CN107746429A - A kind of end symmetrical antibacterial peptide PP and its preparation method and application - Google Patents
A kind of end symmetrical antibacterial peptide PP and its preparation method and application Download PDFInfo
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- CN107746429A CN107746429A CN201711098665.0A CN201711098665A CN107746429A CN 107746429 A CN107746429 A CN 107746429A CN 201711098665 A CN201711098665 A CN 201711098665A CN 107746429 A CN107746429 A CN 107746429A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The present invention provides a kind of end symmetrical antibacterial peptide PP and its preparation method and application.Antibacterial peptide PP sequence is as shown in sequence table SEQ ID No.1.Preparation method：The β-bend position amino acid sequence CRRRFC for being embedded in using two symmetrical α screw unions and wherein PG 1 forms it into end symmetrical antibacterial peptide, designs a kind of end symmetrical antibacterial peptide PP.Applications of the antibacterial peptide PP in the medicine for the treatment of gram-positive bacteria or gram positive bacterial infection disease is prepared, antibacterial peptide PP have higher bacteriostatic activity, hypotoxicity, and its therapeutic index is 80 times of PG 1, therefore has higher cell selective.The pathogen in organism can be more purposefully killed, improves the development potentiality and biological value of antibacterial peptide.
Description
Technical field
The invention mainly relates to a kind of end symmetrical antibacterial peptide PP and its preparation method and application.
Background technology
All the time, antibiotic is widely used in animal husbandry, can promote growth of animals or poultry as a kind of feed addictive, is improved
Animal husbandry production efficiency.However, the long-term abuse of antibiotic causes antibody-resistant bacterium breeding and medicament residue, this not only limits herding
The development of industry more damages human health.It is current mankind urgent problem to be solved that new no resistance, which is screened, without defective material antibacterials.
Antibacterial peptide is a kind of micromolecule polypeptide, have broad spectrum antibiotic activity, antifungal activity, antiviral activity, antiprotozoal activity and
The features such as being not likely to produce the resistance to the action of a drug.Unlike antibiotic, antibacterial peptide mainly acts on cytoplasma membrane, makes its permeabilization, finally
Cause bacterial cell membrane rupture content outflow and it is dead.Antibacterial peptide activity is strong, and function is extensive, in livestock application study
With potential important value, there is increasing researcher to begin to focus on this focus.Antibacterial peptide is also in pharmaceuticals industry
Have broad application prospects, be expected to turn into antibacterium, antiviral, cancer therapy drug new sources.
Antibacterial peptide is widespread in nature, and can be divided into 4 classes according to the amino acid the Nomenclature Composition and Structure of Complexes feature of antibacterial peptide, i.e.,
Alpha-helix type antibacterial peptide, beta sheet type antibacterial peptide, extended pattern antibacterial peptide and ring-like antibacterial peptide.Natural antibacterial peptide is mainly by animal body
Various Tissues produce, and are played an important role in Noninvasive pathogenic microorganism (including bacterium, virus, parasite and cancer cell).
Beta sheet type antibacterial peptides of the pig derived antimicrobial peptide Protegrin-1 (PG-1) containing 18 amino acid, have compared with high antibacterial activity, its
Hemolytic activity and higher to the toxicity of mammalian somatic cell, cell selective is low.Therefore, natural antibacterial peptide is killing bacterium
While toxic side effect is also produced to animal somatic cell, design brand-new antibacterial peptide or transformation natural antibacterial peptide, filter out cell toxicant
The low high selectivity antibacterial peptide of property is urgent problem.
The content of the invention
Based on above weak point, it is an object of the invention to provide a kind of end symmetrical antibacterial peptide PP and preparation method thereof
And application, the antibacterial peptide activity is higher and cytotoxicity is relatively low.
The technology used in the present invention is as follows：A kind of end symmetrical antibacterial peptide PP, its amino acid sequence such as SEQ ID No.1
It is shown.
The present invention also has following technical characteristic：
1st, a kind of end symmetrical antibacterial peptide PP preparation method, step are as follows：
(1) by being embedded in β-corner structure in two symmetrical α screw unions, brand-new end symmetrical antibacterial is designed
Peptide；The amino acid of different types of structure, including hydrophobic amino acid Ile, Phe and Trp are selected first；Positively charged amino acid
His, Lys and Arg, α spiral of the design with high hydrophobicity and perfect amphiphilic structure combine hexa-atomic residue IHKFWR, then,
Two alpha-helix monomer insertion pig derived antimicrobial peptide Protegrin-1 β-rotation angle position amino acid sequence CRRRFC forms it into two
It is the end symmetrical antibacterial peptide of β-corner structure to hold as αhelix, centre；
(2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method, obtained peptide resin is cut by TFA
After cutting, this polypeptide is obtained；
(3) after RPLC purifying and Mass Spectrometric Identification, that is, the preparation of the polypeptide is completed.
2nd, a kind of end symmetrical antibacterial peptide PP as described above is preparing treatment gram-positive bacteria or Gram-negative bacteria
Application in the medicine of infectious diseases.
Antibacterial and hemolytic activity detection are carried out to antibacterial peptide PP, finds PP to Escherichia coli, Pseudomonas aeruginosa, golden yellow grape
Coccus, MRSE, salmonella typhimurium, Salmonella gallinarum, eight kinds of bacillus subtilis, streptococcus fecalis strains have
Obvious inhibitory action, and hemolytic activity is relatively low.Its therapeutic index is 80 times of pig derived antimicrobial peptide Protegrin-1, therefore
With higher cell selective.In summary, PP is a kind of antibacterial peptide with higher application value.
Brief description of the drawings
Fig. 1 is antibacterial peptide PP mass spectrogram.
Fig. 2 antibacterial peptides PP hemolytic activity figure.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
Embodiment 1
The design of antibacterial peptide
By being embedded in β-corner in two symmetrical α screw unions, symmetrical end antibacterial peptide is designed.Select first not
With the amino acid of structure type, including hydrophobic amino acid Ile, Phe and Trp；Positively charged amino acid His, Lys and Arg, if
α spiral of the meter with high hydrophobicity and perfect amphiphilic structure combines hexa-atomic residue (IHKFWR), then, in two alpha-helix monomers
β-rotation angle position amino acid sequence CRRRFC of embedded pig derived antimicrobial peptide Protegrin-1 (PG-1) forms it into end symmetrical and resisted
Bacterium peptide.
The amino acid sequence of the end symmetrical antibacterial peptide of table 1
PP charge numbers are+7, and hydrophobic value is respectively 0.49.By two pig derived antimicrobial peptide Protegrin-1 (PG-1), PP peptides
Terminal-carboxy amidation to improve a positive charge and increase the stability of peptide.Antibacterial peptide sterilization work is being improved by this method
Property and reduce its hemolytic activity, antibacterial peptide is improved the potentiality of cell selective and substitute antibiotics.
Embodiment 2
Solid-state chemical reaction method method synthesizes PP.
1st, the preparation of antibacterial peptide is carried 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 each second amino acid of antibacterial peptide C-terminal)；According to
This program is synthesized to N-terminal from C-terminal successively, until synthesis finishes, obtains sloughing the resin of the side chain protected of Fmoc groups；
2nd, in peptide resin obtained above, cutting reagent is added, reacts 2h under 20 DEG C of lucifuges, is filtered；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, separate out white powder thing, and 10min is centrifuged with 2500g, collect precipitation, then washed and sunk with absolute ether
Form sediment, vacuum drying, obtain polypeptide, wherein cutting reagent by TFA, water and TIS (tri isopropyl chlorosilane) according to mass ratio 95:
2.5:2.5 mix；
3rd, 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, the anti-phase normal pressure posts of C18, using gradient elution, (eluant, eluent is methanol and aqueous sodium persulfate solution according to volume ratio
For 30:70~70:30 mixing), flow velocity 1mL/min, detection ripple is 220nm, collects main peak, is freezed；Recycle anti-phase C18 posts
It is further purified, eluent A is the 0.1%TFA/ aqueous solution；Eluent B is 0.1%TFA/ acetonitrile solutions, wash-out concentration 25%
B~40%B, elution time 12min, flow velocity 1mL/min, then main peak is ibid collected, freeze；
4th, the identification of antibacterial peptide：Antibacterial peptide obtained above is analyzed by electron spray mass spectrometry, shown in mass spectrogram
Molecular weight (as shown in Figure 1) and the theoretical molecular in table 1 are basically identical, and the purity of antibacterial peptide is more than 95%.
Embodiment 3：
The measure of antibacterial peptide antibacterial activity
1st, the measure of antibacterial activity：Peptide is configured as certain storing liquid in case using.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
The antibacterial peptide solution of graded series is configured successively.Take the above-mentioned μ l of solution 100 to be placed in 96 porocyte culture plates, then add respectively
Isometric bacterium solution to be measured (~105Individual/ml) in each hole.Positive control (antibacterial peptide is not contained containing bacterium solution) is set respectively
With negative control (both without bacterium solution or be free of peptide).37 DEG C of incubated 20h, with visually have no bottom hole portion have research of chaotic phenomenon i.e.
For minimal inhibitory concentration.Testing result such as table 2.
The bacteriostatic activity of the antibacterial peptide of table 2
It can be seen from Table 2 that PP shows different degrees of bacteriostatic activity for Gram-negative and positive bacteria, especially
PP is significantly higher than PG-1, table for the bacteriostatic activity of high drug-fast bacteria Pseudomonas aeruginosa (B.pyocyaneum ATCC 27853)
Show that antibacterial peptide activity can be effectively improved by being embedded in β-corner in two symmetrical α screw unions.
2nd, the measure of hemolytic activity：The new blood 1mL of people is gathered, is dissolved into after anticoagulant heparin in 2mL PBS solutions,
1000g centrifuges 5min, collects red blood cell；Washed 3 times with PBS, then be resuspended with 10mL PBS；Take 50 μ L red cell suspensions and 50 μ L
It is well mixed with the antibacterial peptide solution of the various concentrations of PBS dissolvings, the constant-temperature incubation 1h in 37 DEG C of incubators；Taken out after lh, 4
DEG C, 1000g centrifugation 5min；Take out supernatant ELIASA light-metering absorption value at 570nm；Every group is averaged, and is compared 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 L 0.1%Tritonx-100 conducts
Positive control.Minimum hemolytic concentration is antibacterial peptide concentration when antibacterial peptide causes 5% hemolysis rate.Testing result is shown in Table 3.
The measure of the antibacterial peptide hemolytic activity of table 3
It can be seen from Table 3 that PP has no hemolytic activity in the range of detectable concentration, and PG-1 shows in low concentration
Go out obvious hemolytic activity, PP therapeutic index is 80 times of PG-1.
Result above shows, by the way that PG-1 β-corner sequence is combined with two αhelix, can to improve antibacterial peptide thin
Born of the same parents' selectivity.Its therapeutic index can obtain according to the bacteriostatic activity of antibacterial peptide and hemolytic activity to evaluate the biologic valence of antibacterial peptide
Value.As can be seen from Table 3, PP therapeutic index is 80 times of PG-1, and this explanation PP has compared with high bacteriostatic activity and hypotoxicity, has
The development potentiality of substitute antibiotics.
Sequence table
<110>Northeast Agricultural University
<120>A kind of end symmetrical antibacterial peptide PP and its preparation method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Ile His Lys Phe Trp Arg Pro Gly Arg Trp Phe Lys His Ile
1 5 10
Claims (3)
1. a kind of end symmetrical antibacterial peptide PP, it is characterised in that its amino acid sequence is as shown in SEQ ID No.1.
2. a kind of end symmetrical antibacterial peptide PP according to claim 1 preparation method, it is characterised in that step is as follows：
(1) by being embedded in β-corner structure in two symmetrical α screw unions, brand-new end symmetrical antibacterial peptide is designed；
The amino acid of different types of structure, including hydrophobic amino acid Ile, Phe and Trp are selected first；Positively charged amino acid His,
Lys and Arg, α spiral of the design with high hydrophobicity and perfect amphiphilic structure combines hexa-atomic residue IHKFWR, then, at two
Alpha-helix monomer insertion pig derived antimicrobial peptide Protegrin-1 β-rotation angle position amino acid sequence CRRRFC, forms it into both ends
For αhelix, centre is the end symmetrical antibacterial peptide of β-corner structure；
(2) peptide resin is obtained by Peptide synthesizer using solid-state chemical reaction method method, obtained peptide resin is cut by TFA
Afterwards, this polypeptide is obtained；
(3) after RPLC purifying and Mass Spectrometric Identification, that is, the preparation of the polypeptide is completed.
3. a kind of end symmetrical antibacterial peptide PP according to claim 1, preparing treatment gram-positive bacteria and gram
Application in the medicine of negative microbial infections disease.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283252A (en) * | 2019-07-12 | 2019-09-27 | 东北农业大学 | Pig source heterozygous antibacterial peptide PP-1 and its preparation method and application |
| CN111423492A (en) * | 2020-03-30 | 2020-07-17 | 东北农业大学 | β hairpin antibacterial peptide containing D-type proline and glycine corner and preparation method thereof |
| CN111454330A (en) * | 2020-03-30 | 2020-07-28 | 东北农业大学 | β hairpin antibacterial peptide with tryptophan and histidine interaction across chains and preparation method thereof |
| CN111484546A (en) * | 2020-03-30 | 2020-08-04 | 东北农业大学 | β hairpin antibacterial peptide containing asparagine and glycine corner and preparation method thereof |
| CN111533786A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Beta-hairpin antibacterial peptide with tryptophan and arginine cross-chain interaction and preparation method thereof |
| CN111533789A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Tryptophan and lysine chain-crossing interaction beta-hairpin antibacterial peptide and preparation method thereof |
| CN111533788A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Cell-penetrating antibacterial peptide targeting streptococcus agalactiae and preparation method and application thereof |
| CN114989254A (en) * | 2022-06-17 | 2022-09-02 | 中山大学 | Polypeptide, design method thereof and application of polypeptide in preparation of fusobacterium nucleatum inhibiting product or colorectal cancer preventing medicine |
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| CN1398897A (en) * | 2002-09-02 | 2003-02-26 | 上海高科联合生物技术研发有限公司 | Serial synthetic antibacterial peptide |
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2017
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1398897A (en) * | 2002-09-02 | 2003-02-26 | 上海高科联合生物技术研发有限公司 | Serial synthetic antibacterial peptide |
Non-Patent Citations (1)
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283252A (en) * | 2019-07-12 | 2019-09-27 | 东北农业大学 | Pig source heterozygous antibacterial peptide PP-1 and its preparation method and application |
| CN111423492A (en) * | 2020-03-30 | 2020-07-17 | 东北农业大学 | β hairpin antibacterial peptide containing D-type proline and glycine corner and preparation method thereof |
| CN111454330A (en) * | 2020-03-30 | 2020-07-28 | 东北农业大学 | β hairpin antibacterial peptide with tryptophan and histidine interaction across chains and preparation method thereof |
| CN111484546A (en) * | 2020-03-30 | 2020-08-04 | 东北农业大学 | β hairpin antibacterial peptide containing asparagine and glycine corner and preparation method thereof |
| CN111533786A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Beta-hairpin antibacterial peptide with tryptophan and arginine cross-chain interaction and preparation method thereof |
| CN111533789A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Tryptophan and lysine chain-crossing interaction beta-hairpin antibacterial peptide and preparation method thereof |
| CN111533788A (en) * | 2020-03-30 | 2020-08-14 | 东北农业大学 | Cell-penetrating antibacterial peptide targeting streptococcus agalactiae and preparation method and application thereof |
| CN111423492B (en) * | 2020-03-30 | 2021-12-14 | 东北农业大学 | Beta-hairpin antibacterial peptide containing D-type proline and glycine corner and preparation method thereof |
| CN111533786B (en) * | 2020-03-30 | 2022-02-08 | 东北农业大学 | Beta-hairpin antibacterial peptide with tryptophan and arginine cross-chain interaction and preparation method thereof |
| CN111533788B (en) * | 2020-03-30 | 2022-02-08 | 东北农业大学 | Cell-penetrating antibacterial peptide targeting streptococcus agalactiae and preparation method and application thereof |
| CN114989254A (en) * | 2022-06-17 | 2022-09-02 | 中山大学 | Polypeptide, design method thereof and application of polypeptide in preparation of fusobacterium nucleatum inhibiting product or colorectal cancer preventing medicine |
| CN114989254B (en) * | 2022-06-17 | 2023-11-03 | 中山大学 | Polypeptide, design method thereof and application of polypeptide in preparation of medicines for inhibiting Fusobacterium nucleatum products or preventing colorectal cancer |
| WO2023241675A1 (en) * | 2022-06-17 | 2023-12-21 | 中山大学 | Polypeptide and design method therefor, and application thereof in preparing drug for inhibiting fusobacterium nucleatum product or preventing colorectal cancer |
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