CN107699554A - A kind of preparation method of pig blood fibrin ferment - Google Patents
A kind of preparation method of pig blood fibrin ferment Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of pig blood fibrin ferment, it is big to solve fibrin ferment extraction operation difficulty, the problem of being unfavorable for industrialized production, and its drip irrigation device comprises the following steps：Citric acid three sodium solution is added in pig blood and obtains pig blood trisodium citrate mixed liquor；The centrifugation layering of pig blood trisodium citrate mixed liquor is obtained into pig blood blood plasma；Add anionite into pig blood blood plasma, elution post is loaded after stirring and adsorbing, after anionite is washed；NaCl trisodium citrates mixed solution is prepared as eluent, is eluted, collects the eluent containing factor；By eluent desalination, solvent is removed, the electrical conductivity of the eluent eluted from desalting column is monitored, obtains the desalinization liquor containing factor；Add CaCl2Solution obtains the activation liquid containing fibrin ferment, after virus removal processing, obtains thrombin solution, can effectively reduce the impurity such as foreign protein in fibrin ferment, virus, improve fibrin ferment purity.
Description
Technical field
The present invention relates to fibrin ferment extraction, more particularly to a kind of preparation method of pig blood fibrin ferment.
Background technology
Fibrin ferment is an important clotting factor for participating in body coagulation process, is deposited in vivo in the form of factor
.20th century, mid-term started people by fibrin ferment applied to clinic, and because its is evident in efficacy, application is also more and more extensive.Afterwards,
Deng states of the U.S. and Britain isolate fibrin ferment from ox blood slurry and are applied to clinic, and prove do not occur animal fibrin ferment applied to human body
Fibrin ferment antigenicity, the oral and topical outer used time is without allergic reaction and other adverse reactions etc..That is extracted from animal blood plasma is solidifying
Hemase can be applied to clinic, as the choice drug of local hemostasis, have the advantages of hemostasis is fast, has no side effect.Meanwhile fibrin ferment
And the main component of fibrinogen immue quantitative detection reagent box, it is applied to the clinical inspection of coagulation disorders and thrombotic diseases
Survey.
At present, the thrombin product clinically applied, the separation and Extraction mainly from the animal bloods such as people's blood, pig, ox, its
In, because the source of pig blood is abundant, the factors such as cost of material is low, so most of fibrin ferment is the separation and Extraction from pig blood,
It is partially the separation and Extraction from the blood such as people's blood and ox blood.
Application publication number is that CN103160486A patent discloses a kind of preparation method of pig thrombiase, this method bag
Include：Swine plasma is separated first, and blood plasma is subjected to chemical method inactivation of virus；1.5% according to Swine plasma gross weight, which adds ion, hands over
The absorption that resin carries out factor is changed, filter-cloth filtering collection of ions exchanger resin is used after adsorbing 40~60min at room temperature, washes
Ion exchange resin is washed, discards cleaning solution, the ion exchange resin for then washing scrubbed liquid, is eluted with eluent,
Eluent is collected, the eluent of collection is dialysed with molecular cut off for the ultrafilter of 10000 dalton, it is unnecessary to remove
Salts substances, prothrombin solution is obtained, the filter that prothrombin solution is 20nm with aperture is filtered, collect filtrate
Carry out the lyophilized preservation that prothrombin activation prepares fibrin ferment.But the preparation method is only being carried out at virus removal at first
Reason, this processing mode need to remain that product is under germ-free condition in the later stage, increase operation difficulty, be unfavorable for industry
Metaplasia is produced, and easily limits the clinical practice of thrombin product.
The content of the invention
It is an object of the invention to provide a kind of preparation method of pig blood fibrin ferment, simplifies production technology, reduces pig blood and coagulate
The operation difficulty of hemase extraction, makes it be applied to industrial production.
The present invention above-mentioned technical purpose technical scheme is that：
A kind of preparation method of pig blood fibrin ferment, comprises the following steps：
Step1. it is 0.2~0.3g/ that concentration of the citric acid three sodium solution to trisodium citrate in mixed liquor is added in pig blood
L, stir, pH is between 6.5-7.5, obtaining pig blood-trisodium citrate mixed liquor for regulation；
Step2. pig blood-trisodium citrate mixed liquor obtained by Step1 is centrifuged into layering at 4 DEG C, filters off residue, obtain pig blood blood
Slurry；
Step3. the anionite of dry powder-shaped is added into pig blood blood plasma obtained by Step2, after 40~90min of stirring and adsorbing,
Supernatant is filtered off, obtains being adsorbed with the anionite of factor, elution post is loaded after being washed；
Step4. compound concentration is 2~3mol/L NaCl-0.01~0.03mol/L trisodium citrates mixed solution as elution
Liquid, eluent is added in the elution post equipped with anionite into Step3, the anion exchange to being adsorbed with factor
Agent is eluted, and collects the eluent containing factor；
Step5. SEPHAROSE G-50 are loaded into desalting column after pretreatment, by the elution containing factor obtained by Step4
Liquid adds desalting column desalination, removes solvent, obtains being adsorbed with the desalting column of factor；
It is adsorbed with obtained by Step6 to Step5 in the desalting column of factor and adds NaCl solution elution, is monitored and washed from desalting column
The electrical conductivity of the eluent taken off, and electrical conductivity is collected in 0.3~0.4x104Desalinization liquor between us/cm, obtain containing blood coagulation
The desalinization liquor of proenzyme；
Step7 adds CaCl in the desalinization liquor that Step6 is collected into2Solution is to CaCl2Solution concentration is diluted to 0.03~
0.08mol/L, stand to interface stability, obtain the activation liquid containing fibrin ferment；
Step8 centrifuges the activation liquid containing fibrin ferment obtained by Step7 to activation liquid layering, after filtering off residue, to filtering off residue
Activate liquid and carry out virus removal processing, obtain thrombin solution.
By using above-mentioned technical proposal, citric acid three sodium solution is added in pig blood, is complexed with the calcium ion in pig blood,
Anticoagulation can be played, avoids pig blood from condensing blocking, and fibrin ferment is existed in the form of factor, then filtering, is obtained
To the supernatant containing factor, factor is adsorbed by DEAE-sepharose A-50, with NaCl solution to DEAE-
Sepharose A-50 are cleaned, and to improve the purity of factor, are entered afterwards by NaCl- trisodium citrate mixed solutions
Row elution, factor is eluted, obtains the eluent containing factor, by by eluent desalination, electrical conductivity 0.3~
0.4x104Desalinization liquor between us/cm is un-activation prothrombin solution, passes through CaCl2Solution is by PROTHROMBIN ACTIVATOR, i.e.,
Thrombin solution after being activated, by carrying out virus removal processing to activation liquid, you can obtain the higher fibrin ferment of purity
Product, by above-mentioned steps, the impurity such as foreign protein in fibrin ferment, virus can be effectively reduced, fibrin ferment purity is improved, simplifies
Production technology, the operation difficulty of pig blood fibrin ferment extraction is reduced, it is applied to industrial production.
Preferably, a kind of preparation method of pig blood fibrin ferment, comprises the following steps：
Step1. it is 0.29g/ that concentration of the 38g/L citric acid three sodium solutions to trisodium citrate in mixed liquor is added in pig blood
L, stir, pH is between 6.5-7.5, obtaining pig blood-trisodium citrate mixed liquor for regulation；
Step2. pig blood-trisodium citrate mixed solution obtained by Step1 is centrifuged into layering at 4 DEG C, filters off residue, obtain pig blood
Blood plasma；
Step3. preferred DEAE-sepharose A-50 are as anionite, at 0.2mol/L NaCl solution immersions
After managing 20~40min, drain surface NaCl solution and lyophilized obtain the DEAE-sepharose A-50 of dry powder-shaped；
Step4. the DEAE-sepharose A-50 of 1.7g dry powder-shapeds ratio is added according to 1L pig bloods blood plasma by obtained by Step3
DEAE-sepharose A-50 add Step2 obtained by pig blood blood plasma in, after stirring and adsorbing 45min, stand 30min, then
Pour into sand core funnel and filter off supernatant, obtain being adsorbed with the DEAE-sepharose A-50 of factor；
Step5. 0.2mol/L NaCl solutions are selected as cleaning solution, the pig blood that each dosage of cleaning solution is measured by Step4
The 1/40 of Plasma volumes, the DEAE-sepharose A-50 for being adsorbed with factor are washed twice, it is solidifying by being adsorbed with after washing
The former DEAE-sepharose A-50 of hemase load elution post；
Step6. 2mol/L NaCl-0.01mol/L trisodium citrates mixed solutions are prepared as eluent, are equipped with into Step3
DEAE-sepharose A-50 elution post is eluted twice, collects eluent twice；
Step7. by SEPHAROSE G-50 after 20~40min of 3g/L NaCl solution immersion treatments, desalting column is loaded, will
The eluent that Step6 is collected adds desalting column desalination, removes solvent, monitors the conductance of the eluent eluted from desalting column
Rate, and electrical conductivity is collected in 0.3~0.4x104Desalinization liquor between us/cm, obtain the desalinization liquor containing factor；
Step8. 1mol/L CaCl are added in the desalinization liquor that Step7 is collected into2Solution is to CaCl2Concentration in total solution
0.05mol/L is diluted to, 40 DEG C of standing 20min, obtains the activation liquid containing fibrin ferment；
Step9 filters off after the activation liquid centrifugation layering containing fibrin ferment obtained by Step8 residue, the activation liquid for filtering off residue is gone
Virus treated, obtain thrombin solution.
By using above-mentioned technical proposal, it was found from the data that fibrin ferment obtains repeatedly are prepared, adjusted according to above-mentioned content
Preparation method, the fibrin ferment of maximum level on the premise of ensureing than living, purity, can be extracted.
Preferably, being centrifuged in Step2 from horizontal centrifuge, centrifugal rotational speed is set as 4020rpm, centrifugation time
For 15min.
By using above-mentioned technical proposal, factor can be reduced in centrifugal process by setting above rotating speed and centrifugation time
The amount mixed with sediment, so as to improve the content of factor in supernatant.
Preferably, the centrifugate completed in Step2 after centrifugation is filtered with the absorbent gauze of some stackings, it is described de-
The number of plies of fat gauze is between 2~4 layers.
By using above-mentioned technical proposal, 2~4 layers of absorbent gauze is designed on the premise of filtering traffic is ensured,
Filter net residue.
Preferably, with the 0.2mol/L that quality is 10 times of anionite after anionite elution in Step4
After NaCl solution washing, it is soaked in 0.2mol/L NaCl solutions, can be preserved at 0 DEG C 20~30 days.
By using above-mentioned technical proposal, anionite is soaked in 0.2mol/L NaCl solutions and can be used in
The preservation of DEAE media, reduce loss.
Preferably, anionite can preserve 20 in the case where not carrying out Step5 in 4 DEG C of environment in Step4
~30 days.
By using above-mentioned technical proposal, eluent is placed in 4 DEG C of preservations, the composition transfer of eluent is nearly constant, energy
Enough make in preparation process more flexibly, to prepare without disposable.
Preferably, 10~15% that applied sample amount volume is desalination column volume in Step5 are kept in Step5.
By using above-mentioned technical proposal, between 10~15% resin can be enable fully to adsorb applied sample amount control
Factor.
Preferably, centrifugal rotational speed is set in Step8 as 8000rpm, centrifugation time 30min.
By using above-mentioned technical proposal, solid-liquid can be kept completely separate by setting above-mentioned centrifugal rotational speed, avoid virus removal
Centrifugate blocks filter membrane in journey.
Preferably, progress S/D methods are prevented or cure a disease after the processing method of virus removal includes 20nm filtering membrane filtrations in Step8
Poison.
By using above-mentioned technical proposal, milipore filter combination S/D methods can improve the clearance of virus, and then improve blood coagulation
The purity of enzyme.
Preferably, the S/D methods include S/D reagents, the S/D reagents include Tween-80 and TNBP.
By using above-mentioned technical proposal, Tween-80 and TNBP are S/D reagents, and its effect to virus is preferable.
In summary, the invention has the advantages that：
The preparation method of the pig blood fibrin ferment is chromatographed by DEAE-sepharose A-50 and SEPHAROSE G-50 desalinations, and
By 20nm filter membranes and SD method virus removals, by above-mentioned steps, it is miscellaneous can effectively to reduce foreign protein in fibrin ferment, virus etc.
Matter, fibrin ferment purity is improved, simplify production technology, reduce the operation difficulty of pig blood fibrin ferment extraction, it is applied to industry
Production, while yield is ensured, there is higher enzyme activity potency, reduce production cost.
Embodiment
Embodiment one
Pig blood pre-processes
Step1 compound concentrations are 38g/L citric acid three sodium solution；
Glass container clean Step2 collects the fresh pig blood that isolated time is less than 10min, will while pig blood is collected
The citric acid three sodium solution that Step1 is prepared is added in fresh pig blood while stirring, controls pig blood and citric acid three in glass container
The ratio of sodium maintains pig blood volume ratio：Trisodium citrate volume ratio=400:Between 2.5~3.0；
After the completion of Step3 collects to 40L pig bloods, citric acid three sodium solution is continuously added, to citric acid three sodium solution in pig blood-lemon
Concentration in lemon acid trisodium mixed liquor is 0.29g/L, and measurement pH is 6.8；
Pig blood-trisodium citrate mixed liquor is placed under 4 DEG C of environment and refrigerates 4h by Step4, is put into after taking-up in horizontal centrifuge, if
It is 4020rpm to determine centrifugal rotational speed, and centrifuging temperature is 4 DEG C, centrifugation time 15min, is led to the supernatant in centrifugate after centrifugation
Cross double-deck absorbent gauze to be filtered, filter off residue and obtain pig blood blood plasma；
Absorb-elute
After DEAE-sepharose A-50 are immersed in 0.2mol/LNaCl solution 40min by Step5, NaCl solution is filtered to remove,
And the NaCl solution on DEAE-sepharose A-50 surfaces is drained, DEAE-sepharose A-50 is molten in lyophilized surface NaCl
The DEAE-sepharose A-50 of dry powder-shaped are obtained after liquid；
The pig blood Plasma volumes that Step6 measurements obtain in Step4, the DEAE-sepharose of 1.7g dry powder-shapeds is added according to every 1L
A-50 ratio adds the DEAE-sepharose A-50 of dry powder-shaped in pig blood blood plasma, after stirring and adsorbing 45min, stands
30min, the mixture of pig blood blood plasma and DEAE-sepharose A-50 is poured into sand core funnel, filter off pig blood blood plasma, stay suction
DEAE-sepharose A-50 with factor；
The DEAE-sepharose A-50 that Step6.1 residues do not carry out Step5 but have been subjected to Step5 processing are immersed in
In 0.2mol/LNaCl solution, it is put into 4 DEG C of environment and is preserved, can be preserved 20~30 days, in use, draining surface 0.2mol/
LNaCl solution, is lyophilized into powder；
Step7 washs the DEAE-sepharose A-50 that factor is adsorbed with Step6 with 0.2mol/L NaCl solutions, washes
Wash twice, each dosage is calculated according to every 1L blood plasma using 25mL0.2mol/L NaCl solutions, will be adsorbed with fibrin ferment after washing
Former DEAE-sepharose A-50 load elution post；
Step8 prepares 2mol/L NaCl-0.01mol/L trisodium citrates mixed solutions as eluent, to being equipped with Step7
DEAE-sepharose A-50 elution post is eluted twice, collects eluent twice；
Step8.1 can be cleaned to the DEAE-sepharose A-50 after elution with 0.2mol/L NaCl solutions, 0.2mol/
The dosage of L NaCl solutions is loads 10 times of DEAE-sepharose A-50 mass in post, after cleaning, by DEAE-
Sepharose A-50 are soaked in 0.2mol/L NaCl solutions, can be preserved at 0 DEG C 20~30 days；
Desalination virus removal
SEPHAROSE G-50 after 3g/L NaCl solution immersion treatments 40min, are loaded desalting column, by Step8 by Step9
The eluent of collection removes solvent by desalting column desalination in applied sample amount volume 10%, monitors washing of being eluted from desalting column
The electrical conductivity of de- liquid, and electrical conductivity is collected in 0.3~0.4x104Desalinization liquor between us/cm, obtain the desalination containing factor
Liquid；
Step10 adds 1mol/L CaCl in the desalinization liquor that Step9 is collected into2Solution is to CaCl2Solution concentration is diluted to
0.05mol/L, 40 DEG C of standing 20min, obtains the activation liquid containing fibrin ferment；
Activation liquid containing fibrin ferment obtained by Step10 is placed under 4 DEG C of environment and refrigerates 4h by Step11, adds in horizontal centrifuge, if
It is 8000rpm to determine centrifugal rotational speed, and centrifuging temperature is 4 DEG C, centrifugation time 30min, after centrifugation layering, filters off residue；
The activation liquid that Step12 filters off residue to Step11 carries out filtering off virus treated by 20nm filter membrane, and filter membrane elects nitre as
Acid cellulose filter membrane or cellulose acetate sheets, fibrin ferment final mean annual increment solution is obtained, addition afterwards accounts for activation liquid total content 3%
Tween-8 and the TNBP progress chemical sterilizations for accounting for activation liquid total content 1%, obtain thrombin solution after purification.
Embodiment one to embodiment six operation with embodiment one
Thrombin titer measure is carried out to one~embodiment of embodiment six：
1st, accurately weighed fibrinogen 30mg, dissolved with 0.9% sodium chloride solution 1.5ml, add fibrin ferment 0.1ml (3 unit),
Quickly shake up, room temperature places about 1 hour to complete solidification, takes out coagulum, is washed with water to eluate and adds silver nitrate test solution not produce
It is raw muddy, dried 3 hours at 105 DEG C, weigh weight, calculate the content (%) containing coagulum in fibrinogen.Then use
The fibrinogen solution containing 0.2% coagulum is made in 0.9% sodium chloride solution, is adjusted with 0.05mol/L disodium phosphate solns
Section pH value is diluted to the fibrinogen solution containing 0.1% coagulum to 7.0~7.4, then with 0.9% sodium chloride solution, standby；
2nd, thrombin standard product are taken, are respectively prepared with 0.9% sodium chloride solution in every 1ml containing 5.0 units, 6.4 units, 8.0 lists
Position, the standard solution of 10.0 units；Another 4, test tube for taking internal diameter 1cm, long 10cm, each accurate fibre for adding 0.1% coagulum
Fibrillarin original solution 0.9ml, put in 37 DEG C of ± 0.5 DEG C of water-baths and be incubated 5 minutes, then the accurate mark for measuring above-mentioned 4 kinds of concentration respectively
Quasi- each 0.1ml of product solution, is rapidly added in above-mentioned each test tube, timing immediately, shakes up, and puts in 37 DEG C of ± 0.5 DEG C of water-baths, and observation is fine
The presetting period of fibrillarin, every kind of concentration are surveyed 5 times, and averaging, (difference of the maxima and minima of 5 measure must not exceed flat
The 10% of average, is otherwise resurveyed)；The concentration of standard solution should control setting time to be advisable at 14~60 seconds；Sat in double-log
On millimeter paper, with every actual potency of pipe Plays product (unit) for abscissa, setting time (second) is ordinate, and it is bent to draw standard
Line；
3rd, fibrin ferment final mean annual increment solution 0.1mL made from every embodiment is taken, experiment the data obtained is diluted to and falls into standard curve, essence
Close absorption 0.1ml, by the preparation method parallel determination 5 times of standard curve, obtaining the average value of setting time, (error requirements are the same as marking
Directrix curve), potency (unit) is tried to achieve on standard curve, is calculated as follows：Thrombin titer (U/mL)=setting time is corresponding
Fibrin ferment final mean annual increment solution volume ÷ 1 after actual potency × 10 of standard items × dilution.
Design parameter content and thrombin titer see the table below：
Table one：The parameter content of one~embodiment of embodiment six and thrombin titer table
This specific embodiment is only explanation of the invention, and it is not limitation of the present invention, and those skilled in the art exist
The modification of no creative contribution can be made after this specification to the present embodiment as needed by reading, but as long as in the present invention
Right in all protected by Patent Law.
Claims (10)
1. a kind of preparation method of pig blood fibrin ferment, it is characterised in that comprise the following steps：
Step1. it is 0.2 ~ 0.3g/L that concentration of the citric acid three sodium solution to trisodium citrate in mixed liquor is added in pig blood,
Stir, pH is between 6.5-7.5, obtaining pig blood-trisodium citrate mixed liquor for regulation；
Step2. pig blood-trisodium citrate mixed liquor obtained by Step1 is centrifuged into layering at 4 DEG C, filters off residue, obtain pig blood blood
Slurry；
Step3. the anionite of dry powder-shaped is added into pig blood blood plasma obtained by Step2, after 40 ~ 90min of stirring and adsorbing, filter
Supernatant is removed, obtains being adsorbed with the anionite of factor, elution post is loaded after being washed；
Step4. compound concentration be 2 ~ 3mol/L NaCl-0.01 ~ 0.03mol/L trisodium citrates mixed solutions as eluent,
Eluent is added in the elution post equipped with anionite into Step3, the anionite to being adsorbed with factor
Eluted, collect the eluent containing factor；
Step5. SEPHAROSE G-50 are loaded into desalting column after pretreatment, by the elution containing factor obtained by Step4
Liquid adds desalting column desalination, removes solvent, obtains being adsorbed with the desalting column of factor；
Step6. to addition NaCl solution elution in the desalting column of factor is adsorbed with obtained by Step5, monitor from desalting column
The electrical conductivity of the eluent eluted, and electrical conductivity is collected in 0.3 ~ 0.4x104Desalinization liquor between us/cm, obtain containing solidifying
The former desalinization liquor of hemase；
Step7. CaCl is added in the desalinization liquor that Step6 is collected into2Solution is to CaCl2Solution concentration is diluted to 0.03 ~
0.08mol/L, stand to interface stability, obtain the activation liquid containing fibrin ferment；
Step8 centrifuges the activation liquid containing fibrin ferment obtained by Step7 to activation liquid layering, after filtering off residue, to filtering off residue
Activate liquid and carry out virus removal processing, obtain thrombin solution.
2. the preparation method of a kind of pig blood fibrin ferment according to claim 1, it is characterised in that comprise the following steps：
Step1. it is 0.29g/ that concentration of the 38g/L citric acid three sodium solutions to trisodium citrate in mixed liquor is added in pig blood
L, stir, pH is between 6.5-7.5, obtaining pig blood-trisodium citrate mixed liquor for regulation；
Step2. pig blood-trisodium citrate mixed solution obtained by Step1 is centrifuged into layering at 4 DEG C, filters off residue, obtain pig
Blood blood plasma；
Step3. preferred DEAE-sepharose A-50 are as anionite, at 0.2mol/L NaCl solution immersions
After managing 20 ~ 40min, drain surface NaCl solution and lyophilized obtain the DEAE-sepharose A-50 of dry powder-shaped；
Step4. the DEAE-sepharose A-50 of 1.7g dry powder-shapeds ratio is added according to 1L pig bloods blood plasma by obtained by Step3
DEAE-sepharose A-50 add Step2 obtained by pig blood blood plasma in, after stirring and adsorbing 45min, stand 30min, then
Pour into sand core funnel and filter off supernatant, obtain being adsorbed with the DEAE-sepharose A-50 of factor；
Step5. 0.2mol/L NaCl solutions are selected as cleaning solution, the pig blood that each dosage of cleaning solution is measured by Step4
The 1/40 of Plasma volumes, the DEAE-sepharose A-50 for being adsorbed with factor are washed twice, it is solidifying by being adsorbed with after washing
The former DEAE-sepharose A-50 of hemase load elution post；
Step6. 2mol/L NaCl-0.01mol/L trisodium citrates mixed solutions are prepared as eluent, are equipped with into Step3
DEAE-sepharose A-50 elution post is eluted twice, collects eluent twice；
Step7. by SEPHAROSE G-50 after 20 ~ 40min of 3g/L NaCl solution immersion treatments, desalting column is loaded, will
The eluent that Step6 is collected adds desalting column desalination, removes solvent, monitors the conductance of the eluent eluted from desalting column
Rate, and electrical conductivity is collected in 0.3 ~ 0.4x104Desalinization liquor between us/cm, obtain the desalinization liquor containing factor；
Step8. 1mol/L CaCl are added in the desalinization liquor that Step7 is collected into2Solution is to CaCl2Concentration in total solution is dilute
Release to 0.05mol/L, 40 DEG C of standing 20min, obtain the activation liquid containing fibrin ferment；
Step9 filters off after the activation liquid centrifugation layering containing fibrin ferment obtained by Step8 residue, the activation liquid for filtering off residue is gone
Virus treated, obtain thrombin solution.
3. the preparation method of a kind of pig blood fibrin ferment according to claim 1, it is characterised in that from horizontal in Step2
Centrifuge is centrifuged, and sets centrifugal rotational speed as 4020rpm, centrifugation time 15min.
4. the preparation method of a kind of pig blood fibrin ferment according to claim 1, it is characterised in that centrifugation is completed in Step2
Centrifugate afterwards is filtered with the absorbent gauze of some stackings, and the number of plies of the absorbent gauze is between 2 ~ 4 layers.
5. the preparation method of a kind of pig blood fibrin ferment according to claim 1, it is characterised in that anion is handed in Step4
Change after agent elutes after being washed with the 0.2mol/L NaCl solutions that quality is 10 times of anionite, be soaked in 0.2mol/L
In NaCl solution, it can be preserved at 0 DEG C 20 ~ 30 days.
6. the preparation method of a kind of pig blood fibrin ferment according to claim 1, it is characterised in that anion is handed in Step4
Agent is changed in the case where not carrying out Step5, can be preserved in 4 DEG C of environment 20 ~ 30 days.
7. the preparation method of a kind of pig blood fibrin ferment according to claim 1, it is characterised in that loading is kept in Step5
Measure 10 ~ 15% that volume is desalination column volume in Step5.
8. the preparation method of a kind of pig blood fibrin ferment according to claim 1, it is characterised in that centrifugation is set in Step8
Rotating speed is 8000rpm, centrifugation time 30min.
A kind of 9. preparation method of pig blood fibrin ferment according to claim 1, it is characterised in that virus removal in Step8
Processing method carries out S/D method virus removals after including 20nm filtering membrane filtrations.
10. the preparation method of a kind of pig blood fibrin ferment according to claim 9, it is characterised in that the S/D methods include
S/D reagents, the S/D reagents include Tween-80 and TNBP.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711147686.7A CN107699554A (en) | 2017-11-17 | 2017-11-17 | A kind of preparation method of pig blood fibrin ferment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711147686.7A CN107699554A (en) | 2017-11-17 | 2017-11-17 | A kind of preparation method of pig blood fibrin ferment |
Publications (1)
| Publication Number | Publication Date |
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| CN112354002A (en) * | 2020-11-12 | 2021-02-12 | 广东深蓝生物科技有限公司 | Hemostatic sealant and preparation method thereof |
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