CN107674868A - A kind of method that fibrin ferment is extracted from pig blood - Google Patents
A kind of method that fibrin ferment is extracted from pig blood Download PDFInfo
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Abstract
The invention discloses a kind of method that fibrin ferment is extracted from pig blood, it is related to fibrin ferment production technical field, including the following steps：Pig blood is carried out supernatant anti-freezing blood plasma is collected by centrifugation, the factor in supernatant anti-freezing blood plasma is adsorbed using DEAE sepharose A 50, after sodium chloride solution I washs to factor, eluted with sodium chloride calcium chloride mixed solution and activate factor, obtained efflux is blood coagulation enzyme extract, again with 724 ion-exchange resin purifications, collect the fibrin ferment refining liquid at thrombin activity peak, to desalination and concentration after fibrin ferment refining liquid removal virus, finally freeze and obtain fibrin ferment finished product, the present invention accelerates the activation efficiency of factor, simplify preparation technology.
Description
Technical field
The present invention relates to fibrin ferment production technical field, more particularly to a kind of method that fibrin ferment is extracted from pig blood.
Background technology
Fibrin ferment is the natural constituents in body blood coagulation system, by precursor prothrombin through thrombokinase activate and
Into.Fibrin ferment is made up of two peptide chains, and peptide chain element is connected with disulfide bond, is a kind of serine protease with high specific.
4 Arg-Gly peptide bonds in fibrin ferment energy hydrolysis of fibrin original, make fibrinogen become insoluble fibrin, from
And make blood clotting.Mainly prepared both at home and abroad from animal blood plasma and human plasma at present, domestic pig industry scale is big, region
Extensively, pig blood resource is compared with horn of plenty, and fibrin ferment is clinically widely used, and fibrin ferment is extracted from pig blood, not only effectively sharp
With pig blood resource, also solves the ring pollution problem caused by pig blood is discarded.
Application publication number is that CN105624134A Chinese invention patent discloses a kind of preparation side of restricted fibrin ferment
Method, factor is adsorbed from Swine plasma using anion-exchange chromatography medium, anion chromatography post is eluted, activation is solidifying
Hemase is former, obtains the thick solution of fibrin ferment, through being concentrated by ultrafiltration, obtains fibrin ferment concentrate, then adsorbed and coagulated with cation-exchange chromatography medium
Hemase, cation chromatographic column is eluted, obtains thrombin solution, through desalination and concentration by ultrafiltration, finally freeze to obtain restricted blood coagulation
Enzyme finished product.
Above-mentioned preparation method in actual mechanical process, using trisodium citrate as pig blood in extract fibrin ferment elution
Agent, and trisodium citrate can chelate with calcium ion, form the soluble citrate compound for being difficult to dissociate so that calcium ion subtracts
It is few, conversion of prothrombin is prevented into fibrin ferment, and then enough calcium ions need to be supplemented in the later stage, and the calcium ion that the later stage adds leads to
Chang Xianyu trisodium citrates chelate, after trisodium citrate is chelated completely remaining calcium ion promote again conversion of prothrombin into
Fibrin ferment, and then reduce the efficiency that factor is activated.
The content of the invention
It is an object of the invention to provide it is a kind of from pig blood extract fibrin ferment method so that the elution of factor and swash
Synchronous progress living, accelerates the efficiency that factor is activated.
The present invention above-mentioned technical purpose technical scheme is that：
A kind of method that fibrin ferment is extracted from pig blood, comprises the following steps：
(1) toward citric acid three sodium solution is added in pig blood, supernatant is collected after mixed liquor is centrifuged to layering, supernatant is double-deck
Supernatant anti-freezing blood plasma is obtained after absorbent gauze filtering；
(2) the supernatant anti-freezing blood plasma obtained by step (1) is chromatographed with A-50 chromatographic columns, the fibrin ferment in supernatant anti-freezing blood plasma
Original is adsorbed on A-50 chromatographic columns, and A-50 chromatographic columns are washed with sodium chloride solution I, is then mixed with sodium chloride-calcium chloride molten
Liquid elutes and activated the fibrin ferment being adsorbed on A-50 chromatographic columns, obtains blood coagulation enzyme extract；
(3) the blood coagulation enzyme extract obtained by step (2) is chromatographed with resin chromatography post, the fibrin ferment in blood coagulation enzyme extract
It is adsorbed on resin chromatography post, the fibrin ferment being adsorbed on resin chromatography post is eluted with sodium chloride solution II, obtains fibrin ferment
Refining liquid；
(4) desalination and concentration is carried out after the fibrin ferment refining liquid obtained by step (3) being removed into virus, obtains fibrin ferment concentrate；
(5) the fibrin ferment concentrate obtained by step (4) is freezed, produces the powdery fibrin ferment finished product of white.
Using such scheme, the condensation that citric acid three sodium solution prevents blood is added in pig blood, pig blood causes after centrifugation
Haemocyte and blood plasma layering, are easy to the collection of supernatant anti-freezing serum by double-deck absorbent gauze, A-50 chromatographic columns are to supernatant anti-freezing
Factor in blood plasma is adsorbed, and sodium chloride solution I carries out the blood plasma of the other compositions residued on A-50 chromatographic columns
Cleaning, while wash away remnants citric acid three sodium solution；Sodium chloride in sodium chloride-calcium chloride mixed solution strengthens as ion
Agent, the factor for promoting to be adsorbed in A-50 chromatographic columns are eluted, and calcium chloride elutes as activator in factor
While activated, eluted and the synchronous of activation is carried out with realizing, directly obtain blood coagulation enzyme extract；Subsequent fibrin ferment slightly carries
Liquid is purified using A-50 chromatographic columns, obtains the fibrin ferment refining liquid of high-purity, and to its virus removal, desalination and concentration and jelly
It is dry, influence of the trisodium citrate to PROTHROMBIN ACTIVATOR is prevented with this, the efficiency that factor is activated is accelerated, simplifies solidifying
Technique prepared by hemase, while add the storage time of fibrin ferment.
More preferably：
The dosage of the citric acid three sodium solution added in step (1) in pig blood is that 7.0-8.0mL citric acid is added per 1L pig bloods
Three sodium solutions；
The concentration of the sodium chloride solution I of washing A-50 chromatographic columns is 0.1-0.3M/L in step (2)；
The concentration for being eluted in step (2) and activating the sodium chloride of the sodium chloride-calcium chloride mixed solution of factor is 1.5-
2.5M/L, the concentration of calcium chloride is 1.0-2.0M/L；
The concentration of the sodium chloride solution II of elution fibrin ferment is 2.0-3.0g/L in step (3).
Using such scheme, found through studying (experiment), used reagent is obtained in the range of above-mentioned parameter to coagulate
Hemase has higher potency and protein concentration, is easy to be applied in actual production process.
More preferably：
The dosage of the citric acid three sodium solution added in step (1) in pig blood is that 7.5mL trisodium citrate is added per 1L pig bloods
Solution；The concentration of the sodium chloride solution I of washing A-50 chromatographic columns is 0.2M/L in step (2)；
The concentration for being eluted in step (2) and activating the sodium chloride of the sodium chloride-calcium chloride mixed solution of factor is 2.0M/L,
The concentration of calcium chloride is 1.5M/L；
The concentration of the sodium chloride solution II of elution fibrin ferment is 2.5g/L in step (3).
Using such scheme, found through studying (experiment), the fibrin ferment obtained by used reagent above-mentioned parameter
Potency and protein concentration highest.
More preferably：Washing process in step (2) is carried out twice, the dosage of sodium chloride solution I when washing every time
24-26mL sodium chloride solution I is added for every 1L supernatants anti-freezing blood plasma.
Using such scheme, find through studying (experiment), the factor of upper prop is cleaned in batches, and on every 1L
When clear anti-freezing blood plasma adds 24-26mL sodium chloride solution I, sodium chloride solution I is preferable to the cleaning performance of factor, is easy to
The protein concentration of the fibrin ferment of raising.
More preferably：The process of washing in step (2) is carried out at twice, every time sodium chloride solution I during washing
Dosage is that 25mL sodium chloride solution I is added per 1L supernatant anti-freezings blood plasma.
Using such scheme, find through studying (experiment), the factor of upper prop is cleaned in batches, and on every 1L
When clear anti-freezing blood plasma adds 25mL sodium chloride solution I, sodium chloride solution I is optimal to the cleaning performance of factor, is easy to carry
The protein concentration of high fibrin ferment.
More preferably：Eluted in step (2) and activate the dosage of the sodium chloride-calcium chloride mixed solution of factor
18mL-20mL sodium chloride-calcium chloride mixed solution is added for every 100mL supernatants anti-freezing blood plasma.
Using such scheme, found through studying (experiment), 18mL-20mL chlorine is added in every 100mL supernatant anti-freezing blood plasma
When changing sodium-calcium chloride mixed solution, the protein concentration of fibrin ferment is higher.
More preferably：Eluted in step (2) and activate the dosage of the sodium chloride-calcium chloride mixed solution of factor
19mL sodium chloride-calcium chloride mixed solution is added for every 100mL supernatants anti-freezing blood plasma.
Using such scheme, found through studying (experiment), 19mL sodium chloride-chlorine is added per 100mL supernatant anti-freezings blood plasma
When changing calcium mixed solution, the protein concentration highest of fibrin ferment.
More preferably：The operation of elution and activation in step (2) is carried out at twice, the behaviour for eluting and activating every time
Corresponding efflux is obtained, the summation of efflux is blood coagulation enzyme extract twice.
Using such scheme, it was found from the data that fibrin ferment obtains repeatedly are prepared, blood coagulation is eluted and activated in batches
During proenzyme, the protein concentration highest of fibrin ferment.
More preferably：The filter membrane that fibrin ferment refining liquid in step (4) is 20nm with aperture removes virus.
Using such scheme, the filter membrane for being 20nm with aperture removes virus, relative to Pasteur's virus inactivation technology, reduced
To the loss of activity of fibrin ferment.
In summary, the invention has the advantages that：
1st, by that will elute and activate synchronous progress, reduce the processing step for preparing fibrin ferment, avoid factor from activating
When plunder of the anti-coagulants to calcium ion, shorten the cycle of technique, while improve the yield of fibrin ferment；
2nd, sodium chloride has good soil washing, does not produce toxicity, reduce production cost as ion reinforcing agent；
3rd, the filter membrane for being 20nm with aperture removes virus, reduces the loss of activity of fibrin ferment, and the ratio for adding fibrin ferment is lived.
Embodiment
Pretreatment：
1st, the preparation of citric acid three sodium solution：
38g trisodium citrate particles are weighed, are dissolved in distilled water so that its concentration is 38g/L, with 1M/L HCl solution
Or 1M/L NaOH solution adjusts its pH to 7.0-7.4；
2nd, DEAE Sepharose A-50 (abbreviation A-50) balance：
A-50 is suspended in distilled water, stable to solution interface, supernatant and the A-50 floated in supernatant are removed in 1h hypsokinesis,
Remaining A-50 is soaked in 0.5mol/L NaOH solution, 30min is stood after stirring, A-50 is cleaned with distilled water, to from
The pH of the efflux of A-50 outflows is 7, then the NaH with 0.1mol/L2PO4Solution is same as above operating process processing；Finally use
0.01mol/L, pH=6.5 Na2HPO4-NaH2PO4Buffer solution (PB) balances so that is reached from the pH of the efflux of A-50 outflows
To stationary value, decompressing and extracting, save backup；
3rd, the balance of 724 ion exchange resin (abbreviation resin)：
First resin is embathed with distilled water 5-6 times, can be gently agitated in embathing and (be sure not vigorous agitation, be destroyed to prevent resin)；Leaching
5% HCl solution (dosage of this HCl solution is twice of resin volume) immersion 10min of complete resin is washed, with distillation
Water rinse resin, the pH to the efflux from resin outflow are 6；Then with 5%NaOH solution, (dosage is the two of resin volume
Times) immersion resin 10min, with distilled water flushing resin, the pH to the efflux from resin outflow is 9；It is molten with 5% HCl again
Liquid (dosage of this HCl solution is three times of resin volume) immersion more than resin 1h, distilled water is used after resin is converted into H types
Rinse, the pH to the efflux flowed out from resin is 7, and dress post is standby.
Embodiment 1：
1st, the collection of supernatant anti-freezing blood plasma
1.1st, it is less than 30min fresh pig blood with the glass container collection isolated time of cleaning, adds what is prepared into pig blood
38g/L citric acid three sodium solutions, 6.5mL citric acid three sodium solution is added per 1L pig bloods, stir 30min, be placed under 4 DEG C of environment
Refrigeration obtains pig blood treatment fluid to constant temperature；
1.2nd, the parameter for adjusting centrifuge is 4 DEG C, 4020rpm, and pig blood treatment fluid is placed in a centrifuge into centrifugation to being layered, collected
Supernatant；
1.3rd, supernatant is filtered with double-deck absorbent gauze, collects to obtain supernatant anti-freezing blood plasma.
2nd, the collection of blood coagulation enzyme extract
2.1st, it is solidifying in supernatant anti-freezing blood plasma by the supernatant anti-freezing blood plasma loading of collection into the good A-50 chromatographic columns of planned balance
Hemase original is adsorbed on A-50 chromatographic columns；
2.2nd, the sodium chloride solution I that the factor that will attach on A-50 chromatographic columns is 0.2M/L with concentration washes twice, often
The dosage of sodium chloride solution I is that 25mL sodium chloride solution I is added per 1L supernatant anti-freezings blood plasma during secondary washing；
2.3rd, eluted at twice with sodium chloride-calcium chloride mixed solution and activate the factor on A-50 chromatographic columns, washed every time
The operation for taking off and activating obtains corresponding efflux, collects efflux twice and obtains blood coagulation enzyme extract, wherein sodium chloride-chlorination
The concentration of sodium chloride in calcium mixed solution is 2.0M/L, and the concentration of calcium chloride is 1.5M/L, and sodium chloride-calcium chloride mixing is molten
The total dosage of liquid twice is that 19mL sodium chloride-calcium chloride mixed solution is added per 100mL supernatant anti-freezings blood plasma.
3rd, the collection of fibrin ferment refining liquid
3.1st, blood coagulation enzyme extract is injected in the good resin chromatography of planned balance, the fibrin ferment in blood coagulation enzyme extract is adsorbed
In on resin chromatography post；
3.2nd, the sodium chloride solution II for being 2.5g/L with concentration elutes resin, while obtains chromatogram using high performance liquid chromatograph
Figure, collect the efflux for reaching and being flowed out during thrombin activity peak from resin, as fibrin ferment refining liquid.
4th, the preparation of fibrin ferment concentration
4.1st, the filter membrane that fibrin ferment refining liquid is 20nm with aperture is filtered, removes virus；
4.2nd, desalination and concentration then is carried out with the milipore filter in 10kD apertures, and adds glycine and make protective agent, obtain fibrin ferment concentration
Liquid.
5th, fibrin ferment is freeze-dried
5.1st, fibrin ferment concentrate is dispensed, and normal temperature enters cabinet；
5.2nd, baffle temperature control is down to -40 DEG C in 40min, keeps 3h；
5.3rd, vavuum pump is opened, vacuum degree control is in 7-15Pa；
5.4th, dividing plate is warming up to -30 DEG C after 2h, keeps 3h；
5.5th, dividing plate is warming up to -20 DEG C after 4h, keeps 6h；
5.6th, dividing plate is warming up to 0 DEG C after 8h, keeps 20h；
5.7th, dividing plate is warming up to 30 DEG C after 10h, keeps 6h；
5.8th, vacuum degree control keeps 1h in below 5Pa；
5.9th, vacuum tamponade is offerd for sale, and obtains fibrin ferment finished product.
Embodiment 2：It is with the difference of embodiment 1：7.0mL citric acid three sodium solution is added per 1L pig bloods.
Embodiment 3：It is with the difference of embodiment 1：7.5mL citric acid three sodium solution is added per 1L pig bloods.
Embodiment 4：It is with the difference of embodiment 1：8.0mL citric acid three sodium solution is added per 1L pig bloods.
Embodiment 5：It is with the difference of embodiment 1：8.5mL citric acid three sodium solution is added per 1L pig bloods.
Embodiment 6：It is with the difference of embodiment 3：The concentration of sodium chloride solution I is 0.05M/L.
Embodiment 7：It is with the difference of embodiment 3：The concentration of sodium chloride solution I is 0.1M/L.
Embodiment 8：It is with the difference of embodiment 3：The concentration of sodium chloride solution I is 0.3M/L.
Embodiment 9：It is with the difference of embodiment 3：The concentration of sodium chloride solution I is 0.4M/L.
Embodiment 10：It is with the difference of embodiment 3：23mL sodium chloride solution is added per 1L supernatant anti-freezings blood plasma
Ⅰ。
Embodiment 11：It is with the difference of embodiment 3：24mL sodium chloride solution is added per 1L supernatant anti-freezings blood plasma
Ⅰ。
Embodiment 12：It is with the difference of embodiment 3：26mL sodium chloride solution is added per 1L supernatant anti-freezings blood plasma
Ⅰ。
Embodiment 13：It is with the difference of embodiment 3：27mL sodium chloride solution is added per 1L supernatant anti-freezings blood plasma
Ⅰ。
Embodiment 14：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.0M/L, the concentration of calcium chloride is 0.5M/L.
Embodiment 15：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.0M/L, the concentration of calcium chloride is 1.0M/L.
Embodiment 16：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.0M/L, the concentration of calcium chloride is 1.5M/L.
Embodiment 17：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.0M/L, the concentration of calcium chloride is 2.0M/L.
Embodiment 18：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.0M/L, the concentration of calcium chloride is 2.5M/L.
Embodiment 19：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.5M/L, the concentration of calcium chloride is 0.5M/L.
Embodiment 20：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.5M/L, the concentration of calcium chloride is 1.0M/L.
Embodiment 21：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.5M/L, the concentration of calcium chloride is 1.5M/L.
Embodiment 22：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.5M/L, the concentration of calcium chloride is 2.0M/L.
Embodiment 23：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 1.5M/L, the concentration of calcium chloride is 2.5M/L.
Embodiment 24：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.0M/L, the concentration of calcium chloride is 0.5M/L.
Embodiment 25：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.0M/L, the concentration of calcium chloride is 1.0M/L.
Embodiment 26：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.0M/L, the concentration of calcium chloride is 2.0M/L.
Embodiment 27：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.0M/L, the concentration of calcium chloride is 2.5M/L.
Embodiment 28：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.5M/L, the concentration of calcium chloride is 0.5M/L.
Embodiment 29：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.5M/L, the concentration of calcium chloride is 1.0M/L.
Embodiment 30：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.5M/L, the concentration of calcium chloride is 1.5M/L.
Embodiment 31：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.5M/L, the concentration of calcium chloride is 2.0M/L.
Embodiment 32：It is with the difference of embodiment 3：Sodium chloride concentration in sodium chloride-calcium chloride mixed solution
For 2.5M/L, the concentration of calcium chloride is 2.5M/L.
Embodiment 33：It is with the difference of embodiment 3：Every 100mL supernatant anti-freezings blood plasma addition 17mL sodium chloride-
Calcium chloride mixed solution.
Embodiment 34：It is with the difference of embodiment 3：Every 100mL supernatant anti-freezings blood plasma addition 18mL sodium chloride-
Calcium chloride mixed solution.
Embodiment 35：It is with the difference of embodiment 3：Every 100mL supernatant anti-freezings blood plasma addition 20mL sodium chloride-
Calcium chloride mixed solution.
Embodiment 36：It is with the difference of embodiment 3：Every 100mL supernatant anti-freezings blood plasma addition 21mL sodium chloride-
Calcium chloride mixed solution.
Embodiment 37：It is with the difference of embodiment 3：The concentration of sodium chloride solution II is 1.5g/L.
Embodiment 38：It is with the difference of embodiment 3：The concentration of sodium chloride solution II is 2.0g/L.
Embodiment 39：It is with the difference of embodiment 3：The concentration of sodium chloride solution II is 3.0g/L.
Embodiment 40：It is with the difference of embodiment 3：The concentration of sodium chloride solution II is 3.5g/L.
Fibrin ferment in above-described embodiment is subjected to potency and protein concentration detection：
Respectively according to《Pharmacopoeia of People's Republic of China》Second the 1561-1562 pages of relevant thrombin titer in 2015 editions
" the second method Forint phenol method (Lowry methods) " in standard detecting method and the 4th protein determination of general rule 0731.
Test result see the table below：
It is preferred embodiment to select embodiment 3, is compared test.
Comparative example 1：Embodiment 1, embodiment 2, embodiment 4 and embodiment 5
Compared to above-described embodiment 3, this comparative example 1 is to be prepared in the case of above-described embodiment 3 changes citric acid three sodium solution dosage
Fibrin ferment, compare and show that trisodium citrate the dosage potency of fibrin ferment and protein concentration in 7.0-8.0mL/L pig bloods are higher.
Comparative example 2：Embodiment 6, embodiment 7, embodiment 8 and embodiment 9
Compared to above-described embodiment 3, this comparative example 2 is solidifying to be prepared under the change concentration levels of sodium chloride solution I of above-described embodiment 3
Hemase, compare and show that the concentration of sodium chloride solution I potency of fibrin ferment and protein concentration in 0.1-0.3M/L are higher.
Comparative example 3：Embodiment 10, embodiment 11, embodiment 12 and embodiment 13
Compared to above-described embodiment 3, this comparative example 3 is solidifying to be prepared in the case of the change dosage of sodium chloride solution I of above-described embodiment 3
Hemase, compare draw the dosage of sodium chloride solution I in 24-26mL/L supernatant anti-freezing serum the potency of fibrin ferment and protein concentration compared with
It is high.
Comparative example 4：Embodiment 14 is to embodiment 32
Compared to above-described embodiment 3, this comparative example 4 is that above-described embodiment 3 changes sodium chloride-calcium chloride mixed solution concentration levels
The fibrin ferment of lower preparation, compare and draw using fibrin ferment during 1.5-2.5M/L sodium chloride -1.0-2.0M/L calcium chloride mixed solutions
Potency and protein concentration it is higher.
Comparative example 5：Embodiment 33, embodiment 34, embodiment 35 and embodiment 36
Compared to above-described embodiment 3, this comparative example 5 is the elution amount that above-described embodiment 3 changes sodium chloride-calcium chloride mixed solution
In the case of the fibrin ferment for preparing, compare and show that total dosage of sodium chloride-calcium chloride mixed solution resists for 18-20mL/100mL supernatants
When blood coagulation is clear, the potency and protein concentration of fibrin ferment are higher.
Comparative example 6：Embodiment 37, embodiment 38, embodiment 39 and embodiment 40
Compared to above-described embodiment 3, this comparative example 7 is that above-described embodiment 3 changes what is prepared under the concentration levels of sodium chloride solution II
Fibrin ferment, compare and show that the concentration of sodium chloride solution II potency of fibrin ferment and protein concentration in 2.0-3.0g/L are higher.
By above-mentioned it is demonstrated experimentally that passing through trisodium citrate dosage, the concentration of sodium chloride I, the dosage of sodium chloride solution I, chlorination
Sodium-calcium chloride mixed solution concentration, the elution amount of sodium chloride-calcium chloride mixed solution and the concentration of sodium chloride solution II can shadows
Ring the potency and protein concentration of fibrin ferment.Wherein, it is using the trisodium citrate of 7.5mL/L pig bloods, concentration 0.2M/L, dosage
The sodium chloride solution I of 25mL/L supernatant anti-freezing serum, concentration 2.0M/L-1.5M/, total dosage are 19mL/100mL supernatant anti-freezings
When the sodium chloride-calcium chloride mixed solution and concentration of blood plasma are 2.5g/L sodium chloride solution II, the potency and egg of protein
White concentration is higher.
The fibrin ferment prepared by the method, in the case where ensureing thrombin titer and protein concentration, improve fibrin ferment
Former activation efficiency, simplifies preparation technology.
This specific embodiment is only explanation of the invention, and it is not limitation of the present invention, people in the art
Member can make the modification of no creative contribution to the present embodiment as needed after this specification is read, but as long as at this
All protected in the protection domain of invention by Patent Law.
Claims (9)
- A kind of 1. method that fibrin ferment is extracted from pig blood, it is characterised in that comprise the following steps：（1）Citric acid three sodium solution is added into pig blood, supernatant is collected after mixed liquor is centrifuged to layering, supernatant is double-deck Supernatant anti-freezing blood plasma is obtained after absorbent gauze filtering；（2）By step（1）The supernatant anti-freezing blood plasma of gained DEAE sepharose A-50 chromatographic columns（Referred to as A-50 is chromatographed Post）Chromatographed, the factor in supernatant anti-freezing blood plasma is adsorbed on A-50 chromatographic columns, and A- is washed with sodium chloride solution I 50 chromatographic columns, the factor being adsorbed on A-50 chromatographic columns is then eluted and activated with sodium chloride-calcium chloride mixed solution, is obtained To blood coagulation enzyme extract；（3）By step（2）724 ion exchange chromatography posts of the blood coagulation enzyme extract of gained（Referred to as resin chromatography post）Enter Row is chromatographed, and the fibrin ferment in blood coagulation enzyme extract is adsorbed on resin chromatography post, and tree is adsorbed in the elution of sodium chloride solution II Fibrin ferment on fat chromatographic column, obtain fibrin ferment refining liquid；（4）By step（3）The fibrin ferment refining liquid of gained carries out desalination and concentration after removing virus, obtains fibrin ferment concentrate；（5）By step（4）The fibrin ferment concentrate of gained freezes, and produces the powdery fibrin ferment finished product of white.
- A kind of 2. method that fibrin ferment is extracted from pig blood according to claim 1, it is characterised in that：Step（1）The dosage of citric acid three sodium solution in middle addition pig blood is that 7.0-8.0mL citric acid is added per 1L pig bloods Three sodium solutions；Step（2）The concentration of the sodium chloride solution I of middle washing A-50 chromatographic columns is 0.1-0.3M/L；Step（2）The concentration of the sodium chloride of the middle sodium chloride-calcium chloride mixed solution for eluting and activating factor is 1.5- 2.5M/L, the concentration of calcium chloride is 1.0-2.0M/L；Step（3）The concentration of the sodium chloride solution II of middle elution fibrin ferment is 2.0-3.0g/L.
- A kind of 3. method that fibrin ferment is extracted from pig blood according to claim 2, it is characterised in that：Step（1）The dosage of citric acid three sodium solution in middle addition pig blood is that 7.5mL trisodium citrate is added per 1L pig bloods Solution；Step（2）The concentration of the sodium chloride solution I of middle washing A-50 chromatographic columns is 0.2M/L；Step（2）The concentration of the sodium chloride of the middle sodium chloride-calcium chloride mixed solution for eluting and activating factor is 2.0M/L, The concentration of calcium chloride is 1.5M/L；Step（3）The concentration of the sodium chloride solution II of middle elution fibrin ferment is 2.5g/L.
- A kind of 4. method that fibrin ferment is extracted from pig blood according to Claims 2 or 3, it is characterised in that：Step（2）In Washing process carry out twice, the dosage of sodium chloride solution I is to add 24-26mL's per 1L supernatant anti-freezings blood plasma during washing every time Sodium chloride solution I.
- A kind of 5. method that fibrin ferment is extracted from pig blood according to claim 4, it is characterised in that：Step（2）In The process of washing is carried out at twice, and the dosage of sodium chloride solution I is to add 25mL's per 1L supernatant anti-freezings blood plasma during washing every time Sodium chloride solution I.
- A kind of 6. method that fibrin ferment is extracted from pig blood according to Claims 2 or 3, it is characterised in that：Step（2）In The dosage for eluting and activating the sodium chloride-calcium chloride mixed solution of factor is to add 18mL- per 100mL supernatant anti-freezings blood plasma 20mL sodium chloride-calcium chloride mixed solution.
- A kind of 7. method that fibrin ferment is extracted from pig blood according to claim 6, it is characterised in that：Step（2）In wash The dosage for taking off and activating the sodium chloride-calcium chloride mixed solution of factor is to add 19mL's per 100mL supernatant anti-freezings blood plasma Sodium chloride-calcium chloride mixed solution.
- A kind of 8. fibrin ferment preparation method according to Claims 2 or 3, it is characterised in that：Step（2）In elution and swash Operation living is carried out at twice, and the operation for eluting and activating every time obtains corresponding efflux, and the summation of efflux is solidifying twice Hemase crude extract.
- A kind of 9. method that fibrin ferment is extracted from pig blood according to Claims 2 or 3, it is characterised in that：Step（4）In Fibrin ferment refining liquid with aperture be 20nm filter membrane remove virus.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104328101A (en) * | 2014-10-27 | 2015-02-04 | 长春远大国奥制药有限公司 | Preparation method of thrombin |
| CN105624134A (en) * | 2016-02-26 | 2016-06-01 | 福建华灿制药有限公司 | Method for preparing restrictive thrombin |
| CN106474461A (en) * | 2015-08-24 | 2017-03-08 | 上海洲跃生物科技有限公司 | The method preparing freeze dried human zymoplasm |
| CN107058270A (en) * | 2017-05-08 | 2017-08-18 | 湖南格制药有限公司 | The preparation method and its production system of pig thrombiase |
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| CN104328101A (en) * | 2014-10-27 | 2015-02-04 | 长春远大国奥制药有限公司 | Preparation method of thrombin |
| CN106474461A (en) * | 2015-08-24 | 2017-03-08 | 上海洲跃生物科技有限公司 | The method preparing freeze dried human zymoplasm |
| CN105624134A (en) * | 2016-02-26 | 2016-06-01 | 福建华灿制药有限公司 | Method for preparing restrictive thrombin |
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