CN1314362A - Anti-fungus peptide and its preparing method and use - Google Patents

Anti-fungus peptide and its preparing method and use Download PDF

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CN1314362A
CN1314362A CN 00114941 CN00114941A CN1314362A CN 1314362 A CN1314362 A CN 1314362A CN 00114941 CN00114941 CN 00114941 CN 00114941 A CN00114941 A CN 00114941A CN 1314362 A CN1314362 A CN 1314362A
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peptide
fungus
chitin
present
protein
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CN1121412C (en
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龚蓁蓁
黄旭
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Shanghai Institute of Biochemistry
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Abstract

The anti-fungus peptide of the present invention is prepared with ginkgo leaf as raw material and through salting out, affinity chromatography with chitin, separation with molecular sieve and HPLC purification. The application of the anti-fungus peptide in preparing anti-fungus medicine is also provided.

Description

Anti-fungus peptide and preparation method thereof and purposes
The present invention relates to anti-fungus peptide, Preparation Method And The Use.Specifically, be that separation and purification obtains new peptide from ginkgo.
The many little antibacterial proteins that are rich in halfcystine/glycine of plant utilization are resisted the cause of disease invasion and attack.Thionin (Bohlmann, H. and Apel, K. (1991) Annu.Rev.Plant Physiol.Mol.Biol.42,227-240), plant protecting chemical (Broekaert etc., 1995, Plant Physiol.108,1353-1358), Ac-AMPs (Broekaert, W.F., Marien, W., Terras, F.R.G., De Bolle, M.F.C., Proost, P., VanDamme, J., Dillen, L., Claeys, M., Rees, S.B., Vanderleyden, J. and Cammue, B.P.A. (1992) Biochemistry 31,4308-4314), henein class and knottin class antibacterial peptide (Cammue etc., 1992, J.Biol.Chem.267,2228-2233) all belong to the little peptide (29-54 residue) that is rich in halfcystine, but their primary structure is totally different, anti-microbial activity is also different.Wherein, hevein class peptide and Ac-AMPs are the chitin binding peptides, and they have a hevein district or one section homologous sequence.
At present the molecular mechanism for the antimycotic and/or antibacterial activity of many antibacterial peptides it be unclear that.VanParijs etc. think that anti-mycotic activity and its protein molecular weight size of hevein and Urtica dioica lectin (UDA) have certain relation.These albumen are very little, arrive plasmalemma so that can see through fungal cell wall, act on into avtive spot (the Van Parijs of cytolemma then there, J., Broekaert, W.F., Goldstein, I.J. and Peumans, W.J. (1991) Planta (Heidelberg) 183,258-264).Thionin then forms the hole on cytolemma, cause the cytolemma seepage, cause microbial death (Florack, D.E.A. and Stiekema, W.J. (1994) Plant Mol.Biol.26,25-37).
China is a large agricultural country, yet, annual all cause massive losses on the agricultural because of fungus-caused disease.Therefore, for farm crop being improved and develop the meaning that the new anti-mycotic agent that using value is arranged just has particularly important.
For this reason, the invention provides a kind of new anti-fungus peptide.Aminoacid sequence to N-terminal has carried out the NIHBLAST retrieval, finds no and its height homologous sequence, proves that this is to separate the new peptide that obtains first.The present invention also provides from natural plant material Ginkgo Leaf separation and purification to obtain the method for this anti-fungus peptide.Described peptide has significant anti-mycotic activity, so the present invention also provides described peptide to be used to prepare the purposes of antifungal drug.
In order to obtain new anti-mycotic agent, we begin screening operation in plant.We find that although contain abundant polysaccharide in the Ginkgo Leaf, they seldom are subjected to the invasion and attack of fungi; Simultaneously, we find that also many antifungal protein belong to chitin in conjunction with family.Therefore, we select the material of Ginkgo Leaf as the separation and purification anti-mycotic agent, with ammonium sulfate salting-out process extracting protein; The chitin purifying; Molecular sieving; With the HPLC purifying, obtain new anti-fungus peptide.
Described new anti-fungus peptide has aminoacid sequence SEQ ID NO.1:
DPTCSVLGDFKCNPGRCCSKINYCGACYQWLAR, molecular weight are 4244.0Da.
In order to keep activity of proteins, all operations all should be carried out at 0 ℃-4 ℃.
In order to keep the activity of peptide, avoid irreversible denaturation, we adopt ammonium sulfate salting-out process to carry out the protein extracting.For the enrichment target peptide, reduce foreign protein or assorted peptide as far as possible simultaneously, the concentration of ammonium sulfate is advisable with 40 (w/w) % to 80 (w/w) %; 80 (w/w) % preferably.
The present invention adopts chitin chromatography separate targets peptide from the protein crude extract.In order to improve separation efficiency, keep the activity of peptide simultaneously, ionic strength and pH are important parameters wherein.So the chitin affinity chromatography that the present invention adopts comprises: use 20mM NaHCO 3, pH7.3-8.3 washes post, is good with pH7.8; Use the 20mM sodium acetate, pH5.1-6.1 washes post, is good with pH5.6; Use 20mM acetate, the pH3.1-3.5 wash-out is good with pH3.3, and collects elutriant.Ionic strength wherein is advisable with 200mM, in view of the above, can select other damping fluid with suitable ionic strength for use, and this is not difficult to those skilled in the art.
Utilizing molecular sieving albumen is routine techniques well known in the art, the FPLC on the Sepherose post for example, Sephdex column chromatography and PAGE electrophoresis etc.Condition is, the method for selecting for use is target peptide inactivation in addition not.Therefore, the preferred Sepherose12 post of the present invention FPLC.
The HPLC purifying protein is the common practise of this area.The present invention uses conventional commercially available HPLC workstation (C18 post).
The present invention comprises also anti-fungus peptide of the present invention is used to prepare antifungal drug that it is disease-resistant to be used for agricultural, animal doctor and human clinical.
(Biochem.Biophys.Acta 1986,880,161-170) measure the anti-mycotic activity of peptide of the present invention according to Roberts and Selitrennikoff method.Found that it has tangible resistance to rice banded sclerotial blight (Pellicularia sasakii Ito), gibberella saubinetii (Fusarium graminearum Schw), the red star of tobacco (Alternaria alterinate (Fries) Keissler), rice bakanae disease (Fusarium moniliforme).Minimal effective concentration is 100 μ g/ml.
Accompanying drawing 1A-D is the result of the antimycotic analysis of the present invention.Fungi among the A is the rice banded sclerotial blight bacterium; Fungi among the B is the rice bakanae disease bacterium; Fungi among the C is a gibberella saubinetii; Fungi among the D is the red star of tobacco.In each culture dish, the scraps of paper 1 contain pure water, the scraps of paper 2 contain bSA and (are dissolved in the 100mM citrate buffer solution, pH5.0), the scraps of paper 3 contain 1 μ g anti-fungus peptide of the present invention and (are dissolved in the 100mM citrate buffer solution, pH5.0), the scraps of paper 4 contain 2 μ g anti-fungus peptide of the present invention and (are dissolved in the 100mM citrate buffer solution, pH5.0).
Fig. 2 is FPLC chromatography and SDS/PAGE electrophoresis result.Swimming lane M is the molecular weight marker thing; Swimming lane 1 is the extract of saltouing; Swimming lane 2 is chitin affinitive layer purification things; Swimming lane A is the A peak of FPLC.
Fig. 3 is the HPLC chromatography.Wherein the B peak is an anti-fungus peptide of the present invention.
Below be indefiniteness embodiment of the present invention.If not specify that various operations are all carried out at 4 ℃.
The purification of the isolation and purification crude protein of embodiment 1. anti-fungus peptides
In the citrate buffer solution (30mM, pH5.0,1mM Phenylmethylsulfonyl chloride) of 2-3 times of volume with the abundant homogenate of Ginkgo Leaf.Homogenate is with 3 layers of filtered through gauze, and filtrate is 10, centrifugal 30 minutes of 000g.Get supernatant liquor, add solid ammonium sulfate and reach 80 (w/w) % saturation ratio.Stirring is spent the night, and 38, centrifugal 30 minutes of 000g collects crude protein.The crude protein precipitation heavily is dissolved in 30ml 20mM NaHCO 3, the dialysis membrane of using 3.5kDa is to 20mMNaHCO 3Dialysis.The chitin affinity chromatography
Get the 3.5g chitosan, according to described methods such as Molano (Molano, J., Duran, A. and CabibE. (1977) Anal Biochem.83,648-656) the fresh regeneration chitin of preparation, autoclaving is used 20mMNaHCO 3Balance, the chromatography column of the diameter 2.6cm that packs into, last bed volume are 46ml.
On regeneration chitin post, flow velocity is 0.5ml/min with sample on the protein soln of dialysing.Use earlier 20mM NaHCO 3, the pH7.8 wash-out washes out up to no protein.For the second time, the sodium acetate buffer wash-out with 20mM pH5.6 carried out 90 minutes.Use the acetic acid solution wash-out of 20mM pH3.3 at last.Use the dialysis membrane of 3.5kDa to distill water dialysis immediately elutriant.After the dialysis, with elutriant vacuum concentration drying.FPLC
Protein is dissolved in 200mM NaHCO 3, last sample is with 200mM NaHCO 3On equilibrated Superose 12 posts (Pharmacia Biotech), carry out FPLC (the FPLC system of Pharmacia Biotech).Use 200mMNaHCO 3, with the flow velocity wash-out of 0.4ml/min.Detect protein by measuring the 280nm absorbancy.With SDS/PAGE measure each stream part molecular weight (Laemmli, U.K. (1970) Nature (London) 227,680-685).As described in embodiment 3, each stream part is carried out the anti-mycotic activity detection.
As shown in Figure 2, the elution peak at 19ml place shows very strong anti-mycotic activity.SDS/PAGE shows, only contains anti-fungus peptide of the present invention in this stream part.Show that as the mass spectroscopy of carrying out as described in the embodiment 2 its molecular weight is 4244.0Da.HPLC
Use HP1090 HPLC workstation to be further purified protein.With sample on the above elution peak at the anti-phase C of 0.1% trifluoroacetic acid (TFA) equilibrated 18On the post (ABI).With the flow velocity of 1ml/min, carry out following gradient elution: 0-50min, the 0-50% solution B.Solution B is 0.1% TFA acetonitrile solution.Detect protein by the absorbancy of measuring the 214nm place.As shown in Figure 3, wash out two peaks altogether, A and B.In the anti-mycotic activity test, the B peak shows strong anti-mycotic activity, and the anti-mycotic activity at A peak is then very weak.So the B peak is an anti-fungus peptide of the present invention.
Can make the 4mg purified peptide approximately from the 500g Ginkgo Leaf.
Embodiment 2. proteinic evaluations
On Finnigan LCQ-MS mass spectrograph, carry out the mass spectroscopy of peptide.Above-mentioned purified peptide is dissolved in the water/ethanol that contains 1% (v/v) acetate, and (50: 50, v/v), concentration was 5 μ mol/l, is used for mass spectrograph then.The result records, and the molecular weight of anti-fungus peptide of the present invention is 4244Da.
As (Hunkapiller as described in Hunkapiller etc., M.W., Hewick, R.M., Dreyer.W.J. and Hood, L.E. (1983) Methods Enzymol.91,399-413), on Applied Biosystem 473A protein sequencer, measure the aminoacid sequence of peptide, obtain aminoacid sequence SEQ ID NO.1:DPTCSVLGDFKCNPGRCCSKINYCGACYQWLAR.
The test of embodiment 3. anti-mycotic activities
The present invention has tested the anti-mycotic activity of anti-fungus peptide to 4 kinds of pathomycetes.Testing used phytopathogen is all provided by Jiangsu Academy of Agricultural Sciences.They are rice banded sclerotial blight bacterium, gibberella saubinetii, red star bacterium of tobacco and rice bakanae disease bacterium.As Robert, W.K. etc. described (C.P. is the same for Robert, W.K. and Selitrennikoff) extend inhibition test by mycelium and measure anti-mycotic activity.
Preparation potato solid medium in culture dish.Collect hypha,hyphae from the fungi flat board of active growth, the mycelia piece is placed the substratum center.Cultivated 48 hours for 28 ℃, appoint mycelial growth.Aseptic round filter paper is placed the mycelia forward position, on each filter paper, drip 20 μ l protein and (be dissolved in the 100mM citrate buffer solution, pH5.0).Cultivated 24 hours at 28 ℃, take pictures then.Occur meniscate inhibition zone explanation detected solution around the filter paper and have anti-mycotic activity.
As (Broekaert, W.F., Marein, W. as described in Broekaert etc., Terras, F.R.G., De Bolle, M.F.C., Proost, P., Van Damme, J., Dillen, L., Claeys, M., Rees, S.B., Vanderleyden, J. and Cammue, B.P.A. (1992) Biochemistry 31 4308-4314) measures growth-inhibiting per-cent.As (Duvick, J.P., Rood such as Duvick, T., Gururaj Rao, A. and Marshak, D. (1992) J.Biol.Chem.267 is 18814-20) described, and uses standard method (Singleton, L.L., Mihail, J.D. and Rush, C.M. (1992) Methods for Research on Soilborne Phytopathogenic Fungi, APS press, Minnesota) spore or the mycelium of collection fungi.Containing fungal spore or mycelial 90 μ lPD substratum cultivated 24 hours for 28 ℃.The peptide that adds the various concentration of 10 μ l then.Continue 28 ℃ and cultivated 48 hours, the record growing state.Measure the absorbancy at 595nm place and represent fungi growth situation (Terras, F.R.G., Schoofs, H.M.E., De Bolle, M.F.C., Van Leuven, F., Rees, S.B., Vanderleyden, J., Cammue, B.P.A. and Broekaert, W.F. (1992) J.Biol.Chem.267,15301-15309).
As Fig. 1 and shown in, anti-fungus peptide of the present invention has just shown strong resistance to the rice banded sclerotial blight bacterium at the lower concentration of 100ng/ filter paper.And, other fungi also there is the growth-inhibiting effect.
Therefore, Semen Ginkgo extrac of the present invention and anti-fungus peptide can be used for preparing agricultural chemicals, directly are sprayed on plant, the invasion and attack of opposing pathomycete.

Claims (8)

1. anti-fungus peptide, it has aminoacid sequence SEQ ID NO.1:
DPTCSVLGDFKCNPGRCCSKINYCGACYQWLAR, molecular weight are 4244.0Da.
2. the preparation method of peptide according to claim 1, it comprises:
From Ginkgo Leaf, use ammonium sulfate extracting crude protein;
The chitin affinity chromatography;
Molecular sieve purification;
The HPLC purifying.
3. method according to claim 2, wherein the ammoniumsulphate soln concentration range is 40 (w/w) % to 80 (w/w) %.
4. method according to claim 3, wherein the concentration of ammoniumsulphate soln is 80 (w/w) %.
5. method according to claim 2, described chitin chromatography comprises:
Use 20mM NaHCO 3, pH7.3-8.3 washes post;
Use the 20mM sodium acetate, pH5.1-6.1 washes post;
Use 20mM acetate, the pH3.1-3.5 wash-out, and collect elutriant.
6. method according to claim 5, described chitin chromatography comprises:
Use 20mM NaHCO 3, pH7.8 washes post;
Use the 20mM sodium acetate, pH5.6 washes post;
Use 20mM acetate, the pH3.3 wash-out, and collect elutriant.
7. method according to claim 2, sieve method wherein are Sepherose12 FPLC.
8. the purposes of the described peptide of claim 1, it is used to prepare antifungal medicament.
CN 00114941 2000-03-16 2000-03-16 Anti-fungus peptide and its preparing method and use Expired - Fee Related CN1121412C (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005025587A1 (en) * 2003-09-16 2005-03-24 Chosun University Composition comprising ginkgolides and bilobalide
CN102603882A (en) * 2011-01-24 2012-07-25 中国科学院动物研究所 Antifungal peptide as well as preparation method and application thereof
CN102617716A (en) * 2012-03-23 2012-08-01 中国林业科学研究院林产化学工业研究所 Novel ginkgo protein preparation and characterization method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005025587A1 (en) * 2003-09-16 2005-03-24 Chosun University Composition comprising ginkgolides and bilobalide
CN102603882A (en) * 2011-01-24 2012-07-25 中国科学院动物研究所 Antifungal peptide as well as preparation method and application thereof
CN102603882B (en) * 2011-01-24 2013-07-17 中国科学院动物研究所 Antifungal peptide as well as preparation method and application thereof
CN102617716A (en) * 2012-03-23 2012-08-01 中国林业科学研究院林产化学工业研究所 Novel ginkgo protein preparation and characterization method

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