CN102603882B - Antifungal peptide as well as preparation method and application thereof - Google Patents
Antifungal peptide as well as preparation method and application thereof Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides an antifungal peptide as well as a preparation method and application thereof. The antifungal peptide is the peptide with amino acid sequences shown in SEQ ID NO.2, or a biological activity function fragment, variant or derivative of the peptide. The antifungal peptide has the advantages of relatively wide antifungal spectrum and low cytotoxicity. The antifungal peptide is prepared by a biological or chemical method, can be used for overcoming the defects of low specific activity or the like during the natural polypeptide scale production and application, can be applied in the aquaculture industry, especially in controlling the water fungus and can be used for effectively protecting each stage of the fish growth.
Description
Technical field
The present invention relates to a kind of anti-fungus peptide, Preparation Method And The Use with anti-mycotic activity, belong to biological chemistry and biomedical sector.
Background technology
Antibacterial peptide is that a class extensively is present in the amphiphilic small molecules basic polypeptide in the whole organic sphere, extensively be present in the organism, comprise bacterium, fungi, plant, insect, fish, birds, crustacean, Amphibians and Mammals etc., existing above 750 kinds more than, wherein the overwhelming majority is cationic antibacterial peptide, and a small amount of negatively charged ion antibacterial peptide is also arranged.Because antibacterial peptide shows broad-spectrum anti-microbial activity, and have neutralizing effect to bacterial poison, its mechanism of action uniqueness is difficult for producing resistance, and some antibacterial peptide only works to prokaryotic organism and mutant cell again, and is harmless to eukaryote.Thereby produced under the obvious chemical sproof situation in many pathogenic bacterium, the discovery of antibacterial peptide and further investigation may for developing that brand-new antibacterials provide.
Antibacterial peptide is the key factor of body congenital immunity.The mechanism of action of antibacterial peptide is different with traditional microbiotic, and its target site mainly is the pathogen cells film, and therefore difficult generation resistance, and antibacterial peptide does not almost have toxic side effect to eukaryotic cell, only acts on the eukaryotic cell of prokaryotic cell prokaryocyte and generation pathology.Antibacterial peptide not only acts on gram-positive microorganism and Gram-negative bacteria, and acts on fungi, protozoon, some virus and tumour cell, simultaneously, can also accelerate immunity and wound healing process.Because pathogenic bacteria progressively develops immunity to drugs to microbiotic, antibacterial peptide has been opened up wide prospect for the new antibacterium of exploitation, antimycotic, antiviral and antitumor drug.
Antibacterial peptide is that prokaryotic organism, eukaryote even the mankind can produce by the huge gene family of a coded polypeptide.At present, scientist has obtained the antibacterial peptide of the natural and synthetic of hundreds of kind, but though these antibacterial peptides in size, become to be grouped into, all have a very big-difference on electric charge and the secondary structure.In addition, antibacterial peptide has five kinds of structure types: linear structure, spirane structure often; Be rich in cysteine residues; Form the polypeptide of β-laminated structure; The polypeptide of proline rich; The polypeptide that contains rare or modified amino acid.These features are very important for the target structure of identification microorganism.
About antimycotic albumen and polypeptide, though obtained remarkable progress in recent years.Fungal cell membrane has been proved the desired target as alexin sample polypeptide and plant alexin.But the fungal cell exists some inner polypeptide and outside polypeptide, is fully possible and induce fungal cell's death by the cascade of adjusting intercellular signal after the antibacterial peptide polypeptide effect outside with it.
Summary of the invention
The present invention is based on the analysis to the chemical structure of sharp bladder spiral shell natural polypeptides, chemically modified and synthetic its resulting structure are prepared a kind of novel anti-fungus peptide, in the hope of as traditional antibiotic surrogate, strengthen many-sided advantage of antibacterial peptide.
Therefore, the objective of the invention is to, a kind of novel anti-fungus peptide is provided.
Another object of the present invention is, the Preparation method and use of above-mentioned anti-fungus peptide is provided.
The objective of the invention is to be achieved through the following technical solutions.On the one hand, the invention provides a kind of anti-fungus peptide, described anti-fungus peptide is peptide, its bioactive functions fragment, variant or the derivative with aminoacid sequence shown in the SEQ.ID.NO.2.
Preferably, effective antimycotic concentration of described anti-fungus peptide is for being not less than 1.3 μ M; Preferably described concentration is 1.3-33 μ M.
Preferably, described fungi is selected from Fusarium oxysporum, neurospora crassa and water mo(u)ld.
On the other hand, the present invention also provides the preparation method of described anti-fungus peptide, and described anti-fungus peptide can be by the preparation of biological or chemical method.
The present invention also provides the purposes of above-mentioned anti-fungus peptide in the preparation antifungal medicament.Preferably, described fungi is selected from Fusarium oxysporum, neurospora crassa and water mo(u)ld.
In addition, the present invention also provides a kind of antifungal preparation, and it comprises the above-mentioned anti-fungus peptide of effective anti-fungal amount.Preferably, the concentration of described anti-fungus peptide is for being not less than 1.3 μ M; Preferably described concentration is 1.3-33 μ M.
In a preferred embodiment, the design of anti-fungus peptide provided by the invention and synthetic method may further comprise the steps:
1) the wild sharp bladder spiral shell that catches from the field is in laboratory rearing, and temperature remains on 15 ℃, and salinity remains on 35 ‰.
2) every sharp bladder spiral shell is gathered the blood of 0.5ml, and adds isopyknic anti-freezing damping fluid mixing.
3) processing of blood sample.
4) extract and purifying protein according to Solid-Phase Extraction and HPLC (high performance liquid chromatography) method of routine.
5) be further purified albumen with reversed-phase HPLC.
6) albumen to above-mentioned purifying carries out vacuum lyophilization, obtains sharp bladder spiral shell hemocyte antibacterial peptide.
7) with the molecular weight of SDS-PAGE method mensuration antibacterial peptide, use the composition of Edman sequencing analysis of amino acid.
8) use micrococci, staphylococcus epidermidis, intestinal bacteria, Aeromonas hydrophila, Vibrio parahemolyticus and vibrio cholerae carry out antibacterial activity and measure.
9) use Fusarium oxysporum, neurospora crassa and water mold carry out the mensuration of anti-mycotic activity.
10) adopt CHSE-214 raji cell assay Raji antibacterial peptide cytotoxicity.
11) design of novel antimicrobial peptide Apn: for the hydrophobicity (bringing up to 38% from 25%) that improves antibacterial peptide and cation surface activating (from+1 bring up to+5), begin to delete 10 hydrophilic amino acid: T-1, Y-2, Y-10 from the N-end, N-13, S-15, Y-16, Q-19, N-23, Q-31 and Y-44.Delete 5 electronegative amino acid: E-6, E-7, E-9, D-18 and D-37 simultaneously.With the amphiphilic polypeptide called after Apn that forms, it is a polypeptide that is different from AP, and hydrophobicity brings up to 38% from 25%, cation surface activating from+1 bring up to+5.
12) synthetic and purified polypeptide Ap and Apn with the chemical synthesis of standard.
13) adopt the spectrography of circular dichroism (CD) that polypeptide A p and Apn are analyzed, adopt the ClustalW biosoftware to carry out homology analysis, the analytical standard homology is higher than 30%.
14) carry out secondary structure prediction with information biology software PSIPRED and the polypeptide A p of SOPMA and Apn.
In sum, the anti-fungus peptide with anti-mycotic activity that the invention provides a kind of hemocyte antibacterial peptide primary structure based on sharp bladder spiral shell and design, the method of design of this anti-fungus peptide comprises following step: at first separation and purifying obtain a natural polypeptides from the hemocyte of sharp bladder spiral shell, from its primary structure as seen, this polypeptide contains 47 amino-acid residues, with its called after AP; Secondly, by the synthetic polypeptide that to obtain a molecular weight be 5100.78Da; This polypeptide contains 25% hydrophobic amino acid, and have 1 clean positive charge, this polypeptide and known active antibacterial peptide have portion homologous, based on this sequence information, the design and made up novel polypeptide and a called after Apn with 30 amino-acid residues, it has 5 clean positive charges, molecular weight is 3028Da, have 38% hydrophobic molecule, it has stronger hydrophobicity and cation surface activating; The secondary structure of Apn is to adopt the circular dichroism method to measure, so the N-end has a hydrophobicity epi-position, and the C-end has a wetting ability epi-position, has formed an amphipathic molecule.Proof by experiment, the Apn polypeptide has more activity than AP polypeptide, and is cytotoxicity to the CHSE-214 cell still when reaching 100 μ M.As seen, also can obtain to have more the active antibacterial peptide to the chemically modified of natural polypeptides and the chemosynthesis of resulting structure, the defectives such as low specific activity when also having overcome natural polypeptides large-scale production and application simultaneously.
Anti-fungus peptide provided by the invention has anti-fungus spectra comparatively widely; and it is low to cytotoxicity; can adopt the preparation of biological or chemical method; defectives such as low specific activity when having overcome natural polypeptides large-scale production and application; can be applicable to culture fishery; be particularly useful for controlling water mo(u)ld, can protect each stage of fish growth effectively.
Description of drawings
Below, describe embodiment of the present invention by reference to the accompanying drawings in detail, wherein:
Fig. 1 is the polypeptide pedigree chart of cut among the embodiment 1, and wherein, the left side is the standard protein molecular weight among the protein electrophorese figure on the figure, and the right side is the cut with anti-mycotic activity.
Fig. 2 is the homology analysis figure of antibacterial peptide among the embodiment 4.
Fig. 3 be among the embodiment 4 the circular dichroism spectrometry to the analytical results of polypeptide.
The antibacterial peptide secondary structure model of Fig. 4 for obtaining among the embodiment 4, wherein, left side figure is antibacterial peptide Ap, the right side is antibacterial peptide Apn.
Embodiment
Followingly with reference to specific embodiment the present invention is described.It will be appreciated by those skilled in the art that these embodiment only are used for explanation the present invention, the scope that it does not limit the present invention in any way.
Embodiment 1The extraction of AP polypeptide and purifying
Catch the sharp bladder spiral shell of self-sow from the field, remain on 15 ℃ in temperature, salinity remains on laboratory rearing under 35 ‰ conditions.
Every sharp bladder spiral shell is gathered the blood of 0.5ml, and (buffer formulation is as follows: Citric Acid trisodium 2H to add Alsever ' the s solution anti-freezing damping fluid of isopyknic improvement
2O 8.0g, Citric Acid 0.5g, dextrose anhydrous 20.8g, sodium-chlor 22.3g, EDTA3.36g, distilled water 1000ml, dissolving is filtered, packing, autoclave sterilization, 4 ℃ of refrigerators are preserved standby) mixing, and in 800g, 4 ℃ of following centrifugal 15min; Hemocyte precipitates multigelation 3 times, adds the Tris damping fluid (pH8.7 contains 50mM NaCl) of the 50mM of 5 times of volumes then, uses glass homogenizer homogenate then; In 10000g, 4 ℃ of following centrifugal 20min; The precipitation that will contain organoid is resuspended with the 2M acetic acid of 3 times of volumes, and cooling bath, repeats 3 times; In 10000g, 4 ℃ of following centrifugal 20min, in order to remove cell debris, 4 ℃ of preservations of supernatant liquor are standby.
Solid-Phase Extraction and HPLC (high performance liquid chromatography) method according to routine are extracted and purifying protein.Operate as follows: with the above-mentioned supernatant liquor that contains albumen, be added on the Sep-Pak Plus C-18 pillar, use 5%, 40% and 80% acetonitrile acidifying water elution respectively.Collect elutriant and also detect anti-microbial activity, have only 40% acetonitrile acidifying elutriant to have activity.
Then 40% acetonitrile acidifying elutriant is collected, and be added in the reversed-phase HPLC and be further purified.The specification of chromatographic column is: Sephasil C-18,250 * 4.1mm.And then with the acidifying acetonitrile solution wash-out of 0-60% linear gradient, collect elutriant, freeze-drying and with the dissolving of 0.2ml ultrapure water detects anti-microbial activity then.
Polypeptide pedigree with cut of anti-mycotic activity is distributed in the scope less than 6.5kDa, as shown in Figure 1.
Adopt conventional electrophoresis method, supernatant samples is added in the sample cell of SDS-PAGE gel, adopt the 1-45kDa of Sigma company as the standard protein molecular weight.The voltage that electrophoresis adopts is 80V, and electrophoresis time is 3h.Then albumen is forwarded on the pvdf membrane, with the composition of Edman sequencing analysis of amino acid, the result is as follows: TYMPVEEGEYIVNISYADQPKKNSPFTAKKQPGPKVDLSGVKAYGPG (SEQ.ID.NO.1).
Embodiment 2The design of novel antimicrobial peptide Apn
For the hydrophobicity (bringing up to 38% from 25%) that improves antibacterial peptide and cation surface activating (from+1 bring up to+5), begin to delete 10 hydrophilic amino acid: T-1, Y-2, Y-10, N-13, S-15, Y-16, Q-19, N-23, Q-31 and Y-44 from the N-end.Delete 5 electronegative amino acid: E-6, E-7, E-9, D-18 and D-37 simultaneously.With the amphiphilic polypeptide called after Apn that forms, it is a polypeptide that is different from AP, and sequence is as follows: MPVGIVIAPKKSPFTAKKPGPVLSGVKAGPG (SEQ.ID.NO.2).
Embodiment 3The chemosynthesis of antibacterial peptide and purifying
The chemical synthesis of employing standard (Lu Yingjin, Wang He.The solid phase synthesis of antibacterial peptide, the research of separation and purification and structure activity relationship, biotechnology journal, 1997,13 (1): 35-41) synthetic aforementioned polypeptides Ap and Apn.
After synthetic, extract polypeptide with 10% (v/v) acetic acid bi-distilled water, by the RP-HPLC purified polypeptide, purity is greater than 95% then.Determine that by mass spectrometry method the molecular weight of purified polypeptide Apn is 3.028kDa.
Embodiment 4The homology of antibacterial peptide and structural performance analysis
By choose a large amount of albumen at Protein Data Bank, with the ClustalW biosoftware above-mentioned antibacterial peptide is carried out homology analysis and comparison, the results are shown in Table 1 and Fig. 2.
The homology comparative result of table 1 antibacterial peptide
The result shows that Ap and Apn analytical standard homology are higher than 30%.Natural antibacterial peptide Ap and medfly Ceratotoxin C have higher homology, and novel antibacterial peptide Apn then reaches 39% with the homology of medfly Ceratotoxin C.
Adopt the above-mentioned synthetic polypeptide of circular dichroism (CD) analysis of spectral method.Each sample triplicate is averaged as final measurement result, as shown in Figure 3.With information biology software PSIPRED and SOPMA (derive from website ExPASy web server,
Http:// expasy.org) Ap and Apn are carried out secondary structure prediction, the results are shown in Figure 4.
Embodiment 5The antibacterial activity test
Carry out the antibacterial activity of above-mentioned antibacterial peptide measures with micrococci, staphylococcus epidermidis, intestinal bacteria, Aeromonas hydrophila, Vibrio parahemolyticus and vibrio cholerae.
These bacteriums on the TSB substratum in 37 ℃ of cultivations, when OD to be grown into (620nm) value is 0.2-0.3, get 100 μ l (1: 100) dilution bacterium liquid, add 10 μ l antibacterial peptides, continue to cultivate 24h, whether the absorbance (620nm) that detects bacterium liquid decides bacterial growth to be suppressed.The concentration range of antibacterial peptide is 33 μ M.The results are shown in Table 2.
The antibacterial activity of table 2 antibacterial peptide is measured
Above-mentioned antibacterium test-results shows that two kinds of antibacterial peptides all do not have tangible antibacterial activity.
Embodiment 6The anti-mycotic activity test
With Fusarium oxysporum, neurospora crassa and water mold above-mentioned antibacterial peptide is carried out the anti-mycotic activity test.
With the fungal spore of 80 μ l, concentration is 1 * 10
4Individual spore/ml, tsiklomitsin (10 μ g/ml), Streptomycin sulphate (10 μ g/ml) and 20 μ l antibacterial peptides are added in the micro plate jointly, and room temperature (22 ℃) is cultivated 30min then, detects absorbance (595nm).And then after continuing incubated at room temperature 72h, measuring absorbance (595nm).Judge the inhibition situation of fungal growth by the difference that compares absorbance.Antibacterial peptide working concentration 1.3 μ M.The results are shown in table 3.
The anti-mycotic activity of table 3 antibacterial peptide is measured
N represents there is not activity.
Above-mentioned antibacterium test-results shows that two kinds of antibacterial peptides all have anti-mycotic activity, and Apn has anti-fungus spectra comparatively widely.
Embodiment 7Cell toxicity test
Adopt the cytotoxicity of the above-mentioned antibacterial peptide of CHSE-214 raji cell assay Raji.
At the bottom of the CHSE-214 monolayer cell grows into the confluent culture plate hole 70% the time, wash 3 times with PBS, add antibacterial peptide (concentration range is 1-100 μ M) the effect 3h of different concns then, each concentration arranges 3 holes.Wash 3 times with PBS then, again with containing 0.1% tryptic EDTA effect 30-60s, peptic cell.Expect that with platform blue dyeing process detects cytoactive then.The result is as shown in table 4, and the cellular control unit survival rate is greater than 90%.
The cytotoxic assay result of table 4 antibacterial peptide
Above-mentioned test-results shows, in concentration during less than 10 μ M, and Ap and Apn no significant difference, and in concentration during greater than 33 μ M, the toxicity of Apn is significantly less than Ap.
Claims (10)
1. an anti-fungus peptide is characterized in that, described anti-fungus peptide is that sequence is the peptide of the aminoacid sequence of SEQ.ID.NO.2.
2. anti-fungus peptide according to claim 1 is characterized in that, effective antimycotic concentration of described anti-fungus peptide is for being not less than 1.3 μ M.
3. anti-fungus peptide according to claim 1 and 2 is characterized in that, effective antimycotic concentration of described anti-fungus peptide is 1.3-33 μ M.
4. anti-fungus peptide according to claim 1 and 2 is characterized in that, described fungi is selected from Fusarium oxysporum, neurospora crassa and water mo(u)ld.
5. the preparation method of each described anti-fungus peptide in the claim 1 to 4 is characterized in that, described anti-fungus peptide is by the preparation of biological or chemical method.
In the claim 1 to 4 each described anti-fungus peptide in the purposes of preparation in the antifungal medicament.
7. the described purposes of claim 6 is characterized in that, described fungi is selected from Fusarium oxysporum, neurospora crassa and water mo(u)ld.
8. antifungal preparation, it comprises each described anti-fungus peptide in the claim 1 to 4 of effective anti-fungal amount.
9. antifungal preparation according to claim 8 is characterized in that, the concentration of described anti-fungus peptide is for being not less than 1.3 μ M.
10. antifungal preparation according to claim 9 is characterized in that, the concentration of described anti-fungus peptide is 1.3-33 μ M.
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| Antifungal peptides in marine invertebrates;N Fusetani;《Invertebrate Survival Journal》;20101231;第7卷;第53-66页 * |
| N Fusetani.Antifungal peptides in marine invertebrates.《Invertebrate Survival Journal》.2010,第7卷第53-66页. |
| 昆虫抗菌肽和抗真菌肽结构与功能的关系及分子设计;肖业臣等;《昆虫学报》;20041231;第47卷(第5期);第659-669页 * |
| 肖业臣等.昆虫抗菌肽和抗真菌肽结构与功能的关系及分子设计.《昆虫学报》.2004,第47卷(第5期),第659-669页. |
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