CN106957834A - The preparation method and thrombin of beef of a kind of thrombin of beef - Google Patents
The preparation method and thrombin of beef of a kind of thrombin of beef Download PDFInfo
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- CN106957834A CN106957834A CN201710300156.5A CN201710300156A CN106957834A CN 106957834 A CN106957834 A CN 106957834A CN 201710300156 A CN201710300156 A CN 201710300156A CN 106957834 A CN106957834 A CN 106957834A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6429—Thrombin (3.4.21.5)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21005—Thrombin (3.4.21.5)
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Abstract
The invention discloses a kind of preparation method of thrombin of beef and thrombin of beef, it is related to Protein Extraction technical field.The preparation method of thrombin of beef disclosed by the invention includes：The BaCl that concentration is 0.8 1.1mol/L is added into the anti-freezing ox blood slurry containing sodium citrate2Solution, centrifugation, collection precipitation, BaCl2Solution is (5 7) with the volume ratio that the anti-freezing ox blood is starched：50.The preparation method is starched as raw material with the anti-freezing ox blood containing sodium citrate, add after BaCl2 solution precipitation, by using barium citrate absorption method, clotting factor dependent on vitamin K is precipitated, the substrate acted on thrombin of beef is eliminated simultaneously, the loss of thrombin of beef gross activity is reduced, and the ratio for improving thrombin of beef is lived, it is 6.34U/mg than living, recovery rate is 65.15%.
Description
Technical field
The present invention relates to Protein Extraction technical field, preparation method and ox in particular to a kind of thrombin of beef
Fibrin ferment.
Background technology
Fibrin ferment is a kind of serine protease, is also the main effects protease in blood coagulation cascade reaction, shows
Go out to promote the characteristic of solidifying and anti-freezing.Fibrin ferment is at Xa factor (FXa) by the nonactive factor in PCC
Produced in the presence of the factor by way of protein cleavage.When circulation clotting factor is in exposed extravascular tissue and tissue factor
When being in contact, fibrin ferment can organizationally be assembled.Fibrin ferment is converted into fiber egg by activating blood platelet, catalysis fibre proteinogen
In vain, promote clot stable and have central role in the initiation and propagation of thrombotic diseases.
At present, it is main both at home and abroad that fibrin ferment (thrombin of beef) is prepared for raw material with animal blood plasma such as ox blood slurry, also there is report
Road prepares fibrin ferment from salmon and ostrich blood plasma.Its method extracted mainly has isoelectric point precipitation and magnesium hydrate precipitate
Method.But the fibrin ferment that existing method is extracted is high to process equipment condition requirement and has that the yield of fibrin ferment is low, activity is low
The problems such as.
The content of the invention
It is an object of the invention to provide a kind of preparation method of thrombin of beef, this method is used using ox blood slurry as raw material
Barium citrate absorption method extracts thrombin of beef, and it has higher yield, and the thrombin of beef extracted is higher than work.
Another object of the present invention is to provide a kind of thrombin of beef, as obtained by above-mentioned preparation method, the ox blood coagulation
Enzyme has purity and than high characteristic living.
What the present invention was realized in：
A kind of preparation method of thrombin of beef, it includes：
BaCl is added into the anti-freezing ox blood slurry containing sodium citrate2Solution, centrifugation, collection precipitation；Wherein, BaCl2Solution
Concentration is 0.8-1.1mol/L, BaCl2Solution is (5-7) with the volume ratio that the anti-freezing ox blood is starched：50.
A kind of thrombin of beef, it is according to obtained by the preparation method of above-mentioned thrombin of beef.
Compared with prior art, the beneficial effects of the invention are as follows：
The preparation method of the thrombin of beef of the offer of the present invention, the preparation method is starched with the anti-freezing ox blood containing sodium citrate
For raw material, BaCl is added2After solution precipitation, by using barium citrate absorption method, the clotting factor dependent on vitamin K is sunk
Shallow lake is got off, while eliminating the substrate acted on thrombin of beef, is reduced the loss of thrombin of beef gross activity, is improved thrombin of beef
Ratio live；The ratio of the thrombin of beef prepared by preparation method of the present invention is used to live as 6.34U/mg, recovery rate is 65.15%, is adopted
High vigor and thrombin of beef in high yield can be prepared on a large scale with the preparation method that provides of the present invention.Equipment requirement is simple, suitable
Close large-scale production.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be attached to what is used needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore is not construed as pair
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the SDS-PAGE analysis result figures for the thrombin of beef solution that the embodiment of the present invention 1 is provided.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, are the conventional production that can be obtained by commercially available purchase
Product.
The preparation method and thrombin of beef to a kind of thrombin of beef of the embodiment of the present invention are specifically described below.
On the one hand, the preparation method for a kind of thrombin of beef that the present invention is provided, it comprises the following steps：
S1 blood is separated
Fresh bovine blood is taken, sodium citrate anti-freezing is added, is separated using blood cell seperator, haemocyte is removed, takes ox
Blood plasma.
Wherein, final concentration of the sodium citrate in bovine blood is preferably 3.7-3.9%, preferably 3.8% (m:v).
S2 is precipitated
BaCl is added into the anti-freezing ox blood slurry containing sodium citrate2Solution, centrifugation, collection precipitation；Wherein, BaCl2Solution
Concentration is 0.8-1.1mol/L, BaCl2Solution is (5-7) with the volume ratio that above-mentioned anti-freezing ox blood is starched：50.
Preferably, BaCl2Solution concentration is 1mol/L, BaCl2Solution is 6 with the volume ratio that above-mentioned anti-freezing ox blood is starched：50.
S3 dissolves
Above-mentioned precipitation is dissolved with EDTA solution, centrifuges, take supernatant.
Preferably, EDTA solution concentrations are that 0.18-0.22mol/L, pH are 7.8-8.2.
It is highly preferred that it is 8.0 that EDTA solution concentrations, which are 0.2mol/L, pH,.
S4 dialyses
Supernatant is dialysed with Tris-HCl buffer solutions, obtains thrombin of beef proenzyme liquid.
Preferably, Tris-HCl buffer concentrations are that 0.048-0.052mol/L, pH are 7.1-7.3.
It is highly preferred that it is 7.2 that Tris-HCl buffer concentrations, which are 0.05mol/L, pH,.
S5 is activated
Thrombin of beef proenzyme liquid handled through activation after, activated after thrombin of beef crude enzyme liquid.
Preferably, toward the Ca that final concentration of 0.009-0.012mol/L is added in above-mentioned thrombin of beef proenzyme liquid2+, in 26-
1.9-2.0h is activated at 28 DEG C.
Preferably, toward the Ca that final concentration of 0.01mol/L is added in above-mentioned thrombin of beef proenzyme liquid2+, activated at 27 DEG C
1.95h。
Using the fibrin ferment gross activity obtained by barium citrate absorption method, than living than conventional isoelectric point precipitation and hydrogen-oxygen
Change magnesium precipitate method high.Because most of protein is acidic protein in blood plasma, and factor others in isoelectric precipitation are miscellaneous
Albumen (including few fibers proteinogen) also together precipitates, and the loss of activity of fibrin ferment is larger.
Barium citrate absorption factor has certain selectivity, and simply the clotting factor dependent on vitamin K is sunk
Shallow lake is got off, while eliminating the substrate with thrombin action, is reduced the loss of fibrin ferment gross activity, is improved the ratio of fibrin ferment
It is living.
The preparation method for the thrombin of beef that the present invention is provided, high vigor and ox blood coagulation in high yield can be prepared on a large scale
Enzyme.The preparation method is simple to equipment requirement, be adapted to large-scale production, and technology of preparing reaches advanced international level, is domestic solidifying
Prepared by hemase provides advanced technical support.
Thrombin of beef in ox blood is extracted using preparation method provided by the present invention, the ratio of the thrombin of beef after activation is lived
For 6.34U/mg, recovery rate is：65.15%.
Further, in order to obtain refined thrombin of beef, above-mentioned preparation method may also include：
S6 is purified
After the above-mentioned concentrated processing of thrombin of beef crude enzyme liquid, purified with DEAE column chromatographies, obtain refined ox and coagulate
Hemase solution.
Wherein, concentration is：The ultra-filtration centrifuge tube for being 10KD with molecular cut off carries out concentration.
DEAE column chromatographies, which carry out purifying, to be included：
Elution step：Thrombin of beef crude enzyme liquid 0-1mol/L NaCl solution linear elutions after concentration, control
Flow velocity processed is 0.8-1.1ml/3min, collects eluent, and above-mentioned eluent obtains above-mentioned thrombin of beef solution after desalting processing.
Preferably, coutroi velocity is 1ml/3min.
Preferably, A phases：50mM Tris-HCl PH=7.2；B phases：50mM Tris-HCl+1M Nacl PH=7.2；A-
B phases carry out 20CV gradient elution.
Wherein, desalting processing includes：The Tris-HCl for being 7.1-7.3 with 0.048-0.052mol/L, pH is to above-mentioned elution
Liquid carries out desalination and changes liquid, obtains above-mentioned thrombin of beef solution.
Preferably, carry out desalination with the Tris-HCl that 0.05mol/L, pH are 7.2 and change liquid.
Thrombin of beef crude enzyme liquid is purified after purification by above-mentioned, purification 16.52, the rate of recovery 76.21%.
Through Purity, electrophoretic band is in single band, has reached that electrophoresis is pure, the molecular weight of the thrombin of beef extracted is about
For 37kd.
Further, in order that resulting thrombin of beef is easy to store or transported, above-mentioned preparation method may also include：
S7 is freezed
Above-mentioned thrombin of beef solution is subjected to frozen dried, thrombin of beef freeze-dried powder is obtained.
On the other hand, the present invention also provides a kind of thrombin of beef, and it has obtained by the preparation method of above-mentioned thrombin of beef.
On the other hand, the present invention also provides a kind of thrombin of beef product, and it contains above-mentioned thrombin of beef.
The feature and performance to the present invention are described in further detail with reference to embodiments.
Embodiment 1
The preparation method for the thrombin of beef that the present embodiment is provided, it is comprised the following steps that.
1.1 blood are separated
Take fresh bovine blood, add sodium citrate anti-freezing, sodium citrate in bovine blood final concentration of 3.8%, utilize
Blood cell seperator is separated, and is removed haemocyte, is taken ox blood oar.
1.2 precipitations
1.2.1 toward addition 1mol/L BaCl in above-mentioned 100ml anti-freezings ox blood slurry2Solution, BaCl2The addition of solution is ox
Blood plasma 12%, i.e. BaCl2Solution is 6 with the volume ratio that anti-freezing ox blood is starched:50.
1.2.2 after magnetic agitation 1h, in centrifuging 15min under 4000r/min rotating speed, collecting precipitation, (barium citrate sinks
Form sediment).
1.3 dissolvings
Above-mentioned precipitation is dissolved for 8.0 EDTA solution with 0.2mol/L, pH, 1h, 4000r/min, centrifugation 15min is stirred,
Insoluble matter is discarded, supernatant is taken.
1.4 dialysis
Supernatant is dialysed with 0.05mol/L, pH for 7.2 Tris-HCl buffer solutions, constantly changes dialyzate, to without Ba2+
Untill, obtain thrombin of beef proenzyme liquid.
1.5 activation
Ca is added in thrombin of beef proenzyme liquid2+, make its final concentration of 0.01mol/L, activate 1.95h at 27 DEG C, obtain
Thrombin of beef crude enzyme liquid after activation.
The Enzyme assay of 1.6 thrombin of beef
(1) enzyme standard solution：Thrombin standard product are taken, are dissolved with 0.9%NaCl, being quantitatively diluted to every milliliter respectively contains
5U, 6.4U, 8U and 10U enzyme standard solution.
(2) fibrinogen solution：Fibrinogen standard items are taken, the content of coagulum according to contained by standard items is used
0.9%NaCl dissolves the solution for being configured to the thing of solidifiable containing 1mg/ml.
(3) drafting of standard curve：Take internal diameter 1cm 4, test tube, each accurate addition 0.9ml fibrinogen solutions, 37
DEG C water-bath in preserve 5min, draw each 0.1ml of enzyme standard solution of above-mentioned various concentrations, be quickly adding into each test tube,
Clock, shake up immediately, the presetting period of fibrinogen is observed in 37 DEG C of water-bath, every kind of concentration is surveyed 5 times, averaged
(being resurveyed when the difference of maxima and minima exceedes the 10% of average value).The concentration of enzyme standard solution should control setting time
It is advisable in 14-60s.Using standard items enzyme-activity unit as abscissa, setting time is that ordinate draws standard curve.
(4) measure of sample enzyme activity：It is accurate to draw testing sample 0.1ml, the presetting period is determined as stated above, passes through mark
Directrix curve calculates the enzyme activity of testing sample (unit is U/ml).
(5) than computational methods living：The enzyme activity (U/ml) of thrombin of beef crude enzyme liquid and the ratio of protein concentration (mg/ml)；
The computational methods of recovery rate：The ratio of the total activity of thrombin of beef crude enzyme liquid and the total activity of bovine blood, wherein, total activity=enzyme
(U/ml) × cumulative volume (ml) living.
After testing, it is 6.34U/mg that the ratio of the thrombin of beef crude enzyme liquid after activation, which is lived, and recovery rate is 65.15%.
In order to obtain refined thrombin of beef, on the basis of above-mentioned steps, purified with DEAE column chromatographies, operation is such as
Under.
1.6 purifying
1.6.1 DEAE-52 pretreatment
A certain amount of DEAE-52 (Whatman companies) is weighed with 10ml graduated cylinders, using distilled water immersion 12h, is observed after being swelled
Volume, according to chromatographic column bed volume calculate experiment in DEAE-52 consumptions.
4g DEAE-52 cellulose distilled water immersion 12h accurately are weighed, during which repeatedly change water to remove surface flotation
Grain.Buchner funnel is drained, and is placed in 0.5mol/L HCl solutions and is soaked 1h, and repeatedly rinsing makes its pH to neutrality to ionized water；Put again
1h is soaked in 0.5mol/L NaOH solutions, repeatedly rinsing makes its pH to neutrality to ionized water.Saved backup after being disposed.
1.6.2 post is filled
The chromatographic column (XK 16 (16mm*20mm), GE companies) cleaned up is taken, is vertically fixed on iron stand, layer is opened
Post lower liquid outlet is analysed, standby EDAE-52 is taken, is poured slowly into several times in chromatographic column after mixing, make its natural subsidence, treat pillar height
Stop dress post after up to test requirements document.Notice that section and bubble can not occur in chromatographic column, it is ensured that the dense uniform of filler, it is no
Then, post is filled again.
1.6.3 balance
Constant flow pump is opened, chromatographic column is balanced with 3-5 times of bed volume 0.05mol/L, pH7.2 Tris-HCl buffer solutions,
Coutroi velocity is 1ml/3min.
1.6.4 loading
Above-mentioned thrombin of beef crude enzyme liquid is subjected to concentration through molecular cut off for 10KD ultra-filtration centrifuge tube.
Chromatographic column lower liquid outlet is opened, when liquid level in post is 1mm, by the thrombin of beef crude enzyme liquid after concentration along layer
Analyse post surrounding and surround injection, be loaded again with careful in the middle of post after enzyme liquid reaches certain altitude, unsuitable excessive, the general control of applied sample amount
In the 1%-5% (volume fraction) of bed volume.Sample liquid is fully entered after chromatographic column, with 2-3 times of post bed body 0.05mol/L
PH7.2 Tris-HCl buffer solutions cross post, wash out material not to be adsorbed.
1.6.5 elute
Elution step：Eluted with 0-1mol/L NaCl (0.05mol/L pH7.2 Tris-HCl) linear, control
Flow velocity is 1ml/3min, collects eluent.
A phases：50mM Tris-HCl PH=7.2；B phases：50mM Tris-HCl+1M Nacl PH=7.2；A-B phases are carried out
20CV gradient elution.
1.6.6 desalination
Milipore filter bag 0.05mol/L pH7.2 of the eluent through 10KD T ris-HCl carry out desalination and change liquid, are refined
Thrombin of beef solution.
1.6.7 the enzyme activity of refined thrombin of beef solution is detected.
The computational methods of purification：The ratio that the ratio of thrombin of beef solution is lived with the ratio work of thrombin of beef crude enzyme liquid；Return
The computational methods of yield：The ratio of the total activity of thrombin of beef solution and the total activity of thrombin of beef crude enzyme liquid.
Thrombin of beef crude enzyme liquid is purified after purification by above-mentioned, purification 16.52, the rate of recovery 76.21%.
Through Purity, electrophoretic band is in single band (as shown in figure 1, in figure：M is Maker, and 1 is thrombin of beef crude enzyme liquid, and 2 be pure
Thrombin of beef solution after change), reach that electrophoresis is pure, the molecular weight about 37kd of the thrombin of beef extracted.
Further, in order that resulting thrombin of beef is easy to store or transported, on the basis of above-mentioned steps, this implementation
The preparation method that example is provided also includes：
1.7 freeze
Above-mentioned thrombin of beef solution is put into freeze dryer (SCIENTZ-50ND, Ningbo Xin Zhi companies) to be freezed, obtained
Thrombin of beef freeze-dried powder.
Embodiment 2
The preparation method for the thrombin of beef that the present embodiment is provided, it is comprised the following steps that.
2.1 blood are separated
(be the same as Example 1).
2.2 precipitations
2.2.1 take 100ml anti-freezings ox blood to starch, add 0.8mol/L BaCl2Solution, BaCl2The addition of solution is ox blood
Starch 10%, i.e. BaCl2Solution is 5 with the volume ratio that anti-freezing ox blood is starched:50.
2.2.2 (be the same as Example 1) is centrifuged, collects precipitation；
2.3 dissolvings
Above-mentioned precipitation is dissolved for 7.8 EDTA solution with 0.18mol/L, pH, 1h, 4000r/min, centrifugation 15min is stirred,
Insoluble matter is discarded, supernatant is taken.
2.4 dialysis
Supernatant is dialysed with 0.048mol/L, pH for 7.1 Tris-HCl buffer solutions, constantly changes dialyzate, to without Ba2+
Untill, obtain thrombin of beef proenzyme liquid.
2.5 activation
Ca is added in thrombin of beef proenzyme liquid2+, make its final concentration of 0.009mol/L, activate 1.9h at 26 DEG C, obtain
Thrombin of beef crude enzyme liquid after activation.
2.6 purifying
2.6.1 DEAE-52 pretreatment
(be the same as Example 1)
2.6.2 post is filled
(be the same as Example 1).
2.6.3 balance
(be the same as Example 1).
2.6.4 loading 2
(embodiment 1)
2.6.5 elute
Elution step：Eluted with 0-1mol/L NaCl (0.05mol/L pH7.2 Tris-HCl) linear, control
Flow velocity is 0.8ml/3min, collects eluent.
A phases：50mM Tris-HCl PH=7.2；B phases：50mM Tris-HCl+1M Nacl PH=7.2；A-B phases are carried out
20CV gradient elution.
2.6.6 desalination
Milipore filter bag 0.048mol/L pH7.1 of the eluent through 10KD Tris-HCl carries out desalination and changes liquid, is refined
Thrombin of beef solution.
2.7 freeze
Above-mentioned thrombin of beef solution is put into freeze dryer to be freezed, thrombin of beef freeze-dried powder is obtained.
Embodiment 3
The preparation method for the thrombin of beef that the present embodiment is provided, it is comprised the following steps that.
3.1 blood are separated
(be the same as Example 1).
3.2 precipitations
3.2.1 take 100ml anti-freezings ox blood to starch, add 1.1mol/L BaCl2Solution, BaCl2The addition of solution is ox blood
Starch 14%, i.e. BaCl2Solution is 7 with the volume ratio that anti-freezing ox blood is starched:50.
3.2.2 (be the same as Example 1) is centrifuged, collects precipitation；
3.3 dissolvings
Above-mentioned precipitation is dissolved for 8.2 EDTA solution with 0.22mol/L, pH, 1h, 4000r/min, centrifugation 15min is stirred,
Insoluble matter is discarded, supernatant is taken.
3.4 dialysis
Supernatant is dialysed with 0.052mol/L, pH for 7.3 Tris-HCl buffer solutions, constantly changes dialyzate, to without Ba2+
Untill, obtain thrombin of beef proenzyme liquid.
3.5 activation
Ca is added in thrombin of beef proenzyme liquid2+, make its final concentration of 0.012mol/L, activate 2.0h at 28 DEG C, obtain
Thrombin of beef crude enzyme liquid after activation.
3.6 purifying
3.6.1 DEAE-52 pretreatment
(be the same as Example 1)
3.6.2 post is filled
(be the same as Example 1).
3.6.3 balance
(be the same as Example 1).
3.6.4 loading
(embodiment 1)
3.6.5 elute
Elution step：Eluted with 0-1mol/L NaCl (0.05mol/L pH7.2 Tris-HCl) linear, control
Flow velocity is 1.1ml/3min, collects eluent.
A phases：50mM Tris-HCl PH=7.2；B phases：50mM Tris-HCl+1M Nacl PH=7.2；A-B phases are carried out
20CV gradient elution.
3.6.6 desalination
Milipore filter bag 0.052mol/L pH7.3 of the eluent through 10KD Tris-HCl carries out desalination and changes liquid, is refined
Thrombin of beef solution.
3.7 freeze
Above-mentioned thrombin of beef solution is put into freeze dryer to be freezed, thrombin of beef freeze-dried powder is obtained.
To sum up, the preparation method of the thrombin of beef of offer of the invention, the preparation method is with the anti-freezing containing sodium citrate
Ox blood slurry is raw material, adds BaCl2After solution precipitation, by using barium citrate absorption method, the blood coagulation dependent on vitamin K
The factor precipitates, while eliminating the substrate acted on thrombin of beef, reduces the loss of thrombin of beef gross activity, improves ox
The ratio of fibrin ferment is lived；The ratio of the thrombin of beef prepared by preparation method of the present invention is used to live as 6.34U/mg, recovery rate is：
65.15%, the rate of recovery after purification is up to 76.21%, purification 16.52, uses the preparation method that the present invention provides can be with
High vigor and thrombin of beef in high yield are prepared on a large scale.Equipment requirement is simple, be adapted to large-scale production.The preparation method reaches
Advanced international level, prepares for domestic fibrin ferment and provides advanced technical support.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of thrombin of beef, it is characterised in that it includes：
BaCl is added into the anti-freezing ox blood slurry containing sodium citrate2Solution, centrifugation, collection precipitation；Wherein, BaCl2Solution concentration
For 0.8-1.1mol/L, BaCl2Solution is (5-7) with the volume ratio that the anti-freezing ox blood is starched：50.
2. the preparation method of thrombin of beef according to claim 1, it is characterised in that the preparation method also includes：
The precipitation is dissolved with EDTA solution, centrifuges, take supernatant, is dialysed with Tris-HCl buffer solutions, obtains thrombin of beef enzyme
Stoste, after being handled through activation, activated after thrombin of beef crude enzyme liquid；
After the concentrated processing of thrombin of beef crude enzyme liquid, purified with DEAE column chromatographies, obtain refined thrombin of beef molten
Liquid.
3. the preparation method of thrombin of beef according to claim 2, it is characterised in that the EDTA solution concentrations are 0.18-
0.22mol/L, pH are 7.8-8.2.
4. the preparation method of thrombin of beef according to claim 2, it is characterised in that the Tris-HCl buffer concentrations are
0.048-0.052mol/L, pH are 7.1-7.3.
5. the preparation method of thrombin of beef according to any one of claim 2-4, it is characterised in that the activation processing bag
Include：Final concentration of 0.009-0.012mol/L Ca is added into the thrombin of beef proenzyme liquid2+, activated at 26-28 DEG C
1.9-2.0h。
6. the preparation method of thrombin of beef according to any one of claim 2-4, it is characterised in that with DEAE posts layer
Analysis, which carries out purifying, to be included：Thrombin of beef crude enzyme liquid 0-1mol/L NaCl solution linear elutions after concentration,
Coutroi velocity is 0.8-1.1ml/3min, collects eluent, it is molten that the eluent obtains the thrombin of beef after desalting processing
Liquid.
7. the preparation method of thrombin of beef according to claim 6, it is characterised in that the desalting processing includes：With
0.048-0.052mol/L, pH carry out desalination to the eluent for 7.1-7.3 Tris-HCl and change liquid, obtain the ox blood coagulation
Enzyme solutions.
8. the preparation method of thrombin of beef according to any one of claim 2-4, it is characterised in that the concentration
For：The ultra-filtration centrifuge tube for being 10KD with molecular cut off carries out concentration.
9. the preparation method of thrombin of beef according to any one of claim 2-4, it is characterised in that the preparation method is also
Including：The thrombin of beef solution is subjected to frozen dried.
10. a kind of thrombin of beef, it is characterised in that the preparation of its thrombin of beef according to any one of claim 1-9
Obtained by method.
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| CN201710300156.5A Pending CN106957834A (en) | 2017-05-02 | 2017-05-02 | The preparation method and thrombin of beef of a kind of thrombin of beef |
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| CN (1) | CN106957834A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141951A (en) * | 1996-04-06 | 1997-02-05 | 济南军区生物制品药物研究所 | method for extracting and separating thrombin of beef |
| US20060270014A1 (en) * | 2005-05-26 | 2006-11-30 | Dan Pawlak | Thrombin purification |
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2017
- 2017-05-02 CN CN201710300156.5A patent/CN106957834A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141951A (en) * | 1996-04-06 | 1997-02-05 | 济南军区生物制品药物研究所 | method for extracting and separating thrombin of beef |
| US20060270014A1 (en) * | 2005-05-26 | 2006-11-30 | Dan Pawlak | Thrombin purification |
Non-Patent Citations (1)
| Title |
|---|
| 张阳: "牦牛血凝血酶提取纯化工艺及其稳定性研究", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
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