CN103289981A - A preparation method for lyophilized powder of earthworm fibrinolytic enzymes - Google Patents
A preparation method for lyophilized powder of earthworm fibrinolytic enzymes Download PDFInfo
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- CN103289981A CN103289981A CN201310254843XA CN201310254843A CN103289981A CN 103289981 A CN103289981 A CN 103289981A CN 201310254843X A CN201310254843X A CN 201310254843XA CN 201310254843 A CN201310254843 A CN 201310254843A CN 103289981 A CN103289981 A CN 103289981A
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Abstract
The present invention discloses a preparation method for lyophilized powder of earthworm fibrinolytic enzymes. The preparation method comprises the steps of the preparation of earthworm meal, the preparation of a crude enzyme solution, the purification of crude enzymes, the modification of the earthworm fibrinolytic enzymes, and the preparation of lyophilized powder of the earthworm fibrinolytic enzymes. According to the preparation method for lyophilized powder of earthworm fibrinolytic enzymes of the present invention, on the basis of isolated and purified pure enzymes, activated polyethylene glycol is employed for modifying the earthworm fibrinolytic enzymes, so that the earthworm fibrinolytic enzymes have no direct contact with body fluids and the free amino groups are cleared, thus extending the half-life and eliminating immunogenicity of the earthworm fibrinolytic enzymes in the body.
Description
Technical field
The present invention relates to the biological medicine technology field, relate in particular to a kind of preparation method of earthworm fibrinolysin lyophilized powder.
Background technology
Thrombus disease is the common frdquently encountered disease of serious harm human health, the mortality ratio height, the disability rate height. after thrombus takes place, the most effective methods for the treatment of is exactly thrombolysis, in recent years, (earthworm fibrinolytic enzymes EFEs) is bringing into play important effect clinically for the thrombolysis medicine of main active ingredient, as hundred earthworm fibrinolysin capsules difficult to understand, the multiple capsule of general grace etc. with earthworm fibrinolysin.(earthworm fibrinolytic enzyme EFE) after Japanese Miyazaki medical university report in 1984 extracts, gos deep into research widely China, Japan and Korea S scholar to earthworm fibrinolysin from earthworm.Present proof, earthworm fibrinolysin (EFE) had both had the plasmin activity of direct dissolving fibrin, had the Profibrinolysin activation of similar urokinase again, so claim again earthworm fibrinolysin (lumbrckinase, LK).Simultaneously, have anti-cancer, antitumous effect.
Though differences such as difference is next former, raising bait, extraction solvent, means of purification, EFE has certain difference in physico-chemical property, can be summarized as; A, be one group and have the Ser proteolytic ferment in the earthworm leather bag, belong to the Trypsin enzyme.Have fibrinolytic or plasmin activity; B, relative molecular mass are less, about 28800D; C, pI value are p 3-5; Acidic amino acid is more in the zymoprotein, and basic aminoacids is less; D, stablize, stable at 0～60 ℃, pH4～11 scope inner enzyme vigors, non-inactivation in two years under the room temperature; E, mostly be monomer list composition enzyme, oligomer is also arranged, what have contains glycosyl; F, to ser inhibitor sensitivity, Ca
2+Has the activity effect; G, EFE are substrate divided by protein, can also BAEE be substrate, but ATEE is not had effect.
Pharmacodynamic study is seen in domestic more.Studies show that EFE is the thrombolytic drug of a kind of very advantageous and potentiality, based on the direct hydrolysis fibrin, plasminogen activation is auxilliary, to rabbit and mouse intravenous injection, thrombus weight obviously alleviates, thrombosis inhibiting rate 52%, instillation can improve total plasminogen activator vigor rapidly, significantly improve the cerebrum ischemia state, reduce blood viscosity, energy hydrolytically condensable hemase, reduce thrombogen, VIII, the tool anticoagulation.
EFE has antitumor action, studies show that: a, EFE energy enhancing body cell and humoral immune function; B, EFE can put forward CAT, GSH-Px, SOD activity, thereby improve the activities of antioxidant enzymes of body lipid peroxidation protection system; Some structural protein in c, the hydrolysis tumour cell destroy tumour cell, suppress its growth; D, induced tumor apoptosis, thus realize its antitumous effect.
At present, to the research of earthworm fibrinolysin, great majority still stay in the research to its zymologic property, and its applied research is seemed weak relatively, even but also there is bigger dispute in zymologic property.Though it is clinical to consider that simultaneously medicines such as at present commercially available fiber eliminating enzyme, Ahylysantinfarctase, urokinase have been applied to, poor stability, easy inactivation, residence time weak point in the body, expensive reaching easily causes clinical defectives such as hemorrhage, generally do not applied.Desirable thrombolytic drug requires rapidly thrombus, nontoxic, no side effect such as hemorrhage, convenient drug administration, low price.Seeking effective medicine is one of focus of domestic and international medical circle emphasis tackling key problem.In view of earthworm fibrinolysin energy thrombus, have no side effect (road quintessence, 1986), Orally-administrable, low production cost, people expect earthworm fibrinolysin to be developed to a kind of novel thrombolysis medicament.Current, commercially available thrombolysis capsule, earthworm capsule, " 9.12 " capsule etc. are the earthworm lyophilized powder just, is subject to enzyme, inhibitor etc. in vivo and decomposes destruction, also is difficult for entering focus cell and tissue, obviously remains further to be researched and solved.
Therefore, develop a kind of preparation method of new earthworm fibrinolysin lyophilized powder, will extensively benefit the patient and possess good market outlook.
Summary of the invention
In view of this, technical problem to be solved by this invention is to provide a kind of preparation method of earthworm fibrinolysin lyophilized powder, obtain in separation and purification on the basis of pure enzyme, adopt activated polyethylene glycol (PEG) to modify earthworm fibrinolysin, make it directly not contact with body fluid, remove its free amine group, prolonged the earthworm fibrinolysin transformation period in vivo, eliminated its immunogenicity; And, utilize modifier characteristics that are not only hydrophilic but also lipophilic to make enzyme be easy to enter cell, be convenient to absorb, improve drug effect, to compare with the existing earthworm fibrinolysin product in market, the product that adopts the inventive method to prepare is formed stable, and drug effect is better, can reduce administration number of times.To be expected to substitute earthworm fibrinolysin product of former generation with the product of the inventive method preparation product as the medicine source, have vast market prospect.
The present invention solves by following scheme and solves above-mentioned technical problem:
A kind of preparation of earthworm fibrinolysin lyophilized powder comprises the steps:
The preparation of step 1, earthworm meal:
The earthworm that will live, hungry one day, tell clean silt naturally, washing, then in temperature between 20-35 ℃ ventilation condition drying and make pulverizing, make the earthworm meal by the 100-200 mesh sieve;
The preparation of step 2, thick enzyme solution:
Get the earthworm meal with the 0.1mol/L of 2 times of volumes, pH is 7.8 phosphoric acid buffer PBS homogenate, adds 0.1mol/LNaCl solution, places in 4 ℃ of refrigerators and spends the night, and then with 10000r/min, 4 ℃ of centrifugal 15min abandon precipitation, get supernatant; Supernatant liquor is in 50 ℃ of water bath heat preservation 40min, the flowing water cooling, and 8000r/min again, 4 ℃ of centrifugal 10min get supernatant, add ammonium sulfate and reach 30% saturation ratio in supernatant liquor, leave standstill 8000r/min again, 4 ℃ of centrifugal 10min 3 hours; Collect supernatant liquor and add ammonium sulfate again and reach 70% saturation ratio, left standstill 3 hours, 8000r/min again, 4 ℃ of centrifugal 10min, collecting precipitation; Precipitation is dissolved in the above-mentioned damping fluid, stays the dialysis tubing dialysed overnight of 5KD molecular weight to desalt to above-mentioned damping fluid to carry, again with dialyzate 8000r/min, 4 ℃ of centrifugal 10min get supernatant liquor and are the thick enzyme solution of earthworm fibrinolysin;
The purifying of step 3, thick enzyme:
The thick enzyme solution of earthworm fibrinolysin is crossed Sephadex G-75 gel chromatography, use 0.02mol/L before the upper prop in advance, pH is 7.8 phosphoric acid buffer balance, extremely do not have photoabsorption at the 280nm wavelength with above-mentioned buffer solution elution behind the upper prop, 2 protein peaks appear in elutriant, and wherein enzymic activity peak and the 1st protein peak are overlapping, collect the enzyme liquid of lap, cross DEAE-Cellulose A-52 ion exchange column, crossing and using earlier 0.05mol/L, pH before the post is 7.8 PBS balance; With the PBS ionic strength linear gradient elution that contains 0-0.5mol/L NaCl, 3 protein peaks appear in elutriant then, enzymic activity peak and protein peak the 3rd peak overlapping, and all the other 2 basic non-enzymatic activities of protein peak are collected active part and are the pure enzyme of earthworm fibrinolysin;
The modification of step 4, earthworm fibrinolysin:
Add the PEG of the pure enzyme of earthworm fibrinolysin and activation to 15mL, pH9.2 is in the borate buffer of 0.1mol/L, the weight ratio of pure enzyme and PEG is 1:50, under 30 ℃, insulation reaction 1 hour, reactant is to 0.1mol/L, the phosphoric acid buffer dialysed overnight of pH7.3, dialyzate is again with 10000r/min, and 4 ℃ of centrifugal l0min get supernatant liquor and cross post Sephadex G-75, collect activeconstituents, be the earthworm fibrinolysin modifying enzyme;
The preparation of step 5, earthworm fibrinolysin lyophilized powder:
The earthworm fibrinolysin modifying enzyme is contained in the sample disc, expects the thick 8mm that is no more than, in-35 ℃ freezing 12 hours, under the vacuum tightness of 5pa, be warming up to 30 ℃ then, dry 12 hours; Continue to be warming up to 40 ℃, vacuum-drying 14 hours obtains the lyophilized powder of earthworm fibrinolysin modifying enzyme, and its water content is 7.0%, and protein content is 68%, and enzyme activity is 1700U/mg.
Preferably, in the described step 1, the earthworm that lives tells clean silt naturally, and the process of washing is specially ducks in drink the work earthworm one day, changes water one time in per 12 hours, changes 0.1mol/LNaCl salt solution Rapid Cleaning earthworm again, cleans with purified water at last.
Preferably, also need in the described step 4 in advance PEG to be activated: with Cynuric Chloride 4.4g, anhydrous sodium carbonate 8g, the PEG35g of molecular sieve 5A and molecular-weight average 4000 joins in the 400ml dry-out benzene, stir after 15 hours under the normal temperature, filter, filtrate is the centrifugal anhydrous sodium carbonate of removing again, molecular sieve 5A suspended substance; Liquid is used the dry-out benzene dissolution precipitation of 400mL again with the ether sedimentation of 600mL, and so precipitation, dissolving repeated multiple times are removed responseless cyanuric chloride, and until detect no photoabsorption under the 258nm wavelength, vacuum-drying gets white powder, is the PEG of activation.
Than prior art, the technical scheme of the embodiment of the invention has following beneficial effect at least:
In the technical scheme of the present invention, obtain in separation and purification on the basis of pure enzyme, adopt activated polyethylene glycol (PEG) to modify earthworm fibrinolysin, make it directly not contact with body fluid, remove its free amine group, prolonged the earthworm fibrinolysin transformation period in vivo, eliminated its immunogenicity; And, utilize modifier characteristics that are not only hydrophilic but also lipophilic to make enzyme be easy to enter cell, be convenient to absorb, improve drug effect, to compare with the existing earthworm fibrinolysin product in market, the product that adopts the inventive method to prepare is formed stable, and drug effect is better, can reduce administration number of times.To be expected to substitute earthworm fibrinolysin product of former generation with the product of the inventive method preparation product as the medicine source, have vast market prospect.
Particularly, adopt the earthworm fibrinolysin lyophilized powder product of the inventive method preparation to compare with this based article of the prior art and possesses following advantage: (1) prolongs the transformation period in vivo, and the destruction of acceptor endoproteinase etc. is not subjected to the inhibition of inhibitor yet; (2) immunogenicity in the no body; (3) its amido modified rate reaches more than 70%, is easy to enter human body and carefully organizes, EFE-PEG vigor 7000uU/ML, albumen 1.5mg/ML; (4) beavy metal impurity in the former earthworm fibrinolysin of removal.
Easily by other proteolysis, the free amine group on the enzyme molecule can cause allergic reaction not modified enzyme in vivo, because enzyme is biomacromolecule, also is difficult for entering cell, and curative effect is not high like this, also can cause other incompatible reactions.We once modify earthworm fibrinolysin with PEG-4000, and the zymologic property of EFE-PEG has had partly change than protoenzyme (EFE), changes (table 1) towards useful aspect.From table 1 as seen, EEF-PEG in vivo the transformation period improved 1.62 times, it has also improved 4.1 times to substrate avidity.Experiment is proof also, and with EFF, EFE-PEG respectively behind the quiet notes pig, the former fervescence is not taken food; Latter's physiology, life are acted normally.
The part character of table 1.EFE and EFE-PEG relatively
| ? | EFE | EFE-PEG |
| Action pH | pH5.0-10.0 | pH5.0-10.6 |
| Optimal pH | pH7.8 | pH10.0 |
| Thermostability | 20-55℃ | 20-60℃ |
| Optimum temperuture | 55℃ | 55℃ |
| Transformation period | 240d under 4 ℃ and the normal temperature | 280d under 4 ℃ and the normal temperature |
| The distribution phase transformation period in the body | 0.60h | 0.97h |
| Km/Km app（BAEE） | 1.24×10 -3mol/L | 0.3×10 -3mol/L |
Analyze the good biocompatibility of PEG from principle, tool wetting ability lipophilic amphoteric properties, advantage such as nontoxic, as to have no side effect.Adopt the earthworm fibrinolysin lyophilized powder of method preparation of the present invention, PEG can form a tectum at the enzyme molecular surface, and enzyme is not directly contacted with body fluid, overcome immunogenicity, prolonged its transformation period in vivo, and by the easier cell that enters of its amphoteric properties, the curative effect performance fully.Adopting the vigor of the earthworm fibrinolysin modifying enzyme of the inventive method preparation is without 85～90% of modifying enzyme, and free amine group modification rate reaches 70%, Km (BAEE) and descended 4 times than protoenzyme, and illustrating has increased by 4 times to substrate avidity.Ph stability and the thermostability of EFE and PEG-EFE do not have larger difference, deposit 16 months under 4 ℃ and room temperature, and vigor does not change, and their absorption spectrum does not have considerable change yet.With EFE and the quiet notes pig of PEG-EFE difference, pharmacokinetics proves that the distribution phase transformation period has been improved 1.4 times than EFE in the PEG-EFE body.And not modified EFE control group pig performance is not taken food and is waited severe allergic reaction, and PEG-EFE group pig is taken food as usual, no symptoms of allergic.
Description of drawings
Fig. 1 is the post curve excessively of Sephadex G-75 gel chromatography in the embodiment of the invention step 3.
Fig. 2 is the post curve excessively of DEAE-Cellulose A-52 ion exchange column in the embodiment of the invention step 3.
Embodiment
Further specify principle of the present invention below by embodiment, other aspects of the present invention, feature and advantage thereof will become very clear by this detailed description.
The preparation of embodiment 1 earthworm fibrinolysin lyophilized powder:
The preparation of step 1, earthworm meal:
The earthworm that will live, hungry one day, tell clean silt naturally, washing, then in temperature between 20-35 ℃ ventilation condition drying and make pulverizing, make the earthworm meal by the 100-200 mesh sieve.
Earthworm fibrinolysin mainly exist with the earthworm digestive tube in, therefore, for guaranteeing the integrity of earthworm effect, add and should not remove internal organ man-hour.And must grasp xerophilous condition, if temperature surpasses 70 ℃, easily make the sex change of activeconstituents inactivation; If temperature is low excessively, time of drying is oversize, microbial contamination and go bad easily, and earthworm itself also has autolysis 35～55 ℃ of temperature simultaneously, therefore, when specific implementation, selects 20-35 ℃ ventilation condition drying and makes pulverizing.Wherein, in this implementation step one, the earthworm that lives tells clean silt naturally, and the process of washing is specially ducks in drink the work earthworm one day, changes water one time in per 12 hours, changes 0.1mol/LNaCl salt solution Rapid Cleaning earthworm again, cleans with purified water at last.
The preparation of step 2, thick enzyme solution:
Get the earthworm meal with the 0.1mol/L of 2 times of volumes, pH is 7.8 phosphoric acid buffer PBS homogenate, adds 0.1mol/LNaCl solution, places in 4 ℃ of refrigerators and spends the night, and then with 10000r/min, 4 ℃ of centrifugal 15min abandon precipitation, get supernatant; Supernatant liquor is in 50 ℃ of water bath heat preservation 40min, the flowing water cooling, and 8000r/min again, 4 ℃ of centrifugal 10min get supernatant, add ammonium sulfate and reach 30% saturation ratio in supernatant liquor, leave standstill 8000r/min again, 4 ℃ of centrifugal 10min 3 hours; Collect supernatant liquor and add ammonium sulfate again and reach 70% saturation ratio, left standstill 3 hours, 8000r/min again, 4 ℃ of centrifugal 10min, collecting precipitation; Precipitation is dissolved in the above-mentioned damping fluid, stays the dialysis tubing dialysed overnight of 5KD molecular weight to desalt to above-mentioned damping fluid to carry, again with dialyzate 8000r/min, 4 ℃ of centrifugal 10min get supernatant liquor and are the thick enzyme solution of earthworm fibrinolysin;
The purifying of step 3, thick enzyme:
The thick enzyme solution of earthworm fibrinolysin is crossed Sephadex G-75 gel chromatography, use 0.02mol/L before the upper prop in advance, pH is 7.8 phosphoric acid buffer balance, extremely do not have photoabsorption at the 280nm wavelength with above-mentioned buffer solution elution behind the upper prop, 2 protein peaks appear in elutriant, and wherein enzymic activity peak and the 1st protein peak are overlapping, collect the enzyme liquid of lap, cross DEAE-Cellulose A-52 ion exchange column, crossing and using earlier 0.05mol/L, pH before the post is 7.8 PBS balance; With the PBS ionic strength linear gradient elution that contains 0-0.5mol/L NaCl, 3 protein peaks appear in elutriant then, enzymic activity peak and protein peak the 3rd peak overlapping, and all the other 2 basic non-enzymatic activities of protein peak are collected active part and are the pure enzyme of earthworm fibrinolysin.Cross the post experimental data as illustrated in fig. 1 and 2.
Be the measuring method of substrates enzymes vigor with the casein:
Take by weighing the 1g casein, add 100 milliliters of 0.05mol/L, the pH7.8Tris-HCl damping fluid, little fiery heating for dissolving makes substrate solution be 1% concentration.Draw 2 milliliters of enzyme solution and in 37 ℃ of water-baths, be incubated 10 minutes, add 2 milliliters then rapidly at the substrate solution of 37 ℃ of pre-incubations, shake up to wait and continue insulation 15 minutes, add 2 milliliter of 10% trichloroacetic acid solution stopped reaction at last.Room temperature leaves standstill 30 minutes after-filtration, measures the optical density value of filtrate under 280nm.
Determining the protein quantity method (adopting the Xylene Brilliant Cyanine G method, is standard protein with the bovine serum albumin)
Measure earthworm fibrinolysin content and enzyme activity respectively, numerical value is following table 2
Table 2 earthworm fibrinolysin content and enzyme activity determination result
The modification of step 4, earthworm fibrinolysin:
The PEG activation: with Cynuric Chloride 4.4g, anhydrous sodium carbonate 8g, the PEG35g of molecular sieve 5A and molecular-weight average 4000 joins in the 400ml dry-out benzene, stirs under the normal temperature after 15 hours, filters, and filtrate is the centrifugal anhydrous sodium carbonate of removing again, molecular sieve 5A suspended substance; Liquid is used the dry-out benzene dissolution precipitation of 400mL again with the ether sedimentation of 600mL, and so precipitation, dissolving repeated multiple times are removed responseless cyanuric chloride, and until detect no photoabsorption under the 258nm wavelength, vacuum-drying gets white powder, is the PEG of activation.
Add the PEG of the pure enzyme of earthworm fibrinolysin and activation to 15mL, pH9.2 is in the borate buffer of 0.1mol/L, the weight ratio of pure enzyme and PEG is 1:50, under 30 ℃, insulation reaction 1 hour, reactant is to 0.1mol/L, the phosphoric acid buffer dialysed overnight of pH7.3, dialyzate is again with 10000r/min, and 4 ℃ of centrifugal l0min get supernatant liquor and cross post Sephadex G-75, collect activeconstituents, be the earthworm fibrinolysin modifying enzyme.
Easily by other proteolysis, the free amine group on the enzyme molecule can cause allergic reaction not modified enzyme in vivo, because enzyme is biomacromolecule, also is difficult for entering cell, and curative effect is not high like this, also can cause other incompatible reactions.We once modify earthworm fibrinolysin with PEG-4000, and the zymologic property of EFE-PEG has had partly change than protoenzyme (EFE), changes (table 1) towards useful aspect.From table 1 as seen, EEF-PEG in vivo the transformation period improved 1.62 times, it has also improved 4.1 times to substrate avidity.Experiment is proof also, and with EFF, EFE-PEG respectively behind the quiet notes pig, the former fervescence is not taken food; Latter's physiology, life are acted normally.
The part character of table 1.EFE and EFE-PEG relatively
| ? | EFE | EFE-PEG |
| Action pH | pH5.0-10.0 | pH5.0-10.6 |
| Optimal pH | pH7.8 | pH10.0 |
| Thermostability | 20-55℃ | 20-60℃ |
| Optimum temperuture | 55℃ | 55℃ |
| Transformation period | 240d under 4 ℃ and the normal temperature | 280d under 4 ℃ and the normal temperature |
| The distribution phase transformation period in the body | 0.60h | 0.97h |
| Km/Km app（BAEE） | 1.24×10 -3mol/L | 0.3×10 -3mol/L |
The preparation of step 5, earthworm fibrinolysin lyophilized powder:
The earthworm fibrinolysin modifying enzyme is contained in the sample disc, expects the thick 8mm that is no more than, in-35 ℃ freezing 12 hours, under the vacuum tightness of 5pa, be warming up to 30 ℃ then, dry 12 hours; Continue to be warming up to 40 ℃, vacuum-drying 14 hours obtains the lyophilized powder of earthworm fibrinolysin modifying enzyme, and its water content is 7.0%, and protein content is 68%, and enzyme activity is 1700U/mg.
[sample quality check]
By above prescription and technology this product has been carried out three batches of little trial productions (preparing 500 bottles for every batch), three batch sample quality are all qualified, and assay sees Table 3.
The assay of table 3 sample
The quality of three batches of products all meets Chinese Pharmacopoeia regulation (hereinafter to be referred as up to specification), illustrates that the prescription of this product and technology are feasible.
[influence factor test]
Get for 1 batch of test agent, carry out the influence factor test, respectively at placing 10 days under illumination (4500Lx), high temperature (60 ℃), high humidity (RH92.5%) condition, and respectively at 5 days, the 10 days every detection indexs of sampling investigation, and with 0 day detected result relatively, check clarity, protein, other material, the mensuration content of appearance luster, acidity, moisture, solution, the results are shown in Table 4.
Table 4 influence factor test-results
Above-mentioned test-results shows that this product is all more stable under illumination (4500Lx), high temperature (60 ℃), high humidity (RH92.5%) condition.This presentation of results the feasibility of this product technology.
The method that above-described embodiment discloses obtains on the basis of pure enzyme in separation and purification, adopt activated polyethylene glycol (PEG) to modify earthworm fibrinolysin, make it directly not contact with body fluid, remove its free amine group, prolong the earthworm fibrinolysin transformation period in vivo, eliminated its immunogenicity; And, utilize modifier characteristics that are not only hydrophilic but also lipophilic to make enzyme be easy to enter cell, be convenient to absorb, improve drug effect, to compare with the existing earthworm fibrinolysin product in market, the product that adopts the inventive method to prepare is formed stable, and drug effect is better, can reduce administration number of times.To be expected to substitute earthworm fibrinolysin product of former generation with the product of the inventive method preparation product as the medicine source, have vast market prospect.
More than be preferred implementation of the present invention, should be pointed out that for those skilled in the art that under the prerequisite that does not break away from the principle of the invention, can also make some improvements and modifications, these improvements and modifications also are considered as protection scope of the present invention.
Claims (3)
1. the preparation method of an earthworm fibrinolysin lyophilized powder is characterized in that, comprises the steps:
The preparation of step 1, earthworm meal:
The earthworm that will live, hungry one day, tell clean silt naturally, washing, then in temperature between 20-35 ℃ ventilation condition drying and make pulverizing, make the earthworm meal by the 100-200 mesh sieve;
The preparation of step 2, thick enzyme solution:
Get the earthworm meal with the 0.1mol/L of 2 times of volumes, pH is 7.8 phosphoric acid buffer PBS homogenate, adds 0.1mol/LNaCl solution, places in 4 ℃ of refrigerators and spends the night, and then with 10000r/min, 4 ℃ of centrifugal 15min abandon precipitation, get supernatant; Supernatant liquor is in 50 ℃ of water bath heat preservation 40min, the flowing water cooling, and 8000r/min again, 4 ℃ of centrifugal 10min get supernatant, add ammonium sulfate and reach 30% saturation ratio in supernatant liquor, leave standstill 8000r/min again, 4 ℃ of centrifugal 10min 3 hours; Collect supernatant liquor and add ammonium sulfate again and reach 70% saturation ratio, left standstill 3 hours, 8000r/min again, 4 ℃ of centrifugal 10min, collecting precipitation; Precipitation is dissolved in the above-mentioned damping fluid, stays the dialysis tubing dialysed overnight of 5KD molecular weight to desalt to above-mentioned damping fluid to carry, again with dialyzate 8000r/min, 4 ℃ of centrifugal 10min get supernatant liquor and are the thick enzyme solution of earthworm fibrinolysin;
The purifying of step 3, thick enzyme:
The thick enzyme solution of earthworm fibrinolysin is crossed Sephadex G-75 gel chromatography, use 0.02mol/L before the upper prop in advance, pH is 7.8 phosphoric acid buffer balance, extremely do not have photoabsorption at the 280nm wavelength with above-mentioned buffer solution elution behind the upper prop, 2 protein peaks appear in elutriant, and wherein enzymic activity peak and the 1st protein peak are overlapping, collect the enzyme liquid of lap, cross DEAE-Cellulose A-52 ion exchange column, crossing and using earlier 0.05mol/L, pH before the post is 7.8 PBS balance; With the PBS ionic strength linear gradient elution that contains 0-0.5mol/L NaCl, 3 protein peaks appear in elutriant then, enzymic activity peak and protein peak the 3rd peak overlapping, and all the other 2 basic non-enzymatic activities of protein peak are collected active part and are the pure enzyme of earthworm fibrinolysin;
The modification of step 4, earthworm fibrinolysin:
Add the PEG of the pure enzyme of earthworm fibrinolysin and activation to 15mL, pH9.2 is in the borate buffer of 0.1mol/L, the weight ratio of pure enzyme and PEG is 1:50, under 30 ℃, insulation reaction 1 hour, reactant is to 0.1mol/L, the phosphoric acid buffer dialysed overnight of pH7.3, dialyzate is again with 10000r/min, and 4 ℃ of centrifugal l0min get supernatant liquor and cross post Sephadex G-75, collect activeconstituents, be the earthworm fibrinolysin modifying enzyme;
The preparation of step 5, earthworm fibrinolysin lyophilized powder:
The earthworm fibrinolysin modifying enzyme is contained in the sample disc, expects the thick 8mm that is no more than, in-35 ℃ freezing 12 hours, under the vacuum tightness of 5pa, be warming up to 30 ℃ then, dry 12 hours; Continue to be warming up to 40 ℃, vacuum-drying 14 hours obtains the lyophilized powder of earthworm fibrinolysin modifying enzyme, and its water content is 7.0%, and protein content is 68%, and enzyme activity is 1700U/mg.
2. the preparation method of earthworm fibrinolysin lyophilized powder according to claim 1, it is characterized in that, in the described step 1, the earthworm that lives tells clean silt naturally, the process of washing is specially ducks in drink the work earthworm one day, changed water one time in per 12 hours, and changed 0.1mol/LNaCl salt solution Rapid Cleaning earthworm again, clean with purified water at last.
3. the preparation method of earthworm fibrinolysin lyophilized powder according to claim 1, it is characterized in that, also need in the described step 4 in advance PEG to be activated: with Cynuric Chloride 4.4g, anhydrous sodium carbonate 8g, the PEG35g of molecular sieve 5A and molecular-weight average 4000 joins in the 400ml dry-out benzene, stirs under the normal temperature after 15 hours, filters, filtrate is the centrifugal anhydrous sodium carbonate of removing again, molecular sieve 5A suspended substance; Liquid is used the dry-out benzene dissolution precipitation of 400mL again with the ether sedimentation of 600mL, and so precipitation, dissolving repeated multiple times are removed responseless cyanuric chloride, and until detect no photoabsorption under the 258nm wavelength, vacuum-drying gets white powder, is the PEG of activation.
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| CN105200029A (en) * | 2015-10-27 | 2015-12-30 | 广西大学 | Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof |
| CN106222151A (en) * | 2016-08-31 | 2016-12-14 | 武汉真福医药股份有限公司 | A kind of recombination bacillus subtilis fibrinolysin and the preparation method of enteric coated capsule thereof |
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| CN115736251A (en) * | 2022-11-30 | 2023-03-07 | 山东神乐生物科技有限公司 | Preparation method and application of earthworm freeze-dried powder |
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| CN105012381A (en) * | 2015-08-07 | 2015-11-04 | 高努 | Lipid lowering and thrombolysis combination and preparation method of food and medicine capsule thereof |
| CN105200030A (en) * | 2015-10-27 | 2015-12-30 | 广西大学 | Preparation method of magnetic microspheres for immobilizing earthworm fibrinolytic enzyme |
| CN105200031A (en) * | 2015-10-27 | 2015-12-30 | 广西大学 | Magnetic microsphere for immobilizing earthworm fibrinolytic enzyme and preparation method thereof |
| CN105200029A (en) * | 2015-10-27 | 2015-12-30 | 广西大学 | Magnetic microsphere for earthworm fibrinolytic enzyme immobilization and preparation method thereof |
| CN105200031B (en) * | 2015-10-27 | 2018-04-06 | 广西大学 | A kind of magnetic microsphere of immobilization earthworm fibrinolysin and preparation method thereof |
| CN105200029B (en) * | 2015-10-27 | 2018-10-16 | 广西大学 | A kind of magnetic microsphere and preparation method thereof for earthworm fibrinolysin immobilization |
| CN105200030B (en) * | 2015-10-27 | 2019-03-12 | 广西大学 | A kind of preparation method of the magnetic microsphere of immobilization earthworm fibrinolysin |
| CN106222151A (en) * | 2016-08-31 | 2016-12-14 | 武汉真福医药股份有限公司 | A kind of recombination bacillus subtilis fibrinolysin and the preparation method of enteric coated capsule thereof |
| CN109260231A (en) * | 2017-07-18 | 2019-01-25 | 首都儿科研究所 | The preparation method of the anti-inflammatory antimicrobial extract of cough-relieving apophlegmatic in a kind of earthworm |
| CN109260231B (en) * | 2017-07-18 | 2021-09-03 | 首都儿科研究所 | Preparation method of earthworm extract with cough stopping, phlegm eliminating, anti-inflammatory and antimicrobial functions |
| CN115736251A (en) * | 2022-11-30 | 2023-03-07 | 山东神乐生物科技有限公司 | Preparation method and application of earthworm freeze-dried powder |
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