CN108395475A - A kind of hirudin isolation and purification method based on affinity chromatography - Google Patents
A kind of hirudin isolation and purification method based on affinity chromatography Download PDFInfo
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Abstract
The invention discloses a kind of hirudin isolation and purification method based on affinity chromatography, after physiological saline fed to appetite leech live body, physical impact makes it salivate and collect, by the leech saliva sample of acquisition, precooling trichloroacetic acid extracting solution mixing is added, it extracts, centrifuging and taking supernatant, the acetone mixing that precooling is added after adjusting pH3.0 5.0 is stood overnight, recycle acetone in supernatant, centrifugation, takes precipitation, spare；Distilled water is added in the precipitation, fully dissolves, adjusts pH 6.0 8.0, it is isolated and purified with fibrin ferment Sepharose 4FF affinity columns, high performance liquid chromatography detection leech cellulose content, fetch water leech cellulose content peak value at refined solution, above-mentioned refined solution is dialysed, and it repeats to add water 24 times in the process, continue 10 15h, the supernatant after ultrafiltration is subjected to rotary evaporation, after removing moisture removal 70 80%, it takes concentrate freeze dryer to be lyophilized, obtains hirudin product.The natural hirudin purity of the method for the present invention purifying gained is high, and activity is high, at low cost, easy to operate, easily repeats.
Description
Technical field
The present invention relates to a kind of methods isolating and purifying hirudin, belong to biomedicine and isolate and purify field.
Background technology
The study found that having containing various bioactivators such as hirudins and in medicine in leech salivary gland secretion
There is broad prospect of application.Hirudin is a kind of Acid polypeptide of leeches animal saliva glandular secretion, residual containing 65~66 amino acid
Base mainly has this 3 kinds of isomers of HV1, HV2, HV3, has very high structural homology between them.Hirudin is to find so far
The strongest thrombin inhibitor of effect, hirudin (HV3) protein expression engineering bacteria bacterial activity depend on anticoagulation
Enzyme activity, the stable compound that it is combined closely with equimolar than forming non-covalent bond with fibrin ferment.1984,
Haycraft has found that hirudo extract has blood coagulation resisting function.The isolated this work from Hementaria officianalis for the first time of Jocoby in 1904
Property component, and it is named as hirudin.Nineteen fifty-five, Markwardt successfully isolate and purify out sterling leech from Hementaria officianalis head
Element proceeds by the biochemistry and pharmaceutical research on basis, and in 1970, determining hirudin was that the specificity of fibrin ferment inhibits
Agent.1986, Harvey etc. obtained the cDNA of hirudin, and subsequent hirudin gene is expressed successfully in a variety of hosts.
Hirudin is a kind of highly stable protein, extremely stable in the dry state, and simple temperature increases (100
DEG C water-bath) or the change of pH value (l.47-12.9) do not affect its vigor, in the presence of certain denaturants also very
Stablize, only when temperature and pH value raise simultaneously, its vigor is just begun to decline.Generally hirudin can be stablized in water at room temperature
There are heat 15min at 6 months, 80 DEG C not to be destroyed.The pH value of solution is improved, stability then reduces.At 20 DEG C,
In 0.lmol/LHCI or 0.1mol/LNaOH, 15min can be stablized, through 80 DEG C of processing 15min, that is, complete under conditions of pH=13
Inactivation.
The Markwardt of nineteen fifty-five Germany isolates purer hirudin in career in medicine leech for the first time, the hereafter purifying of hirudin
It is gradually improved with standardized technique.Have many related research reports and patent so far, overwhelming majority work is to utilize Europe
The even entire body in head for curing leech does raw material, through ethanol dehydration, then adds the series of steps such as acetone precipitation and obtains leech
Plain semifinished product.Markwardt in 1970 is using ethanol precipitation, cation exchange, gel filtration and anion exchange
And etc. obtain hirudin sterling.WdsmaM and Marhyardt improves purification process within 1985, using ion exchange and parent
Close the hirudin that chromatography was combined and obtained high-purity.Johmnes in 1986 et al. reports a kind of five step method of purification,
In used high performance liquid chromatography new technology.
The method of extraction hirudin at present is carried with water, ethyl alcohol, ultrafiltration or chromatography after rubbing by leech or is dry
It takes.But the above method has many shortcomings, active ingredient is not high after mainly extracting, activity generally 100 ~
4000ATU/g or so, yield differ between 0.27-4.47%, and according to different extracts activity height, and impurity content is high,
Be not suitable for injecting drug use, some extracting methods are complicated, and some procedures are slower, are not suitable for industrial production, so at present
The extracting method of hirudin also has many people studying, it is desirable to find extracting method efficient, at low cost, that active constituent is high.
Invention content
In view of the above-mentioned problems, the present invention provides a kind of method isolating and purifying hirudin, this method is easy to operate, efficiency
Height, while the hirudin of high-purity, high activity can be obtained.
In order to achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of hirudin isolation and purification method based on affinity chromatography, includes the following steps：
（1）Hirudin coarse extraction：After physiological saline fed to appetite leech live body, physical impact makes it salivate and collect, and will obtain
The leech saliva sample obtained is added 2-4 times（V:V）Trichloroacetic acid extracting solution mixing, extraction 2-6 h, 8000-10000 r/ is pre-chilled
Min centrifuges 10-20 min, and supernatant, the acetone mixings that 4 times of precoolings are added after HCL adjustments PH3.0-5.0 is taken to stand overnight, recycle
Acetone in supernatant, 8000-10000 r/min centrifuge 10-20 min, take precipitation, spare；
（2）It isolates and purifies：4-6 times of distilled water is added in above-mentioned precipitation, is fully dissolved, HCL adjusts PH 6.0-8.0, uses fibrin ferment
Sepharose 4FF affinity columns are isolated and purified, elution, high performance liquid chromatography detection leech cellulose content, leech cellulose content of fetching water
Refined solution at peak value, it is spare；
（3）Desalination and concentration：Above-mentioned refined solution is dialysed, and repeats to add water 2-4 times in the process, continues 10-15 h, will surpass
Supernatant after filter carries out roto-vap operation（30-60 DEG C, 0-180 r/min）, after removing moisture removal 70-80 %, take concentrate
It is spare；
（4）It takes concentrate freeze dryer to be lyophilized, collects product and be sealed up to hirudin product.
Hirudin activity detects：Markwardt fibrin ferment direct titrimetric methods detect its anticoagulant active.
Content in crude product detects：It salivates plus 2 times of distilled water, vibrations mixing, HPLC detects leech cellulose content.
The step（2）Support activator is carbonyl dimidazoles, the load in middle fibrin ferment Sepharose 4FF affinity columns
Body activating solvent includes but not limited to DMSO, dioxane, wherein preferably DMSO.
Fibrin ferment Sepharose 4FF medium preparation methods are：Take the Sepharose 4FF that weight in wet base is 10.0 g in cloth
It in family name's funnel, is washed repeatedly with deionized water, 25%, 50%, 75% each 50 mL of DMSO aqueous solutions is sequentially added into medium, so
Sepharose 4FF are put into the anhydrous DMSO containing 20mL afterwards（Dimethyl sulfoxide (DMSO)）In triangular flask, 0.5-1.0 g CDI are added
（Carbonyl dimidazoles）15 min are shaken under 30 DEG C of 100 r/min rotating speeds of constant temperature oscillator, is washed with deionized water, was both obtained repeatedly
The Sepharose 4FF of activation；Separately 50 mg-100 mg fibrin ferments is taken to be dissolved in 50 mL phosphate buffers（0.2mol/L
K2HPO4- 0.01 mol/L KCL, PH8.0）, and the phosphoric acid containing fibrin ferment is added in the above-mentioned Sepharose 4FF activated
In buffer solution, 4-8 DEG C, 50-80 r/min rotating speeds, after being stirred to react 10-15 h, according to deionized water, 1.0 mol/L KCL,
Deionized water, 0.1 mol/LHCL, deionized water（Whole 4-6 times of volumes）Filtering and washing is carried out, finally places it in phosphoric acid again
4 DEG C of preservations in buffer solution, gained is fibrin ferment Sepharose 4FF media.
The step（2）In, dress column method is：The 1/2-3/4 times of fibrin ferment Sepharose 4FF for detaching column volume is taken to be situated between
Matter is loaded on splitter, and column bed is rinsed with 3-5mL/min flow velocitys with the phosphate buffer of the PH 7.4-8.4 of 15 times of bed volumes,
Washing extremely balances.
The step（2）Eluent used in middle elution is 0.01-0.03 mol/L KCL -0.1-0.3 mol/L HCL,
Elution speed is often 1 ml/3 min of pipe.
The step（2）Middle high performance liquid chromatography detection condition：Chromatographic column：10 × 200mm of C18, detector：UV
210nm, mobile phase：Methanol：Water=3:1, column temperature：25 DEG C, sample size：10 μ L, flow velocity：1mL/min.
The Markwardt fibrin ferments direct titrimetric method detects its anticoagulant active principle：
That highest component of anticoagulant active is typically considered hirudin in leech saliva；Hirudin can be special with fibrin ferment
Property combine, be 1 in conjunction with ratio:1, make the principle of thrombin inactivation, can detect day with Matkwardt fibrin ferment direct titrimetric methods
The activity of right hirudin；The measurement unit of hirudin is international unit, is indicated with ATU, the international unit of thrombin activity is
NIH, i.e. 1 ATU are equal to the hirudin amount for neutralizing 1 NIH fibrin ferment.
The Markwardt fibrin ferments direct titrimetric method detects its anticoagulant active concrete operations：
About 0.1 g of this product powder is taken, accurately weighed, 0.9% normal saline solution, 500 μ L are added in precision, are sufficiently stirred dissolving, match
0.2 g/mL test solutions are made, precision measures 100 μ l of supernatant, sets in 1.5 mL test tubes, and 0.5% pig fibrinogen is added
Trishydroxymethylaminomethane hydrochloride buffer (take 0.2m0l/L tris solutions 25ml and 0.1m0l/L hydrochloric acid
Solution about 40ml adds water to 100ml, adjusts pH value to 7.4) 200 μ l, shakes up, set in water-bath (37 DEG C ± 0.5 DEG C) and slowly drip
Add every 1ml (in) for the thrombin solution (5 μ l/min gently shake up when being added dropwise) containing 40U (unit) to solidifying, record consumption is solidifying
The volume of hemase solution, is calculated as follows：
U=C1V1/C2V2W
In formula：U--- is per 1g units containing thrombin activity, i.e. leech cellulose content, U/g；
The concentration of C1--- thrombin solutions, μ/ml；
The concentration of C2--- test solutions, g/ml；
V1--- consumes the volume of thrombin solution, μ l；
The addition of V2--- test solutions, μ l；
Above-mentioned hirudin detection method of content is also suitable for：Using hirudinaria manillensis as the quality of the various preparations made by primary raw material
Monitoring.
Advantageous effect：
The natural hirudin purity of the method for the present invention purifying gained is high, and activity is high, at low cost, easy to operate, easily repeats, Ke Yijin
Row industrialized production.The obtained natural hirudin product of technique can be used for the original of food, health products, drug or cosmetics
Material, has the effects that anticoagulant and thrombolytic, improves blood circulation, boosts metabolism, and is used clinically for prevention cardiovascular and cerebrovascular disease,
Such as apoplexy, coronary heart disease, hyperlipidemia.
Specific implementation mode
According to following embodiments, the present invention may be better understood.Then, as it will be easily appreciated by one skilled in the art that it is real
It applies specific material ratio, process conditions and its result described in example and is merely to illustrate the present invention, without this should will not be limited
Invention.
Embodiment 1
In the present embodiment, the preparation method of fibrin ferment adsorbent is in fibrin ferment Sepharose 4FF affinity columns：The weight in wet base is taken to be
The Sepharose 4FF of 10.0 g（It is purchased from Shanghai Rongjun Bio-Pharmaceutical Technology Co., Ltd.）In in Buchner funnel, spend from
Sub- water washs repeatedly, 25%, 50% is sequentially added into medium, 75% each 50 mL of DMSO aqueous solutions, then by Sepharose
4FF is put into the anhydrous DMSO containing 20mL（Dimethyl sulfoxide (DMSO)）In triangular flask, 0.5-1.0 g CDI are added（Carbonyl dimidazoles）30℃
15 min are shaken under 100 r/min rotating speeds of constant temperature oscillator, are washed repeatedly with deionized water, the Sepharose both activated
4FF ；Separately take 50 mg-100 mg fibrin ferments（It is purchased from Lanzhou Institute of Biological Products Co., Ltd.）It is dissolved in 50 mL phosphoric acid
In buffer solution（0.2mol/L K2HPO4- 0.01 mol/L KCL, PH8.0）, and by the above-mentioned Sepharose 4FF activated
It is added in the phosphate buffer containing fibrin ferment, 4-8 DEG C, 50-80 r/min rotating speeds, after being stirred to react 10-15 h, according to deionization
Water, 1.0 mol/L KCL, deionized water, 0.1 mol/LHCL, deionized water（Whole 4-6 times of volumes）Filtering and washing is carried out, most
Place it in 4 DEG C of preservations in phosphate buffer again afterwards, gained is fibrin ferment Sepharose 4FF media.
Filling column method is：It is 20 x to take the fibrin ferment Sepharose 4FF media of 2/3 times of separation column volume to be loaded on size
The splitter of 100 mm rinses column bed with 3-5mL/min flow velocitys with the phosphate buffer of the PH 8.0 of 15 times of bed volumes, washes
It washs to balance.
A method of hirudin is isolated and purified, is as follows：
（1）Hirudin coarse extraction：After physiological saline fed to appetite leech live body, physical impact makes it salivate and collect, and will obtain
The leech saliva sample obtained is added 3 times（V:V）Trichloroacetic acid extracting solution mixing is pre-chilled, extracts 2 h, 10000 r/min centrifugations 20
Min, 4 times of acetone mixings being pre-chilled of addition are stood overnight after taking supernatant, HCL to adjust PH 4.0, acetone in recycling supernatant, and 10000
R/min centrifuges 20 min, takes precipitation, spare；
（2）It isolates and purifies：5 times of distilled water are added in above-mentioned precipitation, are fully dissolved, HCL adjusts PH 7.0, uses fibrin ferment
Sepharose 4FF affinity columns are isolated and purified, 0.025 mol/L KCL-0.2 mol/L HCL elutions, the concentration
Not only hirudin activity and affinity media stability can be maintained, but also elution hirudin can be efficiently separated, elution speed is often pipe 1
Ml/3 min, collection liquid high performance liquid chromatography detection leech cellulose content, fetch water leech cellulose content peak value at refined solution, it is spare；
（3）Desalination and concentration：Above-mentioned refined solution to be dialysed, dialysis membrane is the bag filter that molecular cut off is less than or equal to 7000,
And it repeats to add water 4 times in the process, continues 10 h, the supernatant after ultrafiltration is subjected to roto-vap operation（30-60 DEG C, 0-
180 r/min）, after removing 70 % of moisture removal, take concentrate spare；
（4）It takes concentrate freeze dryer to be lyophilized, collects product and be sealed；
（5）Activity determination：Markwardt fibrin ferment direct titrimetric methods detect its anticoagulant active.
（6）Content in crude product detects：It salivates plus 2 times of distilled water, vibrations mixing, HPLC detects leech cellulose content.
The obtained hirudin purified product of this method using Markwardt fibrin ferment direct titrimetric methods through detecting the product
The activity of hirudin is 7350ATU/g, and yield 7.51% obtains the hirudin of high-purity, high activity.
Embodiment 2
The present invention provides the influence that different carriers activator isolates and purifies hirudin, is as follows：
Different support activators is used on the basis of embodiment 1（CDI, cyanogen bromide, epoxychloropropane）Processing activation
Sepharose 4FF。
Obtained by this method：Test the work of obtained hirudin after 0.8 g CDI activation Sepharose 4FF in next step
Property is 6937ATU/g, yield 7.37%；1.0 g cyanogen bromides（Optimal addn）It is carried out after activation Sepharose 4FF next
The activity for the hirudin that step experiment obtains is 4219ATU/g, yield 2.19%；130 μm of ol/L Epichlorohydrin activations（Most preferably
Concentration）The activity for carrying out testing obtained hirudin in next step after Sepharose 4FF is 5231ATU/g, yield 3.51%.
So this experiment used carrier activator CDI is a kind of good activator of function and effect, can be that the activation of Ago-Gel carries
For accurate scientific basis.
Embodiment 3
The present invention provides the influence that different carriers activator solvent isolates and purifies hirudin, is as follows：
Different support-activated solvents is used on the basis of embodiment 1（DMSO, dioxane, acetone, ethyl alcohol, isopropanol）Place
Reason activation Sepharose 4FF.
Obtained by this example：The activity for carrying out testing obtained hirudin in next step after using DMSO as solvent is 7524
ATU/g, yield 7.28%；Carry out testing the activity of obtained hirudin in next step after using dioxane as solvent be
6424 ATU/g, yield 3.18%；It uses acetone as and carries out testing the activity of obtained hirudin after solvent in next step and be
3018 ATU/g, yield 2.31%；Use the activity difference for carrying out testing obtained hirudin after ethyl alcohol and isopropanol in next step
For 4359 ATU/g and 4893 ATU/g, yield is respectively 2.09% and 1.68%.So this experiment used carrier activator dissolves
Agent DMSO is a kind of good activator of function and effect, can provide accurate scientific basis for the activation of Ago-Gel.
Embodiment 4
A kind of method isolating and purifying hirudin provided by the present invention, is as follows
（1）Hirudin coarse extraction：After physiological saline fed to appetite leech live body, physical impact makes it salivate and collect, and will obtain
The leech saliva sample obtained is added 3 times（V:V）Trichloroacetic acid extracting solution mixing is pre-chilled, extracts 2 h, 10000 r/min centrifugations 20
Min takes supernatant, the acetone mixings that 4 times of precoolings are added after HCL adjustments PH 4.0 to stand overnight, acetone in recycling supernatant, and 10000
R/min centrifuges 20 min, takes precipitation, spare；
（2）It isolates and purifies：5 times of distilled water are added in above-mentioned precipitation, are fully dissolved, HCL adjusts PH 6.5, uses fibrin ferment
Sepharose 4FF affinity columns are isolated and purified, 0.025 mol/L KCL-0.2 mol/L HCL elutions, and elution speed is
Often 1 ml/3 min of pipe, collection liquid high performance liquid chromatography detection leech cellulose content, fetch water leech cellulose content peak value at refined solution, it is standby
With；
（3）Desalination and concentration：Above-mentioned refined solution is dialysed, and repeats to add water 4 times in the process, continues 10 h, after ultrafiltration
Supernatant carry out roto-vap operation（30-60 DEG C, 0-180 r/min）, after removing 75 % of moisture removal, take concentrate spare；
（4）It takes concentrate freeze dryer to be lyophilized, collects product and be sealed；
（5）Activity determination：Markwardt fibrin ferment direct titrimetric methods detect its anticoagulant active.
（6）Content in crude product detects：It salivates plus 2 times of distilled water, vibrations mixing, HPLC detects leech cellulose content.
The obtained hirudin purified product of this method using Markwardt fibrin ferment direct titrimetric methods through detecting the product
The activity of hirudin is 7518ATU/g, yield 6.79%, compared with prior art, product yield of the invention and final activity
All it is greatly improved.
Claims (8)
1. a kind of hirudin isolation and purification method based on affinity chromatography, which is characterized in that include the following steps：
1）Hirudin coarse extraction：After physiological saline fed to appetite leech live body, physical impact makes it salivate and collect, and will obtain
Leech saliva sample, extract, centrifugation, take precipitation, it is spare；
2）It isolates and purifies：By step 1）Distilled water is added in the precipitation, fully dissolves, and adjusts pH 6.0-8.0, uses fibrin ferment
Sepharose 4FF affinity columns are isolated and purified, high performance liquid chromatography detection leech cellulose content, at leech cellulose content peak value of fetching water
Refined solution, it is spare；
3）Desalination and concentration：Above-mentioned refined solution is dialysed, and repeats to add water 2-4 times in the process, continues 10-15h, by ultrafiltration
Supernatant afterwards carries out rotary evaporation and takes concentrate spare after removing moisture removal 70-80 %；
4）It takes concentrate freeze dryer to be lyophilized, obtains hirudin product.
2. a kind of hirudin isolation and purification method based on affinity chromatography as described in claim 1, which is characterized in that step 1）
It is middle that pH to 3.0-5.0 is adjusted using HCL, make partial impurities protein inactivation, and do not influence the activity of purpose product.
3. a kind of hirudin isolation and purification method based on affinity chromatography as described in claim 1, which is characterized in that step 1）
Leach step is：Precooling trichloroacetic acid extracting solution mixing is added, extraction, centrifuging and taking supernatant, addition is pre- after adjusting pH3.0-5.0
Cold acetone mixing is stood overnight, and recycles acetone in supernatant, and centrifugation takes precipitation.
4. a kind of hirudin isolation and purification method based on affinity chromatography as described in claim 1, which is characterized in that step 2）
Support activator is carbonyl dimidazoles in fibrin ferment Sepharose 4FF affinity columns.
5. a kind of hirudin isolation and purification method based on affinity chromatography as described in claim 1, which is characterized in that step 2）
Dress column method be：The fibrin ferment Sepharose 4FF media of 1/2-3/4 times of column volume are taken to be loaded in splitter, with 15 times of columns
The phosphate buffer of the PH 7.4-8.4 of bed volume rinses column bed, washing to balance with 3-5mL/min flow velocitys.
6. a kind of hirudin isolation and purification method based on affinity chromatography as described in claim 1, which is characterized in that step 2）
Eluent be（0.01-0.03 ）mol/L KCL—（0.1-0.3 ）Mol/L HCL, elution speed are often 1 ml/3 of pipe
min。
7. a kind of hirudin isolation and purification method based on affinity chromatography as claimed in claim 2, which is characterized in that step
（2）Middle high performance liquid chromatography detection condition：Chromatographic column：10 × 200mm of C18, detector：UV 210nm, mobile phase：Methanol：Water
=3:1, column temperature：25 DEG C, sample size：10 μ L, flow velocity：1mL/min.
8. a kind of hirudin isolation and purification method based on affinity chromatography as described in claim 1, which is characterized in that step 3）
The dialysis membrane used of dialysing is less than or equal to 7000 bag filter for molecular cut off.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110734489A (en) * | 2019-12-05 | 2020-01-31 | 昆明学院 | device and method for extracting hirudin from living leech in laboratory |
| CN112480243A (en) * | 2021-01-06 | 2021-03-12 | 广西科康科技集团有限公司 | Large-scale hirudin separation and purification production process method and equipment |
| CN113072638A (en) * | 2021-03-16 | 2021-07-06 | 宁波博睿瀚达生物科技有限公司 | Method for removing endotoxin in recombinant hirudin protein solution |
| CN115386445A (en) * | 2022-09-23 | 2022-11-25 | 文泽沂 | Preparation method of natural hirudin wine |
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| CN110734489A (en) * | 2019-12-05 | 2020-01-31 | 昆明学院 | device and method for extracting hirudin from living leech in laboratory |
| CN110734489B (en) * | 2019-12-05 | 2021-07-23 | 昆明学院 | Device and method for extracting hirudin from living leeches in laboratory |
| CN112480243A (en) * | 2021-01-06 | 2021-03-12 | 广西科康科技集团有限公司 | Large-scale hirudin separation and purification production process method and equipment |
| CN113072638A (en) * | 2021-03-16 | 2021-07-06 | 宁波博睿瀚达生物科技有限公司 | Method for removing endotoxin in recombinant hirudin protein solution |
| CN115386445A (en) * | 2022-09-23 | 2022-11-25 | 文泽沂 | Preparation method of natural hirudin wine |
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