CN104498464A - Method for extracting agkistrodon snake venom hemocoagulase from gloydius blomhoffi brevicaudus venom - Google Patents
Method for extracting agkistrodon snake venom hemocoagulase from gloydius blomhoffi brevicaudus venom Download PDFInfo
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Abstract
The invention provides a method for extracting agkistrodon snake venom hemocoagulase with a relatively high titer from gloydius blomhoffi brevicaudus venom. The method comprises the following steps: sequentially carrying out DEAE-Sephadex A-50 (diethyl aminoethyl-glucan A-50) ion-exchange column chromatography, refrigeration, basic hydrolysis and Sephadex G-75 gel filtration chromatography on the gloydius blomhoffi brevicaudus venom of the changbai mountain, and then collecting protein with coagulation activity. Compared with the prior art, according to the method disclosed by the invention, the extraction step of the agkistrodon snake venom hemocoagulase is simplified; the production cycle is shortened; the titer and the yield of the product can be improved; and the production cost is greatly reduced.
Description
Technical field
The present invention relates to a kind of method extracting white-browed snake venom blood coagulation enzyme from agkistrodon halys ussuriensis snake venom.
Background technology
Blood coagulation is the physiological process of a more complicated, is substantially divided into three phases: the first stage, and factor X (coagulation factor X, Fx) activates; Subordinate phase, PROTHROMBIN ACTIVATOR; Phase III, Fibrinogen becomes scleroproein.
From snake venom, extract hemostatic drug have a long history.At present, the hemostatic drug Reptilase that clinical application comparatively successfully has Switzerland to produce, i.e. reptilase, it extracts from Brazilian spearhead abdomen calmy poison.The product of domestic imitative Reptilase has from agkistrodon shedaoensis, from the snake venom of circle spot adder, agkistrodon acutus and Zhejiang pallas pit viper, extracts the zymoplasm obtained, these products and reptilase are all the mixtures of Thrombin-like enzyme and a small amount of clauden (a kind of factor X activator), dominant mechanism is that Thrombin-like enzyme can make the monomer crosslinked fibroblast cells of Fibrinogen I, thus forms blood clot realization hemostasis.
Before this, contriver is successful extracts a kind of white-browed snake venom blood coagulation enzyme from agkistrodon halys ussuriensis snake venom, and establishes complete technical scheme (see ZL200710099163.X).In the technical scheme that this patent provides, after first time DEAE-Sephadex A-50, electrophoretic analysis is presented at below white-browed snake venom blood coagulation enzyme principal constituent (Thrombin-like enzyme) electrophoresis band that obtains an interference fringe very nearby, is impurity component.Sephadex G-15 gel permeation chromatography below, DEAE-Sephadex A-50 again chromatography mainly in order to remove this impurity.
Summary of the invention
The object of this invention is to provide a kind of method extracting higher white-browed snake venom blood coagulation enzyme of tiring from agkistrodon halys ussuriensis snake venom.
The method of extraction white-browed snake venom blood coagulation enzyme provided by the present invention, the albumen with blood coagulation activity collected after comprising the steps: Agkistrodon halys ussurriensisEmelianov snake venom to be carried out successively DEAE-Sephadex A-50 (diethylamino ethyl-dextran A-50) ion exchange chromatography, refrigeration, alkaline hydrolysis and Sephadex G-75 gel permeation chromatography.
Concrete steps are as follows:
1) Agkistrodon halys ussurriensisEmelianov snake venom is dissolved in the Tris-HCl damping fluid of pH 7-8,0.04-0.06mol/L, obtains the agkistrodon halys ussuriensis snake venom that concentration is 200-300g/L;
2) to step 1) the Agkistrodon halys ussurriensisEmelianov snake venom that obtains carries out ion-exchange chromatography; In described ion-exchange chromatography, solid phase weighting material is DEAE-SephadexA-50, and elutriant is: NaCl concentration is the Tris-HCl damping fluid of pH 7-8,0.04-0.06mol/L of 0-0.75mol/L, and the type of elution adopted is gradient elution;
3) collect step 2) in elutriant; Under the wavelength of 280nm, UV spectrophotometer measuring is carried out to described elutriant, there is under being collected in the wavelength of 280nm the elutriant of absorption peak, and blood coagulation activity detection is carried out to this elutriant, merge the elutriant with blood coagulation activity;
4) to described have in the elutriant of blood coagulation activity add appropriate bovine serum albumin, then mixed solution is refrigerated 4 hours at 4 DEG C of environment, obtain refrigerate after mixed solution;
5) alkaline hydrolysis: slowly add NaOH in the mixed solution after refrigeration, make the concentration of NaOH be 0.05mol/L, then reacts 15-17 hours in 37.5 ± 0.5 DEG C of environment;
6) freeze-drying: by step 5) reaction after gained solution carry out lyophilize, obtain lyophilized powder;
7) by step 6) the lyophilized powder water for injection that obtains redissolves;
8) redissolution liquid is carried out gel permeation chromatography, in described gel permeation chromatography, solid phase weighting material is Sephadex G-75, and elutriant is water for injection, collects elutriant, therefrom collects the part with blood coagulation activity, obtains white-browed snake venom blood coagulation enzyme stoste.
In the extracting method of above-mentioned hemocoagulase, step 1) in be preferably the Tris-HCl damping fluid of pH 7.5,0.05mol/L for the solvent dissolving Agkistrodon halys ussurriensisEmelianov snake venom; In addition, for obtaining better implementation result, first can remove the impurity in agkistrodon halys ussuriensis snake venom by centrifugal method, centrifugal condition can be selected according to practical situation, as centrifugal 10 minutes at 3,000 rpm.
Step 2) in DEAE-Sephadex A-50 chromatography column before use, first with the Tris-HCl damping fluid balance 15-20 hour of pH 7-8,0.04-0.06mol/L, flow velocity is 0.8-1.0mL/min; During chromatography, move to completely after below glue face until agkistrodon halys ussuriensis snake venom, the damping fluid consistent with balance liquid is added above glue face, then carry out linear gradient wash-out with the Tris-HCl damping fluid of pH 7-8,0.04-0.06mol/L and the Tris-HCl damping fluid of isopyknic pH 7-8,0.04-0.06mol/L containing 0.75mol/L NaCl, flow velocity is 0.8-1.0mL/min; Described step 2) in Tris-HCl damping fluid be preferably the Tris-HCl damping fluid of pH 7.5,0.05mol/L.
Step 3) in collection method can be: with automatic fraction collector collect, often pipe 12-15mL; The detection method with the collection part of blood coagulation activity can be: by equal-volume (preferred 0.5mL) and be preheated to the collection liquid of 37 DEG C and blood coagulation Quality Control blood plasma is mixed be incorporated in 37 DEG C at water-bath, collection unit that blood plasma solidified in 20 seconds can be made to be divided into have blood coagulation activity.
Step 4) in the final concentration of bovine serum albumin that adds be 1.2-1.5mg/ml.
Adding bovine serum albumin in this step is in order to during next step alkaline hydrolysis, containment objective material (Thrombin-like enzyme and clauden) is not destroyed.This is that tertiary structure can change with temperature change because bovine serum albumin has hydrophilic group and hydrophobic grouping, and hydrophilic radical is united and Thrombin-like enzyme and clauden can be protected, from destruction; And glycosylated albumen can not be embedded because wetting ability changes.
Step 5) in add NaOH and must slowly add, limit edged stirs gently, and whole dissolution process can not make water temperature more than 40 DEG C.
Step 8) in Sephadex G-75 chromatography column before use, first with injection water balance 15-20 hour, flow velocity is 0.8-1.0mL/min; During chromatography, move to completely after below glue face until supernatant liquor, jetting of annotating above glue face, then uses injection water wash-out, and flow velocity is 0.8-1.0mL/min; With with step 3) identical method collects the part in elutriant with blood coagulation activity.
The white-browed snake venom blood coagulation enzyme prepared according to the inventive method also belongs to protection scope of the present invention.
Described white-browed snake venom blood coagulation enzyme has following characteristics: (1) is become to be grouped into clauden (molecular weight is 69000D) two kinds by Thrombin-like enzyme class material (molecular weight is 34000D), the content that high performance liquid chromatography records Thrombin-like enzyme material is 84-88%, and the content of clauden is 12-16%; (2) 15 amino acids residue sequence of main component Thrombin-like enzyme class material n end are as shown in the sequence 1 in sequence table.
Described white-browed snake venom blood coagulation enzyme can be used for preparing hemostatic drug and/or treatment hemorrhagic diseases medicine.
Another object of the present invention is to provide the medicine of a kind of hemostasis and/or treatment hemorrhagic diseases.
The activeconstituents of medicine provided by the present invention is above-mentioned white-browed snake venom blood coagulation enzyme.
When needing, can also add one or more pharmaceutically acceptable auxiliary materials in said medicine, described auxiliary material comprises the thinner of pharmaceutical field routine, vehicle, weighting agent, tackiness agent, wetting agent, absorption enhancer, tensio-active agent, lubricant and stablizer etc.
Medicine of the present invention can make the various ways such as lyophilized injectable powder, injection liquid or tourniquet bandage.
The medicine of above-mentioned various formulation all can be prepared according to the ordinary method of pharmaceutical field.
Adult's consumption of said medicine is generally 1-8KU/60kg body weight/day, can use by one or many.
The invention provides a kind of method hemocoagulase extracting higher white-browed snake venom blood coagulation enzyme of tiring from Agkistrodon halys ussurriensisEmelianov snake venom.Contriver is by studying discovery further to the method for 200710099163.X, and below white-browed snake venom blood coagulation enzyme principal constituent (Thrombin-like enzyme) electrophoresis band that it obtains, interference fringe very is nearby glycosylated Thrombin-like enzyme, is glycoprotein; And by complete for the glycosyl of this glycoprotein release, then the Thrombin-like enzyme obtained can also structure activity recovery.Based on this discovery, present invention improves over original technical scheme, simplify extraction step, and make glycoprotein discharge glycosyl by a step alkali lye hydrolysis, add the content of Thrombin-like enzyme.The inventive method simplifies the extraction step of white-browed snake venom blood coagulation enzyme, shortens the production cycle, and can improve tiring and yield of product, reduces the production cost chromatography glue of the selection (can Reusability) significantly.
Accompanying drawing explanation
Fig. 1 is the electrophoretic analysis result of the committed step sampling in the present invention and prior art (ZL200710099163.X) scheme leaching process.
Fig. 2 is bovine serum albumin consumption the selection result figure.
Fig. 3 is the high performance liquid chromatography detected result of Thrombin-like enzyme and clauden content in the white-browed snake venom blood coagulation enzyme prepared of comparative example.
Fig. 4 is the high performance liquid chromatography detected result that embodiment 1 prepares Thrombin-like enzyme and clauden content in white-browed snake venom blood coagulation enzyme.
Embodiment
Below by specific embodiment, method of the present invention is described, but the present invention is not limited thereto.
Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
The Agkistrodon halys ussurriensisEmelianov lyophilized venom used in following embodiment is purchased from Shenyang letter moral snake class cultivation company limited; Bovine serum albumin is purchased from Shanghai one and grinds bio tech ltd, and its specification is that 1g/ props up.
Embodiment 1, extraction white-browed snake venom blood coagulation enzyme
From Agkistrodon halys ussurriensisEmelianov snake venom, extract hemocoagulase by following method, detailed process comprises the following steps:
1) Agkistrodon halys ussurriensisEmelianov lyophilized venom (being purchased from Shenyang letter moral snake class cultivation company limited) 10g is dissolved in the Tris-HCl damping fluid of 40mL pH 7.5,0.05mol/L, obtain the agkistrodon halys ussuriensis snake venom that concentration is 250g/L, again by agkistrodon halys ussuriensis snake venom centrifugal 10 minutes at 3,000 rpm, discard precipitation, retain supernatant liquor.
2) to step 1) the Agkistrodon halys ussurriensisEmelianov snake venom that obtains carries out ion-exchange chromatography, method is: first by DEAE-Sephadex A-50 chromatography column (post specification 100 × 5cm before chromatography, purchased from Guangzhou Shen Hua Bioisystech Co., Ltd) balance 15-20 hour with the Tris-HCl damping fluid of pH 7.5,0.05mol/L, flow velocity is 0.8-1.0mL/min, the glue height of bed >=90cm; During chromatography, move to completely after below glue face until snake venom supernatant liquor, pH7.5,0.05mol/L Tris-HCl damping fluid 50mL is added above glue face, then with the Tris-HCl damping fluid of 1000mL pH7.5,0.05mol/L with isopyknicly carry out linear gradient wash-out containing the pH 7.5 of NaCl 0.75mol/L, the Tris-HCl damping fluid of 0.05mol/L, flow velocity is 1.0mL/min.
3) elutriant is collected with automatic fraction collector, often pipe 12-15mL, under the wavelength of 280nm, UV spectrophotometer measuring is carried out to elutriant, obtain wavelength 280nm and absorb collection of illustrative plates, collect the part with blood coagulation activity, method is: water-bath at the 0.5mL being preheated to 37 DEG C being collected liquid and 0.5mL blood coagulation Quality Control blood plasma (Pacific Ocean Reagent Company of the U.S.) are mixed and being incorporated in 37 DEG C, collection unit that blood plasma solidified in 20 seconds can be made to be divided into have blood coagulation activity.Collect liquid measure and be about 300mL, gross activity about 18000 unit (KU).
4) in collection liquid, add bovine serum albumin 400mg, after dissolving mixing, collection liquid is refrigerated 4 hours at 4 DEG C of environment.
5) alkaline hydrolysis: slowly add NaOH 600mg (making final concentration reach 0.05mol/L) in the solution after refrigeration, slowly add, limit edged stirs gently, allows heat distribute, and attention can not make temperature more than 40 DEG C; Then react 16 hours in 37 DEG C of environment;
6) freeze-drying: collection liquid is put into freeze drier lyophilize, obtains lyophilized powder.
7) by step 6) the lyophilized powder 20mL water for injection that obtains redissolves.
8) to step 7) the redissolution liquid that obtains carries out gel permeation chromatography, method is: before chromatography, first by Sephadex G-75 chromatography column, (specification of post is 100 × 5cm, purchased from Guangzhou Shen Hua Bioisystech Co., Ltd) balance 15-20 hour with injection water, flow velocity is 0.8-1.0mL/min, the glue height of bed >=90cm; During chromatography, move to completely after below glue face until supernatant liquor, annotate jetting 50mL above glue face, then uses 2000mL injection water wash-out, and flow velocity is 1.0mL/min; With with step 3) identical method collects the part in elutriant with blood coagulation activity, obtains about 100mL white-browed snake venom blood coagulation enzyme stoste, gross activity about 15000 unit.
According to the active [determination of 1 unit (KU) white-browed snake venom blood coagulation enzyme: the 1mL white-browed snake venom blood coagulation enzyme solution being preheated to 37 DEG C is mixed with 1mL blood coagulation Quality Control blood plasma (U.S.'s Pacific Ocean reagent is produced), at 37 DEG C, 1mL blood coagulation Quality Control blood plasma can be made to solidify within 60-80 second, and the amount of white-browed snake venom blood coagulation enzyme contained in the 1mL solution before mixing is 1 unit (KU).], with distilled water, stoste is diluted to 40 units (KU)/mL, obtains white-browed snake venom blood coagulation enzyme concentrated solution.
In said process, in step 3), step 5), step 8) after sample respectively, be designated as sample 1, sample 2, sample 3 respectively.
White-browed snake venom blood coagulation enzyme (being set to comparative example) is prepared according to the experimentation of embodiment in ZL200710099163.X 1.
Detailed process comprises the following steps:
1) Agkistrodon halys ussurriensisEmelianov lyophilized venom (being purchased from Shenyang letter moral snake class cultivation company limited) 10g is dissolved in the Tris-HCl damping fluid of 40mL pH 7.5,0.05mol/L, obtain the agkistrodon halys ussuriensis snake venom that concentration is 250g/L, again by agkistrodon halys ussuriensis snake venom centrifugal 10 minutes at 3,000 rpm, discard precipitation, retain supernatant liquor.
2) to step 1) the Agkistrodon halys ussurriensisEmelianov snake venom that obtains carries out ion-exchange chromatography, method is: first by DEAE-Sephadex A-50 chromatography column (post specification 100 × 5cm before chromatography, purchased from Guangzhou Shen Hua Bioisystech Co., Ltd) balance 15-20 hour with the Tris-HCl damping fluid of pH 7.5,0.05mol/L, flow velocity is 1.0mL/min, glue height of bed 90cm; During chromatography, move to completely after below glue face until snake venom supernatant liquor, pH7.5,0.05mol/L Tris-HCl damping fluid 50mL is added above glue face, then carry out linear gradient wash-out by the Tris-HCl damping fluid of 1000mL pH7.5,0.05mol/L and equivalent containing the pH 7.5 of NaCl 0.75mol/L, the Tris-HCl damping fluid of 0.05mol/L, flow velocity is 1.0mL/min.
3) elutriant is collected with automatic fraction collector, often pipe 12-15mL, under the wavelength of 280nm, UV spectrophotometer measuring is carried out to elutriant, obtain wavelength 280nm and absorb collection of illustrative plates, collect the part with blood coagulation activity, method is: water-bath at the 0.5mL being preheated to 37 DEG C being collected liquid and 0.5mL blood coagulation Quality Control blood plasma (Pacific Ocean Reagent Company of the U.S.) are mixed and being incorporated in 37 DEG C, collection unit that blood plasma solidified in 20 seconds can be made to be divided into have blood coagulation activity.Collect liquid measure and be about 300mL, gross activity about 18000 unit (KU).
4) chromatography desalination: to step 3) the collection liquid with blood coagulation activity that obtains carries out gel permeation chromatography, method is: before chromatography, first by Sephadex G-15 chromatography column, (specification of post is 100 × 5cm, purchased from Guangzhou Shen Hua Bioisystech Co., Ltd) balance 15-20 hour with water for injection, flow velocity is 1.0mL/min, glue height of bed 80cm; During chromatography, move to completely after below glue face until supernatant liquor, annotate jetting 50mL above glue face, then uses 2000mL injection water wash-out, and flow velocity is 1.0mL/min; With with step 3) identical method collects the part in elutriant with blood coagulation activity, result obtains collects liquid and is about 180mL, and gross activity is about 15000KU.
5) chromatography again: regulating step 4) pH value to 5.2 with the collection liquid of blood coagulation activity of collecting, then ion-exchange chromatography is carried out to it, method is: before chromatography, first by DEAE-Sephadex A-50 chromatography column, (specification of post is 100 × 5cm, purchased from Guangzhou Shen Hua Bioisystech Co., Ltd) balance 15-20 hour with the Tris-HCl damping fluid of pH5.2,0.05mol/L, flow velocity is 0.8-1.0mL/min, glue height of bed 90cm; During chromatography, due-in liquid collecting moves to after below glue face completely, pH5.2,0.05mol/L Tris-HCl damping fluid 50mL is added above glue face, then carry out linear gradient wash-out with the Tris-HCl damping fluid of the Tris-HCl damping fluid of 1000mL pH5.2,0.05mol/L and pH5.2,0.05mol/L containing NaCl 0.25mol/L of equivalent, flow velocity is 1.0mL/min; With with step 3) identical method collects the part in elutriant with blood coagulation activity, result obtains collects liquid and is about 150mL, and gross activity is about 14000KU.
6) freeze-drying; Collection liquid is put into freeze drier lyophilize, obtains lyophilized powder.
7) by step 6) the lyophilized powder 20mL water for injection that obtains redissolves.
8) to step 7) the redissolution liquid that obtains carries out gel permeation chromatography, method is: before chromatography, first by Sephadex G-75 chromatography column, (specification of post is 100 × 5cm, purchased from Guangzhou Shen Hua Bioisystech Co., Ltd) balance 15-20 hour with injection water, flow velocity is 0.8-1.0mL/min, glue height of bed 90cm; During chromatography, move to completely after below glue face until supernatant liquor, annotate jetting 50mL above glue face, then uses 2000mL injection water wash-out, and flow velocity is 1.0mL/min; With with step 3) identical method collects the part in elutriant with blood coagulation activity, obtains about 100mL white-browed snake venom blood coagulation enzyme stoste, gross activity about 12000 unit.
Operate in strict accordance with aforesaid method, in step 3), step 5), step 8) after sample respectively, be designated as sample 4, sample 5, sample 6 respectively.
Above-mentioned sample 1-sample 6 is conventionally done SDS-polyacrylamide gel electrophoresis analysis, and gained electrophoresis result as shown in Figure 1.
As can be seen from electrophorogram, after first time DEAE-Sephadex A-50 chromatography, an assorted band (see sample 1 and sample 4) is had above the principal constituent of 34000D, this assorted band then removes (see sample 2) through alkali lye hydrolysis by the present invention, and also this assorted band can be removed (see sample 5) after second time DEAE-Sephadex A-50 chromatography in the scheme that patent 200710099163.X provides.Sample 2 occurs that at 65000D place the band had more is the bovine serum albumin added.
Embodiment 2 bovine serum albumin consumption screens
According to the scheme revision test in embodiment 1, fix other conditions, change the consumption of bovine serum albumin, to determine optimum amount, result as shown in Figure 2.
Test-results shows, ox blood albumin consumption is too high or too low, all can affect the yield of snake venom blood coagulation enzyme, and optimum concn is 1.2-1.5mg/ml.
The determination of activity of embodiment 3, white-browed snake venom blood coagulation enzyme
One, blood coagulation Quality Control PCT measures
The white-browed snake venom blood coagulation enzyme concentrated solution (40 units (KU)/mL) embodiment 1 obtained is diluted with distilled water into different concentration (white-browed snake venom blood coagulation enzyme content be respectively 5,2,1,0.8,0.6 and 0.4KU/mL), then the diluent 0.2mL of each concentration of preheating at 37 DEG C is got respectively, mix with preheating blood coagulation Quality Control blood plasma (U.S.'s Pacific Ocean reagent is produced) under 37 DEG C of conditions respectively, under 37 DEG C of conditions, record PCT.Result is as shown in table 1, then with the inverse of white-browed snake venom blood coagulation enzyme content (KU/mL) for X-coordinate, with blood coagulation Quality Control PCT for ordinate zou drawing standard curve, calculates regression equation, Y=62.894X+0.532, R
2=0.995.Above-mentioned detected result shows, white-browed snake venom blood coagulation enzyme can make the blood coagulation Quality Control clotting of plasma, and between 0.4-5KU/mL, the Reciprocals sums human plasma setting time of hemocoagulase concentration has good linear relationship.
The blood coagulation Quality Control PCT that the white-browed snake venom blood coagulation enzyme diluent of table 1 different content records
Two, Thrombin-like enzyme sample determination of activity
Thrombin-like enzyme sample activity is mainly reflected in and can acts on fibrinogen monomer, makes it be cross-linked into scleroproein, forms grumeleuse, and DFP (diisopropylfluorophosphate) can suppress Thrombin-like enzyme sample active.The Thrombin-like enzyme sample detecting the white-browed snake venom blood coagulation enzyme that the present invention extracts by following method is active, and concrete grammar is: agkistrodon halys ussuriensis snake venom blood coagulation enzyme concentrated solution embodiment 1 obtained is with 2 × 10
-3the DFP solution of mol/L is diluted to 0.5KU/mL, 1KU/mL and 2KU/mL respectively, act on after 5 hours, the diluent of different concns respectively gets 1mL, 37 DEG C of water-bath preheating 5min, then the bovine fibrinogen solution 1mL of 0.1% of 37 DEG C of water-bath preheatings is added wherein, record setting time.Simultaneously to replace DFP solution in contrast with distilled water.Experimental result is as shown in table 2, under DFP existent condition, white-browed snake venom blood coagulation enzyme of the present invention cannot make bovine fibrinogen solution solidify, and there is solidification phenomenon in the bovine fibrinogen of distilled water control group, prove that white-browed snake venom blood coagulation enzyme of the present invention has Thrombin-like enzyme sample active, can specific action in Fibrinogen.
The Fibrinogen setting time that the white-browed snake venom blood coagulation enzyme diluent of table 2 different content records
Three, clauden sample determination of activity
Clauden sample activity is mainly reflected in and starts Coagulation test by activating blood coagulation X factor, and EDTA can suppress this clauden sample active.The clauden sample detecting the white-browed snake venom blood coagulation enzyme that the present invention extracts by following method is active, and concrete grammar is: white-browed snake venom blood coagulation enzyme concentrated solution embodiment 1 obtained is with 5 × 10
-3the EDTA solution of mol/L is diluted to 0.5KU/mL, 1KU/mL and 2KU/mL respectively, act on after 5 hours, the diluent of different concns respectively gets 1mL, 37 DEG C of water-bath preheating 5min are as trial-product, then in each trial-product, blood coagulation Quality Control blood plasma and the scarce X factor blood plasma (being U.S.'s Pacific Ocean reagent to produce) of 37 DEG C of water-bath preheatings is added respectively, record setting time.Meanwhile, 2 × 10 are used respectively
-3the DFP solution of mol/L and distilled water replace EDTA solution in contrast.Experimental result is as shown in table 3, and under DFP existent condition, white-browed snake venom blood coagulation enzyme of the present invention can not make the clotting of plasma of the scarce X factor, but can make the normal clotting of plasma, and the blood coagulation activity of its clauden can be subject to the suppression of EDTA.
The clauden sample determination of activity result of the white-browed snake venom blood coagulation enzyme diluent of table 3 different content
In embodiment 4, white-browed snake venom blood coagulation enzyme, Thrombin-like enzyme class material and clauden substances content measure
Two kinds of white-browed snake venom blood coagulation enzyme concentrated solutions embodiment 1 and comparative example obtained are as trial-product, the content of Thrombin-like enzyme class material wherein and clauden material is detected with Japanese Shimadzu high performance liquid chromatograph and TKS-G2000swxI gel chromatographic columns (purchased from bird with red feathers nanometer (Tianjin) development in science and technology company limited), with acetonitrile: trifluoroacetic acid: water (40:0.1:60, v/v/v) be moving phase, same the equipment Inspection of two samples, sample size is 10 μ l, flow velocity is 0.8mL/min, and determined wavelength is 280nm.The high performance liquid chromatography scintigram of gained as shown in Figure 3 and Figure 4, Fig. 3 is the liquid chromatogram of comparative example (ZL200710099163.X) gained concentrated solution, Fig. 4 is the liquid chromatogram of embodiment 1 gained concentrated solution, as shown in the figure, (first peak refers to the peak at retention time 16.37 place to first peak, above be moving phase Interference Peaks) be clauden class material (molecular weight 69000D), second peak is Thrombin-like enzyme sample material (molecular weight is 34000D, retention time 73.85min).In Fig. 3, the peak area ratio at two peaks is 18.2:81.8; The peak area ratio at Fig. 3 two peak is 14.1:86.9.
The-terminal amino acid sequencing of embodiment 5, white-browed snake venom blood coagulation enzyme
Carry out ultrafiltration with the white-browed snake venom blood coagulation enzyme concentrated solution 100mL of ultrafiltration post to the embodiment of the present invention 1 gained of molecular weight cut off 60000D, collect ultrafiltrated freeze-drying, obtain the main component of this product: molecular weight is the Thrombin-like enzyme sample component of 34000D; Get Thrombin-like enzyme sample component 10 μ g as trial-product, adopt ABI Procise 491 type sequenator to carry out-terminal amino acid sequencing, according to PVDF program loading.Sequencing result shows that 15 amino acids residue sequence of white-browed snake venom blood coagulation enzyme N-end of the present invention are as shown in the sequence 1 in sequence table.
Claims (8)
1. from agkistrodon halys ussuriensis snake venom, extract a method for white-browed snake venom blood coagulation enzyme, comprise the steps:
1) Agkistrodon halys ussurriensisEmelianov snake venom is dissolved in the Tris-HCl damping fluid of pH 7-8,0.04-0.06mol/L, obtains the agkistrodon halys ussuriensis snake venom that concentration is 200-300g/L;
2) to step 1) the Agkistrodon halys ussurriensisEmelianov snake venom that obtains carries out ion-exchange chromatography, in described ion-exchange chromatography, solid phase weighting material is DEAE-SephadexA-50, elutriant is: NaCl concentration is the Tris-HCl damping fluid of pH 7-8,0.04-0.06mol/L of 0-0.75mol/L, and the type of elution adopted is gradient elution;
3) collect step 2) in elutriant; Under the wavelength of 280nm, UV spectrophotometer measuring is carried out to described elutriant, there is under being collected in the wavelength of 280nm the elutriant of absorption peak, and blood coagulation activity detection is carried out to this elutriant, merge the elutriant with blood coagulation activity;
4) to described have in the elutriant of blood coagulation activity add bovine serum albumin, then mixed solution is refrigerated 4 hours at 4 DEG C of environment, obtain refrigerate after mixed solution;
5) alkaline hydrolysis: add NaOH in the mixed solution after refrigeration, make the concentration of NaOH be 0.05mol/L, then reacts 15-17 hours in 37.5 ± 0.5 DEG C of environment;
6) freeze-drying: by step 5) reaction after gained solution carry out lyophilize, obtain lyophilized powder;
7) by step 6) the lyophilized powder water for injection that obtains redissolves;
8) redissolution liquid is carried out gel permeation chromatography, in described gel permeation chromatography, solid phase weighting material is SephadexG-75, and elutriant is water for injection, collects elutriant, therefrom collects the part with blood coagulation activity, obtains white-browed snake venom blood coagulation enzyme stoste.
2. method according to claim 1, is characterized in that: described step 1) the Tris-HCl damping fluid of described pH 7-8,0.04-0.06mol/L is the Tris-HCl damping fluid of pH 7.5,0.05mol/L;
Described step 1) also comprise the step removing impurity in described agkistrodon halys ussuriensis snake venom by centrifugal method.
3. method according to claim 1 and 2, it is characterized in that: described step 2) in DEAE-Sephadex A-50 chromatography column before use, first with the Tris-HCl damping fluid balance 15-20 hour of pH 7-8,0.04-0.06mol/L, flow velocity is 0.8-1.0mL/min; During chromatography, move to completely after below glue face until agkistrodon halys ussuriensis snake venom, the damping fluid consistent with balance liquid is added above glue face, then carry out linear gradient wash-out with the Tris-HCl damping fluid that the Tris-HCl damping fluid of pH 7-8,0.04-0.06mol/L and isopyknic NaCl concentration are pH 7-8,0.04-0.06mol/L of 0.75mol/L successively, flow velocity is 0.8-1.0mL/min.
4. the method according to any one of claim 1-3, is characterized in that: described step 3) described in step 2) in the method for elutriant be: collect with automatic fraction collector, often pipe 12-15mL; The method that described blood coagulation activity detects is: by equal-volume and be preheated to 37 DEG C collection liquid and blood coagulation Quality Control blood plasma is mixed be incorporated in 37 DEG C at water-bath, the collection unit that blood plasma solidified in 20 seconds can be made to be divided into the elutriant with blood coagulation activity.
5. the method according to any one of claim 1-4, is characterized in that: step 4) described in mixed solution after refrigeration the concentration of bovine serum albumin be 1.2-1.5mg/ml.
6. the method according to any one of claim 1-5, is characterized in that: step 8) in SephadexG-75 chromatography column before use, first with injection water balance 15-20 hour, flow velocity is 0.8-1.0mL/min; During chromatography, move to completely after below glue face until supernatant liquor, jetting of annotating above glue face, then uses injection water wash-out, and flow velocity is 0.8-1.0mL/min.
7. the white-browed snake venom blood coagulation enzyme for preparing of method according to any one of claim 1-6.
8. white-browed snake venom blood coagulation enzyme according to claim 7, is characterized in that: 15 amino acids residue sequence of described white-browed snake venom blood coagulation enzyme N-end are as shown in the sequence 1 in sequence table.
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| CN114689702A (en) * | 2020-12-25 | 2022-07-01 | 锦州奥鸿药业有限责任公司 | Gel column HPLC fingerprint detection method for snake venom of Agkistrodon blossoms in Changbai mountain |
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