 1 shows the freeze-dried powder sample of DDDY polypeptide
HPLC testing result, Fig. 2 show the MS testing result of the freeze-dried powder sample of DDDY polypeptide.
Embodiment 3
According to the sequence of aforementioned polypeptides, synthesis polypeptide DYDD.
1. calculating the weight of every kind of raw material according to the weight of target polypeptides.
2. 5g resin is put into 150ml reactor, and 50ml DCM is added and impregnates 2 hours
3. washing resin with DMF, then drain, be so repeated four times, resin is drained.
4. weighing 0.02molFmoc-Asp (otbu)-OH (first amino acid of C-terminal)+10ml DCM+5ml DIEA addition
Into reactor, then reactor is placed in 30 DEG C of shaking table and is reacted 2 hours.
5. closing (methanol: DIEA:DCM=1:1:2) half an hour with 50ml methanol solution, then washed four times, is taken out with DMF
It is dry.
6. 50ml20% piperidine solution (piperidines/DMF=1:4) is added into reactor, Fmoc blocking group is sloughed.It has taken off
It is washed four times after protection with DMF, is then drained.
7. a small amount of resin is taken to be detected with ninhydrin method, resin has color, illustrates to be deprotected successfully.
8. weighing 0.06molFmoc-Asp (otbu)-OH (second amino acid of C-terminal)+20ml HOBT+10ml DIC to add
Enter into reactor, then reactor is placed in 30 DEG C of shaking table and is reacted 1 hour.
9. a small amount of resin is taken to detect, detected with ninhydrin method, if resin has color, illustrates condensation not exclusively, the reaction was continued.
If resin be it is colourless, illustrate fully reacting；After complete reaction, it is washed resin four times with DMF, is then drained.
10. a certain amount of 20% piperidines (piperidines/DMF=1:4) is added into reactor, it is placed on decolorization swinging table and rocks
20min sloughs the Fmoc blocking group on resin with this.It is washed four times after having taken off protection with DMF, then drains detection protection
Whether slough.
11. a small amount of resin is taken to be detected with ninhydrin method, resin has color, illustrates to be deprotected successfully.
12. sequentially adding 0.06mol Fmoc-Tyr (otbu)-OH+20ml HOBT+10ml DIC according to step 8-11
With 0.06mol Fmoc-Asp (otbu)-OH+20mlHOBT+10ml DIC raw material, amino acid is connected.
13. with cutting reagent by polypeptide blocking group complete resection, and cutting down from resin, purifying is sent.
14. being separated target peptide fragment with impurity by high performance liquid chromatograph device (HPLC), target peptide fragment DYDD is lyophilized
At powder, and send QC quality inspection.Testing result is shown in Fig. 3 and Fig. 4.Fig. 3 shows the HPLC inspection of the freeze-dried powder sample of DYDD polypeptide
It surveys as a result, Fig. 4 shows that DYDD is lyophilized into the MS testing result of powder sample.
Experimental example
By measuring cecropin D DDY, DYDD minimal inhibitory concentration, to reflect its antibacterial effect, minimal inhibitory concentration is inspection
The minimum sample concentration of bacterial growth is not detected.Using doubling dilution, the specific method is as follows:
1. the culture of strain
1.1 bacterium
(1) bacterium (pseudomonas aeruginosa or Escherichia coli) of slant preservation actication of culture: is inoculated into the TSB of 200mL
In fluid nutrient medium, cultivated for 24 hours in 37 DEG C, the shaking table of 180rmp.
(2) single colonie is obtained: by the microbionation through overactivation in TSA culture medium (plate), in 37 DEG C of constant temperature incubations
It is cultivated for 24 hours in case.
(3) cultivate single colonie: picking single bacterium is fallen in 4ml TSB fluid nutrient medium, is trained in 37 DEG C, the shaking table of 180rmp
Support 14h.
(4) dilute bacterium number: the single colonie that 1ml has been cultivated is inoculated in the TSB fluid nutrient medium for having diluted 100 times,
6h is cultivated in 37 DEG C, the shaking table of 180rmp.
1.2 yeast (Candida albicans)
(1) actication of culture: by the SDB fluid nutrient medium of the microbionation of slant preservation to 200mL, at 35 DEG C,
It is cultivated for 24 hours in the shaking table of 180rmp.
(2) single colonie is obtained: by the microbionation through overactivation in SDA culture medium (plate), in 35 DEG C of constant temperature incubations
It is cultivated for 24 hours in case.
(3) cultivate single colonie: picking single bacterium is fallen in 4mlSDB fluid nutrient medium, is trained in 35 DEG C, the shaking table of 180rmp
Support 14h.
(4) dilute bacterium number: the single colonie that 1ml has been cultivated is inoculated in the SDB fluid nutrient medium for having diluted 100 times,
6h is cultivated in 35 DEG C, the shaking table of 180rmp.
2. the preparation of sample to be tested
Polypeptide made from sample, that is, embodiment by freezing is dissolved as certain concentration with distilled water, with 0.22 micron of needle
Hole filter membrane and pin hole are once filtered, and 1ml are added in first, second centrifuge tube in the sample filtered, the
The TSB fluid nutrient medium that 1ml is added in two centrifuge tubes mixes, and 1ml is drawn in second centrifuge tube, the centrifugation of third root is added
Guan Zhong is added 1mlTSB fluid nutrient medium in third root centrifuge tube and mixes, and so on.
3. drug sensitive experiment
(1) preparation of inoculum: bacterium and yeast are diluted to 1x10 with TSB and SDB respectively6Cfu/ml (OD600 0.1,
Then fungi concentration is 1.5x108cfu/ml)。
(2) preparation of culture dish: 1. pouring into configured solid medium in plate, after waiting culture mediums to solidify, with shifting
Liquid rifle draws the above-mentioned bacterium solution 0.5mL diluted with culture medium, bacterium solution is uniformly coated on spreader consolidating of having solidified
Body media surface.Static 10 minutes, after bacterium solution is fully absorbed by solid medium, 3 Oxford cups are put into training with tweezers
Ware is supported, it is 3-5 minutes static, prevent the Oxford cup in mobile culture dish from sliding.100 microlitres are added to each Oxford cup with liquid-transfering gun
Sample liquid.
2. negative control is arranged: each Oxford cup on Micro-Organism Culture Dish is separately added into 100 microlitres of TSB culture mediums；Yeast
Each Oxford cup on culture dish is separately added into 100 microlitres of SDB culture mediums
3. blank control is arranged: being added without bacterium solution in solid medium, directly placement Oxford cup
(3) it cultivates: the culture dish that bacterium liquid is added is cultivated into 12h at 37 DEG C；The culture dish of yeast juice will be added 35
12h is cultivated at DEG C.
(4) it measures: accurately measuring the diameter of transparent antibacterial circle with vernier caliper.
Table 1: minimal inhibitory concentration table of the cecropin D DDY to each bacterium
| Tested thallus | DDDY MIC(mg/mL) |
| Pseudomonas aeruginosa | 18.08 |
| Escherichia coli | 2.26 |
| Candida albicans | 1.13 |
Table 2: minimal inhibitory concentration table of the cecropin D YDD to each bacterium
| Tested thallus | DYDD MIC(mg/mL) |
| Pseudomonas aeruginosa | 4.44 |
| Escherichia coli | 8.87 |
| Candida albicans | 2.22 |
As can be seen from the above table, polypeptide DDDY is antibacterial to the minimum of pseudomonas aeruginosa, Escherichia coli and Candida albicans
Concentration (MIC) is respectively as follows: 18.08,2.26,1.13mg/mL.
Polypeptide DYDD is respectively as follows: the minimal inhibitory concentration (MIC) of pseudomonas aeruginosa, Escherichia coli and Candida albicans
4.44、8.87、2.22mg/mL。
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>ZhongKai Agriculture Engineering Academy
<120>antibacterial peptide, antibacterials and preparation method
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 4
<212> PRT
<213>artificial sequence
<400> 1
Asp Asp Asp Tyr
1
<210> 2
<211> 4
<212> PRT
<213>artificial sequence
<400> 2
Asp Tyr Asp Asp
1
Claims (10)
1. a kind of antibacterial peptide, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.
2. application of the antibacterial peptide described in claim 1 in preparation antibacterials.
3. a kind of antibacterials, which is characterized in that it contains antibacterial peptide described in claim 1 and pharmaceutically acceptable
Carrier.
4. a kind of preparation method of antibacterial peptide as described in claim 1, characterized in that it comprises: past anti-equipped with resin
It answers and sequentially adds polypeptide shown in the following raw material synthesis SEQ ID NO.1: Fmoc-Tyr (otbu)-OH, Fmoc-Asp in device
(otbu)-OH, Fmoc-Asp (otbu)-OH and Fmoc-Asp (otbu)-OH；
Alternatively, sequentially adding polypeptide shown in the following raw material synthesis SEQ ID NO.2: Fmoc- into the reactor equipped with resin
Asp (otbu)-OH, Fmoc-Asp (otbu)-OH, Fmoc-Tyr (otbu)-OH and Fmoc-Asp (otbu)-OH.
5. the preparation method according to claim 4, which is characterized in that after raw material is added every time, proceed as follows:
Step (a): reactor is placed in shaking table and is reacted；
Step (b): piperidine solution is added into reactor and is washed to slough Fmoc blocking group, then with DMF.
6. preparation method according to claim 5, which is characterized in that in step (a), the temperature control of shaking table is 29-
31 DEG C, reaction time control is 90-150min.
7. preparation method according to claim 5, which is characterized in that in step (b), piperidine solution contains piperidines and DMF.
8. preparation method according to claim 7, which is characterized in that the volume ratio of piperidines and DMF are 1:4.
9. preparation method according to claim 5, which is characterized in that, will when raw material being added into reactor the 1st time
DCM and DIEA is added together with this added raw material into reactor；
The 2nd time into the 4th it is any it is primary raw material is added into reactor when, will HOBT and DIC and this time it is added
Raw material is added into reactor together.
10. preparation method according to claim 9, which is characterized in that after the 1st time is added raw material into reactor,
It proceeds as follows: methanol solution being added into reactor and is closed.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111533781A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Non-specific receptor binding type fungus targeted antibacterial peptide and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1684976A (en) * | 2002-09-25 | 2005-10-19 | 福冈县政府 | Antibacterial substance produced by lactic acid bacterium |
| CN1769456A (en) * | 2005-05-20 | 2006-05-10 | 成都西玛生物科技有限公司 | Recombinant a human peptide production method |
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2018
- 2018-11-27 CN CN201811431760.2A patent/CN109485701B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1684976A (en) * | 2002-09-25 | 2005-10-19 | 福冈县政府 | Antibacterial substance produced by lactic acid bacterium |
| CN1769456A (en) * | 2005-05-20 | 2006-05-10 | 成都西玛生物科技有限公司 | Recombinant a human peptide production method |
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| HUIFAN LIU 等: "Detoxifying effects of ultrafiltration fractions of Dendrobium aphyllum peptides on chemical and AAPH-induced oxidative stress", 《THE ROYAL SOCIETY OF CHEMISTRY》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111533781A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Non-specific receptor binding type fungus targeted antibacterial peptide and preparation method and application thereof |
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