CN1142180C - Giant well bombinator proteinase inhibitor and its prepn. and pharmaceutical application - Google Patents
Giant well bombinator proteinase inhibitor and its prepn. and pharmaceutical application Download PDFInfo
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- CN1142180C CN1142180C CNB001224166A CN00122416A CN1142180C CN 1142180 C CN1142180 C CN 1142180C CN B001224166 A CNB001224166 A CN B001224166A CN 00122416 A CN00122416 A CN 00122416A CN 1142180 C CN1142180 C CN 1142180C
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Abstract
The present invention belongs to the technical field of biomedicine. A giant-web bombinator proteinase inhibitor is a single-chain serine protease inhibitor which is obtained by separating the skin of a giant-web bombinator which is an amphibian animal, the apparent molecular weight on SDS-polyacrylamide gel electrophoresis is 67KDa under a reduction condition, and is 45KDa under a non reduction condition; the isoelectric point is 4.8, the sugar content is form 17 to 19%, and the giant-web bombinator proteinase inhibitor has no enzyme activity. The N-end of the giant-web bombinator proteinase inhibitor comprises the sequence structure of 31 amino acids: DSETHIRFLGEVYKKVDTIDFRGLVLITRAQ. The preparation method comprises the steps that the skin of a giant-web bombinator is taken; after the skin carries out homogenate in physiological saline, the skin is centrifugated for removing deposits; supernatant fluid is collected and is frozen and dried, and then, the supernatant fluid is carries out ion exchange, gel filtration and purification to obtain the giant-web bombinator proteinase inhibitor. The giant-web bombinator proteinase inhibitor has the advantages of strong serine proteinase inhibition activity and antineoplastic activity, and is applied to prepare medicines for treating tumors, gastritis, pancreatitis and other kinds of inflammation.
Description
Technical field:
The invention provides a kind of Bombina maxima proteinase inhibitor and preparation method thereof and the application in pharmacy, belong to biomedical sector.
Background technology:
(serine protease inhibitor serpin) is a class single chain protein matter to serpin, and their basic functions is to prevent proteolysis, regulates the hydrolysising balance of serine protease.By the adjusting to serine protease, serpin has significant effects to many important biochemical reaction and the physiological function in the living organism.Below 14 class biochemical reactions and physiological function be subjected to the adjusting of serpin: blood coagulation, complement formation, fibrinolytic, protein folding, cell migration, cytodifferentiation, cell matrix reconstruction, hormone formation, hormone transhipment, intracellular proteolysis, blood pressure regulation, tumor suppression and virus or the pathogenic formation of parasite.Because so numerous physiological functions is regulated by it, serpin has become the focus of international research.Its research and development progress is containing huge clinical treatment medication preparation and is being worth.
Tumour is a class principal disease of harm humans health, and the medicine scarcity, and malignant tumour only increases 1,600,000 cases every year newly in China at present, and sicken age rejuvenation gradually.Employed medicine in chemotherapy at present, as Zorubicin, endoxan, tumour necrosis factor or the like all have bigger human toxicity, and side effect is very big, thereby the exploitation of anti-tumor medicine is the focus of drug development.Tumour expansion and transfer are one of major reasons of present clinical treatment tumour inefficiency.Serpin maspin has the obvious suppression effect with external to tumour in vivo, and the mechanism of action is to suppress tumor cell invasion and vasculogenesis.
Report that according to domestic and foreign literature serpin such as aprotinin except that being widely used in treatment of diseases such as gastritis, pancreatitis clinically, also are used for anti-fibrinolytic in thoracic surgery, suppress contact activation, anti-inflammatory etc.Trypsinase analogue such as Kallikrein, tryptase plays an important role in rheumatic arthritis and inflammation (as rhinitis, conjunctivitis, asthma, gi tract, cardiovascular systems inflammation) generation, and present its inhibitor of clinical proof is effective medicine.On the other hand, also do not treat the medicine of herpesvirus infection at present clinically effectively, find that recently suppressing the simplexvirus serine protease is a kind of effective treatment means.Above-mentioned these new serpin medicines have been in II, III phase clinical stage and commercialization.
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.On the other hand, the pharmaceutical compositions complicacy that Chinese medicine is traditional and the limitation of processing of medical material method, also be the major reason that causes medicine source material composition better not play a role, thereby the active monomeric compound of the specific pharmacology of searching is one of important content of the modernization of Chinese medicine from these conventional medicaments.Abroad, the searching of the specific pharmacology reactive monomer of batrachians skin compound has been the focus of new drug invention, and the foreign scholar has also reported the polypeptide BSTI that isolates a lower molecular weight tool trypsin inhibition activity in the bell toad (Bombina orientalis) eastwardly.Step-down peptide matters bufokinin that obtains from toad and wood frog skin and ranakinin have vasodilator and hypotensive effect, are demonstrating tempting application prospect aspect the treatment cardiovascular disorder; Recently, a kind of batrachians nuclease has more caused the attention of people to the batrachians active substance to the selective inhibitory of HIV.The nearly more than ten years screen more than 170 kinds of batrachians skin active peptides and analogue, prove that 40 various active peptides have drug development prospect preferably.
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 albumen peptide matters.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 proteinase inhibitor N-terminal sequence of the present invention through Protein Data Bank, finds no any identical and homologous series albumen.
Summary of the invention:
The objective of the invention is based on above-mentioned prior art basis, a kind of intensive serine protease inhibition active (comprising trypsinase, Quimotrase) that has is provided, has stronger growth of tumour cell simultaneously and suppress active Bombina maxima proteinase inhibitor and preparation method thereof and the application in pharmacy.
In order to realize purpose of the present invention, the invention provides following technical scheme:
The Bombina maxima proteinase inhibitor, be that we separate a kind of single chain serine protease inhibitor that obtains from amphibian animal Bombina maxima skin, it is 67KDa under the apparent molecular weight reductive condition on the SDS-polyacrylamide gel electrophoresis, under the non-reduced condition 45KDa, iso-electric point 4.8, sugar content 17-19%, non-enzymatic activity, 31 aminoacid sequence structures of N-end of Bombina maxima proteinase inhibitor: aspartic acid-Serine-L-glutamic acid-Threonine-Histidine-Isoleucine-arginine-Phe-Leu-glycine-glutaminic acid-Xie Ansuan-tyrosine-Methionin-Methionin-Xie Ansuan-aspartic acid-Threonine-Isoleucine-aspartic acid-phenylalanine-arginine-glycine-LEU-VAL-leucine-Isoleucine-Threonine-arginine-L-Ala-glutamine (DSETHIRFLGEVYKKVDTIDFRGLVLITRAQ).
The preparation method of Bombina maxima proteinase inhibitor.Bombina maxima picks up from the mountain area, Yunnan Province, and the live body water cleans up, execution, peeling, and skin is cut into small pieces, is dipped in physiological saline, homogenate, centrifugal (10000rpm, 20 minutes), lyophilize, cryopreservation.Adopt DEAE Sephadex A-50 ion-exchange, the SephadexG-75 gel-filtration, DEAE Sephadex A-50 ion-exchange separation and purification is got the peak I that the 3rd step column chromatography for separation goes out and is got final product.Use high performance liquid chromatography (HPLC) then, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) method is identified its purity, molecular weight determination adopts the SDS-polyacrylamide gel electrophoresis method to measure, isoelectric focusing electrophoresis is measured iso-electric point, measures the n terminal amino acid sequential structure with automatic Protein Sequencer.
The Bombina maxima proteinase inhibitor is treated the application of gastritis, pancreatitis medicine as preparation anti-tumor medicine and preparation.
With the biological chemistry of Bombina maxima proteinase inhibitor of the present invention, The pharmacological results illustrates biological chemistry of the present invention, drug action and beneficial effect below:
1, the Bombina maxima proteinase inhibitor suppresses the effect of serine protease:
The Bombina maxima proteinase inhibitor of different amounts be dissolved in 0.05M Tris-HCl damping fluid with-quantitative trypsin ultimate density is 5nM) and Quimotrase under room temperature, be incubated 15 minutes in 0.05M Tris-HCl damping fluid (ultimate density is 5nM), add chromophoric substrate S-2238 (ultimate density is 0.5mM) at last and start reaction, the Bio-4060 spectrophotometer of producing with Pharmacia (Sweden) company changed 2 minutes in 405nm place monitoring absorption value, the 0.05M Tris-HCl damping fluid and the blank that add same volume suppress constant K
iK by formula
i=[I]/(V
0/ V
I+ 1) calculates.[I] is the volumetric molar concentration of Bombina maxima proteinase inhibitor, V
0During for blank trypsinase and Quimotrase respectively with the speed of response of chromophoric substrate S-2238, V
IAfter adding the Bombina maxima proteinase inhibitor, trypsinase and Quimotrase respectively with the speed of response of chromophoric substrate S-2238.This test repeats six times, averages.
The result as shown in Figure 1, the Bombina maxima proteinase inhibitor is the effectively inhibitor of trypsinase and these two kinds of serine proteases of Quimotrase, under above-mentioned experiment condition, suppress half trypsinase and the needed Bombina maxima proteinase inhibitor of chymotrypsin activity concentration and be respectively 150ng/ml, 960ng/ml.The Bombina maxima proteinase inhibitor is 2 * 10 to tryptic inhibition constant K i
-9M is 1.5 * 10 to the inhibition constant K i of Quimotrase
-7M.
2, the Bombina maxima proteinase inhibitor suppresses the effect of growth of tumour cell:
On the flat Tissue Culture Plate in 96 holes, testing compound is carried out 5 times or 2 times of doubling dilutions with perfect medium, totally six extent of dilution, each extent of dilution is established 3 repeating holes, and every hole 100 μ l are provided with the normal cell contrast simultaneously.Every hole drips 3 * 10
5The C8166 cell of/ml or Molt-4 cell 100 μ l.Put 37 ℃, 5%CO
2Cultivate in the incubator.72 hours (C8166) or 48 hours (Molt-4) measures the toxic action of testing compound pair cell with the MTT method.EC50 is the drug level when 50% host cell is produced cytotoxicity.
Table 1 Bombina maxima proteinase inhibitor suppresses the effect of growth of tumour cell: compd E C50 (μ M)
C8166 Molt 4 Bombina maxima proteinase inhibitor crude products, 0.7 1.7 Bombina maxima proteinase inhibitor (lot number 1) 0.1 0.5 Bombina maxima proteinase inhibitor (lot number 2) 0.2 0.7 Bombina maxima proteinase inhibitor (lot number 3) 0.2 0.6
By table 1 as seen, except that suppressing serine protease, Bombina maxima proteinase inhibitor crude product (the first step DEAE-Sephadex A-50 ion-exchange sample) and purifying Bombina maxima proteinase inhibitor all have the effect that remarkable vitro suppresses growth of human tumor cells.
The beneficial effect that can draw Bombina maxima proteinase inhibitor of the present invention from above-mentioned experimental result is:
Bombina maxima proteinase inhibitor crude product and pure product all have the activity that suppresses trypsinase, Quimotrase consumingly, and the Ki value reaches 2 * 10 respectively
-9M and 1.5 * 10
-7M, the Bombina maxima proteinase inhibitor has the effect that suppresses growth of tumour cell significantly simultaneously, and to C8166, the Molt-4 tumour cell reaches the needed protein concentration of 50% inhibiting rate and is respectively 160nM and 600nM.Therefore, Bombina maxima proteinase inhibitor of the present invention be a kind of brand-new from distinct Chinese characteristics medicinal organism resource, develop have inhibition serine protease, anti-tumor bioactivity albumen.
Description of drawings:
Fig. 1 suppresses trypsinase and chymotrypsin activity figure for Bombina maxima proteinase inhibitor of the present invention.
Fig. 2 is the DEAE-Sephadex A-50 ion exchange chromatography figure of Bombina maxima proteinase inhibitor of the present invention.
Fig. 3 is the Sephadex G-75 gel permeation chromatography figure of Bombina maxima proteinase inhibitor of the present invention.
Fig. 4 is the 3rd step DEAE-Sephadex A-50 ion exchange chromatography figure of Bombina maxima proteinase inhibitor of the present invention.
Embodiment:
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.
Embodiment:
1. prepare the Bombina maxima proteinase inhibitor: Bombina maxima, pick up from the mountain area, Yunnan Province, the live body water cleans up, execution, peeling, skin is cut into small pieces, is dipped in physiological saline, homogenate, centrifugal (10000rpm, 20 minutes), lyophilize, cryopreservation.
The first step, DEAE Sephadex A-50 ion-exchange: obtain starting material Bombina maxima skin homogenate lyophilized powder according to the method described above, lyophilized powder is dissolved in 0.05M Tris-HCl, contain 10mM EDTA, the damping fluid of pH7.3 was loaded on the 10KDa dialysis tubing, in same damping fluid dialysis 12 hours; (500mm is long for DEAE SephadexA-50 (Pharmacia product) anion-exchange column, the 26mm diameter) through 0.05M Tris-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 corresponding activeconstituents peak IV by complete wash-out.
Second step, Sephadex G-75 gel-filtration: the activeconstituents peak IV freeze-drying that the first step obtains, be dissolved in pH7.8 Tris-HCl, 0.1M NaCl, contain 10mM edta buffer liquid, be splined on Sephadex G-75 (Pharmacia product) gel-filtration column (1000mm is long, the 26mm diameter), use same buffer solution elution, collect corresponding activeconstituents peak II.
The 3rd step, DEAE Sephadex A-50 ion-exchange: the activeconstituents peak II that gel-filtration obtains is loaded on the 10KDa dialysis tubing, in 0.05M, pH8.8 Tris-HCl contains in the 10mM edta buffer liquid dialysis 12 hours, last sample is in (300mm is long through same damping fluid equilibrated DEAE Sephadex A-50 (Pharmacia product) anion-exchange column, the 26mm diameter), carry out gradient elution with the damping fluid that contains NaCl again, collect activeconstituents peak I, promptly obtain the Bombina maxima proteinase inhibitor of purifying.
Use high-efficient liquid phase chromatogram HPLC then, the SDS-polyacrylamide gel electrophoresis method is identified its purity, molecular weight determination adopts the SDS-polyacrylamide gel electrophoresis method to measure, and isoelectric focusing electrophoresis is measured iso-electric point, measures the n terminal amino acid sequential structure with automatic Protein Sequencer.
PH8.8,10% polyacrylamide gel electrophoresis carries out purity test, and the result is electrophoresis one band.Reductibility and irreducibility SDS-polyacrylamide gel electrophoresis (10% concentration) show that this albumen is made up of 1 subunit, and it is 67KDa under the apparent molecular weight reductive condition on the SDS-polyacrylamide gel electrophoresis, is 45KDa under the non-reduced condition.
31 amino acid residue sequences of N-end of Bombina maxima protease inhibitors are: aspartic acid-serine-glutamic acid-threonine-His-Ile-Arg-Phe-Leu-glycine-glutaminic acid-valine-tyrosine-lysine-lysine-valine-aspartic acid-threonine-isoleucine-aspartic acid-phenylalanine-arginine-glycine-LEU-VAL-Leu-Ile-threonine-arginine-alanine-glutamine (DSETHIRFLGEVYKKVDTIDFRGLVLITRAQ).
Purifying Bombina maxima proteinase inhibitor when dosage reaches the 500mg/kg body weight, does not still show toxicity to small white mouse.
By the Bombina maxima proteinase inhibitor that aforesaid method obtains, treat the application of gastritis, pancreatitis medicine as preparation anti-tumor medicine and preparation.
Claims (3)
1, the Bombina maxima proteinase inhibitor, it is characterized in that from Chinese amphibian animal Bombina maxima skin, separating a kind of single chain serine protease inhibitor that obtains, it is 67KDa under the apparent molecular weight reductive condition on the polyacrylamide gel electrophoresis, under the non-reduced condition 45KDa, iso-electric point 4.8, sugared content 17-19%, non-enzymatic activity; 31 amino acid residue sequences of N-end of Bombina maxima proteinase inhibitor are: aspartic acid-Serine-L-glutamic acid-Threonine-Histidine-Isoleucine-arginine-Phe-Leu-glycine-glutaminic acid-Xie Ansuan-tyrosine-Methionin-Methionin-Xie Ansuan-aspartic acid-Threonine-Isoleucine-aspartic acid-phenylalanine-arginine-glycine-LEU-VAL-leucine-Isoleucine-Threonine-arginine-L-Ala-glutamine.
2, the preparation method of Bombina maxima proteinase inhibitor is characterized in that getting Bombina maxima skin, and after the homogenate, centrifugal removal precipitates in physiological saline, and after the lyophilize of collection supernatant liquor, through ion-exchange, the gel-filtration purifying can obtain; Wherein:
2.1 ion-exchange is: Bombina maxima skin homogenate lyophilized powder is dissolved in 0.05M Tris-HCl, contains 10mM EDTA, the damping fluid of pH 7.3 was loaded on the 10KDa dialysis tubing, in same damping fluid dialysis 12 hours; DEAE Sephadex A-50 anionresin column length 500mm, diameter are 26mm, 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 corresponding activeconstituents peak IV by complete wash-out;
2.2 gel-filtration is: with the activeconstituents peak IV freeze-drying that obtains, be dissolved in pH 7.8 Tris-HCl, 0.1M NaCl, contain 10mM edta buffer liquid, be splined on Sephadex G-75 gel-filtration column, gel-filtration column length 1000mm, diameter are the 26mm diameter, use same buffer solution elution, collect corresponding activeconstituents peak II;
2.3 ion-exchange is: the activeconstituents peak II that gel-filtration obtains is loaded on the 10KDa dialysis tubing, in 0.05M, pH 8.8 Tris-HCl contain in the 10mM edta buffer liquid dialysis 12 hours, last sample is in the same damping fluid equilibrated DEAE Sephadex A-50 anion-exchange column of warp, anionresin column length 300mm, diameter are 26mm, carry out gradient elution with the damping fluid that contains NaCl again, collect activeconstituents peak I, promptly obtain the Bombina maxima proteinase inhibitor of purifying.
3, the Bombina maxima proteinase inhibitor is controlled the application of gastritis, pancreatitis medicine as preparation anti-tumor medicine and preparation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001224166A CN1142180C (en) | 2000-07-29 | 2000-07-29 | Giant well bombinator proteinase inhibitor and its prepn. and pharmaceutical application |
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| CNB001224166A CN1142180C (en) | 2000-07-29 | 2000-07-29 | Giant well bombinator proteinase inhibitor and its prepn. and pharmaceutical application |
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| CN1142180C true CN1142180C (en) | 2004-03-17 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100581584C (en) * | 2006-05-30 | 2010-01-20 | 中国科学院昆明动物研究所 | Serine protease inhibitor of Rana grahami, and its application |
| CN100522992C (en) * | 2007-02-12 | 2009-08-05 | 中国科学院昆明动物研究所 | Novel ring-shape small-peptide BA and its use |
| CN101342194B (en) * | 2008-08-29 | 2010-10-20 | 陕西东泰制药有限公司 | Traditional Chinese medicine preparation for treating median or late-stage malignant tumors, chronic hepatitis B, and preparation method thereof |
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