Application of the polypeptide compound in preparation antibacterials
Technical field
The present invention relates to biomedicine fields, more particularly to a kind of polypeptide compound answering in preparation antibacterials
With.
Background technique
After separation obtains the bombinin being made of 22 amino acid in 1962 Nian Congling toad skins, Ren Menyi
A variety of antibacterial peptides are had found from amphibian skin.The anti-of first insect source is had found within 1980 in U.S.'s giant silkworm body
Bacterium polypeptide is also found to have antibacterial defence function successively in microorganism, aquatic livestock, vertebrate, mammal later
Peptide molecule.The peptides molecule that pathogenic microorganisms is resisted in organism generation is collectively referred to as " antibacterial peptide ", or referred to as host's " defence
Element "." antibacterial peptide " or " alexin " constitutes the first line of defence that host resists external pathogenic bacterial infection, protects host itself not
By the infringement of pathogenic microorganisms.
Antibacterial peptide (antibacterial peptide) is characterized in the peptide molecule being made of amino acid, amino acid number
Less than 100, positive charge is often carried, there is determining antibacterial activity.The natural antibacterial peptide that presently found organism generates has suppression
The functions such as gram-positive bacteria processed, Gram-negative bacteria, fungi, virus, helminth.Antibacterial peptide the most attractive concern the advantages of
Include: most of antibacterial peptides amino acid number between 15-45 amino acid, action target spot be bacterium cell membrane, to place
Master is non-toxic or low-toxic, has no drug resistance, and has good thermal stability and biological safety.With the probiotics cream of mankind close friend's association
The streptococcus lactis peptide (Nisin) that sour bacterium generates is to study to obtain antibacterial peptide the most clear at present, and Nisin is made of 34 amino acid
Straight chain peptide molecule, it has staphylococcus, streptococcus, the micrococcus luteus in the gram-positive bacteria for causing food spoilage good
Inhibition growth, by the U.S., European Union, China food and medicine Surveillance Authority approval as food additives be applied to food
Product save.
The antibacterial peptide found in the past is the natural antibacterial peptide that nature biotechnology body generates, and is mostly single-stranded form according to report
Peptide molecule, the also even shape for identical or different antibacterial peptide occur and being connected by the disulfide bond between peptide chain in nature
State, but the form of the antibacterial peptide molecule presentation copolymerization multivalence in nature is not reported so far.
Since antibacterial peptide does not generate drug resistance, there is good biological safety, environmental safety etc., to attract section
Scholar carries out the molecular improvement of various modes using natural antibacterial peptide as lead compound, to obtain highly efficient antibacterial effect
Fruit, more permanent stability, more good physiological adaptation, it is easier to be applied to daily or clinical implementation, be more convenient for manually closing
At antibacterial peptide.
Summary of the invention
The purpose of the present invention is being directed to technological deficiency existing in the prior art, it is anti-in preparation to provide a kind of polypeptide compound
Application in bacterium drug, the structural formula of the polypeptide compound are as follows: (poly-Bm)-K-(Cyclic-An), poly-BmIt is straight chain
Peptide chain, K are lysine (Lys, K), K- (Cyclic-An) it is cyclic peptide;Wherein, m poly-BmThe amino that straight chain peptide chain includes
Sour number, n are K- (Cyclic-An) the cyclic peptide amino acid number that is included;Preferably, m 1-15, n 4-15;More preferably
, m 4-15, n 4-15;Further preferably, m 6-12, n 6-12;Most preferably, 8 m, n 8.
The antibacterial is to kill the surface of a wound common infection pathogen and fungi for causing burn or wound or inhibit its growth.
The antibacterial includes bacterium (including coccus, bacillus, spirillum etc. or the gram-negative killed in prokaryotic micro-organisms
Property bacterium and positive bacteria), actinomyces (including mycoplasma, Chlamydia, rickettsia), cyanobacteria (including basketball algae, nostoc, quiver
Algae etc.) or inhibit its growth.
The antibacterial includes fungi (including saccharomycete, mould, Penicillium notatum, slime mould etc.) and the original killed in eukaryotic microorganisms
Raw pathogenic microorganisms (including amoeba amoeba etc.) or inhibit its growth.
The antibacterial is to kill staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, Acinetobacter bauamnnii or suppression
Make its growth;Especially kill enterococcus faecium (Enterococcus faecium), staphylococcus aureus
(Staphylococcus aureus), Friedlander's bacillus (Klebsiella pneumoniae), Acinetobacter bauamnnii
(Acinetobacter baumannii), pseudomonas aeruginosa (Pseudomonas aeruginosa), Escherichia coli
Drug-resistant bacterias and drug resistance fungals such as (Enterobacter species) inhibit its growth.
poly-BmIt is the straight chain peptide chain formed using basic amino acid as chief component, can be made of same monoamino-acid,
Or it is made of different aminoacids;Preferably, the basic amino acid is selected from arginine (Arg R), lysine (Lys K), group ammonia
One or more of sour (His H), more preferable arginine (Arg R).
K-(Cyclic-An) it be by hydrophobic amino acid is chief component, it is initially formed straight chain peptide chain, then its end
The amino of Dde protection is condensed the cyclic peptide structures eventually formed on the side chain of c-terminus and lysine K;Preferably, the hydrophobic ammonia
Base acid is selected from phenylalanine (Phe F), valine (Val V), leucine (Leu L), isoleucine (Ile I), methionine
One of (Met M), tryptophan (Trp W), proline (Pro P), alanine (Ala A), glycine (Gly G) are several
Kind.
The polypeptide compound can be the salt compounds with organic acid or inorganic acid formation;Or
The chemical combination that the hydroxyl that the polypeptide compound is had can form but be not limited to be formed by ether, ester, glycosides or glucoside etc.
Object;Or
The sulfydryl that the polypeptide compound is had can form but be not limited to be formed by thioether, sulphur glycosides, or with half Guang ammonia
Acid or the peptide containing cysteine are formed by the compound containing disulfide bond;Or
The amino that the polypeptide compound is had can form but be not limited to be formed by acylate, hydrocarbonylation object and carbohydrate
Substance is formed by glycoside substance etc.;Or
The carboxyl that the polypeptide compound is had can form but be not limited to be formed by ester, amides compound etc.;Or
The imino group that the polypeptide compound is had can form but be not limited to be formed by glycosides, acylate, hydrocarbonylation object etc.;
Or
The phenolic hydroxyl group that the polypeptide compound is had can form but be not limited to be formed by ester, ether, glycosides, glucoside chemical combination
Object is formed by salt compounds with organic base or inorganic base;Or
The polypeptide compound and metal ion are formed by complex, network and object or chelate;Or
The polypeptide compound is formed by hydrate or solvent object.
The method for preparing the polypeptide compound includes linear peptides poly-Bm-K-Cyclic-AnSynthesis and cyclic peptide K-
(Cyclic-An) preparation;
Preferably, the linear peptides poly-Bm-K-Cyclic-AnSynthesis, first using Manual solid phase Fmoc/tBu close
Cheng Fa is condensed amino to N-terminal from the C-terminal of polypeptide with H-CTC resin (substitution value is about 0.6mmol/g) for starting material one by one
Acid extends peptide chain until completing linear straight chain poly-Bm-K-Cyclic-AnThe synthesis of target peptide chain, amino acid starting material used
It can be L-type amino acid, be also possible to D type amino acid;
Specifically: first with the Fmoc-AA of 1.5 times of equivalentsA1- OH, the diisopropylethylamine and H-CTC resin of 3 times of equivalents
(substitution value is about 0.6mmol/g) carries out grafting and introduces first amino acid residue of C-terminal, washs the reaction solution of the resin containing H-CTC,
Unreacted active site on H-CTC resin is closed with methanol after cleaning;Then, removal N-terminal Fmoc protecting group makes N-terminal
As free amino, Fmoc-AA is usedA2- OH, I-hydroxybenzotriazole and N, N '-diisopropylcarbodiimide connect with H-CTC resin
Branch introduces second amino acid residue of C-terminal, Fmoc-AAA2- OH, I-hydroxybenzotriazole and N, N '-diisopropylcarbodiimide
Equivalent is 3 times of H-CTC resin;It is sequentially connected condensation amino acid Fmoc-AA repeatedlyAn- OH completes linear Cyclic-An
The synthesis of chain;Thereafter it uses the amino acid starting material sequence of synthesis for Fmoc-Lys (Dde)-OH, then carries out poly-BmPeptide chain
Synthesis, Fmoc-AAB1- OH, Fmoc-AAB2- OH ... ... Fmoc-AABm- OH is until complete poly-BmThe synthesis of peptide chain, obtains
poly-Bm-K-Cyclic-An- H-CTC resin.
Cyclic peptide K- (the Cyclic-An) preparation specifically:
poly-Bm-K-Cyclic-AnThe N-terminal of-H-CTC resin is blocked using Boc acid anhydrides, is then removed and is relied at cyclisation
The side chain Dde protecting group of propylhomoserin (K, Lys) residue is to release Lys side-chain amino group;Resin is cut straight chain peptide molecule
It cracks and releases from resin, the active side-chain radical for obtaining N-terminal and other amino acid residues is protected, only depended on
The straight chain peptide molecule poly-B of naked state is presented in the side-chain amino group of propylhomoserin (K, Lys) and the carboxyl of peptide chain C-terminalm-K-Cyclic-
An, the condensation and cyclization reaction of intramolecular is carried out, is then removed the protecting group of side-chain radical, recrystallization obtains target (poly-
Bm)-K-(Cyclic-An) heterocycle peptide crude product, the crude product is finally purified using high performance liquid chromatography (HPLC), obtain purity >
98% polypeptide compound (poly-Bm)-K-(Cyclic-An)。
Polypeptide compound (poly-B provided by the inventionm)-K-(Cyclic-An) it is a kind of antibacterial heterocycle peptide, advantage exists
In:
(poly-Bm)-K-(Cyclic-An) molecule is amphipathic molecule, with the characteristic of hydrogel under a certain concentration,
There is good coating adhesion to the surface of a wound;It is steady that the molecular structure feature of polypeptide compound of the present invention makes it have good molecule
It is qualitative, it is more lasting in the residence time of the surface of a wound;Polypeptide compound of the present invention has the antibiotic property of wide spectrum and does not generate drug resistance,
It can be widely applied to the growth of the inhibition to Gram-positive pathogenic bacterium, gram negative pathogenic bacteria, fungal attack bacterium or kill.
Polypeptide compound of the present invention belongs to pure peptides molecule, does not pollute to physiological environment and natural environment;This hair
Bright polypeptide compound synthesis technology is easy to implement, is easy to industrialization, scale, and the polypeptide compound high-purity being prepared.
Specific embodiment
The present invention relates to design and prepare out a kind of polypeptide compound (poly-B that linear peptides is connected with cyclic peptidem)-K-
(Cyclic-An) and its as antibacterials application.
Wherein, poly-BmIt is the straight chain peptide chain formed using basic amino acid as chief component;Basic amino acid includes
Arginine (Arg R), lysine (Lys K), histidine (His H), can any position in straight chain peptide chain participate in it is any its
The amino acid of its classification, poly-BmPeptide chain can also be all made of the basic amino acid of single kind, or all by not of the same race
The basic amino acid of class forms.M is composition poly-BmThe amino acid number that peptide chain includes, optimum range 1-15, optimum range
For 6-12.
K is represented in the synthesis process using lysine Fmoc-Lys (the Dde)-OH having there are two active amino, end
Fmoc-protected amino and basic amino acid condensation constitute poly-Bm, the amino and Cyclic-A of another side chain Dde protectionnSequence
The c-terminus condensation of column end forms it into cyclic peptide structure.
Cyclic-AnBe by hydrophobic amino acid be chief component, be initially formed straight chain peptide chain, then the carboxyl of its end
End and the amino condensation of Dde protection on the side chain of lysine K eventually form cyclic peptide structures.Hydrophobic amino acid includes: phenylalanine
(Phe F), valine (Val V), leucine (Leu L), isoleucine (Ile I), methionine (Met M), tryptophan (Trp
W), one or more of proline (Pro P), alanine (Ala A), glycine (Gly G).Based on hydrophobic amino acid
Cyclic-AnIn peptide chain composition the amino acid of any other classification can be participated in its any position.Cyclic-AnWhat peptide chain was formed
Cyclic peptide can be all made of the hydrophobic amino acid of single kind, can also all be mixed by the hydrophobic amino acid of any kind
Composition.N is K-Cyclic-AnThe amino acid number that cyclic peptide is included, optimum range 4-15, optimum range 6-12.
Heterocycle peptide molecule provided by the invention have kill or inhibit prokaryotic micro-organisms in bacterium (including coccus, bacillus,
Spirillum etc. or Gram-negative bacteria and positive bacteria), actinomyces (including mycoplasma, Chlamydia, rickettsia), cyanobacteria (packet
Include basketball algae, nostoc, the algae that quivers etc.) growth;Also have kill or inhibit in eukaryotic microorganisms fungi (including saccharomycete,
Mould, Penicillium notatum, slime mould etc.) and primary pathogenic microorganisms (including amoeba amoeba etc.) growth;The present invention illustrates miscellaneous
Not only there is cyclic peptide killing to cause the staphylococcus aureus (Quality Control strain) of trauma surface infestation, escherichia coli (Quality Control strain), verdigris
Pseudomonad (Quality Control strain), acinetobacter calcoaceticus (Quality Control strain) etc. also have and inhibit or kill ESKEPE (Enterococcus
Faecium) enterococcus faecium, Staphylococcus aureus staphylococcus aureus, Klebsiella pneumoniae lung
Scorching Klebsiella, Acinetobacter baumannii Acinetobacter bauamnnii, Pseudomonas aeruginosa verdigris are false
The drug-resistant bacterias such as monad, Enterobacter species Escherichia coli, moreover it is possible to inhibit the growth of these bacterium.
The preparation method of heterocycle peptide molecule of the present invention is divided into two parts, and first part is linear peptides poly-Bm-K-
Cyclic-AnSynthesis, second part be K- (Cyclic-An) cyclic peptide preparation.Heterocycle peptide synthesis process is as follows:
One, linear peptides poly-Bm-K-Cyclic-AnSynthesis:
Manual solid phase Fmoc/tBu synthetic method is used first, is starting with H-CTC resin (substitution value is about 0.6mmol/g)
Raw material adds condensation amino acid from the C-terminal of polypeptide to N-terminal one by one, extends peptide chain until completing linear straight chain poly-Bm-K-
Cyclic-AnThe synthesis of target peptide chain.Amino acid starting material used can be L-type amino acid, be also possible to D type amino acid;Ammonia
The D type of base acid and L-type mirror image isomer each other, i.e., centered on the carbon atom of amino acid, for carboxyl upper, amino is L-type on a left side
Amino acid, amino are D acidic amino acid on the right side, and amino acid existing for nature is L-type amino acid.
First with the Fmoc-AA of 1.5 times of equivalentsA1- OH, 3eq DIPEA (Diiso-propylethylamine, diisopropyl
Base ethamine) it carries out being grafted introducing first amino acid residue of C-terminal with resin, it reacts duration 1 hour.It is washed resin 6 times with DMF,
Active site unreacted on resin is closed with methanol after eluted resin.Then, using 25%PIPE
(Piperidine, hexahydropyridine)/DMF (Dimethyl Fromamide, n,N-Dimethylformamide) (v/v) removes N-terminal
Fmoc protecting group makes N-terminal become free amino (2 times, every time 10 minutes).With the Fmoc-AA of 3 times of equivalentsA2-OH/HOBt(1-
Hydroxybenzotriazole, I-hydroxybenzotriazole)/DIC (N, N '-Diisopropylcarbodiimide, N, N '-two
Diisopropylcarbodiimide) and resin grafting introducing second amino acid residue of C-terminal.It is sequentially connected condensation amino acid repeatedly
Fmoc-AAAn- OH completes linear Cyclic-AnThe synthesis of chain.Use the amino acid starting material sequence of synthesis for Fmoc-Lys thereafter
(Dde) then-OH carries out poly-BmThe synthesis of peptide chain, Fmoc-AAB1- OH, Fmoc-AAB2- OH ... ... Fmoc-AABm- OH is straight
To completion poly-BmThe synthesis of peptide chain.
Each step amino acid condensation reaction of linear straight chain peptide is washed resin 6 times after completing with pure DMF, and is contracted every time
It closes and condensation efficiency is detected using Kaiser Test after reaction is completed, if amino acid condensation reaction display is not exclusively, repeat to be condensed
Reaction is primary.
Two, K- (Cyclic-An) cyclic peptide preparation:
Linear straight chain peptide poly-Bm-K-Cyclic-AnAfter synthesis is completed, N-terminal is blocked (4eq using Boc acid anhydrides
Boc2O, 8eq DIPEA, 30 minutes), then using the side of lysine (Lys) K residue at 2% hydrazine/DMF (v/v) removal cyclisation
Chain Dde protecting group is to release Lys side-chain amino group (2 times, every time 15 minutes).
Resin is drained after cleaning 6 times with pure DMF, with 1%TFA (Trifloroacetic Acid, trifluoroacetic acid)/DCM
(Dichloromethane, methylene chloride) (v/v) cuts resin to crack straight chain peptide molecule from resin and be released
Come.The straight chain peptide molecule poly-B of acquisitionm-K-Cyclic-AnN-terminal and other amino acid residues active side-chain radical it is equal
It is protected, is in Boc-AABm-K-AAAn, exposed shape is presented in the only side-chain amino group of lysine (Lys) K and the carboxyl of peptide chain C-terminal
State is suitable for carrying out the condensation reaction of intramolecular.Since the higher cyclisation for being directly used in next step of obtained peptide molecule purity is anti-
It answers.
Protection straight chain peptide molecule made above is dissolved with a small amount of DMF, by DCM by its concentration dilution to 10-3M with
Under.After the BOP/HOBT of 1.2eq is added, solution is adjusted to alkalinity with DIPEA, cyclization process starts.Cyclization continues 6-
12 hours are tracked (cyclisation is dehydration, decrease in molecular weight 18Da after cyclisation) to cyclization process with mass spectrum, until anti-
It should carry out completely.After removing solvent by revolving, cutting reagent (trifluoroacetic acid: 1,2- dithioglycol: thioanisole: benzene is used
Phenol: H2O: tri isopropyl silane=68.5:10:5:3.5:1, v/v) peptide side chain protecting group is removed, it is small that 3 are cut at 30 DEG C
When.A large amount of cold anhydrous ethers, which are added, in cutting solution makes polypeptide Precipitation, and centrifugation obtains polypeptide precipitating.Precipitating is washed with ether
Drying to obtain target (poly-B after for several timesm)-K-(Cyclic-An) heterocycle peptide crude product.
The purifying and characterization of heterocycle peptide molecule:
Heterocycle peptide crude product is purified using HP1100 type (Agilent company of the U.S.) rp-hplc.Color
Spectrum column packing: Agela C18 (10 μm,50×250mm).Operation condition of chromatogram: mobile phase A (contains 0.05% trifluoro second
Acid, the aqueous solution of 2% acetonitrile), Mobile phase B (90% acetonitrile/water), flow velocity is 25 milliliters per minute, and ultraviolet detection wavelength is
220nm.Polypeptide eluting peak part is collected, obtains the heterocycle peptide sterling of white puff state, chemical structure after freeze-dried solvent
Characterized by MALDI-TOF mass spectrum, and its purity then by analytic type high performance liquid chromatograph (Agela C18-10 × 250mm,
1 milliliter per minute of flow velocity) detection, confirming its structure is heterocycle peptide of the invention.
The storage of heterocycle peptide molecule:
(the poly-B purified using HPLCm)-K-(Cyclic-An) heterocycle peptide purification sterling (usual purity > 95%)
It is cotton-shaped that white puff is presented after freeze-drying, heterocycle peptide prod is sealed in bottle and is placed in -20 DEG C and is kept in dark place.
Peptide systhesis becomes routine techniques at present.Peptide systhesis and the principle of purifying and operation advocate to compile referring to by Sheng Shu,
The chapter 3 of " present age of polypeptide hormone is theoretical and applies " book published by scientific and technical literature publishing house (1998) be " polypeptide
Chemical synthesis and purifying ".The mode that the present invention is synthetically prepared polypeptide compound is referred to the above synthesis in solid state mode, but not
It is confined to this synthesis mode.
Embodiment 1:(poly-Bm)-K-(Cyclic-An) straight chain peptide fragment in heterocycle peptide
poly-BmIt is the straight chain peptide chain formed using basic amino acid as chief component.Basic amino acid includes smart ammonia
Acid (Arg R), lysine (Lys K), histidine (His H), can participate in any other class in any position in straight chain peptide chain
Other amino acid, poly-BmPeptide chain can also be all made of the basic amino acid of single kind, or all by different types of
Basic amino acid composition.M is composition poly-BmThe amino acid number that peptide chain includes, optimum range 1-15, optimum range 6-
12。
Manual solid phase Fmoc/tBu synthetic method is used first, is starting with H-CTC resin (substitution value is about 0.6mmol/g)
Raw material adds condensation amino acid from the C-terminal of polypeptide to N-terminal one by one, extends peptide chain until completing linear straight chain (poly-Bm)-K-
(Cyclic-An) target peptide chain synthesis.Amino acid starting material used can be L-type amino acid, be also possible to D type amino acid.
First with the Fmoc-AA of 1.5 times of equivalentsA1- OH, 3eq DIPEA and resin carry out grafting and introduce first amino of C-terminal
Sour residue reacts duration 1 hour.It is washed resin 6 times with DMF, with methanol to active sites unreacted on resin after eluted resin
Point is closed.Then, using 25%PIPE/DMF (v/v) removal N-terminal Fmoc protecting group make N-terminal become free amino (2 times,
10 minutes every time).With the Fmoc-AA of 3 times of equivalentsA2- OH/HOBt/DIC and resin grafting introduce second amino acid residue of C-terminal.
It is sequentially connected condensation amino acid Fmoc-AA repeatedlyAn- OH completes linear Cyclic-AnThe synthesis of chain.Thereafter using synthesis
Amino acid starting material sequence is Fmoc-Lys (Dde)-OH, then carries out poly-BmThe synthesis of peptide chain, Fmoc-AAB1- OH, Fmoc-
AAB2- OH ... ... Fmoc-AABm- OH is until complete poly-BmThe synthesis of peptide chain.
Each step amino acid condensation reaction of linear straight chain peptide is washed resin 6 times after completing with pure DMF, and is contracted every time
It closes and condensation efficiency is detected using Kaiser Test after reaction is completed, if amino acid condensation reaction display is not exclusively, repeat to be condensed
Reaction is primary.
Straight chain peptide fragment of the invention is obtained according to the method described above, and partially synthetic obtained straight chain peptide fragment is listed in table 1,
And measure its mass spectroscopy molecular amount.
1 straight chain peptide fragment poly-B of tablemEach group list
Table 1 the result shows that, the error of linear peptides fragment mass spectra molecular weight and theoretical molecular weight that the present invention synthesizes is thousand
Within the scope of/mono- (‰), illustrate that straight chain peptide fragment confirmation is the straight chain peptide fragment of corresponding embodiment.
This embodiment part is for the content of open straight chain peptide fragment, is not limitation of the present invention, practical to synthesize
When can illustrate to carry out by following embodiment.
Embodiment 2:(poly-Bm)-K-(Cyclic-An) K- (Cyclic-A in heterocycle peptiden) cyclic peptide segment
K-Cyclic-AnBe by hydrophobic amino acid be chief component, be initially formed straight chain peptide chain, then the carboxylic of its end
The amino condensation of Dde protection eventually forms cyclic peptide structures on the side chain of cardinal extremity and lysine K.Hydrophobic amino acid includes: phenylpropyl alcohol ammonia
Acid (Phe F), valine (Val V), leucine (Leu L), isoleucine (Ile I), methionine (Met M), tryptophan
One or more of (Trp W), proline (Pro P), alanine (Ala A), glycine (Gly G).By hydrophobic amino acid
Based on Cyclic-AnIn peptide chain composition the amino acid of any other classification can be participated in its any position.Cyclic-AnPeptide chain
The cyclic peptide of formation can be all made of the hydrophobic amino acid of single kind, can also be all by the hydrophobic amino of any kind
Acid-mixed is combined into.N is K- (Cyclic-An) the cyclic peptide amino acid number that is included, optimum range 4-15, optimum range 6-
12。
Linear straight chain peptide K-Cyclic-AnAfter synthesis is completed, N-terminal is blocked (4eq Boc using Boc acid anhydrides2O,
8eq DIPEA, 30 minutes), then using the side chain Dde of lysine (K, Lys) residue at 2% hydrazine/DMF (v/v) removal cyclisation
Protecting group is to release Lys side-chain amino group (2 times, every time 15 minutes).
Resin is drained after cleaning 6 times with pure DMF, is cut resin by straight chain peptide molecule with 1%TFA/DCM (v/v)
It cracks and releases from resin.The straight chain peptide molecule K-Cyclic-A of acquisitionnN-terminal and other amino acid residues it is active
Side-chain radical is protected, is in Boc-K-AAAn, the only side-chain amino group of lysine (K, Lys) and the carboxyl of peptide chain C-terminal be presented
Naked state is suitable for the condensation reaction for carrying out intramolecular.Due to the higher ring for being directly used in next step of obtained peptide molecule purity
Change reaction.
Protection straight chain peptide molecule made above is dissolved with a small amount of DMF, by DCM by its concentration dilution to 10-3M with
Under.After the BOP/HOBT of 1.2eq is added, solution is adjusted to alkalinity with DIPEA, cyclization process starts.Cyclization continues 6-
12 hours are tracked (cyclisation is dehydration, decrease in molecular weight 18Da after cyclisation) to cyclization process with mass spectrum, until anti-
It should carry out completely.After removing solvent by revolving, cutting reagent (trifluoroacetic acid: 1,2- dithioglycol: thioanisole: benzene is used
Phenol: H2O: tri isopropyl silane=68.5:10:5:3.5:1, v/v) peptide side chain protecting group is removed, it is small that 3 are cut at 30 DEG C
When.A large amount of cold anhydrous ethers, which are added, in cutting solution makes polypeptide Precipitation, and centrifugation obtains polypeptide precipitating.Precipitating is washed with ether
Drying to obtain target K- (Cyclic-A after for several timesn) heterocycle peptide crude product.
Cyclic peptide of the invention is obtained according to the method described above, and partially synthetic obtained cyclic peptide segment is wherein only listed in table 2
Amino acid sequence, since the K in cyclic peptide is and Cyclic-AnThe amino acid c-terminus of end is condensed to be formed, and K is and linear peptides
poly-BmTie point, therefore K is defaulted as to the starting point of cyclic peptide, and measure its mass spectroscopy molecular amount.
2 cyclic peptide segment K- (Cyclic-A of tablen) each group list
Table 2 the result shows that, the error of cyclic peptide fragment mass spectra molecular weight and theoretical molecular weight that the present invention synthesizes is at thousand points
One of within the scope of (‰), illustrate the cyclic peptide segment confirmation be corresponding embodiment cyclic peptide segment.
This embodiment part is for the content of open cyclic peptide segment, is not limitation of the present invention, when reality synthesizes
It can illustrate to carry out by following embodiment.
In order to preferably compare and illustrate the bacteria resistance function of heterocycle peptide, above list respectively is synthetically prepared the straight of heterocycle peptide
Chain part and cyclic peptide part also have detected each section peptide fragment of composition heterocycle peptide to prove the effect of heterocycle peptide of the present invention
Independent fungistatic effect.
Embodiment 3:(poly-Bm)-K-(Cyclic-An) heterocycle peptide
Heterocycle peptide general molecular formula (the poly-B illustrated according to the present inventionm)-K-(Cyclic-An), heterocycle peptide is synthetically prepared
For mode referring to the synthesis step of specification statement, linear peptides and cyclic peptide segment are shown in Table 3.
3 heterocycle peptide general molecular formula (poly-B of tablem)-K-(Cyclic-An) each group list
Embodiment |
poly-Bm |
K-Cyclic-An |
Molecular weight |
Purity |
Embodiment 3-1 |
KRHRRHHK |
KPPVLFFFAIMMW |
2745.38 |
> 98% |
Embodiment 3-2 |
RRRHRR |
KM(M)MM |
1571.04 |
> 98% |
Embodiment 3-3 |
HRR |
KF(F)PAGMM |
1359.68 |
> 98% |
Embodiment 3-4 |
KKKKRRHRRHHH |
KVLPPMWWWGAAM |
3240.92 |
> 98% |
Embodiment 3-5 |
HHHHHHHH |
KFFFFF |
1961.16 |
> 98% |
Embodiment 3-6 |
RHKR |
KVLVWLVVVG |
1671.09 |
> 98% |
Embodiment 3-7 |
RRRKHHRRRKKH |
KVWLVV |
2457.99 |
> 98% |
Embodiment 3-8 |
RRRRRRRR |
KVWLVVVG |
2130.61 |
> 98% |
Embodiment 3-9 |
RRRRRRRRRRRRR |
KVVVVWWW |
3113.75 |
> 98% |
Embodiment 3-10 |
RRRRKRRRRH |
KFVLPWLPVG |
2652.22 |
> 98% |
Remarks: the amino acid abbreviations letter in bracket indicates that the amino acid is D type amino acid
Experimental example one: bacteriostasis of the polypeptide compound of the present invention to gram-positive bacteria staphylococcus aureus
Using the polypeptide compound listed in the polypeptide compound and table 4 listed including table 3 in the present embodiment 3 to gram
Positive common pathogen staphylococcus aureus (Staphylococcus aureus, the Quality Control bacterium from ATCC29213) carries out
The detection of minimal inhibitory concentration (MIC):
1, each polypeptide compound (is listed in Table 4 below) freeze-dried powder 10mg/ pipe, 10ml culture solution is added and is made into 1mg/ml mother
Liquid;Culture solution is LB culture solution, configuration method are as follows: take tryptone (Tryptone) 10g, yeast extract (Yeast
Extract) 5g, NaCl 10g, adjust pH to 7.4,121 DEG C high pressure steam sterilization 20 minutes.
2, Aseptic sterilisation culture tube 5 are taken, 2ml culture solution, number #1, #2, #3, #4, #5 are separately added into;
3, it takes the mother liquor 2ml of step 1 to be added in #1 culture tube, takes 2ml to be added in #2 culture tube after mixing again and mix, with this
Analogize and do double of dilution: respectively obtaining concentration is 1000 μ g/ml, 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml,
The sample of 31.25 μ g/ml;
4, it is placed in the bacterium colony of a little Escherichia coli of oese picking of the calcination on alcolhol burner flame containing 1ml physiology
After mixing in the culture tube of salt water, bacterium solution is obtained, it is each more to be separately added into containing for various concentration with 100 μ l bacterium solutions of sample injector absorption
In the culture tube of peptide compounds (in the sample that i.e. step 3 obtains);
5, it is cultivated 24 hours in 37 DEG C of constant incubators, observes the inhibition situation in each culture tube to bacterium, with culture
In limpid Cmin for the minimal inhibitory concentration (MIC) of the polypeptide compound, the polypeptide compound with embodiment 3-8 is liquid
Example, testing result are shown in Table 4.
Minimal inhibitory concentration (MIC) of each polypeptide compound of table 4 to Staphylococcus aureus
Number |
Molecular structure and sequence |
MIC//sterilization |
#1 |
K- (Cyclic- (V) W (L) V (V)) writes a Chinese character in simplified form: cyclic peptide C5 |
>500μg/ml |
#2 |
K- (Cyclic- (V) W (L) V (V) VG) writes a Chinese character in simplified form: cyclic peptide C7 |
>500μg/ml |
#3 |
K- (Cyclic-VL (V) W (L) V (V) VG) writes a Chinese character in simplified form: cyclic peptide C9 |
>500μg/ml |
#4 |
RRRR writes a Chinese character in simplified form: straight chain R4 |
>1000μg/ml |
#5 |
RRRRRRRR writes a Chinese character in simplified form: straight chain R8 |
>1000μg/ml |
#6 |
RRRRRRRRRRRR writes a Chinese character in simplified form: straight chain R12 |
>1000μg/ml |
#7 |
Embodiment 3-8 polypeptide compound is write a Chinese character in simplified form: R8-C8 |
<62.5μg/ml |
As can be seen from Table 4, individual straight-chain polypeptide molecule and individual ring type polypeptide molecule are to staphylococcus aureus
Minimal inhibitory concentration it is equal > 500 μ g/ml, substantially without bacteriostasis.Polypeptide compound of the invention is by straight chain peptide fragment and ring
After shape peptide molecule combines, minimal inhibitory concentration (MIC) is no more than 62.5 μ g/ml, has bacteriostasis.
#7 polypeptide compound in the minimal inhibitory concentration (MIC) and table 4 of the polypeptide compound that table 3 is listed in the present embodiment 3
Without significant difference, will not repeat them here.
Experimental example two: polypeptide compound of the present invention is antibacterial to Gram-negative bacteria pseudomonas aeruginosa (Pseudomonas aeruginosa)
Effect
1, nutrient agar solidification in culture plate is layered on to drip in each culture plate for bacterial solids culture use
Add the sample that step 3 obtains in experimental example one, and mark trying to get to the heart of a matter, each concentration makes 2 culture detection plates;
2, oese is dipped containing pseudomonas aeruginosa on alcolhol burner flame after calcination cooling (from ATCC27853
Quality Control bacterium) culture solution, uniform smearing is done on solid culture disk, be then placed in 37 DEG C of constant incubators be incubated for culture 24
Hour, the growth of pseudomonas aeruginosa the results are shown in Table 5 by taking the polypeptide compound of embodiment 3-8 as an example in sighting disk.
Minimal inhibitory concentration (MIC) of each polypeptide compound of table 5 to pseudomonas aeruginosa (Pseudomonas aeruginosa)
Number |
Molecular structure and sequence |
Minimal inhibitory concentration (MIC) |
#1 |
K- (Cyclic- (V) W (L) V (V)) writes a Chinese character in simplified form: cyclic peptide C5 |
>500μg/ml |
#2 |
K- (Cyclic- (V) W (L) V (V) VG) writes a Chinese character in simplified form: cyclic peptide C7 |
>500μg/ml |
#3 |
K- (Cyclic-VL (V) W (L) V (V) VG) writes a Chinese character in simplified form: cyclic peptide C9 |
>500μg/ml |
#4 |
RRRR writes a Chinese character in simplified form: straight chain R4 |
>1000μg/ml |
#5 |
RRRRRRRR writes a Chinese character in simplified form: straight chain R8 |
>1000μg/ml |
#6 |
RRRRRRRRRRRR writes a Chinese character in simplified form: straight chain R12 |
>1000μg/ml |
#7 |
Embodiment 3-8 polypeptide compound is write a Chinese character in simplified form: R8-C8 |
<62.5μg/ml |
As can be seen from Table 5, individual straight-chain polypeptide molecule and individual ring type polypeptide molecule are to pseudomonas aeruginosa
Minimal inhibitory concentration it is equal > 500 μ g/ml, substantially without bacteriostasis.Polypeptide compound of the invention is by straight chain peptide fragment and ring
After shape peptide molecule combines, minimal inhibitory concentration (MIC) is no more than 62.5 μ g/ml, has bacteriostasis.
#7 polypeptide compound in the minimal inhibitory concentration (MIC) and table 4 of the polypeptide compound that table 3 is listed in the present embodiment 3
Without significant difference, will not repeat them here.
Experimental example three: inhibiting effect of the polypeptide compound of the present invention to drug-fast bacteria
Experiment one,
By taking the polypeptide compound of embodiment 3-8 as an example, its growth inhibition effect to various drug-fast bacterias is detected.
1, nutrient agar solidification in culture plate is layered on to drip in each culture plate for bacterial solids culture use
Add the sample that step 3 obtains in experimental example one, and mark trying to get to the heart of a matter, each concentration (by gradient dilution obtain 1000 μ g/ml,
The sample of 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml, 31.25 μ g/ml) 2 culture detection plates of production;
2, oese dips various drug-fast bacteria (the wherein Escherichia coli that table 6 is listed on alcolhol burner flame after calcination cooling
There is drug resistance, staphylococcus aureus and Acinetobacter bauamnnii to have drug resistance to vancomycin penicillin and vancomycin
Property) culture solution, do uniform smearing in solid culture panel surface, be then placed in 37 DEG C of incubators be incubated for culture 24 hours, see
The growth for examining various drug-fast bacterias in disk, the results are shown in Table 6.
Minimal inhibitory concentration (MIC) of the polypeptide compound of 6 embodiment 3-8 of table to drug-fast bacteria
Table 6 the result shows that, polypeptide compound of the invention is to drug resistant staphylococcus aureus, Escherichia coli, Bao Man
Acinetobacter calcoaceticus also has the function of inhibiting its production.
Experiment two,
Antibody-resistant bacterium are as follows: Escherichia coli Escherichia coli ATCC 700415TIt is (Gram-negative bacteria), golden yellow
Staphylococcus Staphylococcus aureus ATCC 12600T(gram-positive bacteria), Acinetobacter bauamnnii
Acinetobacter baumannii ATCC 19606T(Gram-negative bacteria).
Experimental material: drug sensitive test paper (diameter 6mm) is commercially available: 10 μ g/ piece of penicillin (Penicillin), mould through the ages
Plain (Vancomycin) 30 μ g/ piece.
LB solid culture based formulas (1L): contain tryptone (Tryptone) 10g, yeast extract (Yeast
Extract) 5g, NaCl 10g, pH 7.4,121 DEG C of steam sterilizing under high pressure, 20min.
Experimental procedure:
1, it the preparation of drug-fast bacteria: (1), by three kind drug-fast bacterias from conservation pipe is activated, is sequenced through 16S, is determined as pure bacterium
It after strain, is transferred on LB solid medium, is placed into 37 DEG C of constant incubators, cultivate 1-2 days, for use.(2), to three kinds of drug resistances
Bacterial strain carries out susceptibility (10 μ g/ piece of penicillin, 30 μ g/ piece of vancomycin) resistance assay, to determine the drug resistance of drug-fast bacteria.(3),
Cultured three kinds of drug-fast bacterias are taken out from incubator, bacteria suspension turbidity is adjusted to 0.5, are uniformly applied to fresh LB solid training
It supports on base.
2, laboratory sample: polypeptide compound 8000r/min listed by table 7 is centrifuged 5 minutes, is then carried out with sterilizing pure water
Dissolution and gradient dilution, the sample of indissoluble can be added 30 μ l DMSO and be dissolved as cosolvent before being dissolved with pure water.
3, the measurement of MIC value: (1), sample successively by sterile pure water be diluted to concentration be 1.0mg/ml, 0.5mg/ml,
0.25mg/ml,0.125mg/ml,0.1mg/ml,0.05mg/ml,0.025mg/ml.(2), each sample diluted is drawn into 2 μ
L is added drop-wise to respectively in the LB solid medium for being coated with drug-fast bacteria.(3), excellent LB solid medium will be put and is steadily placed in 37
DEG C constant incubator in cultivate, cultivate 2 days, the results are shown in Table 7.
Minimal inhibitory concentration (MIC, mg/ml) of the polypeptide compound of the present invention of table 7 to drug-fast bacteria
Sample |
Acinetobacter bauamnnii |
Staphylococcus aureus |
KVWLVVVG:C8 |
>1.0 |
0.5 |
RRRR:R4 |
>1.0 |
>1.0 |
HHHHHHHH-KVWLVVVG:H8-C8 |
>1.0 |
>1.0 |
RRRRRRRR-KVWLVVVG:R8-C8 |
0.1 |
0.05 |
RRRRRRRRRRRRRRR-KVWLVVVG:R15-C8 |
0.25 |
0.25 |
RRRR-KVWLVVVG:R4-C8 |
0.5 |
0.05 |
RRRRRRRR-KVVPWLVGVVPVVLG:R8-C15 |
0.5 |
1.0 |
RRRRRRRR:R8 |
1.0 |
>1.0 |
RRRRRRRRRRRRRRR:R15 |
0.25 |
1.0 |
RRRRRRRR-KVWLV:R8-C5 |
0.25 |
0.5 |
As can be seen from Table 7, heterocycle peptide of the invention will be good and poor than the fungistatic effect of individual cyclic peptide or linear peptides
It is different significant.Such as the good antimicrobial effect of heterocycle peptide R8-C8, R15-C8, R4-C8 of the invention than individual cyclic peptide C8;With it is independent
Cyclic peptide C8 compare, although heterocycle peptide R8-C8 of the present invention is poor to the fungistatic effect of Escherichia coli, to Acinetobacter bauamnnii and
The fungistatic effect of staphylococcus aureus is very excellent, hence it is evident that is better than C8.Such as heterocycle peptide R8-C8, R8-C15, R8- of the invention
Good antimicrobial effect of the C5 than individual linear peptides R8;Compared with individual linear peptides R8, heterocycle peptide R8-C8 of the present invention is although right
The fungistatic effect of Escherichia coli is suitable with R8, but very excellent to the fungistatic effect of Acinetobacter bauamnnii and staphylococcus aureus
It is different, hence it is evident that be better than R8.
Linear peptides and cyclic peptide described above combine the fungistatic effect that can reinforce polypeptide compound, than individual linear peptides
It will be got well with the fungistatic effect of cyclic peptide.
Experimental example four: bacteriostasis of the polypeptide compound of the present invention to fungi
1, nutrient agar is layered on to solidify in culture plate and is grown for fungi, the bacterial strain of Candida albicans is from liberation
The 4th medical center Burn ward of hospital general, army is from being clinically separated the bacterial strain obtained and Candida albicans Quality-control strains
ATCC90028.Calibrating experiment using LS1809 candida albicans colour developing plate (Guangzhou Di Jing microorganism Science and Technology Ltd. produce,
Lot number: CCP-81101D, 2-8 DEG C of preservations).The sample that step 3 obtains in experimental example one is added dropwise in each culture plate, and in disk
Bottom is marked, and each concentration of every kind of bacterial strain makes 2 culture detection plates;
2, oese dips the culture solution of fungi, does in solid culture panel surface on alcolhol burner flame after calcination cooling
It uniformly smears, is then placed in 37 DEG C of incubators and is incubated for culture 24 hours, the growth of fungi in sighting disk, with embodiment 3-8's
For polypeptide compound, it the results are shown in Table 8 (from being clinically separated the Candida albicans and Quality Control Candida albicans ATCC90028 obtained
As a result identical).
Minimal inhibitory concentration (MIC) of each polypeptide compound of table 8 to fungi
Number |
Molecular structure and sequence |
Minimal inhibitory concentration (MIC) |
#1 |
K- (Cyclic- (V) W (L) V (V)) writes a Chinese character in simplified form: cyclic peptide C5 |
>500μg/ml |
#2 |
K- (Cyclic- (V) W (L) V (V) VG) writes a Chinese character in simplified form: cyclic peptide C7 |
>500μg/ml |
#3 |
K- (Cyclic-VL (V) W (L) V (V) VG) writes a Chinese character in simplified form: cyclic peptide C9 |
>500μg/ml |
#4 |
RRRR writes a Chinese character in simplified form: straight chain R4 |
>1000μg/ml |
#5 |
RRRRRRRR writes a Chinese character in simplified form: straight chain R8 |
>1000μg/ml |
#6 |
RRRRRRRRRRRR writes a Chinese character in simplified form: straight chain R12 |
>1000μg/ml |
#7 |
Embodiment 3-8 polypeptide compound is write a Chinese character in simplified form: R8-C8 |
<125μg/ml |
As can be seen from Table 8, individual straight-chain polypeptide molecule and individual ring type polypeptide molecule to Candida albicans most
Small Mlc is equal > 500 μ g/ml, substantially without bacteriostasis.Polypeptide compound of the invention is by straight chain peptide fragment and cyclic peptide
After molecule combines, minimal inhibitory concentration (MIC) is no more than 125 μ g/ml, has bacteriostasis.
#7 polypeptide compound in the minimal inhibitory concentration (MIC) and table 8 of the polypeptide compound that table 3 is listed in the present embodiment 3
Without significant difference, will not repeat them here.
Experimental example five: the biological safety of polypeptide compound of the present invention
Test one: intact skin irritant test
1, experimental material and source
Rabbit (comes from the 4th medical center Animal Lab. of PLA General Hospital), sample: peptide in table 3 of the present invention
Polypeptide compound in table 3 of the present invention (is dissolved in deionized water, be configured to the solution that concentration is 10mg/ml) by object solution respectively;
2, experimental procedure
(1) before the test for 24 hours, the hair of family's rabbit back backbone two sides is removed with depilatory agent, does not damage skin.Unhairing model
It encloses, left and right each about 3cm × 3cm.
(2) the directly drop of polypeptide compound solution (concentration 10mg/ml) in table 3 of the present invention is by next day in area respectively
On the side skin of unhairing of 2.5cm × 2.5cm, or drop on an equal amount of 2 layers of -4 layers of gauze and apply in side unhaired hide
Then skin surface is covered with one layer of non-stimulated plastic foil or oilpaper, then with non-stimulated immobilization with adhesive tape.Other side skin of unhairing conduct
Blank control (or solvent control).The application time is 4h.After the test, residual sample is removed with warm water or nonirritant solvent
Product.
(3) it is reacted respectively at removal sample 1h, for 24 hours with observation local skin after 48h, and carries out stimulate the reaction by table 9 and comment
Point.
9 skin wound repair standards of grading of table
By the standards of grading of table 9, the processed all rabbit scorings of polypeptide compound solution are 0 point in table 3 of the present invention,
It has no erythema or oedema, shows that polypeptide compound of the present invention is nonirritant for animal intact skin.
Test two: damaged skin irritant tests
1, experimental material and source
Rabbit (comes from the 4th medical center Animal Lab. of PLA General Hospital), sample: peptide in table 3 of the present invention
Polypeptide compound in table 3 of the present invention (is dissolved in deionized water, be configured to the solution that concentration is 10mg/ml) by object solution respectively;
2, experimental procedure
(1) before applying sample, on the skin of unhairing of 2.5cm × 2.5cm, with the cleaning of 75% alcohol, exposed skin is sterilized, to
After alcohol volatilization, with the damaged wound of sterilizing blade or injection needle standardized " well " shape in dermatotome, and in the breakage dermatotome
Interior contamination, skin injury only up to epidermis, does not injure corium.
(2) smearing of Skin sensitization test, sample before applying sample and the observation of local dermoreaction, methods of marking is the same as experiment
One.It needs to identify the difference of infection and primary stimulate the reaction during observation, if there is infection suspicious, carries out retest.
It in each observing time point, scores according to erythema of the table 9 to animal with oedema formational situation, and respectively
Temporally o'clock the scoring of 3 animals is added, divided by number of animals, obtains the skin wound repair integral mean value of different time points
(stimulus index).Wherein highest skin irritation index is taken, evaluates the sample to the rank of animal skin stimulus intensity by table 10.
10 skin irritatin strength grading of table
Skin irritation index |
Stimulus intensity rank |
0~0.5 |
It is nonirritant |
0.5~2.0 |
Subexcite |
2.0~6.0 |
Medium irritation |
6.0~8.0 |
Strong and stimulating |
By the strength grading of table 10, the processed all rabbit scorings of polypeptide compound solution are 0 in table 3 of the present invention
Point, it has no erythema or oedema, shows that polypeptide compound of the present invention is nonirritant for animal damaged skin.
Experiment three: acute eye irritation test
1, experimental material and source
Rabbit (comes from the 4th medical center Animal Lab. of PLA General Hospital), and pretest inspection rabbit eyes have different
Normal person cannot be used for testing.
Sample: polypeptide compound in table 3 of the present invention (is dissolved in deionization by polypeptide compound solution respectively in table 3 of the present invention
In water, it is configured to the solution that concentration is 10mg/ml);
2, experimental procedure
(1) pipette samples 0.1ml, instillation rabbit side eye conjunctiva is intracapsular, and another branch hole drop is using physiological saline as normally
Control.
(2) after dripping sample, normal saline flushing is used after eye is passively closed 30s.In eye drip 1h, for 24 hours, 48h, 72h, 7d,
After 14d and 21d, damage and the recovery situation of rabbit eye conjunctiva, iris and cornea are visually observed.If do not stimulated in 72h
Reaction or 7d or 14d, eye irritation fully reacting restore, and can shift to an earlier date termination test.When necessary, with 2% fluorescein sodium
Solution or slit-lamp, lens examination cornea and iris variation.
3, evaluation regulation
It scores by table 11 the acute irritation reaction of rabbit cornea, iris and conjunctiva, and calculates separately every and move
Object in three different observing times (for 24 hours, 48h and 72h), in corneal injury, iris damage, conjunctival congestion and chemosis four directions
" average score " (i.e. every animal for 24 hours, the sum of 48h and 72h scoring be divided by number of observation 3) in face.Respectively with animal canthus
Film, iris and conjunctival congestion, the average score of oedema and recovery time carry out, and react grade scale by table 12,13 Eye irritation of table
Determine sample to the stimulus intensity of eyes.
The standards of grading of 11 Rabbits with Acute Eye irritation of table reaction
12 eye irritation of table reacts grade scale
13 eye irritation of table reacts grade scale
By the grade scale of table 11-13, the processed all rabbit scorings of polypeptide compound solution are in table 3 of the present invention
It 0 point, has no erythema or oedema, shows polypeptide compound of the present invention for rabbit cornea, iris and conjunctiva without Acute irritation test.
The above is only a preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
Also it should be regarded as protection scope of the present invention.