CN104357430B - A kind of production method of Effect of Agkistrodon acutus Enzyme - Google Patents
A kind of production method of Effect of Agkistrodon acutus Enzyme Download PDFInfo
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- CN104357430B CN104357430B CN201410706763.8A CN201410706763A CN104357430B CN 104357430 B CN104357430 B CN 104357430B CN 201410706763 A CN201410706763 A CN 201410706763A CN 104357430 B CN104357430 B CN 104357430B
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Abstract
The present invention relates to the production method of a kind of Effect of Agkistrodon acutus Enzyme, comprise the following steps: (1) agkistrodon acutus venom lyophilized powder is dissolved in excess 0.05mol/L, pH value be 8.5 Tris HCl buffer in；(2) by DEAE Sepharose Fast Flow anion-exchange column on described agkistrodon acutus venom solution, eluting separation is carried out；(3) SOURCE 30Q gel column on solution step (2) obtained, chromatographs；Then phenyl sepharose high performance column chromatography for separation is gone up；(4) Hiload 26/60Superdex75PG post on solution step (3) obtained, collects second protein peak with thrombin-like enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.The Effect of Agkistrodon acutus Enzyme solution that the production method of the Effect of Agkistrodon acutus Enzyme of the present invention prepares is one-component, has the advantage that purity is high.
Description
Technical field
The present invention relates to field of biological pharmacy, particularly to the production method of a kind of Effect of Agkistrodon acutus Enzyme.
Background technology
Effect of Agkistrodon acutus Enzyme is a kind of thrombin-like enzyme isolated and purified from Agkistrodon acutus venom, is serine-type albumen water
Solve enzymatic mixture.Effect of Agkistrodon acutus Enzyme has thrombin-like enzyme activity, and its molecular weight is 24000～30000 dalton,
Isoelectric point, IP is 4.5～5.0.Effect of Agkistrodon acutus Enzyme preparation Acutobin Injection can be used for treating acute cerebral infarction and various thrombosis
Property disease, clinically use intravenous drip be administered.
About the existing document of Effect of Agkistrodon acutus Enzyme production technology, the patent of Chinese patent application Publication No. CN1229136A
The open Effect of Agkistrodon acutus Enzyme of file and production technology thereof, with sephadex G 75 post on known buffer solution, receive
Collection has A, B peak of thrombin-like enzyme activity, with DEAE polydextran gel A 50 column chromatography, collects respectively
There is the V of thrombin-like enzyme activity, VI, VII peak.By collect have the V of thrombin-like enzyme activity, VI,
VII peak respectively with CM polydextran gel C 50 column chromatography, collect respectively have thrombin-like enzyme activity CM1,
CM2, CM3 peak.CM1, CM2, CM3 peak having thrombin-like enzyme activity collected is used respectively
Superdex75PG post filters, and each gets a, b two peak, collects the b peak with high thrombin-like enzyme activity, mixed
It is combined into molecular weight 24000～30000 dalton and the Effect of Agkistrodon acutus Enzyme of isoelectric point, IP 4.5～5.0.Prepared by this production technology
The purity of Effect of Agkistrodon acutus Enzyme needs to be improved further, and production process is complicated, the production cycle is long, is not easy to produce
Industryization is promoted.
Prior art there is also employing tentatively chromatograph through gel filtration chromatography, then chromatograph through ion exchange chromatography
The purpose component of gained, this component detected through gel electrophoresis is a protein component, its be really molecular weight and
Protein that isoelectric point, IP is close or the mixture of polypeptide, need to be further purified, i.e. there is the Effect of Agkistrodon acutus Enzyme of acquisition
The not high enough problem of purity.Additionally, existing Effect of Agkistrodon acutus Enzyme production technology generally to there is the production technology cycle long
Problem.
Summary of the invention
The technical problem to be solved is: provide the Effect of Agkistrodon acutus Enzyme that a kind of purity is high, with short production cycle
Production method.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is:
The production method of a kind of Effect of Agkistrodon acutus Enzyme, comprises the following steps:
(1) under the conditions of temperature is 4～10 DEG C, agkistrodon acutus venom lyophilized powder is dissolved in 0.04～0.06mol/L, pH
Value is in the Tris HCl buffer of 8～9, stands 60min, centrifugal acquisition supernatant under the conditions of 0～10 DEG C,
The Tris HCl buffer that supernatant 0.01mol/L, pH value are 8～9 is dialysed, it is thus achieved that agkistrodon acutus venom is molten
Liquid；
(2) by DEAE Sepharose Fast Flow anion-exchange column on described agkistrodon acutus venom solution, wash
De-separation, collects the protein peak in protein peak with thrombin-like enzyme activity, and the protein peak that will collect merges,
Then dialyse with the Tris HCl buffer that 0.01mol/L, pH value are 7～8.
(3) solution step (2) obtained is after 0.22 μm filtering with microporous membrane, upper SOURCE 30Q
Gel column, chromatographs, and collects the protein peak with thrombin-like enzyme activity, the protein peak solution that will collect
After merging, upper phenyl sepharose high performance chromatographic column, chromatographs, has after chromatography
The protein peak of high thrombin-like enzyme activity, concentrates below vacuum 0.08MPa, under the conditions of 5～10 DEG C,
Then dialyse with the Tris HCl buffer that 0.01mol/L, pH value are 7～8, molten by obtain after dialysis
Liquid is below vacuum 0.08MPa, and concentrating under reduced pressure under the conditions of 5～10 DEG C, concentrated solution is filtered through 0.22um micropore
Membrane filtration；
(4) Hiload 26/60Superdex75PG post on solution step (3) obtained, collection has solidifying
Second protein peak of hemase sample enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
The beneficial effects of the present invention is:
(1) the Effect of Agkistrodon acutus Enzyme solution prepared is one-component, and purity is single band and through height on gel electrophoresis
Effect Liquid Detection be one unimodal, purity be more than 98%；
(2) with short production cycle, production technology favorable reproducibility.
Accompanying drawing explanation
Fig. 1 is the agkistrodon acutus venom DEAE Sepharose Fast of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention
The chromatogram of Flow column chromatography；
Fig. 2 be the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention collect there is thrombin-like enzyme activity
VIII and the Ⅸ protein peak chromatogram of SOURCE 30Q column chromatography；
Fig. 3 be the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention collect there is thrombin-like enzyme activity
S2 peak and the S3 peak chromatogram of phenyl sepharose high performance column chromatography；
Fig. 4 be the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention collect there is thrombin-like enzyme activity
The P3 peak chromatogram of Hiload 26/60Superdex75PG column chromatography；
Fig. 5 is that Effect of Agkistrodon acutus Enzyme solution (lot number 01) subunit of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention divides
The thin slice scan figure of son amount；
Fig. 6 is Effect of Agkistrodon acutus Enzyme solution (lot number 01) electrophoresis method of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention
The thin slice scan figure of purity test；
Fig. 7 is Effect of Agkistrodon acutus Enzyme solution (lot number 01) high-efficient liquid of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention
The chromatogram that chromatography purity checks.
Detailed description of the invention
By describing the technology contents of the present invention in detail, being realized purpose and effect, below in conjunction with embodiment also
Accompanying drawing is coordinated to be explained.
The design of most critical of the present invention is: by dissolving agkistrodon acutus venom lyophilized powder, be centrifuged, dialysing, through post
The compounding practice of chromatography, dialysis etc., prepares highly purified Effect of Agkistrodon acutus Enzyme solution.
The production method of a kind of Effect of Agkistrodon acutus Enzyme of the present invention, comprises the following steps:
(1) under the conditions of temperature is 4～10 DEG C, agkistrodon acutus venom lyophilized powder is dissolved in 0.04～0.06mol/L, pH
Value is in the Tris HCl buffer of 8～9, stands 60min, centrifugal acquisition supernatant under the conditions of 0～10 DEG C,
The Tris HCl buffer that supernatant 0.01mol/L, pH value are 8～9 is dialysed, it is thus achieved that agkistrodon acutus venom is molten
Liquid；
(2) by DEAE Sepharose Fast Flow anion-exchange column on described agkistrodon acutus venom solution, egg is collected
White peak has the protein peak of thrombin-like enzyme activity, will collect protein peak merge, then with 0.01mol/L,
PH value be 7～8 Tris HCl buffer dialyse.
(3) solution step (2) obtained is after 0.22 μm filtering with microporous membrane, upper SOURCE 30Q
Gel column, chromatographs, and collects the protein peak with thrombin-like enzyme activity, is merged by the protein peak collected
Rear upper phenyl sepharose high performance chromatographic column, chromatographs, has Gao Ning by after chromatography
The protein peak of hemase sample enzyme activity, concentrates under the conditions of 5～10 DEG C below vacuum 0.08MPa, then uses
0.01mol/L, pH value be 7～8 Tris HCl buffer dialyse, will after dialysis the solution that obtains very
Below reciprocal of duty cycle 0.08MPa, concentrating under reduced pressure under the conditions of 5～10 DEG C, concentrated solution is through 0.22um filtering with microporous membrane；
(4) Hiload 26/60Superdex75PG post on solution step (3) obtained, collection has solidifying
Second protein peak of hemase sample enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
The technology design of the present invention: the protein ingredient contained by agkistrodon acutus venom is complicated, the production technology that Effect of Agkistrodon acutus Enzyme is original
SephadexG 75, DEAE SephadexA 50 and SephadexG 75 is used to carry out isolated and purified successively,
Obtain three isozymes.The production technology using this patent can effectively remove non-targeted protein component and antibacterial
Endotoxin, obtains single protein component, and the purity of product is high, and with short production cycle, technical process is easy
In control, favorable reproducibility.
Knowable to foregoing description, the beneficial effects of the present invention is:
(1) the Effect of Agkistrodon acutus Enzyme solution prepared is one-component, and purity is single band and through height on gel electrophoresis
Effect liquid phase chromatogram be detected as one unimodal, purity be more than 98%；
(2) with short production cycle, production technology favorable reproducibility.
Further, agkistrodon acutus venom described in step (1) is dissolved in after Tris HCl buffer in 0～10 DEG C of condition
Under be centrifuged, the supernatant of gained again with Tris HCl buffer dialyse.
Seen from the above description, the described centrifugal effect with further remove impurity.
Further, described dialysis uses bag filter to carry out, and the molecular cut off of described bag filter is 7000 roads
Er Dun.
Further, the liquid after dialysis treatment in step (1) is filtered with 0.22 μm microporous filter membrane.
Seen from the above description, with 0.22 μm microporous filter membrane carry out being filtered into the liquid after dialysis preliminary
Filtration treatment, can facilitate the carrying out of subsequent operation.
Further, agkistrodon acutus venom solution described in step (1) is dissolved in Tris HCl buffer, then in
Being centrifuged under the conditions of 0～10 DEG C, the supernatant Tris HCl buffer of gained is dialysed.
Further, by the protein solution with thrombin-like enzyme activity of collection in step (3) prior to vacuum
Concentrate under the conditions of degree below 0.08M Pa, 5～10 DEG C, then dialyse with Tris HCl buffer.
Further, by the solution after dialysis in step (3) below vacuum 0.08MPa, 5～10 DEG C
Under the conditions of concentrate.
Further, Hiload 26/60Superdex75PG post on the concentrated solution that step (3) is obtained, receive
Collection has second protein peak of thrombin-like enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
Embodiments of the invention one are:
Step 1: at temperature is 4～10 DEG C, agkistrodon acutus venom lyophilized powder is dissolved in excess 0.04～
0.06mol/L, pH value are in the Tris HCl buffer of 8～9, stand more than 60min, centrifugal at 0 10 DEG C
Obtain supernatant, take supernatant and put (molecular cut off 7000 dalton) in bag filter, with 0.01mol/L,
PH value is the Tris HCl buffer dialysis of 8～9, to remove salt and biological micromolecule impurity；Will be through dialysing
Agkistrodon acutus venom solution with 0.22 μm filtering with microporous membrane.
Step 2: solution DEAE Sepharose Fast Flow anion-exchange column step 1 obtained is washed
De-separation, to remove containing hemorrhage poisonous substance matter.Fig. 1 is the chromatograph of DEAE Sepharose Fast Flow column chromatography
Figure, its abscissa is time (min), and vertical coordinate is response value (mAU).As it is shown in figure 1, obtain 11
Protein peak, collects respectively and has VIII peak of thrombin-like enzyme activity and the first half peak of Ⅸ, merge VIII peak and Ⅸ
First half peak collect liquid, then with concentration be 0.01mol/L, pH value be 7～8 Tris HCl buffer carry out
Dialysis, the molecular cut off of bag filter used is 7000 dalton, to remove salt and aminoacid.This step
DEAE Sepharose Fast Flow post disengaging time, only with 6～7 hours, has the advantage that efficiency is high.
Step 3: by VIII peak with thrombin-like enzyme activity dialysed and the first half peak of Ⅸ, use SOURCE
30Q column chromatography, Fig. 2 is the chromatogram of SOURCE 30Q column chromatography, and its abscissa is time (min), vertical
Coordinate is response value (mAU).As in figure 2 it is shown, obtain multiple protein peaks, collect respectively and there is thrombin-like enzyme
S2, S3 protein peak of vigor；S2, S3 protein peak mixing one with thrombin-like enzyme activity that will collect
Reinstate phenyl sepharose high performance chromatography, to remove the impurity in S2, S3.Figure
3 is the chromatogram of phenyl sepharose high performance column chromatography, and its abscissa is time (min),
Vertical coordinate is response value (mAU).As it is shown on figure 3, collect the P3 protein peak with thrombin-like enzyme activity
After, rotated vaporizer, below vacuum 0.08MPa, concentrating under reduced pressure under the conditions of 5～10 DEG C.Concentrated solution fills
Entering in bag filter, the molecular cut off of bag filter is 7000 dalton.It is 78 with 0.01mol/L, pH value
The dialysis of Tris HCl buffer, desalination, then below vacuum 0.08MPa, carry out under the conditions of 5～10 DEG C
Concentrating under reduced pressure, concentrated solution is through 0.22um filtering with microporous membrane.
Step 4: the P3 protein peak Hiload 26/60Superdex75PG post after filtering filters,
To remove depyrogenation and macromole impurity.Fig. 4 is the chromatogram of Hiload 26/60Superdex75PG column chromatography,
Its abscissa is time (min), and vertical coordinate is response value (mAU).As shown in Figure 4, collect highly purified
After there is second protein peak of thrombin-like enzyme activity, through the 0.22 aseptic sucking filtration of μm microporous filter membrane, it is
Effect of Agkistrodon acutus Enzyme solution.
The quality analysis of Effect of Agkistrodon acutus Enzyme:
1, Rate activity measures
1.1 titration
The preparation of reference material solution
Take Effect of Agkistrodon acutus Enzyme solution reference material, add 0.01mol/L TRIS buffer and (take trihydroxy methyl
Aminomethane 0.121g, sodium chloride 0.878g, add water appropriate dissolving, regulates pH with 0.5mol/L hydrochloric acid solution
To 7.4, add water to 100ml and prepare) to be respectively prepared concentration be single containing 0.4,0.6,0.8,1.0 in every 1ml
The solution of position.
Algoscopy
Take 4, test tube (10mm × 100mm, internal diameter should be 8.0mm ± 0.2mm), each addition people's fiber
Proteinogen solution 0.1ml, puts insulation 2min in 37 DEG C ± 0.5 DEG C water-bath, is sequentially added into 4 kinds of concentration
Reference material solution 0.4ml, shakes up timing immediately, observes fibrinous initial set in 37 DEG C ± 0.5 DEG C water-bath
Time.Every kind of concentration measures 5 times, and averaging, (difference that 5 times measure maximal and minmal value must not exceed averagely
The 10% of value, otherwise resurveys).With the logarithm of reference material concentration as abscissa, the logarithm in presetting period is sat for vertical
Mark, draws standard curve or calculates regression equation (correlation coefficient r >=0.990).Described human fibrinogen is molten
Liquid is: take human fibrinogen 1, adds above-mentioned buffer (pH7.4) and makes the fiber egg Han people in every 1ml
The solution of white former 4mg, puts 37 DEG C of water-baths 30～within 60 minutes, makes fully to dissolve.Take this product to survey as stated above
Fixed, standard curve or regression equation try to achieve units.
1.2 protein contents: it is appropriate that precision measures this product, with 0.9% sodium chloride solution make in every 1ml containing about
The solution of 0.1mg albumen is as need testing solution, and precision measures need testing solution 1.0ml, with Forint phenol method (in
State's pharmacopeia (version two in 2010) annex VII M protein determination the second method) measure protein content.
1.3 Rate activity: be calculated as follows
Albumen mg number in Rate activity=every 1ml test sample potency unit number/every 1ml test sample
Table 1 is that Effect of Agkistrodon acutus Enzyme solution Rate activity measures table.
Table 1
As shown in Table 1,01,02 and 03 3 batch of Rate activity of Effect of Agkistrodon acutus Enzyme solution lot number be respectively 14.8u/mg,
13.4u/mg and 13.6u/mg.
2, molecular weight subunit measures:
Take this product appropriate, with SDS polyacrylamide gel electrophoresis (Chinese Pharmacopoeia (version two in 2010)
Annex V F electrophoresis method the 5th method) measure its molecular weight subunit.Standard substance are SDS polyacrylamide gel electricity
Swimming low-molecular-weight standard protein, composition includes: rabbit phosphorylase B (molecular weight: 97400 dalton),
Bovine serum albumin (molecular weight: 66200 dalton), rabbit actin (molecular weight: 43000), cattle carbon
Anhydride enzyme (molecular weight: 31000 dalton), trypsin inhibitor (molecular weight: 20100 dalton),
Hen's egg-white lysozyme (molecular weight: 14400 dalton).By the gel slab after electrophoresis at Shimadzu dual wavelength flying spot
The upper scanning of thin-layer chromatogram scanner (CS 9301PC type), reference wavelength 700nm, measure wavelength 580nm, obtain
Three batches of molecular weight subunits of Effect of Agkistrodon acutus Enzyme solution (lot number 01,02 and 03) are respectively 14327.166,14263.507,
14184.903.The thin slice scan figure of Effect of Agkistrodon acutus Enzyme solution lot number 01 molecular weight subunit is shown in the abscissa of Fig. 5, Fig. 5
For exhibition away from (mm), vertical coordinate is absorbance.
3, purity test
3.1SDS polyacrylamide gel electrophoresis
Taking this product, according to SDS polyacrylamide gel electrophoresis, (Chinese Pharmacopoeia (version two in 2010) is attached
Record V F electrophoresis method the 5th method) check, point sample 20ul, by the gel slab after electrophoresis at Shimadzu dual wavelength flying spot
The upper scanning of thin-layer chromatogram scanner (CS 9301PC type), reference wavelength 700nm, measure wavelength 580nm, by peak
Area normalization method calculates, and obtains three batches of electrophoresis method master tape content of Effect of Agkistrodon acutus Enzyme solution (lot number 01,02 and 03)
It is respectively 99.276%, 99.576%, 98.294%, is all higher than 98%.Fig. 6 is Effect of Agkistrodon acutus Enzyme solution lot number 01
Electrophoresis method purity test thin slice scan figure, the abscissa of Fig. 6 is for exhibition away from (mm), and vertical coordinate is absorbance.
3.2 high performance liquid chromatography
It is filler (TSK gel G3000SW, 7.5mm × 300mm) with gel, with 0.02mol/L phosphoric acid
Salt buffer (take disodium hydrogen phosphate 4.37g and sodium dihydrogen phosphate 1.22g, be dissolved in 1000ml water, regulation
PH value is 7.0 to prepare) the 0.1mol/L metabisulfite solution made is flowing phase；Flow velocity is 1.0ml per minute,
Detection wavelength is 280nm.Effect of Agkistrodon acutus Enzyme solution (lot number 01,02 and 03) three batches detects through high performance liquid chromatography,
Purity is respectively 98.908%, 99.341%, 98.623%, is all higher than 98%.Effect of Agkistrodon acutus Enzyme solution lot number 01 batch
High-efficient liquid phase chromatogram see Fig. 7, abscissa is time (min), and vertical coordinate is response value (uV)；
In sum, the Effect of Agkistrodon acutus Enzyme solution that the production method of the Effect of Agkistrodon acutus Enzyme that the present invention provides prepares is one-component,
Purity on gel electrophoresis for single band and through high performance liquid chromatography be detected as one unimodal, purity is more than
98%；This production method with short production cycle, production technology favorable reproducibility.
The foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, every profit
The equivalents made by description of the invention and accompanying drawing content, or directly or indirectly it is used in relevant technology
Field, is the most in like manner included in the scope of patent protection of the present invention.
Claims (4)
1. the production method of an Effect of Agkistrodon acutus Enzyme, it is characterised in that comprise the following steps:
(1) under the conditions of temperature is 4～10 DEG C, agkistrodon acutus venom lyophilized powder is dissolved in 0.04～0.06mol/L, pH
Value is in the Tris-HCl buffer of 8～9, stands 60min, centrifugal acquisition supernatant under the conditions of 0～10 DEG C,
The Tris-HCl buffer that supernatant 0.01mol/L, pH value are 8～9 is dialysed, it is thus achieved that agkistrodon acutus venom
Solution；
(2) by DEAE-Sepharose Fast Flow anion-exchange column on described agkistrodon acutus venom solution, carry out
Eluting separates, and collects the protein peak in protein peak with thrombin-like enzyme activity, is merged by the protein peak collected,
Then dialyse with the Tris-HCl buffer that 0.01mol/L, pH value are 7～8；
(3) solution step (2) obtained is after 0.22 μm filtering with microporous membrane, upper SOURCE 30Q
Gel column, chromatographs, and collects the protein peak with thrombin-like enzyme activity, is merged by the protein peak collected
Rear upper phenyl sepharose high performance chromatographic column, has thrombin-like enzyme activity by collect
Protein solution concentrates prior to vacuum-0.08M below Pa, under the conditions of 5～10 DEG C, then with 0.01mol/L,
PH value be 7～8Tris-HCl buffer dialysis, will dialysis after solution in below vacuum-0.08MPa, 5～
Concentrate under the conditions of 10 DEG C；
(4) upper Hiload 26/60 after the solution that step (3) obtains being filtered with 0.22 μm microporous filter membrane
Superdex75PG post, collects second protein peak with thrombin-like enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
The production method of Effect of Agkistrodon acutus Enzyme the most according to claim 1, it is characterised in that described dialysis uses
Bag filter is carried out, and the molecular cut off of described bag filter is 7000 dalton.
The production method of Effect of Agkistrodon acutus Enzyme the most according to claim 1, it is characterised in that by step (1)
Liquid after middle dialysis treatment filters with 0.22 μm microporous filter membrane.
The production method of Effect of Agkistrodon acutus Enzyme the most according to claim 1, it is characterised in that by step (3)
Solution after middle dialysis filters with 0.22 μm microporous filter membrane.
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