CN106754839A - A kind of preparation method of human urinary kallidinogenase crude product - Google Patents
A kind of preparation method of human urinary kallidinogenase crude product Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of human urinary kallidinogenase crude product, comprises the following steps：Adsorbent is collected, wash-out collects Urine proteins eluting peak；It is concentrated by ultrafiltration, regulation pH is 4~5.8, and electrical conductivity is 20~70mS/cm, obtains concentrate；Loading metal chelate affinity chromatography post, equilibrium liquid rinses metal chelate affinity chromatography post, is then eluted using water as eluent, collects human urokinase-type peptidase eluting peak；Ammonium sulfate is slowly added to, is stirred, stood, collect precipitation, obtain human urinary kallidinogenase crude product.The invention belongs to technical field of chromatography separation, the present invention improves human urinary kallidinogenase crude product yield, reduces production cost, and the human urinary kallidinogenase crude product good product quality of acquisition, KN is higher than work.
Description
Technical field
The invention belongs to technical field of chromatography separation, more particularly to a kind of preparation method of human urinary kallidinogenase crude product.
Background technology
Human urokinase-type peptidase (Urinary Kallidinogenase, KN), be from human urine separation and Extraction by 238
The glycoprotein of amino acid composition, isoelectric point is in 4 or so, molecular weight about 54000D.Human urokinase-type peptidase can activate human plasma and swash
Peptide former is converted into kassinin kinin, so as to exercise a series of physiological functions, for example, playing expansion capillary, relax vascular smooth muscle, changes
Kind microcirculation, increases the effect such as CBF, anti-freezing, thrombus dissolving, and polarity cerebral infarction is waited indefinitely with significant curative effect.
Human urine trypsin inhibitor (Urinary trypsininhibitor, UTI) is isolated and purified from human urine
The glycoprotein being made up of 143 amino acid, 2 or so, molecular weight about 65000D, human urine trypsin inhibitor has isoelectric point
Suppress the activity of the enzymes such as multiple protein enzyme, carbohydrase and lipase, so as to suppress the excessive release of inflammatory mediator, suppress systemic inflammatory
Reaction, improves microcirculation and perfused tissue, plays the effects such as protection internal organs.
The main production stage of Urine proteins product includes：Urine egg is prepared by steps such as the absorption of Urine proteins, wash-out, precipitations
White semifinished product, then Urine proteins semifinished product process is refined, obtains Urine proteins highly finished product.Adsorbent, eluent, equilibrium liquid etc. all can
Yield and purity on Urine proteins semifinished product produce influence.
Chinese patent application CN102660525 discloses a kind of method for preparing human urinary kallidinogenase crude product, including such as
Lower step：Using anion exchange resin as adsorbent, after collecting the Urine proteins in urine, the NaCl of 0.5-1.0M is used
Solution carries out concentration wash-out, and eluent loading metal chelate affinity chromatography is lived, and metal chelate affinity chromatography column equilibration liquid is
0.01-0.2M phosphate buffers, NaCl concentration is 0-2M, pH6.0-9.0, uses the 0.01-0.2M of pH4.5-2.8
Acetic acid-sodium acetate solution as wash-out solution, so as to realize the separation of KN and UTI.With it, can quick adsorption KN simultaneously
Central system, improves the advantage of the stability of KN, but the method is in anion exchange resin and metal chelate affinity chromatography post
KN yields during wash-out are all relatively low, are unfavorable for industrialized production.
Chinese patent application CN105754977 discloses a kind of method for preparing human urinary kallidinogenase crude product, including such as
Lower step：Using anion exchange resin as adsorbent adsorbing urine protein, eluted with NaCl solution, collected eluent；Will
Eluent is concentrated by ultrafiltration, and the pH value that regulation is concentrated by ultrafiltration liquid is 3.2-4.2, and conductance is 0.05-8Ms/cm, then loading is
The cationic ion-exchange resin balanced with equilibrium liquid, rinses, and equilibrium liquid is the Acetic acid-sodium acetate buffer solution of 0.01-0.5mol/L,
Conductance is 0.05-8Ms/cm, and pH value is 3.2-4.2, with elution, collects eluent, and 65% sulphur is added in eluent
Sour ammonium powder stirring, collects precipitation, obtains human urinary kallidinogenase crude product.KN yield of the method when resin anion (R.A.) is eluted compared with
It is low, it is unfavorable for industrialized production.
Therefore, the preparation method tool of simple to operate, high income, purity human urinary kallidinogenase crude product higher is researched and developed
There is important industrialization meaning.
The content of the invention
To solve there are problems that yield is dissatisfactory in the prior art, the present invention provides human urinary kallidinogenase crude product
Preparation method, is optimized to anion exchange resin, the eluent of metal chelate affinity chromatography post and equilibrium liquid etc., improves
Human urinary kallidinogenase crude product yield, reduces production cost, the human urinary kallidinogenase crude product good product quality of acquisition, KN
It is higher than work.
The present invention provides a kind of preparation method of human urinary kallidinogenase crude product, comprises the following steps：
S1 collects the adsorbent after adsorbing urine protein, and wash-out collects Urine proteins eluting peak；
The Urine proteins eluting peak that S2 obtains step S1 is concentrated by ultrafiltration, regulation pH be 4~5.8, electrical conductivity be 20~
70mS/cm, obtains concentrate；
The concentrate loading metal chelate affinity chromatography post that S3 obtains step S2, equilibrium liquid rinses the affine layer of metal-chelating
Analysis post, is then eluted using water as eluent, collects human urokinase-type peptidase eluting peak；The equilibrium liquid by 0.5~
The phosphate buffer of 2mol/L and the acetum of 0.01~0.1mol/L are with 3~5：1 volume ratio composition, the pH of equilibrium liquid
It is 4~5.8；
Ammonium sulfate is slowly added in the human urokinase-type peptidase eluting peak that S4 is obtained to step S3, is stirred, stood, collect heavy
Form sediment, obtain human urinary kallidinogenase crude product.
Using above-mentioned technical proposal, it is possible to achieve efficiently collect and KN and UTI is efficiently separated, improve human urine kassinin kinin
Protoenzyme semifinished product yield, reduces production cost, and the semifinished product good product quality of acquisition, KN is higher than work.
Preferably, in the step S2, regulation pH is 4.5~5.5, and electrical conductivity is 25~60mS/cm.
Preferably, in the step S3, equilibrium liquid by the phosphate buffer of 0.8~1.2mol/L and 0.03~
The acetum of 0.5mol/L is with 3~4：1 volume ratio composition, the pH of equilibrium liquid is 4.2~5.0.
Preferably, in the step S3, equilibrium liquid by 1.0~1.2mol/L phosphate buffer and 0.1~0.3mol/
The acetum of L is with 4：1 volume ratio composition, the pH of equilibrium liquid is 4.4~4.8, and electrical conductivity is 40~55mS/cm.
Preferably, in the step S1, adsorbent is anion exchange resin.It is highly preferred that anion exchange resin is
Strong basic type anion-exchange resin, such as：Macroporous type strong anion exchange resin, strong base anion exchange column Q Sepharose
H.p、Q Sepharose F.F、Q Sepharose 4F.F、Q Sepharose XL、QAE Sephadex A-25、QAE
Sephadex A-50 or STREAMLINE.The macroporous type strong anion exchange resin CAS is 9037-24-5.
Preferably, in the step S1, eluent is the phosphate buffer of 0.3~2mol/L, and pH is 4~6, NaCl's
Concentration is 0.01~0.45mol/L.
Preferably, the metal ion of the metal chelate affinity chromatography post is Cu2+、Zn2+Or Ni2+。
Preferably, the metal ion of the metal chelate affinity chromatography post is Cu2+。
Compared with prior art, the beneficial effects of the invention are as follows：By the present invention in that with 0.3~2mol/L that pH is 4~6
Phosphate buffer as anion exchange resin eluent, it is possible to achieve KN from the abundant wash-out in adsorbent, wash-out
Yield is improved；By using the phosphate buffer of 0.5~2mol/L and the acetum of 0.01~0.1mol/L with 3~5：1
Volume ratio constitute metal chelate affinity chromatography post equilibrium liquid, water is used as eluent, it is possible to achieve KN and UTI efficiently point
From, human urinary kallidinogenase crude product yield is improve, KN and UTI subsequently refined difficulty is greatly reduced, reduce and be produced into
This, the human urinary kallidinogenase crude product good product quality of acquisition, KN is higher than work.
Brief description of the drawings
The HPLC of the human urokinase-type peptidase eluting peak in Fig. 1 embodiment of the present invention one through affinity chromatography schemes.
Specific embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.
The preparation of the human urinary kallidinogenase crude product of embodiment one
The preparation method of human urinary kallidinogenase crude product comprises the following steps：Collect the moon after adsorbing urine protein and water flushing
Ion exchange column Q Sepharose F.F 100kg, wash-out, eluent is the phosphate buffer of 0.5mol/L that pH is 4.5,
The concentration of NaCl is 0.3mol/L, collects Urine proteins eluting peak；The Urine proteins eluting peak that will be obtained is concentrated by ultrafiltration, and adjusts pH
It is 4.5, electrical conductivity is 40mS/cm, obtains concentrate；The Cu that the concentrate loading that will be obtained has been balanced2+Metal-chelating is affine
Chromatographic column (Chelating sepharose Fast Flow, purchased from General Electric's Medical Group (GE companies, GE
Healthcare), equilibrium liquid rinse metal chelate affinity chromatography post, equilibrium liquid by 1.0mol/L phosphate buffer and
The acetum of 0.15mol/L is with 4：1 volume ratio composition, the pH of equilibrium liquid is 4.5, and electrical conductivity is 40mS/cm, is then used
Deionized water is eluted as eluent, collects human urokinase-type peptidase eluting peak, carries out HPLC detections, as a result as shown in Figure 1；
To ammonium sulfate is slowly added in the human urokinase-type peptidase eluting peak for obtaining, stirring to saturation stands, and adds diatomite 5g, collects
Precipitation, obtains human urinary kallidinogenase crude product (KN semifinished products) 41g.Collection loading penetrates liquid and flushing penetrates liquid, is slowly added to
Ammonium sulfate, stirring to saturation stands, and adds diatomite 20g, collects precipitation, obtains human urine trypsin inhibitor semifinished product
(UTI semifinished products) 303g.
From fig. 1, it can be seen that the human urokinase-type peptidase eluting peak that the equilibrium liquid provided through the present embodiment and elution are collected
Human urokinase-type peptidase content be up to 69.02%, and the content of human urine trypsin inhibitor (corresponding retention time is 8.5)
It is low.
The preparation of the human urinary kallidinogenase crude product of embodiment two
The preparation method of human urinary kallidinogenase crude product comprises the following steps：It is big after collection adsorbing urine protein and water flushing
Pass strong anion exchange resin 100kg, wash-out, eluent is the phosphate buffer of 1mol/L that pH is 4, the concentration of NaCl
It is 0.1mol/L, collects Urine proteins eluting peak；The Urine proteins eluting peak that will be obtained is concentrated by ultrafiltration, and regulation pH is 4.5, conductance
Rate is 40mS/cm, obtains concentrate；The Cu that the concentrate loading that will be obtained has been balanced2+Metal chelate affinity chromatography post
(Chelating sepharose Fast Flow, purchased from General Electric's Medical Group (GE companies, GE Healthcare)), puts down
Weighing apparatus liquid rinses metal chelate affinity chromatography post, and equilibrium liquid is molten by the phosphate buffer of 1.0mol/L and the acetic acid of 0.15mol/L
Liquid is with 4：1 volume ratio composition, the pH of equilibrium liquid is 4.5, and electrical conductivity is 40mS/cm, then using deionized water as wash-out
Liquid is eluted, and collects human urokinase-type peptidase eluting peak；To being slowly added to ammonium sulfate in the human urokinase-type peptidase eluting peak for obtaining,
Stirring stands to saturation, adds diatomite 5g, collects precipitation, obtains human urinary kallidinogenase crude product 47g.Loading is collected to penetrate
Liquid and flushing penetrate liquid, are slowly added to ammonium sulfate, and stirring to saturation stands, and adds diatomite 20g, collects precipitation, obtains human urine
Trypsin inhibitor semifinished product 327g.
The preparation of the human urinary kallidinogenase crude product of comparative example one
The preparation method of human urinary kallidinogenase crude product comprises the following steps：Collect the moon after adsorbing urine protein and water flushing
Ion exchange column Q Sepharose F.F 100kg, wash-out, eluent is the NaCl solution of 0.8mol/L, collects Urine proteins and washes
De- peak；The Urine proteins eluting peak that will be obtained is concentrated by ultrafiltration, and regulation pH is 4.5, and electrical conductivity is 40mS/cm, obtains concentrate；
The Cu that the concentrate loading that will be obtained has been balanced2+Metal chelate affinity chromatography post (Chelating sepharose Fast
Flow, purchased from General Electric's Medical Group (GE companies, GE Healthcare)), equilibrium liquid rinses metal chelate affinity chromatography
Post, equilibrium liquid is by the phosphate buffer of 1.0mol/L and the acetum of 0.15mol/L with 4：1 volume ratio composition, balance
The pH of liquid is 4.5, and electrical conductivity is 40mS/cm, is then eluted using deionized water as eluent, collects human urine kininogen
Enzyme eluting peak；To ammonium sulfate is slowly added in the human urokinase-type peptidase eluting peak for obtaining, stirring to saturation stands, and adds diatom
Native 5g, collects precipitation, obtains human urinary kallidinogenase crude product 22g.Collection loading penetrates liquid and flushing penetrates liquid, is slowly added to sulphur
Sour ammonium, stirring to saturation stands, and adds diatomite 20g, collects precipitation, obtains human urine trypsin inhibitor semifinished product 282g.
Comparative example one is with the difference of embodiment one：The NaCl that the eluent of anion-exchange column is changed to 0.8mol/L is molten
Liquid.
The preparation of the human urinary kallidinogenase crude product of comparative example two
The preparation method of human urinary kallidinogenase crude product comprises the following steps：It is big after collection adsorbing urine protein and water flushing
Pass strong anion exchange resin 100kg, wash-out, eluent is the phosphate buffer of 0.5mol/L that pH is 4.5, NaCl's
Concentration is 0.3mol/L, collects Urine proteins eluting peak；The Urine proteins eluting peak that will be obtained is concentrated by ultrafiltration, and regulation pH is 8.0,
Electrical conductivity is 2.2mS/cm, obtains concentrate；The Cu that the concentrate loading that will be obtained has been balanced2+Metal chelate affinity chromatography post
(Chelating sepharose Fast Flow, purchased from General Electric's Medical Group (GE companies, GE Healthcare)), puts down
Weighing apparatus liquid rinses metal chelate affinity chromatography post, and the pH of equilibrium liquid is 8.0, phosphate buffer and 0.2mol/ containing 0.02mol/L
The NaCl solution of L, is then eluted using the Acetic acid-sodium acetate buffer solution of the 20mmol/L of pH3.8 as eluent, is received
Collection human urokinase-type peptidase eluting peak；To ammonium sulfate is slowly added in the human urokinase-type peptidase eluting peak for obtaining, stir to saturation, it is quiet
Put, add diatomite 5g, collect precipitation, obtain human urinary kallidinogenase crude product 24g.Collection loading penetrates liquid and flushing is penetrated
Liquid, is slowly added to ammonium sulfate, and stirring to saturation stands, and adds diatomite 20g, collects precipitation, obtains human urinary trypsin suppression
Agent semifinished product 296g.
Comparative example two is with the difference of embodiment one：The Urine proteins eluting peak that will be obtained is concentrated by ultrafiltration, and adjusts pH
It is 8.0, electrical conductivity is 2.2mS/cm, obtains concentrate；The pH of equilibrium liquid is 8.0, phosphate buffer containing 0.02mol/L and
The NaCl solution of 0.2mol/L；Using pH3.8 20mmol/L Acetic acid-sodium acetate buffer solution as eluent.
Test example：The detection of human urinary kallidinogenase crude product and human urine trypsin inhibitor semifinished product
Embodiment one, embodiment two, comparative example one, KN semifinished products obtained in comparative example two and UTI semifinished products are made respectively
Detected with fluorescence method, as a result as shown in table 1.
The KN semifinished products and UTI semifinished product testing result tables of the different embodiments of table 1
As known from Table 1, embodiment one, the ratio of KN is lived as the ratio of UTI in 5.8, UTI semifinished products is lived as 389 in KN semifinished products
Embodiment two, the ratio of KN is lived for the ratio work of UTI in 6.2, UTI semifinished products is 426 comparative examples one, in KN semifinished products in KN semifinished products
It is 139 comparative examples two that the ratio of KN is lived and lived for the ratio of UTI in 0.8, UTI semifinished products, and the ratio of KN is lived as 1.1, UTI is thick in KN semifinished products
It is 152 that the ratio of UTI is lived in product
Therefore, the preparation method of the human urinary kallidinogenase crude product that the present invention is provided can realize the efficient wash-out of KN, realize
KN semifinished products and UTI semifinished products are efficiently separated, and UTI is concentrated in UTI semifinished products substantially, and KN concentrates on KN semifinished products substantially
In, refined beneficial to KN semifinished products and the follow-up of UTI semifinished products.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
Specific implementation of the invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should be all considered as belonging to of the invention
Protection domain.
Claims (9)
1. a kind of preparation method of human urinary kallidinogenase crude product, it is characterised in that：Comprise the following steps：
S1 collects the adsorbent after adsorbing urine protein, and wash-out collects Urine proteins eluting peak；
The Urine proteins eluting peak that S2 obtains step S1 is concentrated by ultrafiltration, and regulation pH is 4~5.8, and electrical conductivity is 20~70mS/
Cm, obtains concentrate；
The concentrate loading metal chelate affinity chromatography post that S3 obtains step S2, equilibrium liquid rinses metal chelate affinity chromatography
Post, is then eluted using water as eluent, collects human urokinase-type peptidase eluting peak；The equilibrium liquid is by 0.5~2mol/
The phosphate buffer of L and the acetum of 0.01~0.1mol/L are with 3~5：1 volume ratio composition, the pH of equilibrium liquid for 4~
5.8；
Ammonium sulfate is slowly added in the human urokinase-type peptidase eluting peak that S4 is obtained to step S3, is stirred, stood, collect precipitation, obtained
To human urinary kallidinogenase crude product.
2. the preparation method of human urinary kallidinogenase crude product according to claim 1, it is characterised in that：The step S2
In, regulation pH is 4.5~5.5, and electrical conductivity is 25~60mS/cm.
3. the preparation method of human urinary kallidinogenase crude product according to claim 1, it is characterised in that：The step S3
In, equilibrium liquid is by the phosphate buffer of 0.8~1.2mol/L and the acetum of 0.03~0.5mol/L with 3~4：1 body
Than composition, the pH of equilibrium liquid is 4.2~5.0 to product.
4. the preparation method of human urinary kallidinogenase crude product according to claim 3, it is characterised in that：The step S3
In, equilibrium liquid is by the phosphate buffer of 1.0~1.2mol/L and the acetum of 0.1~0.3mol/L with 4：1 volume ratio
Composition, the pH of equilibrium liquid is 4.4~4.8, and electrical conductivity is 40~55mS/cm.
5. the preparation method of human urinary kallidinogenase crude product according to claim 1, it is characterised in that：The step S1
In, adsorbent is anion exchange resin.
6. the preparation method of human urinary kallidinogenase crude product according to claim 5, it is characterised in that：The step S1
In, adsorbent is macroporous type strong anion exchange resin.
7. the preparation method of human urinary kallidinogenase crude product according to claim 1, it is characterised in that：The step S1
In, eluent is the phosphate buffer of 0.3~2mol/L, and pH is 0.01~0.45mol/L for the concentration of 4~6, NaCl.
8. the preparation method of human urinary kallidinogenase crude product according to claim 1, it is characterised in that：The metal-chelating
The metal ion of affinity column is Cu2+、Zn2+Or Ni2+。
9. the preparation method of human urinary kallidinogenase crude product according to claim 8, it is characterised in that：The metal-chelating
The metal ion of affinity column is Cu2+。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109091667A (en) * | 2018-09-18 | 2018-12-28 | 广东天普生化医药股份有限公司 | Purposes and combinations thereof of the human urokinase-type peptidase in preparation treatment migraine remedy |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660525A (en) * | 2012-05-15 | 2012-09-12 | 扬州艾迪生物科技有限公司 | Method for preparing human urinary kallidinogenase crude product |
| CN105754977A (en) * | 2016-04-05 | 2016-07-13 | 广东天普生化医药股份有限公司 | Method for preparing crude human urine kininogenase product |
-
2016
- 2016-12-02 CN CN201611101691.XA patent/CN106754839A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660525A (en) * | 2012-05-15 | 2012-09-12 | 扬州艾迪生物科技有限公司 | Method for preparing human urinary kallidinogenase crude product |
| CN105754977A (en) * | 2016-04-05 | 2016-07-13 | 广东天普生化医药股份有限公司 | Method for preparing crude human urine kininogenase product |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109091667A (en) * | 2018-09-18 | 2018-12-28 | 广东天普生化医药股份有限公司 | Purposes and combinations thereof of the human urokinase-type peptidase in preparation treatment migraine remedy |
| CN109091667B (en) * | 2018-09-18 | 2020-05-05 | 广东天普生化医药股份有限公司 | Application of human urinary kallidinogenase in preparing medicine for treating migraine and composition thereof |
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Application publication date: 20170531 |
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| RJ01 | Rejection of invention patent application after publication |