CN106518999B - Antibacterial peptide WW and its preparation method and application based on micro peptide strategy - Google Patents
Antibacterial peptide WW and its preparation method and application based on micro peptide strategy Download PDFInfo
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- 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
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Abstract
The present invention provides a kind of antibacterial peptide WW and its preparation method and application based on micro peptide strategy, and sequence is as shown in sequence table SEQ ID No.1.The present invention obtains the crucial physical parameter with female peptide using the strategy of micro peptide theory and raising bioactivity using the dermaseptin family peptides of amphibian frog skin secretion as template, and length foreshortens to the sequence of 13 amino acid.The bacteriostatic activity of WW is slightly below melittin, but therapeutic index is up to 69.8, is 698 times of melittin.The risk that immunogenicity and heterologous repellency are theoretically avoided by this method occurs, and improves selectivity of the antibacterial peptide between bacterial cell and mammalian cell, and improving it becomes the development potentiality of antibiotic substitute.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of antibacterial peptide WW and its preparation side based on micro peptide strategy
Method and application.
Background technology
It is tight to result in antibiotic residue etc. in unbalance intestinal microecology, the generation of drug resistance flora, livestock products for the abuse of antibiotic
The consequence of weight, double risks are brought to human health and environmental protection.Various countries have had been carried out pole to antibiotic residue in livestock products
For severe limit standard, or even disabling.This brings great risk to the production of China's livestock products, such as in " green trade wall
Build " under, frequency is now shut out processing, and economic loss is huge.Therefore, there is an urgent need for find novel green, safe and efficient antibiotic replacement
Product.
Antibacterial peptide (Antimicrobial peptides, AMPs), is prevalent in the defence of nearly all viable organism
In system.There is antibacterial peptide participation in ancient, nonspecific innate immune system, is the starting stage of organism infection
Main preventing mechanism.Antibacterial peptide is usually made of 12-50 amino acid residue, and molecular weight is less than 10kDa, most of antibacterial peptides
Containing positively charged amino acid, there is amphipathic and cationic, to various bacteria, fungi, virus, parasite even cancer
Cell has inhibiting effect, and very low to the toxicity of body itself, and extremely strong stability is conducive to large-scale industry metaplasia
Production.Antibacterial peptide has different antimicrobial mechanisms from antibiotic, the former interferes the performance of a variety of biological sexual functions with " mild formula ", and
Non- high degree of specificity target site point makes its loss of function, and the latter's tendency acts on the single target spot of thalline.And antibacterial peptide exists
Noresidue in animal body, it is environmentally safe, meet the needs of livestock product safety production, is suitble to make in feed preparation
With with the potential quality as new generation of green feed addictive.Currently, the exploitation and application to antibacterial peptide are in husbandry sector
Carry out extensively with pharmaceutical sanitary field.
But compared with conventional antibiotic, natural antibacterial peptide and production application there is also gap, activity is low, cytotoxicity is high,
The defects of production cost height, half-life short, can not be ignored.Therefore engineer's antibacterial peptide or molecular modification have natural antibacterial peptide
It is one of the effective way of high-efficiency antimicrobial preparation development.But in fact about antibacterial peptide, there are two can not be ignored using upper
Problem：On the one hand the antibacterial peptide after engineered still retains the trace of female peptide, easily there is immunogenic response, can if being used for a long time
The secretion that can inhibit animal body subjective antisepsis peptide, causes immune system dysfunction；On the other hand for the anti-of brand-new design
Bacterium peptide has no to be associated with, can theoretically have rejection, be also easy to produce allergic reaction, it is strong to be unfavorable for livestock and poultry with by livestock and poultry animal is raised
Health and raising production performance.
In consideration of it, how to evade above-mentioned risk, need to attach the importance.Using species taxonomy and systematic evolution tree as foundation,
It is motherboard to select the antibacterial peptide of the species of affiliation farther out, carries out micro and appropriate reconstruction, with strong, the haemolysis that obtains antibacterial activity
The low novel antimicrobial peptide of property, becomes an effective approach.
Invention content
Based on the above shortcoming, the purpose of the present invention is to provide a kind of antibacterial peptide WW based on micro peptide strategy and its
Preparation method and application；The antibacterial peptide activity is higher and cytotoxicity is relatively low.
The purpose of the present invention is realized by following technology：A kind of antibacterial peptide WW based on micro peptide strategy, sequence such as sequence
Shown in table SEQID No.1.
The present invention also has following technical characteristic：
1, a kind of antibacterial peptide WW based on micro peptide strategy as described above, preparation method are as follows：
(1) based on the dermaseptin families of amphibian frog skin secretion, using micro peptide strategy, retain and close
Key physical parameter is foreshortened to the antibacterial peptide of 13 amino acid lengths by long peptide；Activity to improve peptide uses central symmetry knot
The strategy design such as structure, the cation-π effect of Trp and its anchoring effect positioned at headgroup, C-terminal amidation has obtained antibacterial peptide
WW, sequence is as shown in sequence table SEQ No.1；
(2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method, obtained peptide resin is cut by TFA
After cutting, two polypeptides are obtained；
(3) after reversed-phase high performance liquid chromatography purifying and Mass Spectrometric Identification, that is, the preparation of polypeptide is completed.
2, a kind of antibacterial peptide WW based on micro peptide strategy as described above is preparing treatment gram-positive bacteria or leather orchid
Application in family name's negative microbial infections disease medicament.
Experimental technique by the antibacterial peptide of this method preparation is simple, and antibacterial and hemolytic activity are carried out to obtained antibacterial peptide
Detection finds WW to Escherichia coli, Pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermis, salmonella typhimurium, chicken
Salmonella, the strains such as bacillus subtilis, streptococcus fecalis have apparent inhibiting effect, and have very low hemolytic activity.
In conclusion WW is a kind of antibacterial peptide with higher application value.
Description of the drawings
Fig. 1 is the mass spectrogram of antibacterial peptide WW.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
The design of antibacterial peptide
Based on the dermaseptin families of amphibian frog skin secretion, using micro peptide strategy,
Dermaseptin families are made of 55 peptides, are come out by APD database retrievals, and it is micro- that bacteriostatic activity is not less than 100
It rubs；The design according to some parameters of dermaseptin families, is designed first, while necessary carry is additionally added in WW
The design of high activity such as introduces two ends and Trp, symmetrical structure etc. is added, though WW is derived from this family, with 55 female peptides
Characteristic is compared, and there has been apparent difference, then compared respectively with itself and 55 female peptides, then without comparativity.It is active in order to illustrate it, I
With common antibacterial peptide melittin as a contrast, it may be said that the design of bright WW is successful.
It is as shown in table 1 to retain critical physical parameter, the antibacterial peptide of 13 amino acid lengths is foreshortened to by long peptide；To improve peptide
Activity use centrosymmetric structure, the cation-π effect of Trp and its anchoring effect positioned at headgroup, C-terminal amidation
WW antibacterial peptides are obtained Deng strategy design, sequence is as shown in table 2.
The physical parameter of 1 separate sources peptide of table
The amino acid sequence of 2 derived peptide of table
The charge number of WW is+3, and hydrophobic value is -0.32.By the terminal-carboxy amidation of peptide WW to improve a positive charge simultaneously
Increase the stability of peptide.
Embodiment 2
Solid-state chemical reaction method method synthesizes WW antibacterial peptides
1, the preparation of antibacterial peptide carries out one by one from C-terminal to N-terminal, is completed by Peptide synthesizer.First by Fmoc-X (X
It is first amino acid of C-terminal of each antibacterial peptide) Wang resins are linked into, obtain X-Wang trees after then sloughing Fmoc groups
Fat；Again by Fmoc-Y-Trt-OH (9- fluorenes methoxy carboxyl-trimethyl-Y, Y is second amino acid of each antibacterial peptide C-terminal)；According to
This program is synthesized to N-terminal from C-terminal successively, until synthesis finishes, obtains the resin for sloughing the side chain protection of Fmoc groups；
2, in peptide resin obtained above, cutting reagent is added, 20 DEG C are protected from light lower reaction 2h, filtering；Precipitate TFA (three
Fluoroacetic acid) washing, washing lotion is mixed with above-mentioned filtrate, Rotary Evaporators concentration, the precooling for adding 10 times or so volumes is anhydrous
Ether, -20 DEG C precipitate 3h, and white powder object is precipitated, and 10min is centrifuged with 2500g, collect precipitation, then washed and sunk with anhydrous ether
Form sediment, vacuum drying obtains polypeptide, wherein cutting reagent by TFA, water and TIS (tri isopropyl chlorosilane) according to mass ratio 95:
2.5:2.5 mixing；
3, column equilibration 30min is carried out using 0.2mol/L sodium sulphate (phosphoric acid is adjusted to pH7.5), with 90% acetonitrile solution
Polypeptide is dissolved, filtering, C18 reverse phase normal pressure columns, using gradient elution, (eluant, eluent is methanol and aqueous sodium persulfate solution according to volume ratio
It is 30:70~70:30 mixing), flow velocity 1mL/min, detection wave is 220nm, collects main peak, freeze-drying；Recycle reverse phase C18 columns
It is further purified, eluent A is 0.1%TFA/ acetonitrile solutions；Eluent B is 0.1%TFA/ aqueous solutions, wash-out concentration 50%
~90%B, elution time 30min, flow velocity 1mL/min, then main peak is ibid collected, freeze-drying；
4, the identification of antibacterial peptide：Antibacterial peptide obtained above is analyzed by electron spray mass spectrometry, is shown in mass spectrogram
Molecular weight (as shown in Figure 1) and the theoretical molecular weight in table 2 are almost the same, and the purity of antibacterial peptide is more than 95%.
Embodiment 3：The measurement of antibacterial peptide antibacterial activity
1, the measurement of antibacterial activity：Peptide is configured as certain storing liquid in case using.It is surveyed using micro broth dilution method
The minimal inhibitory concentration of fixed several antibacterial peptides.Using 0.01% acetic acid (containing 0.2%BSA) as dilution, doubling dilution is used
Configure in order the antibacterial peptide solution of graded series.It takes 100 μ L of above-mentioned solution to be placed in 96 porocyte culture plates, then adds respectively
Isometric bacterium solution to be measured (~105A/mL) in each hole.Positive control (antibacterial peptide is not contained containing bacterium solution) is respectively set
It (had both been free of bacterium solution with negative control or had been free of peptide).37 DEG C of constant temperature incubation 20h, with visually have no hole bottom have research of chaotic phenomenon i.e.
For minimal inhibitory concentration.Testing result is as shown in table 3.
The bacteriostatic activity of 3 antibacterial peptide of table
It can be seen from Table 3 that WW shows Gram-negative and positive bacteria different degrees of bacteriostatic activity, with bee
Toxin ME26 is compared, and WW bacteriostatic activities are slightly below ME26, illustrates that the design of WW succeeds, and theoretically can avoid exempting from
The risk of epidemic focus and heterologous repellency.
2, the measurement of hemolytic activity：The new blood 1mL of people is acquired, is dissolved into 2mL PBS solutions after anticoagulant heparin,
1000g centrifuges 5min, collects red blood cell；It is washed 3 times with PBS, then is resuspended with 10mL PBS；Take 50 μ L red cell suspensions and 50 μ L
It is uniformly mixed with the antibacterial peptide solution of the various concentration of PBS dissolvings, the constant-temperature incubation 1h in 37 DEG C of incubators；It is taken out after l h, 4
DEG C, 1000g centrifuge 5min；It takes out supernatant microplate reader and surveys absorbance value at 570nm；Every group is averaged, and compares point
Analysis.Wherein 50 μ L red blood cells add 50 μ L PBS as negative control；50 μ L red blood cells add 50 μ L0.1%Tritonx-100 conducts
Positive control.Minimum hemolytic concentration is antibacterial peptide concentration when antibacterial peptide causes 5% hemolysis rate.Testing result is as shown in table 4.
The measurement of 4 antibacterial peptide hemolytic activity of table
It can be seen from Table 4 that WW does not show hemolytic activity in detection range, and ME26 is compareed in 0.25 μM of concentration
When just show high hemolytic activity.Illustrate that WW is suitable for the addition in livestock and poultry.
The above results show that WW is obviously shortened length, cost is reduced, antibacterial activity is strong, and hemolytic is low.Comprehensive analysis
The antibacterial and hemolytic activity of antibacterial peptide can more fully be commented by therapeutic index (ratio of hemolytic concentration and Mlc)
The biological activity of each antibacterial peptide of valence.As can be seen from Table 4, WW has higher therapeutic index, shows the WW that design obtains
Antibacterial peptide has the development potentiality of higher substitute antibiotics.
<110>Northeast Agricultural University
<120>Antibacterial peptide WW and its preparation method and application based on micro peptide strategy
<160> 1
<210> 1
<211> 13
<212> PRT
<213>Artificial sequence
<400> 1
Trp Trp Lys Ala Ala Ala Lys Ala Ala Ala Lys Trp Trp-NH2
1 5 10 13
Claims (3)
1. a kind of antibacterial peptide WW based on micro peptide strategy, which is characterized in that sequence is as shown in sequence table SEQ ID No.1.
2. a kind of antibacterial peptide WW based on micro peptide strategy according to claim 1, which is characterized in that preparation method is such as
Under：
(1) based on the Magainin antibacterial peptide family of amphibian frog skin secretion, using micro peptide strategy, retain crucial object
Parameter is managed, the antibacterial peptide of 13 amino acid lengths is foreshortened to by long peptide；For improve peptide activity use centrosymmetric structure,
Cation-π the effects and its anchoring effect positioned at headgroup, C-terminal amidation of Trp has obtained antibacterial peptide WW, sequence such as sequence
Shown in table SEQ ID No.1；
(2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method, obtained peptide resin is cut by TFA
Afterwards, the polypeptide is obtained；
(3) after reversed-phase high performance liquid chromatography purifying and Mass Spectrometric Identification, that is, the preparation of the polypeptide is completed.
3. a kind of antibacterial peptide WW based on micro peptide strategy according to claim 1 prepare treatment gram-positive bacteria or
Application in gram positive bacterial infection disease medicament.
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| CN109553657B (en) * | 2018-11-30 | 2021-10-19 | 东北农业大学 | Non-perfect amphiphilic peptide W4 and preparation method and application thereof |
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| CN103467580B (en) * | 2013-09-18 | 2015-07-08 | 刘诚 | Preparation method and application of artificial antimicrobial peptide MA-D4 |
| EP3096616B1 (en) * | 2014-01-24 | 2020-04-29 | The Regents of the University of Colorado, a Body Corporate | Dermaseptin-type and piscidin-type antimicrobial peptides |
| KR101652263B1 (en) * | 2014-06-13 | 2016-08-30 | (주)콜로디스 바이오사이언스 | Adhesive Protein Comprising Antimicrobial Peptide and Antimicrobial Coating Composition Comprising the Same |
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