CN107298707A - One species Bac5 antibacterial peptides and its application - Google Patents
One species Bac5 antibacterial peptides and its application Download PDFInfo
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- CN107298707A CN107298707A CN201710643182.8A CN201710643182A CN107298707A CN 107298707 A CN107298707 A CN 107298707A CN 201710643182 A CN201710643182 A CN 201710643182A CN 107298707 A CN107298707 A CN 107298707A
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- 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
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- 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
Abstract
The invention discloses a species Bac5 antibacterial peptides, its amino acid sequence such as SEQ ID NO.1, its molecular weight is 4480.51Da, and isoelectric point is 12.6.Described class Bac5 antibacterial peptides are using automatic Peptide synthesizer according to conventional polypeptide Solid phase synthesis.The class Bac5 antibacterial peptides of the present invention have efficient antibacterial activity to gram positive bacteria (staphylococcus aureus), gram-negative bacteria (Escherichia coli, detection of Salmonella), and antibacterial action is fast, with low hemolytic, does not produce drug resistance.Therefore, class Bac5 antibacterial peptides of the invention have broad application prospects in terms of anti-infectives, animal husbandry development, human disease treatment is prepared.
Description
Technical field
The invention belongs to biological technical field, and in particular to a species Bac5 antibacterial peptides and its application.
Background technology
The discovery of antibiotic once brings huge welfare to the mankind.However, as a large amount of of antibiotic use and abused, carefully
Bacterium drug resistance is constantly produced, and residue of veterinary drug hazardous events constantly occur, and have seriously endangered human health and animal husbandry development.Find
New antibacterials become more urgent.During searching, sight is gradually focused on antibacterial peptide by people.Antibacterial peptide
(Antimicrobial peptides, AMPs) is the important component of living organism innate immune system, by 10-60 ammonia
Base acid residue is constituted, with the bioactivity such as antibacterium, antiviral, anti parasitic, antitumor, immunological regulation, because of its molecular weight
Small, heat endurance is good, good water solubility, has a broad antifungal spectrum, is difficult to form the characteristics such as drug resistance and enjoy people to pay close attention to, it is considered to be anti-
The best substitute of raw element.
At present, the research of antibacterial peptide and application has become focus in biological technical field, many antibacterial peptides by
Develop into clinical medicine.Cecropin B gene ac5 in 1989 from ox neutrophil leucocyte it is isolated, be made up of 43 amino acid
The antibacterial peptide rich in arginine and proline, with broad spectrum antibiotic activity.However, most natural antibacterial peptide bacteriostatic activity
It is not high, and toxicity, and haemocylolysis are also often produced to normal eukaryotic while fungistatic effect is played, and
Natural antibacterial peptide yields poorly, and this all limits the development and application of antibacterial peptide.According to the structure-activity relationship and mechanism of action of antibacterial peptide,
Using excellent antibacterial peptide as template, the artificial reconstructed and design for carrying out existing antibacterial peptide is the important directions that antibacterial peptide is developed.
Compared with natural antibacterial peptide, engineer's antibacterial peptide has more preferable target cell specificity and antibacterial activity, or even also has low
The characteristic such as hemolytic and hyposensitivity to proteasome degradation.Therefore, the present invention devises simple in construction, chemical close according to Bac5
Into easy, the class Bac5 antibacterial peptides with high antibacterial activity.
The content of the invention
It is an object of the invention to provide a species Bac5 antibacterial peptides and its application.The class Bac5 antibacterial peptides of the present invention are blue to leather
Positive bacteria (staphylococcus aureus), gram-negative bacteria (Escherichia coli, detection of Salmonella) have efficient antibacterial activity, antibacterial action
It hurry up, with low hemolytic, do not produce drug resistance.Therefore, class Bac5 antibacterial peptides of the invention are preparing anti-infectives, animal husbandry
Had broad application prospects in terms of development, human disease treatment.
To realize goal of the invention, the technical solution adopted by the present invention is：
One species Bac5 antibacterial peptides, its amino acid sequence is as follows：
Phe-A taug-Pro-Pro-isoleucine-Arg-Arg-Pro-Pro-different
Leucine-arginine-Pro-Pro-phenylalanine-tyrosine-Pro-Pro-Phe-A taug-dried meat
Propylhomoserin-proline-isoleucine-Arg-Pro-proline-isoleucine-Phe-Pro-Pro-different
Leucine-arginine-Pro-Pro-Phe-A taug-Pro-Pro (SEQ ID NO.1).
According to above-mentioned class Bac5 antibacterial peptides, the class Bac5 antibacterial peptides have 37 amino acid residues, and its molecular weight is
4480.51Da, isoelectric point is 12.6.
Class Bac5 antibacterial peptides of the present invention can be used for preparing treatment gram-positive bacteria and gram positive bacterial infection
Medicine.The medicine includes above-mentioned class Bac5 antibacterial peptides, and is mixed with one or more kinds of pharmaceutically acceptable carriers
And/or additive.
Class Bac5 antibacterial peptides of the present invention can also be used to prepare feed addictive, disinfectant, preservative or cosmetics
Additive.
Class Bac5 antibacterial peptides of the present invention are using automatic Peptide synthesizer according to conventional polypeptide Solid phase synthesis, synthesis
Direction is that from C-terminal to N-terminal, its detailed step synthesized is：
A, swelling resin：Fmoc-Pro-Wang resin resins are added in automatic Peptide synthesizer reactor, and added
Dimethylformamide DMF carries out swelling, and the amount that 1gFmoc-Leu-Wang resinin resins add DMF is about that (DMF should by 12mL
It is totally submerged resin), the swelling time is 10~30min, and swelling is repeatable 1~2 time；
B, deprotection：After resin swelling, by 20% piperidines in storage tank, (20% piperidines is that piperidines is dissolved in into dimethylformamide
It is formulated in DMF) it is added in reactor, (1gFmoc-Pro-Wangresinin resins need to add submergence resin
20% piperidines is about 10mL), the resin after swelling is deprotected, the time is about 30min, then adds dimethylformamide DMF
Washed；
C, condensation reaction：Sequence then according to class Bac5 antibacterial peptide amino acid sequences adds second of amino proline
Fmoc-Pro, and add condensing agent HCTU (6- Chloro-Benzotriazoles -1,1,3,3- tetramethylurea hexafluorophosphoric acid esters) and catalyst
NMM (N- methylmorpholines) or DIEA (N, N diisopropylethylamine) carries out condensation reaction；Reaction adds dimethyl formyl after terminating
Amine DMF carries out cyclic washing, removes unreacted amino acid；The proline and Fmoc-Pro-Wang resinin resins
Mol ratio is 5:1；Mol ratio between the condensing agent HCTU and proline Fmoc-Pro is 1:1；The catalyst n MM or
DIEA is 4 with the mol ratio of proline therebetween:1；
Products therefrom is deprotected according to step b same methods, washed after washing, according to above-mentioned steps c same methods
Sequentially add amino acid Fmoc-Arg, Fmoc-Phe, Fmoc-Pro, Fmoc-Pro, Fmoc-Arg, Fmoc-Ile, Fmoc-Pro,
Fmoc-Pro、Fmoc-Phe、Fmoc-Ile、Fmoc-Pro、Fmoc-Pro、Fmoc-Arg、Fmoc-Ile、Fmoc-Pro、Fmoc-
Pro、Fmoc-Arg、Fmoc-Phe、Fmoc-Pro、Fmoc-Pro、Fmoc-Tyr、Fmoc-Phe、Fmoc-Pro、Fmoc-Pro、
Fmoc-Arg、Fmoc-Ile、Fmoc-Pro、Fmoc-Pro、Fmoc-Arg、Fmoc-Arg、Fmoc-Ile、Fmoc-Pro、Fmoc-
Pro, Fmoc-Arg and Fmoc-Phe carry out condensation reaction, washing, after last condensation reaction, washing, obtain polypeptide product；
Products therefrom is deprotected according to step b method, washed after each condensation reaction, washing, then adds next ammonia
Base acid carries out condensation reaction, washing and (in each condensation reaction, adds amino acid and Fmoc-Pro-Wang resinin resins
Mol ratio be 5:1；Mol ratio between the condensing agent HCTU and every kind of added amino acid is 1:1；It is described to urge
Mol ratio between agent NMM or DIEA and every kind of added amino acid is 4:1)；
D, polypeptide product deprotection and cracking：20% piperidines is added in polypeptide product obtained by step c in reactor,
And polypeptide product (the amount about 10mL of 20% piperidines used in being deprotected to 1g resins) is submerged, it is carried out polypeptide product
Deprotection；After deprotection, sequentially add dimethylformamide DMF, dichloromethane DCM and carry out cyclic washing；After washing, three are added
Fluoroacetic acid TFA is cracked (the volume about 20mL of TFA used in being cracked to 1g resins), is made under polypeptide cracks from resin
Come；Product after cracking is filtered and (filtered with sand core funnel), filtrate is obtained；
E, precipitation synthesis polypeptide：Gained filtrate is washed with cold diethyl ether, centrifuged, to precipitate synthesis polypeptide, its process
For：The cold diethyl ether (pre-saving in 4 DEG C) of its 3 times of volumes is added in the filtrate that step d is obtained, overturns and mixes after sealing, ice
10min is bathed, 10min is centrifuged under 4000r/m, supernatant fluid is abandoned, precipitation is reclaimed；It is resuspended and is precipitated with cold diethyl ether, repeats above-mentioned step
Rapid three times, gained is precipitated dry 12~16h afterwards, obtain synthetic peptide crude product；
F, synthetic peptide crude product purifying：Synthetic peptide crude product was carried out using semipreparative high performance liquid chromatography instrument P2000
Post is purified, and is collected the cut of purity >=95%, that is, is obtained class Bac5 antibacterial peptides after purification, its purity >=95%.
Semipreparative high performance liquid chromatography instrument for P2000 is provided by Beijing Wo Hua Creative Technology Ltd., chromatographic column
For C18 reversed-phase columns (4.6*250mm), (0.1mL trifluoroacetic acids are dissolved in the trifluoroacetic acid acetonitrile solution that mobile phase A is 0.1%
In 100mL acetonitrile solutions), (0.1mL trifluoroacetic acids are dissolved in 100mL deionizations to the trifluoroacetic acid aqueous solution that Mobile phase B is 0.1%
In water), using above-mentioned processing post, after loading in 25min, the ratio of mobile phase A is progressively adjusted to 52% by 27%, Mobile phase B
When ratio is progressively adjusted to 48%, 25.1min by 73%, the ratio of mobile phase A is changed into 100%, and the ratio of Mobile phase B is changed into 0%,
30min separation is carried out with 1mL/min flow velocity, Detection wavelength is 220nm, collect the cut of purity >=95% (such as the institute of accompanying drawing 1
Show)；
The identification of gained class Bac5 antibacterial peptides of the invention：Using liquid chromatography mass combined instrument
(Watersmicromass ZQ-2000) determines the molecular weight of products obtained therefrom antibacterial peptide, after measured：Its molecular weight is
4480.51Da (as shown in Figure 2).High-efficient liquid phase chromatogram condition：Chromatographic column is C18 reversed-phase columns (4.6*250mm), mobile phase A
For 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acids are dissolved in 100mL acetonitrile solutions), Mobile phase B is 0.1%
After trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acids are dissolved in 100mL deionized waters), loading in 25min, the ratio of mobile phase A
50%, when the ratio of Mobile phase B is progressively adjusted to 50%, 25.1min by 75% is progressively adjusted to by 25%, the ratio of mobile phase A becomes
For 100%, the ratio of Mobile phase B is changed into 0%, 30min separation is carried out with 1mL/min flow velocity, Detection wavelength is 220nm.
Mass spectroscopy condition：Using positive ionization mode, its capillary voltage is 3.00KV, capillary outlet voltage 50V, fragmentation voltage
For 5V, dry gas stream amount is 1.5L/min, and it is 350 DEG C to dry temperature degree, and scanning range is 400~1900m/z.
The positive beneficial effect that the present invention has：
The class Bac5 antibacterial peptides of the present invention are made up of 37 amino acid, rich in arginine, to gram positive bacteria (golden yellow Portugal
Grape coccus), gram-negative bacteria (Escherichia coli, detection of Salmonella) and fungi have efficient antibacterial activity, antibacterial action is fast, and antibacterial is lived
Property it is strong, with low hemolytic, do not produce drug resistance.Therefore, class Bac5 antibacterial peptides of the invention are preparing anti-infectives, herding
Had broad application prospects in terms of industry development, human disease treatment.
Brief description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of class Bac5 antibacterial peptides of the present invention.
Fig. 2 is the mass spectrogram of class Bac5 antibacterial peptides of the present invention.
Fig. 3 is the isoelectric point analysis result of class Bac5 antibacterial peptides.
Fig. 4 is class Bac5 antibacterial peptides to Escherichia coli minimal inhibitory concentration measurement result.
Fig. 5 is class Bac5 antibacterial peptides to detection of Salmonella minimal inhibitory concentration measurement result.
Fig. 6 is class Bac5 antibacterial peptides to staphylococcus aureus minimal inhibitory concentration measurement result.
Fig. 7 is the hemolysis rate measurement result of class Bac5 antibacterial peptides.
Embodiment
Below in conjunction with specific embodiment, the present invention is further detailed explanation, but is not intended to limit the model of the present invention
Enclose.
Embodiment 1：
One species Bac5 antibacterial peptides, its amino acid sequence is as follows：
Phe-A taug-Pro-Pro-isoleucine-Arg-Arg-Pro-Pro-different
Leucine-arginine-Pro-Pro-phenylalanine-tyrosine-Pro-Pro-Phe-A taug-dried meat
Propylhomoserin-proline-isoleucine-Arg-Pro-proline-isoleucine-Phe-Pro-Pro-different
Leucine-arginine-Pro-Pro-Phe-A taug-Pro-Pro.
Such Bac5 antibacterial peptide contains 37 amino acid residues, and its molecular weight is 4480.51Da, and isoelectric point is 12.6.Institute
Class Bac5 antibacterial peptides are stated rich in arginine residues (positively charged), the negative electrical charge of bacterium surface can be attracted.
The present embodiment class Bac5 antibacterial peptides are the final conjunction according to conventional polypeptide Solid phase synthesis using automatic Peptide synthesizer
Into class Bac5 antibacterial peptides purity analysis processing, its purity >=95% are carried out through semipreparative high performance liquid chromatography instrument.
The identification of gained class Bac5 antibacterial peptide products of the invention：
(1) measure of molecular weight：Using analytic type liquid chromatography mass combined instrument (Waters micromassZQ-2000)
The molecular weight of gained class Bac5 antibacterial peptides is determined, after measured, its molecular weight is 4480.51Da, as a result as shown in Figure 2.
Specifically testing conditions are：1. chromatographic condition is：Chromatographic column is C18 reversed-phase columns (4.6*250mm), and mobile phase A is
0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acids are dissolved in 100mL acetonitrile solutions), Mobile phase B is the three of 0.1%
After the fluoroacetic acid aqueous solution (0.1mL trifluoroacetic acids are dissolved in 100mL deionized waters), loading in 25min, the ratio of mobile phase A by
25% is progressively adjusted to 50%, when the ratio of Mobile phase B is progressively adjusted to 50%, 25.1min by 75%, and the ratio of mobile phase A is changed into
100%, the ratio of Mobile phase B is changed into 0%, 30min separation is carried out with 1mL/min flow velocity, Detection wavelength is 220nm.②
Mass spectroscopy condition：Using positive ionization mode, its capillary voltage is 3.00KV, capillary outlet voltage 50V, fragmentation voltage
For 5V, dry gas stream amount is 1.5L/min, and it is 350 DEG C to dry temperature degree, and scanning range is 400~1900m/z.
(2) amino acid sequence structure is determined：The amino acid sequence of class Bac5 antibacterial peptides is determined using automatic Protein Sequencer
Array structure.After measured, class Bac5 antibacterial peptides contain 37 amino acid residues, and its amino acid sequence is：Phe-A taug-
Pro-Pro-isoleucine-Arg-Arg-Pro-Pro-isoleucine-Arg-Pro-dried meat
Propylhomoserin-phenylalanine-tyrosine-Pro-Pro-Phe-A taug-Pro-Pro-isoleucine-essence
Propylhomoserin-Pro-Pro-isoleucine-Phe-Pro-Pro-isoleucine-Arg-Pro-dried meat
Propylhomoserin-Phe-A taug-Pro-Pro.
(3) isoelectric point is analyzed：
The EditSeq in DNAStar softwares is opened, file is opened, " new protein ", input class in " new " is clicked on
The amino acid sequence of Bac5 antibacterial peptides, saves as the document of pro forms.Then the Protean in DNAStar softwares is opened again,
File is opened, is clicked on " open ", selection just saves as the document of pro forms, is clicked on " opening ", in selection " Analysis "
" Titration Curve ", you can to obtain isoelectric point data, as a result see Fig. 3.After measured, class Bac5 antibacterial peptides of the present invention
Isoelectric point is respectively 12.6.
Embodiment 2：The application implementation of class Bac5 antibacterial peptides of the present invention
1st, the bacteriostatic activity analysis of class Bac5 antibacterial peptides
The bacteriostatic activity of class Bac5 antibacterial peptides is determined using lysoplate assay, its challenge organisms bacterial strain is：Large intestine
Bacillus ATCC8099, detection of Salmonella CVCC541, staphylococcus aureus ATCC6538, Escherichia coli and staphylococcus aureus purchase
From Nanjing Bian Zhen bio tech ltd, detection of Salmonella purchases Chinese veterinary microorganism culture presevation administrative center.
Above-mentioned 3 kinds of challenge organisms are diluted in 50 DEG C of bottom culture medium (TSB 0.3g, ultrapure agarose respectively
0.1g, plus ultra-pure water is to 10ml, pH 7.4) in, sterilizing flat board, solidification, (aperture about 3mm), alcolhol burner are punched with card punch
Slightly hot carry out back cover, 5 μ L antibacterial peptide samples is added with pipettor, each flat board includes positive control and feminine gender is right into every hole
According to.Positive control：Ampicillin is directed to gram-negative bacteria and gram positive bacteria；Negative control：PBS.Flat board is quiet
1h is put, test fluid is diffused into agarose.Then, then upper strata covering culture medium (TSB 0.3g, ultrapure agarose is added
0.1g, plus ultra-pure water is to 10ml, 7.4,50 DEG C or so of pH).Culture plate is inverted overnight incubation at 37 DEG C, then register hole
The diameter of the transparent circle on periphery, each strain is repeated three times, and calculates average value.
The bacteriostatic activity analysis result of the class Bac5 antibacterial peptides of table 1
Shown by the antibacterial activity testing result of table 1, class Bac5 antibacterial peptides of the invention are to Escherichia coli, detection of Salmonella, golden yellow
Color staphylococcus has stronger antibacterial activity.Therefore, class Bac5 antibacterial peptides of the invention are expected preparing treatment animal and people
There is good application prospect in terms of the medicine of bacteriosis, meanwhile, according to the general characteristic of known antibacterial peptide, class Bac5 resists
Bacterium peptide has good application potential in terms of feed addictive, disinfectant, preservative or cosmetic additive agent.
2nd, the minimal inhibitory concentration (MIC) of class Bac5 antibacterial peptides is determined
Challenge organisms bacterial strain is：Escherichia coli ATCC8099, detection of Salmonella CVCC541, staphylococcus aureus
ATCC6538, Escherichia coli and staphylococcus aureus are purchased from Nanjing Bian Zhen bio tech ltd, and detection of Salmonella purchases China
Veterinary microorganism culture presevation administrative center.
Strain subject is expanded into culture in TSB fluid nutrient mediums, the OD of bacterium solution is surveyed600Value.When OD values are in 0.6-0.8,
Bacterium solution is centrifuged into 10min under the conditions of 6000r/min, supernatant discarding collects bacterial sediment, bacterial sediment is used in equal volume
PBS (0.01M) is resuspended, then bacterial suspension is diluted into 2 × 10 with MH culture mediums6CFU/mL.Diluted with deionized water
Class Bac5 antibacterial peptide samples, it is 0.78-100 μ g/mL to make its concentration.In 96 porocyte culture plates, 50 μ L are added respectively per hole
The class Bac5 antibacterial peptides of various concentrations and the bacterial solution of 50 μ L dilutions, each concentration repeat three holes, are placed in 37 DEG C of culture 16-
18h, the solution in then mixing per hole, determines its OD600Value, using the corresponding class Bac5 antibacterials peptide concentration of OD value suddenly changes as
Its MIC, is as a result shown in Fig. 4, Fig. 5 and Fig. 6.
From Fig. 4, Fig. 5 and Fig. 6, class Bac5 antibacterial peptides are 6.25 μ g/ to the minimal inhibitory concentration (MIC) of Escherichia coli
ML, the minimal inhibitory concentration (MIC) to detection of Salmonella is 6.25 μ g/mL, to the minimal inhibitory concentration (MIC) of staphylococcus aureus
For 12.5 μ g/mL.As a result show, such Bac5 antibacterial peptide reaches Gamma Magnitude to the minimal inhibitory concentration of common bacterium have
There is extremely strong Antibacterial Activity.
3rd, the hemolytic analysis of class Bac5 antibacterial peptides
Fresh rabbit blood is taken, with 3.8% sodium citrate anti-freezing, anticoagulation is centrifuged into 10min through 3000r/min, phosphoric acid is used
Salt buffer (PBS) washing precipitation 3 times, is colourless, transparent to supernatant, then prepare 1% red blood cell with PBS.With PBS by class Bac5
The concentration of antibacterial peptide is adjusted to 400 μ g/mL, 200 μ g/mL, 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ
G/mL, 3.125 μ g/mL, 1.56 μ g/mL, 0.78 μ g/mL, 0.39 μ g/mL, add isometric red cell suspension.Using PBS as
Negative control, using 1%Tritonx-100 as positive control, 37 DEG C effect 1h, 1500r/min centrifugation 10min, by supernatant according to
96 orifice plates of secondary addition, the OD values surveyed with ELIASA at 540nm.Then according to calculation formula hemolysis rate (%)=(detection hole OD values-
Negative hole OD values)/(positive hole OD values-negative hole OD values) × 100% calculates hemolysis rate, and its result is shown in Fig. 7.
As shown in Figure 7, under 400 μ g/mL High Concentration Situation, the hemolysis rate of class Bac5 antibacterial peptides is 4.29%, still low
In 5%, illustrate that class Bac5 antibacterial peptides have preferable security, potential applicability in clinical practice is huge.
In summary, class Bac5 antibacterial peptide products of the present invention do not produce hemolytic, has a broad antifungal spectrum, to Gram-negative bacteria,
Gram-positive bacteria all has efficient antibacterial action.So, product class Bac5 antibacterial peptides of the present invention are preparing treatment gram
Can preferably it be applied in positive bacteria, the medicine of Gram-negative bacteria, and, it may also be used for prepare feed addictive, disappear
Toxic agent, preservative or cosmetic additive agent.
SEQUENCE LISTING
<110>Henan Science and Technology College
<120>One species Bac5 antibacterial peptides and its application
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 37
<212> PRT
<213> Artificial Sequence
<220>
<223> 1
<400> 1
Phe Arg Pro Pro Ile Arg Arg Pro Pro Ile Arg Pro Pro Phe Tyr Pro
1 5 10 15
Pro Phe Arg Pro Pro Ile Arg Pro Pro Ile Phe Pro Pro Ile Arg Pro
20 25 30
Pro Phe Arg Pro Pro
35
Claims (5)
1. a species Bac5 antibacterial peptides, it is characterised in that the amino acid sequence of the class Bac5 antibacterial peptides is as follows：Phenylalanine-
Arg-Pro-proline-isoleucine-Arg-Arg-Pro-Pro-isoleucine-arginine-dried meat
Propylhomoserin-Pro-Phe-Tyr-Pro-Pro-Phe-Arg-Pro-proline-different bright
Propylhomoserin-Arg-Pro-proline-isoleucine-Phe-Pro-Pro-isoleucine-arginine-dried meat
Propylhomoserin-Pro-Phe-Arg-Pro-proline.
2. class Bac5 antibacterial peptides according to claim 1, it is characterised in that：The class Bac5 antibacterial peptides have 37 amino acid
Residue, its molecular weight is 4480.51Da, and isoelectric point is 12.6.
3. the purposes of class Bac5 antibacterial peptides described in a kind of claim 1, it is characterised in that the purposes is：Class Bac5 antibacterial peptides
Application in the medicine for the treatment of gram-positive bacteria and gram positive bacterial infection is prepared.
4. purposes according to claim 3, it is characterised in that the medicine includes the class Bac5 antibacterials in claim 1
Peptide, and it is mixed with one or more kinds of pharmaceutically acceptable carriers and/or additive.
5. the class Bac5 antibacterial peptides described in a kind of claim 1 are in feed addictive, disinfectant, preservative or cosmetic additive agent
In application.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690667A (en) * | 2020-06-05 | 2020-09-22 | 广州颜如玉生物科技有限公司 | Method for improving antibacterial activity of antibacterial peptide BSN-37 |
| CN115819499A (en) * | 2022-07-05 | 2023-03-21 | 浙江大学 | Antibacterial nonapeptide and application thereof |
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| RAINER W.FRANK: "Amino Acid Sequences of Two Proline-rich Bactenecins", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 赵昆等: "牛抗菌肽Bac7-Bac5融合基因在大肠杆菌中的过表达，纯化及抑菌活性", 《中国生物工程杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690667A (en) * | 2020-06-05 | 2020-09-22 | 广州颜如玉生物科技有限公司 | Method for improving antibacterial activity of antibacterial peptide BSN-37 |
| CN115819499A (en) * | 2022-07-05 | 2023-03-21 | 浙江大学 | Antibacterial nonapeptide and application thereof |
| CN115819499B (en) * | 2022-07-05 | 2024-01-05 | 浙江大学 | Antibacterial nonapeptide and application thereof |
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| Publication number | Publication date |
|---|---|
| CN107298707B (en) | 2020-05-08 |
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