CN105837674A - Bombina orientalis polypeptide, and preparation method and application thereof - Google Patents
Bombina orientalis polypeptide, and preparation method and application thereof Download PDFInfo
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- CN105837674A CN105837674A CN201610131481.9A CN201610131481A CN105837674A CN 105837674 A CN105837674 A CN 105837674A CN 201610131481 A CN201610131481 A CN 201610131481A CN 105837674 A CN105837674 A CN 105837674A
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- abp27
- polypeptide
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- amino resins
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- QEWYKACRFQMRMB-UHFFFAOYSA-N fluoroacetic acid Chemical compound OC(=O)CF QEWYKACRFQMRMB-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229960004130 itraconazole Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000012503 pharmacopoeial method Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 239000011814 protection agent Substances 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000012109 statistical procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940043263 traditional drug Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Cosmetics (AREA)
Abstract
The invention relates to the field of polypeptide drugs, specifically to a Bombina orientalis polypeptide, and a preparation method and application thereof. The Bombina orientalis polypeptide ABP27 has an amino acid sequence as shown in SEQ ID No. 1. Experimental results show that the ABP27 has good antibacterial activity to Staphylococcus aureus, Escherichia coli DH5alpha, Bacillus acnes, animal pathogenic Escherichia coli, Candida albicans and Saccharomyces sake. Moreover, the ABP27 has good corrosion resistance and can used as an antiseptic substitute. The ABP27 is capable of inhibiting inflammatory response of Propionibacterium acnes. The Bombina orientalis polypeptide ABP27 provided by the invention can be used for preparing antibacterial drugs or cosmetics, has a wide application scope and produces good economic and social effect.
Description
Technical field
The present invention relates to polypeptide drugs field, be specifically related to a kind of Bombina orientalis polypeptide and preparation method and application.
Background technology
Recently as the abuse of antibiotic, drug resistance bacterium gets more and more, and bacterial drug resistance is the most serious.According to data,
The S. aureus L-forms being separated on U.S. clinical has 95% pair of Penicillin-resistant, and more than 50% to methicillin resistance (Frindkin
SK,Gaynes RP.Antimicrobial resistance in intensive care units.Clin Chest
Med.1999,20(2):303-316).Within 2012, China's Clinical detection finds, first in S. aureus L-forms and coagulase negative staphylococcus
The recall rate in oxygen XiLin persister (MRSA and MRCNS) is averagely respectively 47.9% and 77.1%, and (within 2012, Chinese CHINET is thin
Bacterium Monitoring of drug resistance Chin J Infect Chemother, 2013,13 (5): 321-330).People in this situation serious threat
The productive life of class, thus cause awakening and the raising of food safety rank consciousness, the people that this potential threat realized by people
In the urgent need to environmental protection, safety, the antibiotic of new generation of overriding resistance, to substitute existing antibiotic.
Antibacterial peptide is the important component part of immune defense system in organism, is small molecule peptide, is referred to as safety
Natural antibiotics, have has a broad antifungal spectrum, have no drug resistance, the advantage such as thermally-stabilised good, water solublity is high, nontoxic residue-free.According to statistics,
China still has the antibiotic that up to 20 kinds people and animals share or poultry are special to make feed additive at present, in addition, and antibacterial peptide quilt
It is widely used in cosmetics, food preservative, medicine, transgene expression etc. central with human being's production life.Antibacterial peptide product is opened
Send out into the new product replacing conventional antibiotic, the problem serious by effectively solving existing antibiotic bacterial drug resistance.
Amphibian animal skin is exposed, soft and moisten, and body surface is rich in body of gland, and its skin secretion contains and substantial amounts of has
The complicated and diversified bioactive molecule of special molecular structure, function.From amphibian oneself separated to include angiotensin sample
Skin, thyroliberin skin, frog coffee skin, the sharp many skins of skin sample of releiving, speed swash the 14 many skins of class such as the many skins of skin sample, tree toad skin sample polypeptide
Family and biogenic amine alkaloid etc..Bombina orientalis (Bombina orientalis), another name fire abdomen bell toad, smelly Oviductus Ranae, red tripe
Skin Oviductus Ranae, belongs to Discoglossidae, genus bombina, is a kind of pole medicinal salientian of having potentiality to be exploited, in being distributed mainly on
The northeast of state and Russia, Korea and Japan, lives under pond or streams, mountain area stone more.Its skin body of gland is flourishing, irriate
The mucus rich in active substance can be secreted in a large number, be the Excellent sources of isolating bioactive molecules.
Summary of the invention
In view of this, it is an object of the invention to provide a kind of Bombina orientalis polypeptide and preparation method and application.
For realizing the purpose of the present invention, the present invention adopts the following technical scheme that
A kind of Bombina orientalis polypeptide, it has the aminoacid sequence shown in SEQ ID NO:1.
Present invention also offers in the aminoacid sequence of described Bombina orientalis polypeptide, replace, lack or add one or many
The polypeptide of the aminoacid sequence obtained by individual amino acid residue.
Present invention provides the DNA molecular encoding polypeptide of the present invention.
Present invention also offers the preparation method of described Bombina orientalis polypeptide, comprise the following steps:
1) solid phase synthesis ABP27-amino resins；
2) cracking of ABP27-amino resins obtains the thick peptide of ABP27；
3) after the thick peptide of ABP27 dissolves with 1% acetum, gel filtration chromatography isolated ABP27 fine peptide.
Preferably, described solid phase synthesis be Fmoc-Arg (Pbf)-OH and amino resins be synthesized Fmoc-Arg (Pbf)-
Amino resins, then uses other aminoacid of the mode coupling Fmoc blocking group of coupling one by one to obtain ABP27-amino tree
Fat.
Wherein, described amino resins is preferably Rink Amide resin.
Preparation method step 2 of the present invention) decomposition agent of described cracking is Fluoroethanoic acid, tri isopropyl silane and phenol
Mixture.
Preferably, described trifluoroacetic acid, tri isopropyl silane are 95:2.5:2.5 with the volume ratio of phenol.
Therefore, the invention provides described polypeptide A BP27 application in preparing antibacterial medicine.
Further, present invention also offers described polypeptide A BP27 application in preparing antibacterial cosmetics.
As shown from the above technical solution, the invention provides a kind of Bombina orientalis polypeptide and preparation method and application.This
Invent described Bombina orientalis polypeptide A BP27 and there is the aminoacid sequence shown in SEQ ID NO:1, do not contain rare amino acid with outer
Source chemical composition, has the advantages such as immunogenicity little, good water solubility, broad-spectrum sterilization, is resistant to protease and peptidase in gastrointestinal tract
Degraded.The most in acid condition, heating even high temperature high pressure process does not affect its antibacterial activity, Ke Yi
Product persistently plays its function during preserving, keep product quality, extends the shelf life.Test result indicate that of the present invention
ABP27 is to staphylococcus aureus, bacillus coli DH 5 alpha, Propiobacterium, animal pathogenic escherichia coli, Candida albicans, rice wine
Yeast all has good antibacterial activity, and anti-corrosive properties are good, can be the most traditional antibiosis as the additive of a new generation
Element and preservative succedaneum, effectively can must solve the residual problem with pathogenic bacteria resistance to drugs of humans and animals medicine, and can become novel
Safety preservative succedaneum.ABP27 has the inflammatory reaction effect of suppression propionibacterium acnes simultaneously.East of the present invention
Side bell toad polypeptide A BP27 may be used for preparing antibacterial medicine or cosmetics, has wide range of applications, has good economy and society
Can effect.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described.
Fig. 1 shows that embodiment 2-in-1 one-tenth cecropin A BP27 to staphylococcus aureus, Propiobacterium, bacillus coli DH 5 alpha and moves
The antibacterial experiment result of thing pathogenic escherichia coli；In each figure, 1 represents the antibacterial activity of ampicillin, and 0 is negative control
ddH2O, 2,3,4 is the antibacterial activity of artificial synthetic antimicrobial peptide ABP27；
Fig. 2 embodiment 2-in-1 one-tenth cecropin A BP27 antibacterial experiment result to Candida albicans and saccharomyces sake bacterium；Each figure
In 1 represent itraconazole antibacterial activity；0 is negative control ddH2O, 2,3,4 is the antibacterial work of artificial synthetic antimicrobial peptide ABP27
Property.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, all
Belong to the scope of protection of the invention.
For realizing the purpose of the present invention, the present invention adopts the following technical scheme that
A kind of Bombina orientalis polypeptide, it has the aminoacid sequence shown in SEQ ID NO:1.
Bombina orientalis polypeptide particular sequence of the present invention is GIGGKLLTAGKTAKKGLAKGLK EHFAN, and molecular weight is about
MW [AV]: 3kDa, named ABP27, do not contain rare amino acid and exogenous chemical components, has that immunogenicity is little, water solublity
The advantages such as good, broad-spectrum sterilization, are resistant to protease and the degraded of peptidase in gastrointestinal tract.The most in acid condition, heating is very
To being that its antibacterial activity is not affected by high temperature high pressure process, can persistently play its function during product preserves, keep
Product quality, extends the shelf life.
Present invention also offers and replace in the aminoacid sequence of described Bombina orientalis polypeptide A BP27, lack or add one
The polypeptide of the aminoacid sequence obtained by individual or multiple amino acid residue.Described aminoacid replacement, lack or add, Ke Yi
Any site of aminoacid sequence is carried out, as long as the polypeptide of improved aminoacid sequence has antibacterial activity.It is similar to,
The amino acid whose number being replaced, lack or adding also is arbitrary, as long as the polypeptide of improved aminoacid sequence has anti-
Bacterium activity.
Present invention provides the DNA molecular encoding polypeptide of the present invention.Due to the degeneracy of codon, can exist
The a variety of nucleotide sequences that can encode specific polypeptide of the present invention.Divide for encoding the DNA of polypeptide of the present invention
Son, those skilled in the art can utilize existing known method manufacture synthesis easily.Such as, by selecting corresponding to structure
The codon of the amino acid residue of the aminoacid sequence described in one-tenth, can be readily determined and provide the ammonia corresponding to described polypeptide
The DNA molecular of base acid sequence.
Present invention also offers the preparation method of described Bombina orientalis polypeptide A BP27, comprise the following steps:
1) solid phase synthesis ABP27-amino resins；
2) cracking of ABP27-amino resins obtains the thick peptide of ABP27；
3) after the thick peptide of ABP27 dissolves with 1% acetum, gel filtration chromatography isolated ABP27 fine peptide.
Wherein, preparation method step 1 of the present invention) use Fmoc/tBu synthesis strategy, with the amino of suitable substitution degree
Resin is carrier, and the aminoacid solid phase synthesis of coupling Fmoc blocking group obtains ABP27-amino resins one by one.
Preferably, described solid phase synthesis be Fmoc-Arg (Pbf)-OH and amino resins be synthesized Fmoc-Arg (Pbf)-
Amino resins, then uses other aminoacid of the mode coupling Fmoc blocking group of coupling one by one to obtain ABP27-amino tree
Fat.
Wherein, described amino resins is preferably Rink Amide resin.Further, described in preparation method of the present invention
Other aminoacid of the Fmoc blocking group of coupling are followed successively by one by one:
Gly-Ile-Gly-Gly-Lys-Leu-Leu-Thr-Ala-Gly-Lys-Thr-Ala-Lys-Lys-Gly-Leu-
Ala-L ys-Gly-Leu-Lys-Glu-His-Phe-Ala-Asn (that is: glycine-Isoleucine-glycine-glycine-rely
Propylhomoserin-Leu-Leu-threonine-Ala-Gly-lysine-threonine-alanine-lysine-lysine-sweet
Propylhomoserin-Leu-Ala-LYS-GLY-leucine-LYS-GLU-HIS-PHE-alanine-
Agedoite).
Wherein, the aminoacid of above-mentioned Fmoc blocking group can be bought by channel of goods distribution, it is possible to utilizes commodity
The raw material changed prepares according to method known to those skilled in the art.
In some detailed description of the invention, step 1) described in coupling one by one mode in each amino acid whose coupling method
Specifically include following steps:
A, washing: alternately washing 2 to 3 times of DMF, DCM and methanol；
B, deprotection: the piperidines with 20%/DMF solution room temperature treatment 2-3 time, each 5-15min, slough Fmoc-protection
Agent, the detection reaction of Kaiser reagent is the most complete；
C, washing: alternately washing 2 to 3 times of DMF, DCM and methanol；
D, amino acid couplings: after Fmoc-protected amino acid, HOBt, DIC being dissolved with appropriate DMF, add in reaction column,
The most complete with mixed with resin stirring 2-4h, Kaiser reagent detection reaction.
Preparation method step 2 of the present invention) crack ABP27-amino resins by decomposition agent, obtain the thick peptide of ABP27.Its
In, the decomposition agent of described cracking is the mixture of trifluoroacetic acid (TFA), tri isopropyl silane (Tis) and phenol (Phenol).
Preferably, described trifluoroacetic acid, tri isopropyl silane are 95:2.5:2.5 with the volume ratio of phenol.
Preparation method step 3 of the present invention) the thick peptide of ABP27 is with after 1% acetum dissolving, and gel filtration chromatography separates
To ABP27 fine peptide.
In certain embodiments, described gel filtration chromatography separates and is specially with 1% acetum eluting, monitors at 220nm,
Applied sample amount 2.00g/ time, flow velocity is 5mL min-1, collect the component having absorption.The refined liquid decompression distillation that will collect, controls matter
Amount concentration is at 100mg mL-1Lyophilization during left and right.
In order to improve the purity of prepared ABP27 polypeptide, in some embodiments, preparation method step of the present invention
3) ABP27 peptide purification step is also included.
Preferably, described purification is reversed-phase high-performance liquid chromatography purification.
Reversed-phase high-performance liquid chromatography, English name reversed phase high performance liquid
Chromatography, is called for short, RP-HPLC, the liquid chromatographic system being made up of non-polar stationary phase and polarity flowing phase.
It is just contrary with the liquid chromatographic system (normal-phase chromatography) being made up of polar stationary phase and low pole flowing phase.RP-HPLC
The topmost clastotype of current liquid chromatograph, may be used with nearly all can be dissolved in polarity or weak polar solvent organic
Thing isolated and purified.
Step 3 of the present invention) flowing of described reversed phase high-performance liquid chromatography can be trifluoroacetic acid aqueous solution, formic acid water mutually
Solution or aqueous acetic acid.
In certain embodiments, step 3) mobile phase A of described reversed phase high-performance liquid chromatography is 0.1% trifluoro second
Aqueous acid, Mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution.
Step 3 of the present invention) filler used by described reversed phase high-performance liquid chromatography can be octadecylsilane bonded silica
Glue (C18), butylsilane bonded silica gel (C4) or eight alkyl silane bonded silica gel (C8), preferably octadecylsilane bonded silicas
Glue.
In a certain detailed description of the invention, step 3 of the present invention) chromatographic condition of described reversed-phase high-performance liquid chromatography purification
For: prepare post: C18Prepare post (50 × 80mm)；Flow velocity: 100ml/min；Wavelength: 220nm；Flowing phase: 22-28%B in
10min, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, Mobile phase B: 0.1% trifluoroacetic acid acetonitrile solution.
In a certain specific embodiment, the present invention uses plating method that synthesis cecropin A BP27 is carried out antimicrobial spectrum
Measuring, it is to staphylococcus aureus, bacillus coli DH 5 alpha, Propiobacterium, animal pathogenic escherichia coli, Candida albicans, clear
Brewer yeast bacterium all has good antibacterial activity.Use the ABP27 solution of the method preparation variable concentrations of doubling dilution, join training
Survey OD630 value after bacteria liquid is cultivated 16h, determine synthetic ABP27 minimal inhibitory concentration MIC to different strains.
Further, the present invention uses the antimicrobial preservation efficacy test method of American Pharmacopeia, to adding 0.01%ABP27's
Cream carries out preservative efficacy test, and cosmetic sample total number of bacteria, mycete and the ferment of the preservative free of ABP27 is added in result display
Female bacterium sum all meets the requirement of " American Pharmacopeia USP32<51>", illustrates that these antibacterial peptide anti-corrosive properties are good, can replace as preservative
Use for product.
In another specific embodiment, the cream adding variable concentrations ABP27 is entered by the present invention by mice Acne Model
The detection of the inflammatory reaction that row suppression Propiobacterium causes, result display ABP27 basic, normal, high dosage group and tretinoin emulsifiable paste group are big
The ear swelling rate of Mus substantially reduces, and compared with same period model control group, has significant difference (P < 0.05, P < 0.01), shows
ABP27 has the inflammatory reaction effect of suppression propionibacterium acnes.
Therefore, the invention provides described polypeptide A BP27 application in preparing antibacterial medicine
Polypeptide A BP27 directly or indirectly addition of the present invention can be prepared different dosage form time institute by those skilled in the art
The pharmaceutically acceptable various conventional adjuvants needed, such as filler, disintegrating agent, lubricant, binding agent etc., with traditional drug formulations
Method, makes common dosage forms such as tablet, capsule, injection, unguentum etc..Wherein, filler such as starch, lactose, sucrose, Fructus Vitis viniferae
Sugar, mannitol and silicic acid；Disintegrating agent such as agar, calcium carbonate, potato starch or tapioca, alginic acid, some silicate and carbon
Acid sodium, low-substituted hydroxypropyl cellulose；Lubricant such as Pulvis Talci, calcium stearate, magnesium stearate, solid polyethylene glycol, Laurel sulfur
Acid sodium；Binding agent such as carboxymethyl cellulose, alginate, gelatin, polyvidon, sucrose and arabic gum.
Further, present invention also offers described polypeptide A BP27 application in preparing antibacterial cosmetics.
Polypeptide A BP27 of the present invention can directly or indirectly be added the different cosmetics time institute of preparation by those skilled in the art
The raw material needed, with cosmetics preparation method, makes conventional cosmetics such as emulsion, facial cream, essence etc..
As shown from the above technical solution, the invention provides a kind of Bombina orientalis polypeptide and preparation method and application.This
Invent described Bombina orientalis polypeptide A BP27 and there is the aminoacid sequence shown in SEQ ID NO:1, do not contain rare amino acid with outer
Source chemical composition, has the advantages such as immunogenicity little, good water solubility, broad-spectrum sterilization, is resistant to protease and peptidase in gastrointestinal tract
Degraded.The most in acid condition, heating even high temperature high pressure process does not affect its antibacterial activity, can produce
Product persistently play its function during preserving, keep product quality, extend the shelf life.Test result indicate that of the present invention
ABP27 is to staphylococcus aureus, bacillus coli DH 5 alpha, Propiobacterium, animal pathogenic escherichia coli, Candida albicans, rice wine
Yeast all has good antibacterial activity, and anti-corrosive properties are good, can be the most traditional antibiosis as the additive of a new generation
Element and preservative succedaneum, effectively can must solve the residual problem with pathogenic bacteria resistance to drugs of humans and animals medicine, and can become novel
Safety preservative succedaneum.ABP27 has the inflammatory reaction effect of suppression propionibacterium acnes simultaneously.East of the present invention
Side bell toad polypeptide A BP27 may be used for preparing antibacterial medicine or cosmetics, has wide range of applications, has good economy and society
Can effect.
In order to be further appreciated by the present invention, below in conjunction with specific embodiment, the present invention will be described in detail.Wherein, except spy
Do not mentionlet alone bright outside, described in preparation method of the present invention, reagent is to well known to a person skilled in the art, can pass through commercial sources
It is commercially available.
Embodiment 1: the preparation of polypeptide A BP27
1, the preparation of Fmoc-Arg (pbf)-amino resins
By substitution degree 0.84mmol/g amino resins Fmoc-Rink Amide 10.03g, join in solid phase reactor,
Add DMF to soak so that it is abundant swelling 30min, drain, add 20% piperidines/DMF solution and take off Fmoc blocking group 2 times, for the first time
Reaction 5min, second time reaction 15min, by DMF, DCM, methanol washes clean.Take 11.1g Fmoc-Arg (Pbf)-OH,
2.24gHOBt, 6.14gHBTU DMF joins balance 5min in reactor after dissolving, add 6.0mLDIPEA room temperature reaction 1
Hour, drain, successively respectively wash 3 times with DMF, DCM, methanol, be dried Fmoc-Arg (pbf)-Rink Amide resin of weighing to obtain
17.12g, detection substitution value is 0.67mmol/g.
2, the preparation of ABP27 amino resins
Above-mentioned resin is put in and connects in peptide bottle, add 20% piperidines/DMF solution and take off Fmoc blocking group 2 times, the most anti-
Answering 5min, second time reaction 15min, by DMF, DCM, methanol washes clean..Then by sequence
The order of GIGGALLSAGKSALKGLAKGLAEHFAN-NH2 connects the aminoacid of Fmoc protection successively, and reaction system mol ratio is
Fmoc-AA:DIC:HOBt presses 1:1:1 and throws in, and Fmoc-AA is by 3 times of additions of amount of resin in this instance, and each DIC adds 4.10g
Left and right, HOBt adds about 4.27g.Period 20% piperidines successively/DMF removing Fmoc blocking group 2 times, first set reaction 5min,
Second time reaction 15min；Each step amino acid condensation reacts one time 2-4 hour, whether all uses Kaise Test detection reaction
Condensation completely, repeats to be condensed this aminoacid as chromogenic reaction is positive, the last directly removing Fmoc blocking group of sequence condensation,
DMF, DCM, methanol wash several times.
3, the preparation of ABP27 crude product
The ABP27 amino resins mixed solution of TFA:Tis:Phenol 95:2.5:2.5 by volume processes peptide resin 3-
4 hours, peptide scaled off from resin and excises Side chain protective group, after being concentrated to dryness, separating out solid, centrifugal drying with ice ether
Obtain crude product 29.51g.
4, the preparation of ABP27 finished product
After ABP27 crude product dissolves with 1% acetum, centrifuging and taking supernatant crosses G10 post, with 1% acetum eluting,
220nm monitors, and applied sample amount 2.00g/ time, flow velocity is 5mL min-1, collect the component having absorption.The refined liquid decompression that will collect
Distillation, controls mass concentration at 100mg mL-1Lyophilization during left and right.Collect cecropin A BP27 peak concentrate drying and obtain finished product
17.71g.Purity is more than 60%, and total recovery reaches 60%.
The mensuration of embodiment 2:ABP27 antimicrobial spectrum
From YPD flat board, picking PCR is accredited as the staphylococcus aureus of the positive, bacillus coli DH 5 alpha, acne bar respectively
Bacterium, animal pathogenic escherichia coli, Candida albicans, saccharomyces sake bacterium list colony inoculation are in 100mlBMGY culture medium, 30 DEG C of vibrations
Cultivating to OD600 2～6, remove supernatant, add the resuspended thalline of 20mlBMMY culture medium, 30 DEG C of shaken cultivation 96 hours, every 24
Hour take 1mL sample in Eppendorff pipe, take supernatant and detect for antibacterial activity.
Agar culture medium is added in a water bath heat fusing, is cooled to about 50 DEG C, draw the life of 60 μ L by aseptic manipulation
Survey bacterium (OD600=0.3), add in 20mL agar culture medium, mix rapidly, pour in the aseptic plane ware of a diameter of 9cm,
Horizontal positioned is to be solidified.Agar is beaten the circular hole of a diameter of 2.7mm, respectively addition phosphate buffered saline in hole
Synthesis ABP27, sterilized water and 100mg/mL Amplicin.Plate is inverted in 37 DEG C of overnight incubation, next day observed result, as
Shown in Fig. 1 and Fig. 2
Result shows, staphylococcus aureus, bacillus coli DH 5 alpha, Propiobacterium, animal are caused by the ABP27 of synthetic
Sick escherichia coli, Candida albicans, saccharomyces sake bacterium all have good antibacterial activity.
Embodiment 3: the mensuration of synthetic ABP27 minimal inhibitory concentration (MIC)
MIC assay method: by each for embodiment 2 test strain cultivate to exponential phase bacterium solution dilute be about 5 ×
106CFU/mL, adds in 96 well culture plates, and the every hole of instrument connection adds bacterium solution 90 μ L, is subsequently adding the variable concentrations of doubling dilution
Synthetic antimicrobial peptide solution, 10 μ L/ holes.Positive control is the bacterium solution in 100 μ L/ holes, and negative control is that corresponding 100 μ L cultivate
Base.Then shake slowly in 37 DEG C and cultivate about 16h, survey OD630 by microplate reader.The least concentration of bacteria growing inhibiting is MIC, result
It is shown in Table 1.
The MIC of table 1 artificial synthetic antimicrobial peptide ABP27
| Bacterial strain | MIC(μg/mL) |
| Staphylococcus aureus | 125 |
| Propiobacterium | 125 |
| Bacillus coli DH 5 alpha | 2.5 |
| Escherichia coli (animal is caused a disease) | 2.5 |
| Candida albicans | 500 |
| Saccharomyces sake bacterium | 500 |
Embodiment 4: the preservative of synthetic ABP27 substitutes experiment
Laboratory reference cosmetics, toilet articles and fragrance association (CTFA) and the antimicrobial preservation efficacy test of American Pharmacopeia
Method, will join in cosmetics by a certain amount of microorganism suspension, detects micro-life with colony counting method at regular intervals
The survival condition of thing, judges the antiseptic effect of preservative with this.
Training method is pure culture.The preservative free cosmetic sample that weighing 20g has prepared respectively is in aseptic bottle, pure
Degree is 0.01% for the addition of 95%ABP27.Every bottle is separately added into every kind of bacteria suspension 0.2mL, fully after concussion mixing, in
Room temperature is deposited.In the 0th, 1,7,14,21,28 days growing states with colony counting method detection microorganism.
Evaluation criterion reference " American Pharmacopeia USP32<51>": total number of bacteria is from initially to the bacterial population logarithm minimizing of 14 days
Value less than 2.0 and can not be able to not increase from 114 days to 28 day bacterial populations；Mycete and yeast sum from initial by 14 days and 28
My god, mycete can not increase with yeast, it is judged that good for preservative antiseptic effect.Experimental result is shown in Table 2.
The survival condition of table 2 microorganism
From experimental result it will be seen that add the cosmetic sample total number of bacteria of the preservative free of ABP27 from initial to 14
It log reduction is 2.98, and is more than 6.60 to 28 days reduced value, mycete and yeast sum from initially by 14 days and
28 days, log reduction was 0.99 and 1.48, therefore met the requirement of " American Pharmacopeia USP32<51>", illustrated that this antibacterial peptide is prevented
Corruption is good, can use as preservative succedaneum.
Embodiment 5: mice Acne Model is tested
Choose healthy SD rat 50, female, 200 ± 20g.Quarantine qualified after, be randomly divided into 7 groups, be respectively normal
Matched group, bare substrate group, positive drug tretinoin emulsifiable paste group, model control group, ABP27 ointment basic, normal, high dosage group is (i.e. toward empty
Bai Jizhi adds the ABP27 of basic, normal, high concentration), often 6 rats of group, remaining gives over to preliminary experiment, only often organizes 1-2, for Cuo
Skin ulcer propionibacterium checkings etc. are tested.
First in anaerobic culture box, propanoic acid Propiobacterium amplification culture is carried out before experiment.The propionibacterium lyophilized powder that will buy
After redissolving with normal saline, under anaerobic, increase bacterium amplification culture with sulfur Sodium ethylate liquid culture medium, until exponential phase of growth
Time, first use brine 3 times, it is 60,000,000/mL that last normal saline adjusts propionibacterium acnes concentration, makes acne
Propionibacterium reserve liquid, standby after 95 DEG C of water-bath inactivation 15min.
When experiment starts, after first rat being anaesthetized with the urethane of 12.5%, Normal group and bare substrate group rat
Auricle intradermal injection normal saline 50 μ L, remaining is respectively organized rat auricle intradermal injection propionibacterium acnes bacterium solution 50 μ L, and is noting
The place of penetrating makes marks, and in preparation the 2nd day, from 1 checking rat of each group of extraction, with aseptic syringe needle picking auricle edema position, is coated with ware,
Anaerobic jar is cultivated 48h, it was demonstrated that infect for propionibacterium acnes.After modeling success, each group rat auricle is administered respectively, the most right
Only being coated with normal saline according to group, bare substrate group gives isopyknic preservative free substrate, and other is respectively organized and presses table 3 dosed administration, even
Continuous administration 15 days, every day 2 times, is administered 0.2g every time, is applied in the ear swelling position of rat.After injection, measured 1 every 24 hours
Secondary rat auricle thickness, continuous 5d, calculate each Mus auricle edema rate, observe the change of rat auricle structure.By each group of experimental result
Carry out statistical procedures, and compare group difference.Experimental result is shown in Table 4 and table 5.
Auricle thickness before auricle edema rate (%)=(auricle thickness before auricle thickness-injection after injection) ÷ injection
The adding medicine scale of table 3 Acne Model experiment
| Group | Be equivalent to multiple of being grown up | Sample concentration (μ g/g) | Smear weight (g) | Dosage (μ g) |
| ABP27 low dose group | 5 | 2.5 | 0.4 | 2 |
| Dosage group in ABP27 | 10 | 5 | 0.4 | 4 |
| ABP27 high dose group | 30 | 7.5 | 0.4 | 7 |
| Tretinoin emulsifiable paste group | - | 0.025% | 0.4 | 0.1 |
Before table 4 rat injection propionibacterium acnes and after injection continuously 5d, 14d auricle thickness results (N=
6)
Before table 5 rat injection propionibacterium acnes and after injection continuously 5d, 14d auricle edema rate (N=
6)
Note: △ p>0.05, * p<0.05, * * p<0.01, * * * p<0.001 (t inspection) compared with blank group.
Can obtain from table 4 table 5 experimental result, 5d ear obvious tumefaction after rat injection propionibacterium acnes, until being administered
14d, auricle edema rate compared with same period Normal group, has significant difference (P < 0.001), points out propionibacterium acnes
Ear injection modeling success.The auricle edema rate of bare substrate group rat is close with Normal group, and prompting bare substrate is without antiinflammatory
Effect, is only used as substrate existence of making up a prescription.After ABP27 basic, normal, high dosage group rat auricle injection seat skin ulcer propionibacterium, occur bright
Aobvious swelling phenomenon, but calculate swelling rate compared with same period model control group, tool there was no significant difference (P > 0.05)；It is being administered the
During 14d, the ear swelling rate of ABP27 basic, normal, high dosage group and tretinoin emulsifiable paste group rat substantially reduces, with the model control group same period
Compare, there is significant difference (P < 0.05, P < 0.01), show that ABP27 given the test agent has certain suppression acne propanoic acid bar
The inflammatory reaction effect of bacterium, the effect of tretinoin emulsifiable paste is consistent with clinical practice.It will be seen that ABP27 can be expected to answer
The inflammatory reaction caused for Propiobacterium.
Above content is the further description combining concrete preferred implementation to the present invention, it is impossible to assert this
Bright being embodied as is confined to these explanations.Without departing from the inventive concept of the premise, the technical field of the invention
Technical staff can make some deduction or replace, is regarded as protection scope of the present invention.
Claims (10)
1. a Bombina orientalis polypeptide, it is characterised in that it has the aminoacid sequence shown in SEQ ID NO:1.
2. have by replace, lack or add in the aminoacid sequence of Bombina orientalis polypeptide described in claim 1 one or
The polypeptide of the aminoacid sequence obtained by multiple amino acid residues.
3. the DNA molecular of polypeptide described in coding claim 1 or 2.
4. the preparation method of polypeptide described in claim 1, it is characterised in that comprise the following steps:
1) solid phase synthesis ABP27-amino resins；
2) cracking of ABP27-amino resins obtains the thick peptide of ABP27；
3) after the thick peptide of ABP27 dissolves with 1% acetum, gel filtration chromatography isolated ABP27 fine peptide.
Preparation method the most according to claim 4, it is characterised in that step 1) described solid phase synthesis is Fmoc-Arg
(Pbf)-OH and amino resins are synthesized Fmoc-Arg (Pbf)-amino resins, then use the mode coupling of coupling one by one
Other aminoacid of Fmoc blocking group obtain ABP27-amino resins.
Preparation method the most according to claim 5, it is characterised in that described amino resins is Rink Amide resin.
Preparation method the most according to claim 4, it is characterised in that step 2) decomposition agent of described cracking is trifluoro second
The mixture of acid, tri isopropyl silane and phenol.
Preparation method the most according to claim 7, it is characterised in that described trifluoroacetic acid, tri isopropyl silane and phenol
Volume ratio be 95:2.5:2.5.
9. the application in preparing antibacterial medicine of the polypeptide described in claim 1 or 2.
10. the application in preparing antibacterial cosmetics of the polypeptide described in claim 1 or 2.
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Cited By (2)
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| CN110684098A (en) * | 2019-10-09 | 2020-01-14 | 江苏医药职业学院 | Method for preparing oriental fire-bellied toad antibacterial peptide bombinin and its application |
| CN114989246A (en) * | 2022-04-15 | 2022-09-02 | 兰州大学 | FK3 polypeptide analogs and their applications |
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