CN102731629B - Antibacterial peptide and application thereof - Google Patents
Antibacterial peptide and application thereof Download PDFInfo
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- CN102731629B CN102731629B CN201210157559.6A CN201210157559A CN102731629B CN 102731629 B CN102731629 B CN 102731629B CN 201210157559 A CN201210157559 A CN 201210157559A CN 102731629 B CN102731629 B CN 102731629B
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- GSXRBRIWJGAPDU-BBVRJQLQSA-N tyrocidine A Chemical compound C([C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCCN)C(=O)N[C@H](C(N[C@H](CC=2C=CC=CC=2)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N1)=O)CC(C)C)C(C)C)C1=CC=C(O)C=C1 GSXRBRIWJGAPDU-BBVRJQLQSA-N 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention discloses an antibacterial peptide and an application thereof. The amino acid sequence of the antibacterial peptide is Ac-LysL-TrpL-LysL-SerL-PheL-LeuL-LysL-ThrL-PheL-LysL-SerL-LeuD-LysL-LysL-ThrL-ValL-LeuL-HisL-ThrL-LeuD-LeuL-LysL-AlaL-IleL-SerL-SerL-NH2 (SQ-1), and the derivative sequence is Ac-LysL-TRPL-LysL-SERL-PheL-LeuL-LysL-thrL-PheL-LysL-SerL-LeuL-LysL-LysL-ThrL-ValL-LeuL-HisL-ThrL-LeuL-LeuL-LysD-AlaL-IleL-SerL-SerL-NH2 (SQ-25). According to the present invention, a solid phase polypeptide synthesis technology is adopted to synthesize a polypeptide capable of adopting as an antimicrobial agent and related compounds of the polypeptide, such that the increasingly serious problem of drug resistance can be solved, and distresses on majority of patients due to stubborn infection can be solved. The antimicrobial peptide of the present invention can be applicable for various stubborn infectious diseases and ordinary infections, and can be adopted as a superior alternative drug or an auxiliary drug of the existing antibiotics.
Description
Technical field
The present invention relates to a kind of novel antibacterial peptide and the application of this antibacterial peptide in the medicine composition for the preparation of control infected by microbes thereof, belong to technical field of polypeptide.
Background technology
Microbiotic is the medicine resisting pathogenic microorganism, is a class maximum in antibiosis anti-inflammatory drug.Microbiotic is the material produced in life process by bacterium, fungi or other microorganisms, has the effect of the pathogenic microorganisms such as suppression or kill bacteria, fungi, spirochete, mycoplasma, chlamydozoan, therefore as anti-infectives.The antibiotics-treatable malignant tumour also had.Antibiotics is widely used in various infectious diseases, and it is various in style.Cationic antibacterial peptide can represent the novel microbiotic of a class, although the binding mode of cationic antibacterial peptide is determined not yet completely, but all cationic amphiphilic antibacterial peptides all can interact with cytolemma, cytolemma is the major target class of antibacterial peptide, and the gathering of antibacterial peptide molecule on cytolemma can cause the increase of permeability and make cytolemma lose its barrier function.Produce the material alterations that this resistance needs microorganism cells film fat composition, therefore, it is almost impossible that the antibacterial peptide for these film activities develops immunity to drugs.
-spiral type and
-folded form antibacterial peptide is topmost two large cationoid antibacterial peptides.
-folded form antibacterial peptide comprises the circular polypeptides fixed by intramolecular disulfide bond, such as defensin and protection element, and has the polypeptide of N-terminal to the covalent linkage of C-terminal, such as Gramicidin S and tyrocidine.With
-folded form antibacterial peptide is different,
-spiral type antibacterial peptide is more linear molecule, and it exists with disordered structure in water medium, but they are by interacting with hydrophobic cell membranes, such as, in Amphiphilic helix state, cecropins, and magainin and mellitin.
Biological study shows
the pharmacologically active of-spiral type antibacterial peptide and hemolytic activity generally to the hydrophobicity of antibacterial peptide, amphipathic and spirality is relevant.In most cases, the amino acids formed antibacterial peptide of D-type aminoacid replacement L-type, its helicity can reduce, thus its toxicity (hemolytic activity) also decreases.And stronger anti-microbial activity can be had with the polypeptide that the amino acid that the aminoacid replacement hydrophobicity that hydrophobicity is higher is lower is formed.By the hydrophobic or hydrophilic amino-acid residue utilizing a series of selected D-or L-type amino acid to replace specific position on the nonpolar of these amphiphile, amphiphilic molecules and polar surface, we have devised has higher antibacterial specific antibacterial peptide.
Summary of the invention
The object of the present invention is to provide a kind of antibacterial peptide, can as the polypeptide of anti-microbial agents and related compound thereof by the method preparation of synthetic and Solid-phase synthesis peptides, the misery that the day by day serious resistance problems of solution and obstinate infection cause extensive patients.Its anti-microbial effect onset is stablized, nontoxicity, is easy to human body and accepts.
Another object of the present invention is to provide the application of a kind of antibacterial peptide in the medicine composition for the preparation of control infected by microbes, various obstinate infectious diseases and common infection can be applied to, as existing antibiotic excellent alternative medicine or ancillary drug.
Technical scheme of the present invention is: a kind of antibacterial peptide, is characterized in that derived from Ac-Lys
l-Trp
l-Lys
l-Ser
l-Phe
l-Leu
l-
Lys
l-Thr
l-Phe
l-Lys
l-Ser
l-Ala
l-Lys
l-Lys
l-Thr
l-Val
l-Leu
l-His
l-Thr
l-Ala
l-Leu
l-Lys
l-Ala
l-Ile
l-Ser
l-Ser
l-NH
2(code is NK
l) a kind of novel polypeptide of sequence, its amino acid whose sequence is: Ac-Lys
l-Trp
l-Lys
l-Ser
l-
Phe
l-Leu
l-Lys
l-Thr
l-Phe
l-Lys
l-Ser
l-Leu
d-Lys
l-Lys
l-Thr
l-Val
l-Leu
l-His
l-Thr
l-Leu
d-Leu
l-Lys
l-Ala
l-Ile
l-Ser
l-Ser
l-NH
2(called after SQ-1), in sequence, lower footnote represents that this position amino acid is L-type or D-type amino acid respectively, and Ac represents N-and holds acetylize, NH
2represent C-and hold amidation.SQ-1 is at NK
lin sequence basis, by Ala lower for the hydrophobicity on hydrophobic surface the 12nd and 20
lbe substituted by the Leu that hydrophobicity is higher
d, because Leu hydrophobicity is higher than Ala, thus make its anti-microbial activity increase, replace L-type amino acid by D-type, again reduce the helicity of SQ-1 sequence, thus its hemolytic activity is reduced, finally reach the object optimized, obtain relative NK
lthe preferred polypeptide that sequence-specific is higher, anti-microbial activity strengthens, and cytotoxicity (hemolytic activity) reduces simultaneously.The replacement mode of the following stated SQ-1 series polypeptide all derives based on this design concept.
Such as, polypeptide analog SQ-25 aminoacid sequence is: Ac-Lys
l-Trp
l-Lys
l-Ser
l-Phe
l-Leu
l-Lys
l-Thr
l-Phe
l-
Lys
l-Ser
l-Leu
l-Lys
l-Lys
l-Thr
l-Val
l-Leu
l-His
l-Thr
l-Leu
l-Leu
l-Lys
d-Ala
l-Ile
l-Ser
l-Ser
l-NH
2, be by by the Leu on SQ-1 hydrophobic surface 12 and 20
dbe substituted by Leu
l, simultaneously by the Lys on hydrophilic surface the 22nd
lchange Lys into
d, this replacement is to obtain the stronger peptide sequence of specificity.
Preferred polypeptide SQ-1 of the present invention and polypeptide analog SQ-25 thereof, it contains aminoacid sequence below.
Table 1 polypeptide and aminoacid sequence thereof
Here, in table, amino acid subscript letter D below represents this amino acid is D-type amino acid; Identical therewith, it is L-type amino acid that subscript L represents this amino acid.Ac represents that the N end of polypeptide is acetylation, NH
2represent that C holds amidation.
Leu in described antibacterial peptide aminoacid sequence is by Ile, Val, and nor-leucine, in norvaline, arbitrary amino-acid residue replaces.
Phe in described antibacterial peptide aminoacid sequence is by Trp, Tyr, Leu, Ile, Val, and nor-leucine, in norvaline, arbitrary amino-acid residue replaces.
Trp in described antibacterial peptide aminoacid sequence is by Phe, Tyr, Leu, Ile, Val, and nor-leucine, in norvaline, arbitrary amino-acid residue replaces.
Any 1-2 amino acid of described antibacterial peptide and corresponding Dui Showing-that-momentum-turning isomer can by L-type or D-type aminoacid replacement.
Replace wherein 1-2 amino acid in described antibacterial peptide sequence by different way or extend peptide sequence and brachymemma peptide sequence 1-2 amino acid whose mode obtain similar sequence and related compound.
Antibacterial peptide controls the application in the pharmaceutical composition of infected by microbes in preparation.
In described pharmaceutical composition, the dosage range of antibacterial peptide is: injection 0.1-50mg/kg; External preparation 1/10000-10%/.
Positively effect of the present invention is through improved SQ-1 series antibacterial peptide comparatively source sequence NK
lthere is higher anti-microbial activity, or toxicity reduces, or antibacterial specificity is stronger.Human medicine and/or livestock medicine, veterinary drug or conduct agricultural, food and industrial compounds effective reagent can be used as.
We find that the certain physical characteristics of antibacterial peptide is vital for its anti-microbial activity.These characteristics comprise: with suitable charge number under hydrophilic pH value, there is hydrophobic residue and alkaline residue, that hydrophobic residue and alkaline residue are separated is amphipathic simultaneously, derivable or preformed secondary structure (
-spiral or
-folding).
In clinical practice, infected by microbes comprises bacterium, virus, fungi or the infection caused by one or more pathogenic agent protozoic.Such as, the infection etc. that simultaneously causes of two kinds of different bacteriums.But clinical detection is the process of which kind of infection and determines treatment plan, relatively loaded down with trivial details.By a kind of administering mode (antibacterial peptide), complexity and drug-resistant type pathogenic infection can be treated.
The technology that from the beginning Solid-phase Polypeptide designs is utilized, at NK in the present invention
lsequence basis is optimized, and synthesis obtains SQ-1 and analogue thereof.These polypeptide have very strong anti-microbial activity, simultaneously very low to human body cell toxicity.On sequence composition, the amino acid identity of these polypeptide and SQ-1 all (can be made up of 24-28 homologous amino acid, sequence information table in detailed in Example 1) more than 90%.
Peptide molecule of the present invention is certain secondary structure (such as spirane structure) in hydrophobic environment.We have utilized circular dichroism spectrum (CD) to monitor antibacterial peptide molecule in 50% trifluoroethanol (simulation of cytolemma hydrophobic environment)
-spirane structure.
The preferred antibacterial peptide of the present invention is the spiral analogue with potential biologic activity, is detected by circular dichroism spectrum, and this antibacterial peptide (as contained 100 mM Repone K, the 50 mM phosphoric acid buffers of pH7) in hydrophilic environment has little
-spirane structure.This constitutional features may be significant to antibacterial peptide activity mechanism, such as: a) be reduced in hydrophilic environment and form polymeric ability, i.e. and self-interaction ability; B) allow antibacterial peptide molecule to more easily pass cell walls and arrive microbial cell film.Further, right in hydrophilic environment
the destruction of-spirane structure, can not to the electrostatic attraction effect of antibacterial peptide (positive polarity) with microbial cell wall (electronegativity); But, lacking of ad hoc structure can reduce the affinity interaction (hydrophobic interaction of hydrophobic grouping in cell walls and polypeptide hydrophobic surface) of cell wall to antibacterial peptide, thus allow antibacterial peptide to be easier to pass through cell walls, enter into the critical surface of the parent of cytolemma/hydrophobic, on this region antibacterial peptide and film surface in parastate.In film, antibacterial peptide can be induced into by the hydrophobic environment of cytolemma
-spirane structure.Due to this
-spirane structure, we guess that the non-polar plane of antibacterial peptide can interact with the hydrophobic part of cytolemma, and the group of polar group in its polar surface and positively charged can interact with phosphatide polar head (electronegativity) on surface of cell membrane.
When antibacterial peptide is
during-spirane structure, antibacterial peptide molecule presents band clean positive electricity and amphipathic.Such as,
-spiral type antibacterial peptide has nonpolar or hydrophobic surface in the side of molecule, has polarity or positively charged surface, i.e. amphipathic molecule at molecule opposite side.
By the Temperature monitoring technology of high performance liquid chromatography reversed-phase column RP-HPLC, in the scope of 5 DEG C ~ 80 DEG C, the interactional ability of some antibacterial peptide analogues oneself is in the solution assessed.Oneself's ability of interacting of antibacterial peptide weighs another important indicator of its anti-microbial activity and hemolytic activity.Above-mentioned antibacterial peptide and composition thereof can be prepared as any one medically can bio-carrier or dosage form to catch patient.
Bulk drug preferred formulation dosage range of the present invention is 0.01-500mg weight part.
Prepare injection formulations of the present invention to commonly use auxiliary material and comprise: disodium ethylene diamine tetraacetate, tween 80, N.F,USP MANNITOL, glycerine, propylene glycol.
The auxiliary material preparing oral solid formulation of the present invention conventional comprises: Microcrystalline Cellulose, L-hydroxypropyl cellulose, polyvinylpyrrolidone, micropowder silica gel, starch, dextrin, sucrose, lactose, talcum powder, Magnesium Stearate, sodium starch glycolate, cross-linked polyvinylpyrrolidone, pregelatinized Starch etc.
Prepare conventional auxiliary material described in external preparation of the present invention to comprise: N.F,USP MANNITOL, Tween-80, polyoxyethylene glycol, s6, glycerine, carbomer, trolamine, ethanol, polyvinylpyrrolidone, tartrate, sodium bicarbonate, polyvinyl alcohol, Sodium Benzoate, Microcrystalline Cellulose, Vltra tears etc.
Prepare oral liquid auxiliary material of the present invention to comprise: ethanol, ethylparoben, methyl hydroxybenzoate, Tween-80, Sodium Benzoate, Sorbic Acid, honey, sucrose, sodium bisulfite, Sulfothiorine, xitix, thiocarbamide, disodium ethylene diamine tetraacetate, phosphoric acid, Citric Acid, glycerine, lactose etc.
Above-mentioned raw materials component can match with a certain proportion of common medicinal supplementary material, can be made into the one comprised in injection, tablet, capsule, granule, oral liquid, ointment, ointment, gelifying agent, eye drops, sprays, aerosol, patch, liniment, lotion according to this area ordinary method.
In described antiseptic-germicide, the dosage range of antibacterial peptide is: injection 0.1-500mg/kg; External preparation 1/10000-10%/.
Accompanying drawing explanation
Fig. 1 is the aminoacid sequence of SQ-1 and helix wheel and spiral net schematic diagram.
Fig. 2 is the aminoacid sequence of SQ-25 and helix wheel and spiral net schematic diagram.
Fig. 3 is antibacterial peptide NK
lthe circular dichroism spectrogram compared with SQ-1, SQ-25.
Fig. 4 is antibacterial peptide NK
lwith the comparison of SQ-1, SQ-25 temperature curve and self-interaction ability.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further details.
embodiment 1 is derived from the dependent antimicrobial peptide sequence information of SQ-1
Polypeptide SQ-1 is made up of 26 amino-acid residues, and sequence is Ac-Lys
l-Trp
l-Lys
l-Ser
l-Phe
l-Leu
l-Lys
l-
Thr
l-Phe
l-Lys
l-Ser
l-Leu
d-Lys
l-Lys
l-Thr
l-Val
l-Leu
l-His
l-Thr
l-Leu
d-Leu
l-Lys
l-Ala
l-Ile
l-Ser
l-Ser
l-NH
2, be derived from NK
lthe antibacterial peptide of sequence.SQ-1 is the amphipathic alpha-helix type antibacterial peptide with a polar surfaces and an apolar surfaces, as template peptide in this research.Its polar surfaces is made up of 14 hydrophilic amino acids (6 Methionins, 4 Serines, 3 Threonines, 1 Histidine).By contrast, its apolar surfaces contains 11 hydrophobic amino acids (1 tryptophane, 2 phenylalanines, 5 leucines, 1 α-amino-isovaleric acid, 1 L-Ala, 1 Isoleucine) and 1 hydrophilic amino acid (1 Methionin) composition.
Replaced the mode of wherein some amino acid or prolongation and brachymemma peptide sequence by multitude of different ways, we obtain serial SQ-1 polypeptide analog at screening.Fig. 1 (A) shows that preferred polypeptide SQ-1, Fig. 2 (A) show aminoacid sequence and the helix wheel figure of SQ25 in the present invention, and Fig. 1 (B, C) shows SQ-1 spiral net figure, Fig. 2 (B, C) SQ-25 spiral net figure; Amino acid wherein with four square frames represent be positioned at spiral nonpolar/hydrophobic amino acid of hydrophobic surface, amino acid with circle represents the hydrophilic amino acid be positioned on polarity/water-wetted surface, and the amino acid in triangle frame represents the hydrophilic amino acid be positioned on hydrophobic surface.In helix wheel Fig. 1 (A), water-wetted surface arcly to represent with hollow, and hydrophobic surface arcly to represent with solid.Ac refers to that N holds acetylize, NH
2refer to that C holds amidation.Amino-acid residue represents with one-letter code and transforms further based on this, obtains one group of active basically identical dependent antimicrobial polypeptide.Polypeptide SQ-1 mainly by L-type amino acid form (wherein only the 12nd and the leucine of 20 be D type amino acid), we devise its enantiomer polypeptide SQ-2 primarily of the D-type amino acid of correspondence form (wherein only the 12nd and the leucine of 20 be L-type amino acid).Therefore, SQ-2 is completely contrary with corresponding SQ-1 in stereochemistry.Existing a large amount of research work shows, enantiomer polypeptide is consistent in various physico-chemical property and biological activity, and thus in this text in following embodiment, only choosing has arranged the wherein experimental data of the representative peptide sequence of part.
SQ-3 and SQ-5 is one of polypeptide analog of different charged aminoacid replacement formation, obtains by selecting the performance replacing amino acid change Charged acids in SQ-1 sequence.Selecting the amino acid for replacing to be preferably charged amino acid, particularly net charge is positively charged amino acid.Charged amino acid comprises Lys, Arg, Orn, His, DAB and diaminopropionic acid.Orn has the epsilon-amino that δ-amino carrys out alternative Lys, and namely its side chain reduces by a carbon atom; DAB has γ amino, i.e. its carbon atom fewer than the side chain of Orn; Diaminopropionic acid two carbon atoms fewer than Orn side chain, namely have β amino.SQ-4 and SQ-6 is the enantiomer of above-mentioned two polypeptide.
The polypeptide analog that SQ-7, SQ-9 and SQ-11 is formed for multiple spot replacement SQ-1.The polypeptide (as three sites replace) carrying out multiple spot replacement formation in the different loci of invention polypeptide still can keep active.Multiple spot is replaced to the specific polypeptide formed, such multiple spot at non-polar plane center replaces to replace to I haven't seen you for ages with single-point and has same effect.Consider the amino acid composition of peptide sequence, the particularly importance of hydrophobic amino acid in biological activity, think to there is the polypeptide with the similarity forming more than 90% with the amino acid of SQ-1 sequence good biologic activity.SQ-8, SQ-10 and SQ-12 are the enantiomer of above-mentioned three polypeptide.
SQ-13, SQ-15 and SQ-17 intercept the N end of polypeptide and the amino acid of C end, obtain the polypeptide shorter than invention polypeptide, this removal 1-2, SQ-1 one or both ends amino acid and the polypeptide that produces, its amino acid similarity still more than 90%, thus keeps and the antibacterial activity quite of SQ-1.SQ-14, SQ-16 and SQ-18 are the enantiomer of above-mentioned three polypeptide.
The hydrophobic amino acid of polypeptide surface, by hydrophobic interaction, forms the overall hydrophobic surface of polypeptide.By hydrophobic amino acid W2 in the apolar surfaces of SQ-1, F5, L6, F9, L
d12, V16, L17, L
d20, L21, A23, I24 and a hydrophilic amino acid K13 form.Equally, polar surfaces is made up of K1, K3, S4, K7, T8, K10, S11, K14, T15, H18, T19, K22, S25, S26.To the amino-acid residue forming SQ-1, particularly hydrophobic amino acid is recombinated (can be recombinated by hydrophobic amino acid residue in apolar surfaces, or the polar amino acid residues on polar surfaces is recombinated, or polar surfaces and apolar surfaces carry out amino-acid residue restructuring and can not the amphipathic combination of material change's peptide molecule) polypeptide that forms still has good biologic activity, SQ-19, SQ-21 and SQ-23 is Partial sequence information (have chosen any 2 amino acid and carry out site exchange), this amino acid that is binned in forms in homology the homology maintaining 100% with SQ-1, it is active suitable with SQ-1 anti-microbial activity.SQ-20, SQ-22 and SQ-24 are the enantiomer of above-mentioned three polypeptide.
SQ-25 and SQ-27 is that the interchangeable of SQ-1 same site amino acids L-type and D-type replaces the polypeptide analog formed.This interchangeable replaces the hydrophobicity maintaining amino acid side chain length and cause, but appropriateness reduces hydrophobicity and the helicity of polypeptide entirety, thus can reduce its cytotoxicity at its anti-microbial activity of maintenance simultaneously.SQ-26 and SQ-28 is the enantiomer of above-mentioned two polypeptide.
Table 2 partial polypeptide sequence information summary sheet
A., outside indicating apart from special footnote, other are L-type enantiomer, and it is D-type amino acid that amino acid subscript letter D below represents this amino acid
B., outside indicating apart from special footnote, other are D type enantiomer, and it is L-type amino acid that amino acid subscript letter L below represents this amino acid
Embodiment 1-1. extends the polypeptide analog that SQ-1 is formed
In the present invention, polypeptide is the polypeptide analog by increasing N end or 1-2 the amino-acid residue formation of C end.SQ-29, on SQ-1 hydrophobic surface, (N end) adds a hydrophobic amino acid, increases polypeptide hydrophobic surface; SQ-31, SQ-1 basis extends two hydrophobic amino acids; SQ-33 and SQ-35 increases amino acid in the same way as described above at SQ-1 (C end), improves the positive polarity of polypeptide.This length variations saves the amino acid similarity of more than 90%, has the biologic activity similar to SQ-1.SQ-30, SQ-32, SQ-34 and SQ-36 are the enantiomer of above-mentioned four polypeptide.
Table 3 extends the amino acids formed representative peptide sequence in SQ-1 two ends
A., outside indicating apart from special footnote, other are L-type enantiomer, and it is D-type amino acid that amino acid subscript letter D below represents this amino acid
B., outside indicating apart from special footnote, other are D type enantiomer, and it is L-type amino acid that amino acid subscript letter L below represents this amino acid
The polypeptide analog of the hydrophobic amino acid replacement formation that embodiment 1-2. is similar
In the present invention, more polypeptide is the polypeptide analog formed by the replacement of the similar hydrophobic amino acid residues in single site.Adopt the amino acid with similar hydrophobic side chains usually can produce the polypeptide with biologic activity to the replacement carrying out single hydrophobic amino acid.
Table 4 can be used for the amino-acid residue that homologous amino acid replaces
prepared by embodiment 2 SQ-1 and dependent antimicrobial peptide and correlation parameter detects
Polypeptide in the present invention all adopts Solid-phase synthesis peptides method, uses the method synthesis of N-fluorenylmethyloxycarbonyl protection.It is pointed out that from technical standpoint, the polypeptide in the present invention can adopt other synthesis strategy and synthetic method to carry out synthesizing and producing.The raw peptide of synthesis carries out separation and purification by preparative RPLC, and experiment condition is as follows: Zorbax 300 SB-C
8post (250x9.4mm internal diameter; 6.5 μm of particle diameters, 300 apertures; Agilent company), AB linear eluent gradient (0.2% acetonitrile/min), elution speed is 2ml/min, and wherein, A moving phase is the aqueous solution containing 0.1%TFA, and B moving phase is the acetonitrile containing 0.1%TFA.The sterling peptide prepared adopts analysis mode RPLC RP-HPLC to analyze by the following method.The further qualification of polypeptide product adopts mass spectrometry method and amino acid composition analytical procedure.
The RP-HPLC of polypeptide analyzes---and adopt Agilent 1200 series liquid chromatograph to carry out the analysis of polypeptide product.Experiment condition is as follows: Zorbax 300 SB-C
8post (150 × 4.6 internal diameters; 5 μm of particle diameters; 300 apertures), AB linear eluent gradient (1% acetonitrile/min), elution speed is 1ml/min.Wherein, A moving phase is the aqueous solution containing 0.1%TFA, and B moving phase is the acetonitrile containing 0.1%TFA.
The sign of spirane structure--utilize Jasco J-720 circular dichroism spectrum (CD) instrument, (50 mM KH under 25 ° of C hydrophilic conditions
2pO
4/ K
2hPO
4/ 100 mM KCl, pH 7) and containing 50%
solution (the 50 mM KH of-spiral induction agent 2,2,2 tfifluoroethyl alcohol (TFE)
2pO
4/ K
2hPO
4/ 100 mM KCL, pH7 buffered soln 50%TFE).Measure the average residue molar ellipticity of antibacterial peptide respectively, the polypeptide mother liquor of 750 μMs is joined after 10 times of dilutions in 0.02cm quartz testing tube, obtained the ovality numerical value of product polypeptide by the scanning of 190 to 250 nm.Numerical value is larger, represents that polypeptide helix degree is larger.
In order to measure the polypeptide secondary structure under varying environment, we are at hydrophilic conditions (100 mM KCl, 50 mM KH
2pO
4/ K
2hPO
4, pH 7, is called KP damping fluid) and 50% trifluoroethanol (TFE) in (under the hydrophobic environment condition of analog cell film) utilize circular dichroism spectrometer measure SQ-1, SQ-25 and NK
lcircular dichroism.Accompanying drawing 2 is shown in by circle two collection of illustrative plates of the polypeptide such as SQ-1.As shown in the figure, polypeptide SQ-1 and analogue SQ-25 is basic in KP damping fluid is without sequential structure; But when containing 50%TFE, two sequences polypeptides are all fully folded into
-spirane structure, with NK
lsequence is compared, and it is larger that SQ-1 helicity increases degree, and SQ-25 helicity also increases (table 5) to some extent.
The bio-physical data of the polypeptide such as table 5 SQ-1
A. peptide sequence is in table 1.
B., under 25oC condition, hydrophilic conditions (100mM KCl, 50mM PO is determined at 222nm wavelength place respectively by circular dichroism spectrum
4pH7.0) or containing 50% trifluoroethanol hydrophilic damping fluid in primary amino acid residues molar ellipticity, [θ]
222, (deg.cm
2.dmol
-1).
C. the helical content (percentage ratio) of polypeptide represents comparing of polypeptide analog and the molar ellipticity of SQ-1 in 50% trifluoroethanol.
D. condition determination is 0.5% acetonitrile/min gradient
The retention behavior of RP-HPLC judges the hydrophobic common method of polypeptide.As everyone knows, the hydrophobic binding domain produced by the secondary structure of polypeptide can affect be combineding with each other of polypeptide and reversed-phase column stationary phase, and this phenomenon is particularly evident in amphipathic polypeptide.Due to this preferential binding domain, amphipathic
-helical peptides can than to have the non-amphiphilic polypeptide retention time that same amino acid forms longer with it.In addition, the chromatographic condition (hydrophobic stationary phase, nonpolar eluent) of RP-HPLC also can be induced in the mode similar to spiral inducing solvent trifluoroethanol and stablize spirane structure in potential helical polypeptide.So by different aminoacids replace brought hydrophobic change can direct reaction in the retention time of RP-HPLC.
We utilize high performance liquid chromatography reversed-phase column Temperature monitoring technology to determine the interactional ability of the oneself of SQ-1 and analogue SQ-25 further, and oneself interacts by these Amphiphilic peptides
the apolar surfaces of-spiral interacts and realizes.We utilize the hydrophobic conditions of 0.1%TFA acetonitrile solution and chromatogram reversed-phase column (hydrophobic stationary phase and the hydrophobic organic reagent in moving phase), and the hydrophobic environment of reversed-phase column also can be induced
-spirane structure.RP-HPLC Temperature monitoring technology has been applied on many dissimilar molecules, comprising ring-type from invention so far
-folded polypeptide, monomer
-helical peptides and dimer
the dimer helical peptides of-helical peptides and formation superhelix.The wash-out of polypeptide on chromatogram reversed-phase column is mainly by adsorbing and going adsorption mechanism, even if a polypeptide is combined on hydrophobic stationary phase strongly, when the concentration of the acetonitrile in moving phase reach a certain height, this polypeptide still can distribute between moving phase and stationary phase.On the whole, mechanism is based on 4 kinds of hypothesis: 1) having the ability during low temperature, it is dimeric amphipathic to be formed
-helical molecule, it is bound to form dimer in the aqueous solution (hydrophobicity, apolar surfaces) of reverse-phase chromatography; 2) when high temperature due to dimer be destroyed, monomer-dimeric balance tends to monomer; 3) monomer is only had to exist in the aqueous solution when temperature is enough high; 4) polypeptide can only be attached on chromatographic column fixed phase with the form of monomer, and namely dimer can only be deposited in the solution, only has disrupted dimer could combine with chromatographic column fixed phase.
When utilizing high performance liquid chromatography reversed-phase column monitoring temperature to weigh polypeptide polymerizing power, a polypeptide (peptide C) in disordered structure is by polypeptide in contrast.This sequence is Ac-ELEKGGLEGEKGGKELEK-NH
218 residue peptide, even if at low temperature 5 DEG C and strong
under-spiral inductor 50% trifluoroethanol (TFE) existent condition, still in disordered structure.Due to PEPC in aqueous phase and hydrophobic medium all in monomer state of disarray, it only embodies the impact of temperature on polypeptide retention behavior by the change of retention behavior in 5 DEG C to 80 DEG C, namely peptide retention time linearly reduces along with the rising retention time of temperature, and this is the mass transfer due to the higher solutes accumulation between stationary phase and moving phase caused by high temperature and enhancing.Therefore, after doing standard control with the retention time of peptide C, polypeptide retention behavior only represents polypeptide oneself interaction ability.
p abe worth higher representative self-interaction ability stronger, self-interaction ability is directly related with the hydrophobicity of polypeptide, except due to except above-mentioned temperature action, during raised temperature
-spirane structure is destroyed, increasing of the disordered structure of polypeptide, and retention time declines.
Polypeptide retention time data are as shown in table 6, and it have recorded the retention time of in thetagram 5 DEG C, maximum retention time and the retention time at 80 DEG C.5 DEG C and 80 DEG C is upper temperature limit and the lower limit of RP-HPLC thetagram, be that polymer state exists, and at 80 DEG C because high temperature makes polypeptide sex change, polymer resolves into monomer at 5 DEG C of polypeptide.Represent polypeptide is all transformed into monomer boundary's point by polymer maximum retention time.
Table 6. in RP-HPLC temperature curve, the dependency of the hydrophobicity of polypeptide analog and polypeptide oneself interaction ability
a . represent that the not homopolypeptide measured through RP-HPLC is in the retention time of 5 ° of C and 80 ° C and polypeptide maximum retention time measured in temperature changing process, condition determination is 1% acetonitrile/min gradient.
b . represent at different temperatures (5 ° of C and 80 ° C), the retention time of polypeptide and the difference of retention time contrasting peptide C, represent the relative hydrophobicity of polypeptide analog.
c .
p arepresent in the monitoring of RP-HPLC temperature control, the dissociation constant of often kind of peptide.In range of temperature, with the maximum retention time difference of polypeptide namely ((
t r t-
t r 5helical peptides)-(
t r t-
t r 5control peptide C)) represent, wherein, (t
r t -t
r 5) represent polypeptide specified temp (
t) difference of retention time under condition and its retention time under 5 ° of C conditions.
d . peptide C is the contrast polypeptide of disordered structure, and its retention behavior at RP-HPLC can reflect because temperature variation causes the change of RP-HPLC system.For deducting the chromatographic condition that causes because of temperature variation to the impact of polypeptide retention time, thus only reflect polypeptide change in physical at different temperatures.
The elution time of RP-HPLC is through being commonly used to the relative hydrophobicity weighing polypeptide analog.Therefore the retention time data in table 6 can be used for reacting the hydrophobic difference of polypeptide analog.In table 65 DEG C and 80 DEG C time retention time data retention time at the corresponding temperature compare, can visually see, through improved SQ-1 and SQ-25 polypeptide hydrophobicity apparently higher than NK
lchange, this also just means, the very possible comparatively NK of SQ-1 and SQ-25
lthere is higher anti-microbial activity.
Fig. 3 represents and varies with temperature the retention time change curve of (5 DEG C-80 DEG C) polypeptide in RP-HPLC.The oneself of polypeptide described above interacts and temperature correlation.The distribution of polypeptide in PR-HPLC is in the running balance transformed mutually between polymer-monomer, and under low temperature, polypeptide tends to dimer or multimeric forms existence (oneself interacts).It is realized by the hydrophobic interaction of polypeptide hydrophobic surface that usual oneself interacts, and cause the binding ability of polymer and chromatographic column fixed phase to die down like this, therefore retention time is relatively low.Along with the rising of temperature, the balance that polymer-monomer transforms mutually moves to the direction being easier to be formed monomer.The monomer distribution on a column of high density add polypeptide and chromatographic column in conjunction with probability, so retention time increases relatively.It should be noted that raised temperature also introduces other effect simultaneously, as the mass transfer etc. reducing moving phase viscosity and be increased between moving phase and stationary phase.As disordered structure control peptide C retention time shown in, along with the increase of temperature, its retention time can reduction linearly.On the contrary, for the polypeptide of polymerization, raised temperature can destroy polymer and convert monomer to, and monomer is strong in conjunction with the ability of chromatographic column fixed phase, and such retention time can reach maximum value.On this critical temperature, the retention time that we can observe along with temperature continuation rising polypeptide starts to decline.This mainly causes caused by peptide molecule sex change owing to reducing moving phase viscosity and increase mass transfer and high temperature.The contrast peptide C of the disordered structure of the Temperature monitoring technology introducing of RP-HPLC, its retention behavior is used in reflection temperature changing process, the changing condition of chromatographic column condition, thus removes because of the impact of chromatographic condition change on polypeptide retention behavior.
NK in this research
land SQ-1, SQ-25 peptide sequence shows different oneselfs in aqueous and to interact ability (Fig. 3).In figure, retention time difference changes ((polypeptide
t r t-
t r 5)-(peptide C
t r t-
t r 5)) greatest measure be defined as polypeptide oneself interaction coefficient (
p a), be used for quantizing polypeptide and form polymeric ability in aqueous.As seen from the figure, the oneself of SQ-25 interacts ability the most by force, and SQ-1 takes second place, all higher than NK
lsequence, shows that SQ-1 and SQ-25 series polypeptide may have comparatively NK in aqueous
lhigher antibacterial specificity.
embodiment 3:SQ-1 series represents the sensitivity test of antibacterial peptide anti-bacterial drug
Choice experiment room is preserved reference culture and is separated Resistant strain 20 strain with clinical, adopts plate doubling dilution and Denlay multi-point inoculator to carry out drug sensitive experiment, and test organisms nutrient broth and brain heart infusion increase bacterium.Become various desired concn with the doubling dilution of MH meat soup after medicine dissolution, add appropriate in plate respectively.Multi-point inoculator inoculation test bacterium (10 is used after culture medium solidifying in plate
4cFU/ point), put 35 DEG C of constant temperature culture observations after 18 hours.The concentration that in the plate of asepsis growth, contained drug is minimum is minimum inhibitory concentration (MIC).A large amount of research work shows, enantiomer polypeptide is consistent in various physico-chemical property and biological activity, and thus in the present embodiment, only choosing has arranged the wherein experimental data of the representative peptide sequence of part.SQ-1 series of samples to the anti-microbial effect of 20 strain bacteriums in table 7.
Table 7 SQ-1 series represents polypeptide minimum inhibitory concentration MIC(μ g/ml)
The above results shows, through the serial polypeptide of this well-designed and after screening SQ-1, anti-microbial activity obviously strengthens, and for the common sensitive bacterial of the overwhelming majority, SQ-1 series antibacterial peptide sterilization effect and NK
lrelatively have great advantage.
embodiment 4:SQ-1 series represents polypeptide hemolytic activity and therapeutic index compares
As shown in table 8, SQ-1 series polypeptide hemolytic activity, has a mind to increase hydrophobic amino acid composition because designing, or with D-type aminoacid replacement L-type amino acid, thus reach the object reducing new transformation sequence hemolytic activity.Show that because there being a large amount of research work enantiomer polypeptide is consistent in various physico-chemical property and biological activity, thus in the present embodiment, only choosing has arranged the wherein hemolytic experiment data of the representative peptide sequence of part.
Table 8 candidate polypeptide hemolytic activity (MHC) detects and compares
Experimental result shows, through above-mentioned sequence alterations, except SQ-25 and NK
lhemolytic activity is substantially suitable, and other serial polypeptide hemolytic activities are all obviously lowered.
In order to evaluate the biologic activity of SQ-1 series polypeptide better, we adopt this specific parameter of representative antibacterials be widely used of therapeutic index to compare.Therapeutic index is by MHC(hemolytic activity) and MIC(anti-microbial activity) ratio calculation, numerical value is larger, represents that polypeptide antibacterial specificity is stronger.As shown in table 9, SQ-1 series polypeptide passes through the well-designed transformation in feature site, and therapeutic index obviously becomes large, and the average multiple that increases is more than 10 times, and SQ-1 can increase to NK for enterococcus faecalis C625 therapeutic index
l106.7 times, can increase to 116 times to its therapeutic index of serratia marcescens 41002, SQ-25 increases to NK for enterococcus faecalis C625 therapeutic index is the highest
l160 times, meaning that antibacterial specificity has obviously increases, and this also further demonstrate that our from the beginning design concept.
Table 9 SQ-1 series polypeptide therapeutic index compares
* before separation line, data represent polypeptide therapeutic index, are drawn by the ratio calculation of MHC and MIC;
After separation line, data are this polypeptide and NK
lthe multiple that comparison therapy index increases
the anti-infective test of embodiment 5:SQ-1/ SQ-25 external composition for skin
S. aureus L-forms ATCC25923 and Pseudomonas aeruginosa ATCC27853 bacterium liquid are diluted to 5 × 10
6cFU/ml, for subsequent use.ICR mouse, by body weight random packet, often organizes 10.Be divided into infection negative control group, trans-pseudomonic acid positive controls, 1 ℅ concentration external preparation group, 1 ‰ concentration external preparations and bare substrate control group.After mouse back is lost hair or feathers, with 60 order sand paperings to oozing of blood.Subcutaneous injection concentration is 5 × 10
6the bacterium liquid 0.1ml of CFU/ml.Except infection negative control group, other respectively organize the external preparation that equal correspondence gives various dose, and dosage is 0.1ml, each external application sooner or later 1 time every day, continuous 4 days.After last administration next day the aseptic skin getting each treated animal infection site, detect viable bacteria counting statistics, detected result is in table 10.
Table 10 SQ-1/SQ-25 external preparation is on skin infections impact (CFU/ ware n=10)
The skin infections that the above results display SQ-1 and SQ-25 external preparation can effectively suppress S. aureus L-forms and Pseudomonas aeruginosa ATCC27853 to cause, and bacteriostasis rate all can be increased to more than 95%, is obviously better than NK
l(its high-concentration and low-concentration is to the equal <50% of bacteriostasis rate of above-mentioned two kinds of pathogenic bacterium).
embodiment 6:SQ-1 injection is to the therapeutic test of bacterium systemic infection animal
Pseudomonas aeruginosa ATCC27853 and streptococcus aureus ATCC29213ICR bacterium liquid are diluted to 5 × 10
7cFU/ml, for subsequent use.ICR mouse, by body weight random packet, is often organized 10, is divided into blank group, negative control group, SQ-1/SQ-25/NK
lhigh, medium and low dosage group.Except blank group, every mouse peritoneal injection bacterium liquid 0.2ml modeling.Immediately by body weight administration after modeling, except blank group and infection negative control group, other respectively organize each injection of equal tail vein injection various dose, 2 times/day, successive administration 5 days, observe 14 days.Observe animal dead situation, record animal survival time.SQ-1 injection refers to table 11-12 to systemic infection mouse survival time effects test-results.
The agent of table 11 SQ-1 injection series is on the impact (n=10) of Pseudomonas aeruginosa systemic infection mouse survival time
Note: compare * P<0.05 with negative control group, * * P<0.01
The agent of table 12 SQ-1 injection series is on the impact (n=10) of S. aureus L-forms systemic infection mouse survival time
Note: compare * P<0.05 with negative control group, * * P<0.01
From table 11 and table 12, until off-test, SQ-1 and SQ-25 high, medium and low dosage group is difference all extremely remarkable (p<0.01) compared with negative control group smaller white mouse Average Survival number of days, NK
lalthough reagent group 1mg/kg dosage group also can effectively extend infection animal survival time, the anti-infectious function of the high, medium and low dosage of SQ-1 and SQ-25 is obviously better than NK
lreagent group, the infection animal survival time compares NK
lgroup extends 2-3 doubly.
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>1
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-1:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>2
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-2:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>3
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-3:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Arg
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>4
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-4:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Arg
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>5
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-5:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-His
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>6
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-6:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-His
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>7
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-7:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Val
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Ile
L-His
L-Thr
L-Leu
D-Val
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>8
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-8:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Val
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Ile
D-His
D-Thr
D-Leu
L-Val
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>9
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-9:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Trp
L-Leu
L-Lys
L-Thr
L-Leu
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>10
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-10:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Trp
D-Leu
D-Lys
D-Thr
D-Leu
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>11
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-11:
Ac-Lys
L-Phe
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>12
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-12:
Ac-Lys
D-Phe
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>13
<211>25
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-13:
Ac-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>14
<211>25
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-14:
Ac-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>15
<211>24
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-15:
Ac-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>16
<211>24
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-16:
Ac-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>17
<211>24
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-17:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>18
<211>24
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-18:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>19
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-19:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Leu
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Phe
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>20
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-20:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Leu
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Phe
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>21
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-21:
Ac-Lys
L-Lys
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Trp
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>22
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-22:
Ac-Lys
D-Lys
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Trp
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>23
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-23:
Ac-Lys
L-Trp
L-Ser
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Lys
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>24
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-24:
Ac-Lys
D-Trp
D-Ser
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Lys
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>25
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-25:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
L-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
L-Leu
L-Lys
D-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>26
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-26:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
D-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
D-Leu
D-Lys
L-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>27
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-27:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
L-Lys
D-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
D-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>28
<211>26
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-28:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
D-Lys
L-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
L-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>29
<211>27
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-29:
Ac-Leu
L-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>30
<211>27
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-30:
Ac-Leu
D-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>31
<211>28
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-31:
Ac-Leu
L-Phe
L-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>32
<211>28
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-32:
Ac—Leu
D-Phe
D-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>33
<211>27
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-33:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-Phe
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>34
<211>27
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-34:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D- Phe
D-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>35
<211>28
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-35:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L- Phe
L- Ser
L-NH
2
<110> Changchun Pu Lai medical biotechnology company limited
<120> antibacterial peptide and application thereof
<160>1
<210>36
<211>28
<212>PRT
<213> artificial sequence
<220>
<223> is for controlling the polypeptide of the medicine group of infected by microbes
<400>1
SQ-36:
Ac-Lys
D-Trp
D-Lys
D-Ser
D-Phe
D-Leu
D-Lys
D-Thr
D-Phe
D-Lys
D-Ser
D-Leu
L-Lys
D-Lys
D-Thr
D-Val
D-Leu
D-His
D-Thr
D-Leu
L-Leu
D-Lys
D-Ala
D-Ile
D-Ser
D-Ser
D- Phe
D- Ser
D-NH
2
Claims (4)
1. an antibacterial peptide, is characterized in that its amino acid whose sequence is SQ-1 or SQ-25:
SQ-1:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
D-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
D-Leu
L-Lys
L-Ala
L-Ile
L-Ser
L-Ser
L-NH
2；
SQ-25:
Ac-Lys
L-Trp
L-Lys
L-Ser
L-Phe
L-Leu
L-Lys
L-Thr
L-Phe
L-Lys
L-Ser
L-Leu
L-Lys
L-Lys
L-Thr
L-Val
L-Leu
L-His
L-Thr
L-Leu
L-Leu
L-Lys
D-Ala
L-Ile
L-Ser
L-Ser
L-NH
2。
2. an antimicrobial compound, it contains described antibacterial peptide according to claim 1, and the dosage of its antibacterial peptide is 0.01-500mg weight part.
3. antimicrobial compound according to claim 2, its formulation is the one in injection, tablet, capsule, granule, oral liquid, ointment, gelifying agent, eye drops, sprays, aerosol, patch, liniment, lotion.
4. antibacterial peptide according to claim 1 is preparing the application in the pharmaceutical composition controlling microbial bacterial and fungi infestation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210157559.6A CN102731629B (en) | 2012-05-21 | 2012-05-21 | Antibacterial peptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210157559.6A CN102731629B (en) | 2012-05-21 | 2012-05-21 | Antibacterial peptide and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102731629A CN102731629A (en) | 2012-10-17 |
| CN102731629B true CN102731629B (en) | 2015-05-20 |
Family
ID=46988000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210157559.6A Active CN102731629B (en) | 2012-05-21 | 2012-05-21 | Antibacterial peptide and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102731629B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102940876B (en) * | 2012-12-12 | 2013-11-06 | 中国科学院昆明动物研究所 | Application of polypeptide GH27 in preparation of medicament for preventing and treating goat diarrhea |
| CN102935223B (en) * | 2012-12-12 | 2013-10-16 | 中国科学院昆明动物研究所 | Application of polypeptide GC25 in preparing anti-infective drug |
| CN105017384B (en) * | 2015-07-13 | 2018-06-19 | 长春普莱医药生物技术有限公司 | A kind of antibacterial peptide and its application |
| CN106478795A (en) * | 2015-09-01 | 2017-03-08 | 山东国际生物科技园发展有限公司 | A kind of sea snake variant antibacterial peptide HC3-5 and its preparation method and application |
| CN107875373A (en) * | 2017-12-07 | 2018-04-06 | 郑州国食科技有限公司 | A kind of antimycotic artificial synthetic polypeptide spraying combination and preparation method thereof |
| CN108148117A (en) * | 2017-12-26 | 2018-06-12 | 张艳雪 | The protein structure characteristic and its research method of a kind of antibacterial peptide |
| JP2024503875A (en) * | 2021-01-19 | 2024-01-29 | 江▲蘇▼普莱医▲藥▼生物技▲術▼有限公司 | Antimicrobial peptide liquid composition and its formulation |
| CN113081953B (en) * | 2021-04-29 | 2023-10-31 | 江苏普莱医药生物技术有限公司 | Topical antibacterial peptide gel and preparation method thereof |
| CN113081977B (en) * | 2021-04-30 | 2022-07-29 | 江苏普莱医药生物技术有限公司 | Anti-infection antibacterial peptide freeze-dried preparation and preparation method thereof |
| CN114907447B (en) * | 2022-02-23 | 2023-07-25 | 湖南大学 | Antibacterial peptide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1751065A (en) * | 2003-01-13 | 2006-03-22 | 基因植物－瓦勒公司 | Resistance gene of cotton aphid (Aphis gossypii) |
| CN101111256A (en) * | 2004-12-15 | 2008-01-23 | 科罗拉多大学 | Antimicrobial peptides and methods of use thereof |
-
2012
- 2012-05-21 CN CN201210157559.6A patent/CN102731629B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1751065A (en) * | 2003-01-13 | 2006-03-22 | 基因植物－瓦勒公司 | Resistance gene of cotton aphid (Aphis gossypii) |
| CN101111256A (en) * | 2004-12-15 | 2008-01-23 | 科罗拉多大学 | Antimicrobial peptides and methods of use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102731629A (en) | 2012-10-17 |
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