CN106366162A - Efficient alpha-helix antibacterial peptide GV and preparation method and application thereof - Google Patents
Efficient alpha-helix antibacterial peptide GV and preparation method and application thereof Download PDFInfo
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- CN106366162A CN106366162A CN201611064519.1A CN201611064519A CN106366162A CN 106366162 A CN106366162 A CN 106366162A CN 201611064519 A CN201611064519 A CN 201611064519A CN 106366162 A CN106366162 A CN 106366162A
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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 invention relates to an efficient alpha-helix antibacterial peptide GV and a preparation method and application thereof. The antibacterial peptide GV is an antibacterial peptide with high cell selectivity and achieves an optimal point of junction of the antibacterial activity and cell toxicity of the antibacterial peptide. A sequence of the antibacterial peptide GV is represented by a sequence table SEQ ID No. 1. The preparation method comprises the following steps: (1) carrying out designing in accordance with alpha-helix peptide GRX2RX3RX2RG serves as a template, so as to obtain a brand-new antibacterial peptide GV, wherein X= V; (2) synthesizing a polypeptide crude product through a polypeptide synthesizer by a solid-phase chemical synthesis method; purifying the synthesized polypeptide by using out-phase high-performance liquid chromatography, and carrying out identification on the synthesized polypeptide by using electrospray mass spectrography, thereby preparing the polypeptide. The antibacterial peptide GV provided by the invention has relatively high bacteriostatic activity and relatively low hemolytic activity and is the highest in therapeutic index, thereby having great development potential.
Description
Technical field
The present invention relates to a kind of efficient α spiral antibacterial peptide gv and its preparation method and application.
Background technology
Antibacterial peptide, also known as antimicrobial peptide or peptide antibiotic, is that the small molecule being produced by antibacterial specific gene coding is many
Peptide, has the biological activitys such as broad-spectrum antiseptic, antiviral, antifungal, parasiticide and antitumor.Antibacterial peptide passes through alkaline positively charged
There is electrostatic attraction with negatively charged group on bacterial cell membrane in the residue of lotus, its hydrophobic amino acid inserts bacterial cell membrane
Lipid bilayer, cause the physical injury of film, lead to cellular content extravasation and dead.The effect of antibacterial peptide is not related to
Specific receptor, it can quickly killing microorganisms and do not produce drug resistance.In addition antibacterial peptide is a kind of polypeptides matter in itself,
There is not residual sex chromosome mosaicism, so the antibacterial peptide optimal substitute that is antibiotic, there is extremely wide market application foreground.
Although but antibacterial peptide has a variety of advantages, at present, the antibacterial peptide of only only a few is applied to the row such as medical science, food, herding
Industry produces.The use of antibacterial peptide is primarily limited to high synthesis cost with it for normal cell, for example, erythrocyte etc. is made
Become haemolysiss, that is, cell selective is low, then, find a kind of method gesture improving antibacterial peptide cell selective must
OK.
Antibacterial peptide is typically made up of 12-60 amino acid residue, and molecular mass is less, and species is various, complex structure, therefore
It is difficult to find unified model and studied representing all antibacterial peptides.In addition, people's blindness using engineered method
Expression antibacterial peptide, without comprehensively being groped to the 26S Proteasome Structure and Function of antibacterial peptide and in vitro tests, leads to expression to obtain
Antibacterial peptide most active not high, or there is hemolytic activity.Antibacterial peptide is divided into following four classes by its secondary structure: 1) α-spiral shell
Rotation antibacterial peptide；2) beta sheet antibacterial peptide；3) annular antibacterial peptide；4) random coil antibacterial peptide.Alpha-helix antibacterial peptide is antibacterial peptide
The most class antibacterial peptide of quantity in family, is the primary structure form of antibacterial peptide.Melittin (melittin) be typical α-
Spiral antibacterial peptide, its molecule carries 6 positive charges, containing 3 lysine residues and 2 arginine residues.Melittin has
There is broad spectrum antibiotic activity, but the cytotoxicity such as haemolysis is also higher.
Content of the invention
The present invention is to obtain the antibacterial peptide that antibacterial activity is higher and cytotoxicity is relatively low, thus providing one kind
Efficiently α spiral antibacterial peptide gv and its preparation method and application.
The technology used in the present invention is as follows: a kind of efficient α spiral antibacterial peptide gv, its sequence such as sequence table seq id
Shown in no.1.
The present invention also has a kind of following technical characteristic: preparation method bag of efficient α spiral antibacterial peptide gv as above
Include following steps:
1) pass through with alpha-helix peptide grx2rx3rx2Rg, x=v, are that stencil design obtains brand-new antibacterial peptide gv；
2) adopt solid-state chemical reaction method method, by Peptide synthesizer synthesis polypeptide crude product；The polypeptide of synthesis is used anti-phase
High performance liquid chromatography carries out purification, and using electrospray mass spectrometry, the polypeptide of synthesis is identified, completes the preparation of polypeptide.
A kind of efficient α spiral antibacterial peptide gv as above, in preparation treatment gram positive bacteria or gram negative bacteria
Application in infectious disease medicament.
The present invention by with alpha-helix grx2rx3rx2rg (x represents w, f, v, i, a and l) obtain for stencil design brand-new
Antibacterial peptide gw, gf, gv, gi, ga, gl, the arg (arginine in template；R), it is a kind of basic amino acid, the positive charge providing can
There is electrostatic attraction effect with the negative charge of bacterium surface.Trp (tryptophan；W), phe (Phenylalanine；F), val (L-Valine；
V), ala (alanine；A), leu (leucine；L) with ile (isoleucine；I) it is all hydrophobic amino acid, they can be with antibacterial
There is hydrophobicity effect in the phospholipid on surface, destroy the immobilized artificial membrane of antibacterial, thus producing biologic activity.Especially contain hydrophobicity ammonia
The antibacterial peptide gv of base acid L-Valine (v) has high cell selective.
The experimental technique of the antibacterial peptide prepared by this method is simple, carries out antibacterial and hemolytic activity to the antibacterial peptide obtaining
Detection, finds gv not only to escherichia coli, Salmonella typhimurium, bacillus pyocyaneus, staphylococcus aureuses, epidermis Fructus Vitis viniferae ball
Bacterium, bacillus subtilises, S. pullonum, eight kinds of strains of streptococcus faecalis have obvious inhibitory action, and have very low
Hemolytic activity.Thus, in general, gv is a kind of antibacterial peptide with higher using value.
Brief description
Fig. 1 is the mass spectrum of the antibacterial peptide gv that design obtains.
Specific embodiment
Embodiment 1
With alpha-helix peptide grx2rx3rx2rg, (x represents w, f, v, i, a and l) obtain brand-new alpha-helix peptide for stencil design
Gw, gf, gv, gi, ga, gl.
Its aminoacid sequence is:
Above-mentioned two antibacterial peptide is synthesized using Peptide synthesizer, method is solid-state chemical reaction method method, concrete steps
For:
1) preparation of antibacterial peptide is carried out to n end one by one from c end, is completed by Peptide synthesizer.First by fmoc-x (x
It is first, the c end aminoacid of each antibacterial peptide) it is linked into wang resin, obtain x-wang tree after then sloughing fmoc group
Fat；Again by fmoc-y-trt-oh (9- fluorenes methoxy carboxyl-trimethyl-y, y is second, each antibacterial peptide c end aminoacid)；According to
This program is synthesized to n end from c end successively, until synthesis finishes, obtains sloughing the resin of the side chain protected of fmoc group；Upper
State in the peptide resin obtaining, add cutting reagent, react 2 hours under 20 DEG C of lucifuges, filter；Precipitation tfa (trifluoroacetic acid) washing,
Washing liquid is mixed with above-mentioned filtrate, Rotary Evaporators concentrate, add the pre-cooling absolute ether of 10 times about volumes, -20 DEG C are sunk
Shallow lake 3h, separates out white powder thing, is centrifuged 10min with 2500g, collects precipitation, then washs precipitation with absolute ether, vacuum drying,
Obtain polypeptide, wherein cutting reagent mixed according to mass ratio 95:2.5:2.5 by tfa, water and tis (tri isopropyl chlorosilane) and
Become；
Carry out column equilibration 30min using 0.2mol/l sodium sulfate (phosphoric acid is adjusted to ph7.5), molten with 90% acetonitrile solution
Solution polypeptide, filters, c18 anti-phase normal pressure post, and using gradient elution, (eluant is methanol and aqueous sodium persulfate solution according to volume ratio is
30:70～70:30 mixes), flow velocity is 1ml/min, and detection ripple is 220nm, collects main peak, lyophilizing；Anti-phase c18 post is recycled to enter
One step purification, eluent a is 0.1%tfa/ aqueous solution；Eluent b is 0.1%tfa/ acetonitrile solution, and wash-out concentration is 25%b
～40%b, elution time is 12min, and flow velocity is 1ml/min, more ibid collects main peak, lyophilizing；
The identification of antibacterial peptide: antibacterial peptide obtained above is analyzed through electron spray mass spectrometry, in mass spectrum, display divides
Son amount is basically identical with the theoretical molecular in table one, and the purity of antibacterial peptide is more than 95%.
The design of table 1 antibacterial peptide
Embodiment 2
It is compared detection to designing and synthesizing the antibacterial peptide obtaining by In Vitro Bacteriostasis and Hemolytic activity assays；
The mensure of antibacterial activity: peptide is configured as certain storing liquid in case using.Using micro-broth dilution method
The minimal inhibitory concentration of several antibacterial peptides.Using 0.01% acetic acid (containing 0.2%bsa) as diluent, using doubling dilution according to
The antibacterial peptide solution of secondary configuration graded series.Above-mentioned solution 100 μ l is taken to be placed in 96 porocyte culture plates, then add respectively etc.
The bacterium solution to be measured (～10 of volume5Individual/ml) in each hole.Be respectively provided with positive control (not containing antibacterial peptide containing bacterium solution) and
Negative control (had not both contained bacterium solution and had not contained peptide).With naked eyes, 37 DEG C of constant temperature culture 20h, have no that there is being of research of chaotic phenomenon in bottom hole portion
Minimal inhibitory concentration.
Testing result is shown in Table 2.It can be seen from Table 2 that, gf, gv and gl show relatively for Gram-negative and positive bacteria
Strong bacteriostatic activity, and ga is almost not detected by bacteriostatic activity in detection range.
The antibacterial and hemolytic activity table of table 2 antibacterial peptide
The mensure of hemolytic activity: the fresh blood 1ml of collection people, it is dissolved into after anticoagulant heparin in 2mlpbs solution, 1000g
Centrifugation 5min, collects erythrocyte；Washed with pbs 3 times, more resuspended with 10ml pbs；Take 50 μ l red cell suspensions and 50 μ l pbs
The antibacterial peptide solution mix homogeneously of the variable concentrations of dissolving, constant-temperature incubation 1h in 37 DEG C of incubators；Take out after l h, 4 DEG C,
1000g is centrifuged 5min；Take out supernatant microplate reader light-metering absorption value at 570nm；Average for every group, and comparative analysiss.
Wherein 50 μ l erythrocyte add 50 μ l pbs as negative control；50 μ l erythrocyte add 50 μ l 0.1%tritonx-100 as sun
Property comparison.Minimum hemolytic concentration is that antibacterial peptide causes antibacterial peptide concentration during 10% hemolysis rate.
Testing result is shown in Table 2.Hemolytic concentration is bigger, shows that hemolytic activity is less；It can be seen from Table 2 that, only gv and
Ga does not have hemolytic activity in detection range, and gf, gw, ga, gl and gi all show higher haemolysis in detection range and live
Property.
Result above shows, with alpha-helix peptide grx2rx3rx2(x represents w to rg, f, v, i, a and l) be stencil design antibacterial peptide
There is higher bacteriostatic activity (except ga), have and there is no hemolytic activity in detection range compared with the ga of high antibacterial activity.Comprehensive
The antibacterial and hemolytic activity of analysis antibacterial peptide, can be come more comprehensively by therapeutic index (ratio of hemolytic concentration and Mlc)
Evaluation each antibacterial peptide cell selective.As can be seen from Table 2, gv has higher therapeutic index, shows that design obtains
Antibacterial peptide gv there is the development potentiality of higher substitute antibiotics.
<110>Northeast Agricultural University
<120>a kind of efficient α spiral antibacterial peptide gv and its preparation method and application
<160> 1
<210> 1
<211> 13
<212> prt
<213>artificial sequence
<400> 1
gly arg val val arg val val val arg val
1 5 10
val arg gly-nh2
11 13
Claims (3)
1. a kind of efficient α spiral antibacterial peptide gv, its sequence is as shown in sequence table seq id no.1.
2. according to claim require 1 described in a kind of efficient α spiral antibacterial peptide gv it is characterised in that preparation method include as
Lower step:
1) pass through with alpha-helix peptide grx2rx3rx2Rg, x=v, are that stencil design obtains brand-new antibacterial peptide gv；
2) adopt solid-state chemical reaction method method, by Peptide synthesizer synthesis polypeptide crude product；The polypeptide of synthesis is used reversed phase high efficiency
Liquid chromatograph carries out purification, and using electrospray mass spectrometry, the polypeptide of synthesis is identified, completes the preparation of polypeptide.
3. a kind of efficient α spiral antibacterial peptide gv according to claim 1 is it is characterised in that treat gram sun in preparation
Application in property bacterium or gram positive bacterial infection disease medicament.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266533A (en) * | 2017-05-02 | 2017-10-20 | 东北农业大学 | A kind of α spirals antibacterial peptide RL and its preparation method and application |
| CN109553657A (en) * | 2018-11-30 | 2019-04-02 | 东北农业大学 | A kind of non-perfect peptide amphiphile W4 and its preparation method and application |
| CN110437305A (en) * | 2019-07-22 | 2019-11-12 | 东北农业大学 | A kind of the α spiral antibacterial peptide GW4A and preparation method and application of tail end anchoring |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851276A (en) * | 2010-04-28 | 2010-10-06 | 东北农业大学 | Preparation method and activity detection for antibacterial peptides |
| CN103788184A (en) * | 2013-12-13 | 2014-05-14 | 东北农业大学 | Antibacterial peptide containing two leucine repeating units as well as preparation method and application |
-
2016
- 2016-11-28 CN CN201611064519.1A patent/CN106366162B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851276A (en) * | 2010-04-28 | 2010-10-06 | 东北农业大学 | Preparation method and activity detection for antibacterial peptides |
| CN103788184A (en) * | 2013-12-13 | 2014-05-14 | 东北农业大学 | Antibacterial peptide containing two leucine repeating units as well as preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| MA Q,等: "Structure-Function Relationship of ValArg-rich Peptides Effects of Net Charge and Pro on Activity", 《CHEM BIOL DRUG DES》 * |
| MA QQ,等: "Biochemical property and membrane-peptide interactions of de novo antimicrobial peptides designed by helix-forming units", 《AMINO ACIDS》 * |
| 王良，等: "利用亮氨酸和赖氨酸设计新型α-螺旋抗菌肽", 《微生物学通报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107266533A (en) * | 2017-05-02 | 2017-10-20 | 东北农业大学 | A kind of α spirals antibacterial peptide RL and its preparation method and application |
| CN107266533B (en) * | 2017-05-02 | 2018-08-28 | 东北农业大学 | A kind of α spirals antibacterial peptide RL and its preparation method and application |
| CN109553657A (en) * | 2018-11-30 | 2019-04-02 | 东北农业大学 | A kind of non-perfect peptide amphiphile W4 and its preparation method and application |
| 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 |
| CN110437305A (en) * | 2019-07-22 | 2019-11-12 | 东北农业大学 | A kind of the α spiral antibacterial peptide GW4A and preparation method and application of tail end anchoring |
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| CN106366162B (en) | 2018-03-13 |
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