CN1336383A - Giant web bombinin and its prepn. and pharmaceutical application - Google Patents
Giant web bombinin and its prepn. and pharmaceutical application Download PDFInfo
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- CN1336383A CN1336383A CN 00122414 CN00122414A CN1336383A CN 1336383 A CN1336383 A CN 1336383A CN 00122414 CN00122414 CN 00122414 CN 00122414 A CN00122414 A CN 00122414A CN 1336383 A CN1336383 A CN 1336383A
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- antibacterial peptide
- bombina maxima
- glycine
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Abstract
The present invention relates to large puling toad antibacterial peptide which is single chain polypeptide with molecular weight 2697.6, isoelectric point 10.2, its fuel full sequence structure is NH2-GIGGVLLSAGKAALKGLAKVLAAKYANN-NH2. Its preparation method is: the large puling toad living body is washed and placed in glass container with cover, drip absolute ethylether, close vessel for 3-5 min, large quantity foam material is secreted from toad skin, collect secretion, centrifugal to remove sediment, freeze dried, ion exchange, gel filtration, high pressure liquid phase inversed phase column chromatography separation and purification to obtain product. The invented toad antibacterial peptide possesses broad spestrum inhibition action against bacteria and fungus.
Description
The invention provides a kind of Bombina maxima antibacterial peptide and preparation method thereof and the application in pharmacy, belong to biomedical sector.
Since the microbiotic invention, the mankind have obtained bigger achievement in control and treatment infected by microbes disease, but lasting use along with medicine, the microorganism resistance has become the significant problem that Clinical microorganism catches and treats at present, to such an extent as to some pathogenetic bacteria has not had a line medicine of clinical treatment.It all is worldwide clinical difficult problems at present that the staphylococcus of vancomycin resistance, faecalis and other Gram-negatives catch.Three classes cause that mainly strong resistance has also appearred in meningitic bacterium clinically, anti-penicillin, paraxin meningococcus, and streptococcus pneumoniae, antibiotic streptococcus pneumoniae also extensively occurs the antagonism cephalosporin.Therefore the antibiotic development of new class has become the task of top priority and international focus.Compare with present widely used microbiotic, peptide antibiotics has lot of advantages: as when the least action concentration, fast and the killing microorganisms of wide spectrum (comprising clinical anti-medicine bacterium at present), fungi also there is restraining effect, the resistant strain generative nature is little, all effective to local infection and systemic infection, just becoming the new class microbiotic, its development is subjected to extensive attention at present.
A lot of amphibian animals belong to traditional Chinese medicine and national medicine and widespread use in China, as Chinese toad (Bufo gargarizans), Bombina maxima (Bombina maxima), Rana nigromaculata (pelophylaxnigromaculata), Rana guentheri (Hylarana guentheri) and rana limnocharis (Euphlyctis limnocharis) etc.The skin of these amphibian animals and internal organ have pharmacologically active and clinical efficacy widely, have reported that pharmacologically active has wide spectrum anti-microbial effect, antitumor, toponarcosis, analgesia, immunomodulatory, cardiovascular systems effect etc.Abroad, the searching of the specific pharmacology reactive monomer of batrachians skin compound has been the focus of new drug invention, according to foreign literature, antibacterial peptide Magainin tool wide spectrum anti-microbial effect from the acquisition of Xenopus laevis (Xenopus laevis) skin juice, has anti-tumor activity simultaneously, got permission as extensive pedigree antibiotic in the U.S., it is clinical that its gene engineering product entered for three phases; Recently, Magainin drugmaker announces, the Xenopus laevis skin antibacterial peptide Magainin analogue MSI-78 that they produce is used for the treatment of 926 routine diabetic subjects and causes that because of various bacteria infects the III phase clinical effectiveness of foot ulcers shows, MSI-78 and ofloxacin have same effect, but produce littler side effect.The antibacterial peptide IB-367 that Intrabiotics company produces has got permission to be used for the treatment of cancer patient and has infected stomatocace one clinical trial phase that causes because of various bacteria; AppliedMicrobiology company and the cooperation of Astra company also obtain good curative effect with the clinical trial of antibacterial peptide nisin treatment stomach ulcer.The foreign scholar has also reported the polypeptides matter of isolating some tool broad-spectrum anti-microbial activities in the bell toad (Bombina orientalis) and Hua Lingchan (Bombina variegata) eastwardly, as Bombinin and BLP-like polypeptide.
There is long history in China to the application of batrachians medicine, but the research of its activeconstituents and pharmacological properties is mainly concentrated on organic molecules such as alkaloid, and is also less to the research of its skin activity peptide matters.Research to batrachians skin antibacterial peptide yet there are no report.Bombina maxima (Bombina maxima) mainly is distributed in Yunnan, the Sichuan Province of China, is one of characteristic resources animal of China, also is the national folk medicine of characteristic.
The contriver searches comparison with Bombina maxima antibacterial peptide complete sequence structure of the present invention through Protein Data Bank, finds no any phase homopolypeptide.
The objective of the invention is based on above-mentioned prior art basis, a kind of have active Bombina maxima antibacterial peptide of wide spectrum antimicrobial (comprising gram positive bacterium, gram negative bacterium, fungi) and preparation method thereof and the application in pharmacy are provided.
In order to realize purpose of the present invention, the invention provides following technical scheme:
The Bombina maxima antibacterial peptide, for we separate a kind of single chain polypeptide that obtains first from Chinese amphibian animal Bombina maxima skin secretion, molecular weight 2697.6, iso-electric point 10.2, polypeptide one-level complete sequence structure is: glycine-Isoleucine-glycine-glycine-Val-Leu-Leu-Ser-Ala-glycine-Methionin-Ala-Ala-leucine-LYS-GLY-leucine-L-Ala-Methionin-Val-Leu-Ala-Ala-Methionin-tyrosine-L-Ala-l-asparagine-amidation l-asparagine (NH
2-GIGGVLLSAGKAALKGLAKVLAAKYANN-NH
2).
The preparation method of Bombina maxima antibacterial peptide, be that live body Bombina maxima water is cleaned up, place Glass Containers with cover, drip anhydrous diethyl ether (1 liter of volumetric capacity adds 1 milliliter of anhydrous diethyl ether), encloses container 3-5 minute, as seen a large amount of foam-like materials is secreted out from Bombina maxima skin, collect secretory product, centrifugal (10000rpm, 20 minutes), after the lyophilize, after DEAE-Sephadex A-50 ion-exchange, collect gained peak I and get peak IV after Sephadex G-50 gel-filtration, peak IV gets the peak II that the 3rd step column chromatography for separation goes out again through CM-Sephadex C-25 ion-exchange, again through the anti-phase C18 column separating purification of high performance liquid chromatography (HPLC), get the peak II that the anti-phase C18 column chromatography for separation of the 4th step HPLC goes out and get final product (shown in Fig. 4 arrow).Identify its purity with the high-efficient liquid phase chromatogram HPLC method then, molecular weight determination adopts fast atom bombardment mass spectroscopy(FABMS) method (Fast atom bombardment mass spectrometry, FAB-MS), isoelectric focusing electrophoresis is measured iso-electric point, measures the aminoacid sequence structure with automatic Protein Sequencer.
The Bombina maxima antibacterial peptide is as the application of preparation wide spectrum antimicrobial drug.
The pharmacological results with Bombina maxima antibacterial peptide of the present invention illustrates drug action of the present invention and beneficial effect below:
1, the effect of Bombina maxima antibacterial peptide bacteria growing inhibiting:
Anti-microbial activity detects, and adopts cylinder plate method, and substratum is the plain agar substratum.The substratum 20ml that injects heating and melting respectively as bottom, makes its even stand cloth at the bottom of the ware, after solidifying in plate, after other gets an amount of heating and melting of substratum, in every ware, add the 5ml bacteria suspension respectively, shake up, make it on bottom, evenly spread out cloth, as the bacterium layer.After the cooling, evenly put into 6 of disinfectant stainless steel cups in the plate moderate distance.First steel bowl adds the testing compound solution 0.1ml of 0.3mg/ml concentration, and all the other steel bowls adopt doubling dilution to add sample liquid, and the inhibition zone size is observed in 37 ℃ of cultivations.Inhibition zone 10mm above as minimal inhibitory concentration (minimal inhibitory concentration, MIC).Bacterial isolates derives from No.1 Hospital Attached to Kunming Medical College, and this test repeats four times, averages result such as table 1.
2, the Bombina maxima antibacterial peptide suppresses the effect of fungal growth:
Anti-mycotic activity detects, and adopts cylinder plate method.Substratum is improvement husky Bao Shi (Sabousand) substratum, the substratum 20ml that injects heating and melting respectively in plate as bottom, make it at the bottom of the ware, evenly spread out cloth, after solidifying, after other gets an amount of heating and melting of substratum, in every ware, add the 5ml bacteria suspension respectively, shake up, make it on bottom, evenly spread out cloth, as the bacterium layer.After the cooling, evenly put into 6 of disinfectant stainless steel cups in the plate moderate distance.First steel bowl adds the testing compound solution 0.1ml of 0.3mg/ml concentration, and all the other steel bowls adopt doubling dilution to add sample liquid, and the inhibition zone size is observed in 37 ℃ of cultivations.Inhibition zone 10mm above as minimal inhibitory concentration (minimal inhibitory concentration, MIC).The fungi strain derives from institute of microbiology of Yunnan University, and this tests triplicate, averages result such as table 2.
Table 1, the effect of Bombina maxima antibacterial peptide bacteria growing inhibiting:
| Bacterial strain | Minimal inhibitory concentration (μ g/ml) |
| Crude samples Bombina maxima antibacterial peptide | |
| Escherichia coli ATCC25922 (Escherichia coli ATCC25922) staphylococcus aureus ATCC2592 (Staphylococcus auneus ATCC2592) Pseudomonas aeruginosa CMCCB10104 (Pseudomonas aeruginosa CMCCB10104) Bacillus megatherium (Bacillus megaterium) shigella dysenteriae (Bacillus dysenteriae) pneumococcus (Klebsiella pneumoniae) | 150?????????1.2 75??????????3.6 75??????????14.4 150?????????1.2 37.5????????1.2 150?????????3.6 |
By table 1 as seen, Bombina maxima antibacterial peptide the first step DEAE-Sephadex A-50 ion-exchange crude product and purifying Bombina maxima antibacterial peptide all have significant inhibition bacterium and (comprise gram positive bacterium, gram negative bacterium) Sheng Chang effect, and compare with other source antimicrobial polypeptides of bibliographical information, the anti-microbial activity mean height 5-20 of Bombina maxima antibacterial peptide is doubly.
Table 2, the Bombina maxima antibacterial peptide suppresses the effect of fungal growth:
| Bacterial strain | Minimal inhibitory concentration (μ g/ml) |
| Crude samples Bombina maxima antibacterial peptide | |
| Candida albicans ATCC2002 (Candida albicans ATCC2002) aspergillus flavus IFFI4015 (Aspergillus flavus sp IFFI4015) mould IFFI2001 (Penicillium sp IFFI2001) Mucor pusillus (Mucor pusillus) yeast: brewer's yeast (Sacharomyces cerervisiae) | 25??????????3.6 75??????????12.8 ＞300???????50 50??????????5.2 80??????????10.4 |
By table 2 as seen, except that bacteria growing inhibiting, Bombina maxima antibacterial peptide the first step DEAE-SephadexA-50 ion-exchange crude product and purifying Bombina maxima antibacterial peptide all have the effect of significant inhibition fungal growth, and compare with other source antimicrobial polypeptides, the on average also high 5-20 of anti-mycotic activity is doubly.
The beneficial effect that can draw Bombina maxima antibacterial peptide of the present invention from above-mentioned experimental result is:
Bombina maxima antibacterial peptide crude product and purifying Bombina maxima antibacterial peptide all have the effect of significant inhibition bacterium and fungal growth, the microorganism used therefor bacterial strain comprises: intestinal bacteria, streptococcus aureus, Pseudomonas aeruginosa, dysentery bacterium, Candida albicans, mould, aspergillus oryzae etc., the result shows that Bombina maxima antibacterial peptide tool broad-spectrum anti-microbial activity (comprises gram positive bacterium, gram negative bacterium, fungi; MIC is in 1-15 μ g/ml scope), and compare with other source antimicrobial polypeptides, the on average also high 5-20 of anti-microbial activity is doubly.Therefore, Bombina maxima antibacterial peptide of the present invention is a kind of biologically active polypeptides of developing from distinct Chinese characteristics medicinal organism resource first with broad-spectrum anti-microbial activity.
Further specify essentiality content of the present invention below in conjunction with accompanying drawing with embodiment, but content of the present invention is not limited thereto.
Fig. 1 is the DEAE-Sephadex A-50 ion exchange chromatography figure of Bombina maxima antibacterial peptide of the present invention.
Fig. 2 is the Sephadex G-50 gel permeation chromatography figure of Bombina maxima antibacterial peptide of the present invention.
Fig. 3 is the CM-Sephadex C-25 ion exchange chromatography figure of Bombina maxima antibacterial peptide of the present invention.
Fig. 4 is the anti-phase C18 column chromatography of the HPLC figure of Bombina maxima antibacterial peptide of the present invention.
Embodiment:
1, preparation Bombina maxima antibacterial peptide:
Live body Bombina maxima water cleans up, Bombina maxima is placed Glass Containers with cover, drip anhydrous diethyl ether (1 liter of volumetric capacity adds 1 milliliter of anhydrous diethyl ether), encloses container 3-5 minute, as seen a large amount of foam-like materials is secreted out from Bombina maxima skin, collect secretory product, centrifugal (10000rpm, 20 minutes), lyophilize, cryopreservation.The first step: DEAE Sephadex A-50 ion-exchange, obtain starting material Bombina maxima secretory product lyophilized powder as stated above, lyophilized powder is dissolved in 0.05M Tris-HCl, contain 10mMEDTA, the damping fluid of pH7.3 was loaded on the 3.5KDa dialysis tubing, in same damping fluid dialysis 12 hours; (500mm is long for DEAESephadex A-50 (Pharmacia product) anion-exchange column, the 26mm diameter) through 0.05MTris-HCl, contain 10mM EDTA, pH7.3 damping fluid balance, the sample upper prop of having dialysed, to penetrating the peak, carry out gradient elution with the damping fluid that contains NaCl through two same damping fluid flushings of column volume again, collect and penetrate peak I by complete wash-out.
In second step, Sephadex G-50 gel-filtration: the activeconstituents that the first step obtains penetrates peak I freeze-drying, is dissolved in 0.1M Na
2HPO
4-NaH
2PO
4, the pH6.0 damping fluid is splined on Sephadex G-50 (Pharmacia product) gel-filtration column (1000mm is long, the 26mm diameter), uses same buffer solution elution, collects peak IV.
In the 3rd step, CM Sephadex C-25 ion-exchange: the activeconstituents peak IV that gel-filtration obtains goes up sample in through 0.1M Na
2HPO
4-NaH
2PO
4, pH6.0 damping fluid equilibrated CH Sephadex C-25 (Pharmacia product) cationic exchange coloum (300mm is long, the 26mm diameter) carries out gradient elution with the damping fluid that contains NaCl again, collects peak II.
The 4th step, reverse phase HPLC (RP-HPLC): the activeconstituents peak II that CM Sephadex C-25 ion-exchange obtains is with water (containing 0.1% trifluoroacetic acid): the elution system that second fine (containing 0.1% trifluoroacetic acid) constitutes is carried out gradient elution, collects high pressure liquid chromatography (HPLC) HPLC anti-phase C18 column chromatography peak (as Fig. 4 arrow mark) and is the Bombina maxima antibacterial peptide.
2, molecular weight determination adopts the fast atom bombardment mass spectroscopy(FABMS) method, and (Fast atom bombardment massspectrometry, FAB-MS), with glycerine: m-nitrobenzyl alcohol: methyl-sulphoxide (1: 1: 1, V: V: V, volume ratio) is a substrate, Cs
+As projectile, electric current is 1 μ A, and emission voltage is 25Kv.
3, the Bombina maxima antibacterial peptide of purifying is identified its purity with the high-efficient liquid phase chromatogram HPLC method, and molecular weight determination adopts the fast atom bombardment mass spectroscopy(FABMS) method, and isoelectric focusing electrophoresis is measured iso-electric point, measures the aminoacid sequence structure with automatic Protein Sequencer.
Bombina maxima antibacterial peptide by method for preparing, be that we separate a kind of single chain polypeptide that obtains first from Chinese amphibian animal Bombina maxima skin secretion, molecular weight 2697.6, iso-electric point 10.2, polypeptide complete sequence primary structure is: glycine-Isoleucine-glycine-glycine-Val-Leu-Leu-Ser-Ala-glycine-Methionin-Ala-Ala-leucine-LYS-GLY-leucine-L-Ala-Methionin-Val-Leu-Ala-Ala-Methionin-tyrosine-L-Ala-l-asparagine-amidation l-asparagine (NH
2-GIGGVLSAGKAALKGLAKVLAAKYANN-NH
2).The Bombina maxima antibacterial peptide has broad-spectrum anti-microbial activity and significant inhibition bacterium and fungal growth effect, controls the application of infected by microbes disease medicament in preparation as the wide spectrum antimicrobial material.
Claims (3)
1, the Bombina maxima antibacterial peptide, it is characterized in that from Chinese amphibian animal Bombina maxima skin secretion, separating a kind of single chain polypeptide that obtains, molecular weight 2697.6, iso-electric point 10.2, polypeptide complete sequence primary structure is: glycine-Isoleucine-glycine-glycine-Val-Leu-Leu-Ser-Ala-glycine-Methionin-Ala-Ala-leucine-LYS-GLY-leucine-L-Ala-Methionin-Val-Leu-Ala-Ala-Methionin-tyrosine-L-Ala-l-asparagine-amidation l-asparagine.
2, the preparation method of Bombina maxima antibacterial peptide, it is characterized in that live body Bombina maxima water is cleaned up, place Glass Containers with cover, drip anhydrous diethyl ether, encloses container 3-5 minute, as seen a large amount of foam-like materials is secreted out from Bombina maxima skin, collect secretory product, after centrifugal removal precipitation, the lyophilize, after ion-exchange, gel-filtration, the separation and purification of high-pressure liquid phase reversed phase column chromatography, promptly obtain.
3, the Bombina maxima antibacterial peptide is controlled the application of infected by microbes disease medicament in preparation as the wide spectrum antimicrobial material.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00122414 CN1336383A (en) | 2000-07-29 | 2000-07-29 | Giant web bombinin and its prepn. and pharmaceutical application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00122414 CN1336383A (en) | 2000-07-29 | 2000-07-29 | Giant web bombinin and its prepn. and pharmaceutical application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133235A (en) * | 2011-03-07 | 2011-07-27 | 哈尔滨工业大学 | Toad peptide antibiotics for preparing medicine for treating phthisis |
| CN101031583B (en) * | 2004-08-18 | 2013-06-05 | 诺瓦生命科学有限公司 | Antimicrobial peptides containing arginine and/or lysine motif |
| CN104223121A (en) * | 2014-09-16 | 2014-12-24 | 重庆馗旭生物科技股份有限公司 | Extracting device for giant salamander skin mucus and extracting method |
| CN105837674A (en) * | 2016-03-08 | 2016-08-10 | 无限极（中国）有限公司 | Bombina orientalis polypeptide, and preparation method and application thereof |
| CN110684098A (en) * | 2019-10-09 | 2020-01-14 | 江苏医药职业学院 | Method for preparing toad antibacterial peptide bombinin and application thereof |
-
2000
- 2000-07-29 CN CN 00122414 patent/CN1336383A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101031583B (en) * | 2004-08-18 | 2013-06-05 | 诺瓦生命科学有限公司 | Antimicrobial peptides containing arginine and/or lysine motif |
| CN103360486A (en) * | 2004-08-18 | 2013-10-23 | 诺瓦生命科学有限公司 | Antimicrobial peptides comprising arginine-and/or lysine-containing motif |
| CN102133235A (en) * | 2011-03-07 | 2011-07-27 | 哈尔滨工业大学 | Toad peptide antibiotics for preparing medicine for treating phthisis |
| CN102133235B (en) * | 2011-03-07 | 2013-03-20 | 哈尔滨工业大学 | Toad peptide antibiotic medicine for treating phthisis |
| CN104223121A (en) * | 2014-09-16 | 2014-12-24 | 重庆馗旭生物科技股份有限公司 | Extracting device for giant salamander skin mucus and extracting method |
| CN105837674A (en) * | 2016-03-08 | 2016-08-10 | 无限极（中国）有限公司 | Bombina orientalis polypeptide, and preparation method and application thereof |
| CN110684098A (en) * | 2019-10-09 | 2020-01-14 | 江苏医药职业学院 | Method for preparing toad antibacterial peptide bombinin and application thereof |
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