CN100506983C - Method for extracting single component batroxobin from Bothrops atrox poison - Google Patents
Method for extracting single component batroxobin from Bothrops atrox poison Download PDFInfo
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- CN100506983C CN100506983C CNB2006100445941A CN200610044594A CN100506983C CN 100506983 C CN100506983 C CN 100506983C CN B2006100445941 A CNB2006100445941 A CN B2006100445941A CN 200610044594 A CN200610044594 A CN 200610044594A CN 100506983 C CN100506983 C CN 100506983C
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Abstract
It' s a method for extracting Batroxobin for defibrination and thrombolysis from venom of snakes, which relates to medicine technologies. The method comprises the steps of soaking, dissolving and centrifuging the venom of the snake; chromatography of the supernatant by an affinity column; chromatography of the eluent that has been desalinated by flowing it through polydextran gel G25; entrapment and concentration of the eluent that containing effective compositions and chromatography of the concentrated sample by a molecular sieve so as to obtain the Batroxobin with sole composition. The method features convenient operation, short production cycle, high purity and is suitable for production in large scale.
Description
Technical field
The present invention relates to a kind of two subspecies (Bothrops moojeni or Bothrops atrox) snake venom to extract and have the method for the batroxobin that falls effect of fibrinolytic bolt or anastalsis, belong to medical technical field from the spearhead pallas pit viper.
Background technology
Contain abundant proteolytic ferment in the snake venom, mainly contain two classes, a class is a snake venom thrombin-like enzyme, causes solidifying of blood external, but is the composition of fine anti-freezing in vivo; Another kind of is nevin fibrinolytic enzyme, the fibrin of can directly degrading, and it is former that major part also can fibrin degradation.Snake venom thrombin-like enzyme be study early, and the class of enzymes of widespread use clinically.
Snake venom thrombin-like enzyme is used for the treatment of the history in existing more than 30 year of thrombotic diseases as medicine, and the purposes of two aspects is arranged clinically.One for falling fine effect, promptly clings to the Qu Keshuan enzyme, as eastern water chestnut enlightening cottonrose hibiscus (coming from Bothrops moojeni), is mainly used in vessel embolism such as cerebral infarction, thromboangiitis obliterans, femoral artery embolism, pulmonary infarction.Crust Qu Keshuan enzyme injection liquid has been widely used in multiple treatment of diseases clinically at present, comprise ischemic cerebrovascular disease, Acute Myocardial Infarction, sudden deafness, acute cerebral infarction, intractable nephrotic syndrome, diabetic peripheral neuropathy, pulmonary heart disease, unstable angina pectoris, high blood viscosity, heart source property cerebral embolism, determined curative effect, untoward reaction is slight; Two is anastalsis, i.e. Ba Qu hemostatic enzyme is as reptilase, also claim snake venom blood coagulation enzyme (coming from Bothrops atrox), what can be used for that multiple reason causes is hemorrhage, particularly uses the invalid hemorrhage of traditional hemostatic drug, determined curative effect is not found tangible untoward reaction.Crust Qu Keshuan enzyme and Ba Qu hemostatic enzyme are referred to as batroxobin.
The Thrombin-like enzyme that extracts in the natural snake venom is most to be acidic protein, so adopts anion-exchange columns to separate more, and method purifying such as attached gel filtration, affinity chromatography and reversed-phase liquid chromatography.The batroxobin preparation method who has reported comprises:
(1) carries out diethyllaminoethyl-dextrane gel A 50 (DEAE-Sephadex A50) anion-exchange chromatography and molecular sieve Sephadex G 100 chromatographic separation after the snake venom pre-treatment
This technology preprocessing process is loaded down with trivial details, causes the loss of enzymic activity composition more, influences the ratio vigor and the yield of product, and use organic solvents such as phenol derivmives blend biology, methyl alcohol in pre-treatment, influences the purifying in later stage, is unsuitable for large-scale production.
(2) carrying out heparin-cyanogen bromide-sepharose 4B (Heparin-CNBr-Sepharose 4B) affinity chromatography after the snake venom pre-treatment separates
Though the more preceding method of this technology pretreatment process is slightly simple, in pre-treatment, use the derivative of phenol equally, and the technology activity yield is lower, is 7.4% only, is unsuitable for large-scale production;
(3) benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) step affinity chromatography is separated
The aglucon benzenyl amidine (Benzamidine) of the used affinity media of this technology is the inhibitor of serine stretch protein enzyme, snake venom contains the isozyme of multiple Thrombin-like enzyme as native protein, all belong to Serine trypsinase, can combine with aglucon simultaneously, therefore, specificity is not high, and the purity of batroxobin is relatively poor in the gained sample.
We test reference literature technology, and the result is as follows:
| Test-results | Document | |
| Electrophoresis purity | 30％～40％ | 85％～90％ |
| Activity yield | Greater than 90% | Greater than 90% |
| Compare vigor | 320～450BU/mg | 1166～1278BU/mg |
According to test-results, think and only use benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) step affinity chromatography to separate the requirement that does not at all reach separation and purification, and the data of bibliographical information do not conform to the actual conditions.
In sum, document technology or be difficult to reach ideal and separate the preparation requirement obtains single composition product, or is difficult to realize the production purpose of mass-producing, thereby can't be used for actual production.
Summary of the invention
The objective of the invention is to: disclose a kind of can be in the method for from the spearhead agkistrodon halyx pallas venom, extracting single composition batroxobin of industrial realization.
Step of the present invention comprises:
(1) snake venom soaks, dissolves and spend the night with damping fluid I, and centrifugal then removal precipitation gets supernatant liquor;
(2) affinity column on the snake venom supernatant liquor carries out wash-out with damping fluid I, reaches baseline to Protein Detection instrument reading, uses damping fluid II wash-out again, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
(3) batroxobin crude extract sephadex G 25 (Sephadex G25) desalting column of directly flowing through is removed NSC 2020 (BenzamidineHCL);
(4) the batroxobin crude extract of removing NSC 2020 is with dialysis membrane or ultra-filtration equipment or desalting column exchange buffering liquid III;
(5) on the batroxobin crude extract through damping fluid III equilibrated cation-exchange chromatography post, use ionic strength gradient buffering liquid or pH value gradient buffering liquid wash-out, nucleic acid-protein detector test sample peak, collection sample solution;
(6) sample solution concentrates through Macrogol 2000 0 (PEG20000) embedding, or through ultrafiltration and concentration, again through molecular sieve chromatography, with damping fluid IV wash-out, promptly gets single component batroxobin.
In above-mentioned steps, damping fluid I is Tris-HCL damping fluid or glycine-NaOH damping fluid or phosphoric acid salt (Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC) damping fluid that pH6.0～10.0 contain 0.1～2.0M NaCL.
Damping fluid II is Tris-HCL damping fluid or glycine-NaOH damping fluid or the phosphate buffered saline buffer that pH6.0～10.0 contain 0.1～0.5M NaCL and 0.1～0.5M NSC 2020 (BenzamidineHCL).
Damping fluid III is that pH5.0～6.5, concentration are phosphate buffered saline buffer or acetate (sodium acetate-acetate) damping fluid or citrate (Sodium Citrate-Citric Acid) damping fluid or the Tris-HCL damping fluid of 0.001～0.05M.
Damping fluid IV is citrate buffer or Tris-HCL damping fluid or phosphate buffered saline buffer or the acetate buffer that pH4.0～8.0 contain 0.01～0.5M NaCL.
Affinity column described in the step (2) is meant benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) or benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) chromatography column;
Cation-exchange chromatography post described in the step (5) is meant sulfopropyl sepharose (SP Sepharose), metilsulfate sepharose (S Sepharose) or carboxymethyl sepharose (CM Sepharose) chromatography column.
Molecular sieve chromatography described in the step (6) is meant propylene dextrane gel S200 (Sephacryl S200) or propylene dextrane gel S100 (Sephacryl S100) or Superdex-75 chromatography column.
Key problem in technology point of the present invention is after using benzenyl amidine sepharose (Benzamidine Sepharose) affinity chromatography; increase cation exchange column chromatography; make the electrophoresis purity of sample can reach 70%～80%; pass through sieve chromatography again; better (electrophoresis purity can be greater than 95% can to obtain purity; meet medicinal standard; see accompanying drawing 1), than the single composition batroxobin of vigor higher (can reach 1200～1600BU/mg albumen); total yield of products can reach 25%～35%, makes the production of batroxobin realize mass-producing fully.
Excellent results compared with prior art of the present invention is as follows:
Technological design of the present invention is reasonable, and technological process is simple, is easy to control, is convenient to operation, good reproducibility; Technological process does not have obvious damage to the activity of target protein, and good separating effect can reach higher sample purity and higher activity yield; Primary treatment snake venom amount is big, is fit to large-scale industrial production.
For obtaining better yield and purity, in process of production, can adopt the concrete operations of following several respects in the such scheme at the same time or separately separately:
1, affinity column in the step (2) and the desalting column in the step (3) can be cascaded or separately according to the design needs in the sample elution process.
2, the ionic strength gradient in the above-mentioned steps (5) or the gradient of pH gradient elution can adopt following proposal: stage gradient or straight line gradient, gradient is: 0～1.0M NaCL or pH value 4.0～9.0.
3, each step of the present invention is operated under 3～8 ℃ environment.
Description of drawings
Accompanying drawing is the electrophoresis purity collection of illustrative plates of gained sample batroxobin of the present invention.
Embodiment
Following examples will further specify the present invention, but not limit the present invention.
Embodiment one:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 3g, be dissolved in the Tris-HCL damping fluid that 60ml pH8.6 contains 1.0M NaCL, soak in 4 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 1.0M NaCL with pH8.6 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 1.0M NaCL with pH8.6 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.2M NaCL and 0.1M NSC 2020 (BenzamidineHCL) with pH8.6, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.01M phosphate buffered saline buffer with the method displacement pH6.0 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.01M phosphate buffered saline buffer balance through pH6.0 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the phosphate buffered saline buffer that contains 0-1.0M NaCL respectively, collect the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the citrate buffer wash-out that contains 0.1M NaCL with pH5.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1500BU/mg albumen than vigor, and activity yield is 31%.
Embodiment two:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in the Tris-HCL damping fluid that 80ml pH9.0 contains 0.6M NaCL, soak in 4 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 0.6M NaCL with pH9.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 0.6M NaCL with pH9.0 behind the end of the sample carries out wash-out, reaches baseline to Protein Detection instrument reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.1M NaCL and 0.2M NSC 2020 (BenzamidineHCL) with pH9.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.02M phosphate buffered saline buffer with the method displacement pH6.2 concentration of dialysis;
6, carboxymethyl sepharose (CM Sepharose F.F.) chromatography column is at least 10 column volumes of 0.02M phosphate buffered saline buffer balance through pH.6.2 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the phosphate buffered saline buffer that contains 0.1-1.0M NaCL respectively, collect the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the Tris-HCL buffer solution elution that contains 0.2M NaCL with pH7.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 96%, is 1250BU/mg albumen than vigor, and activity yield is 28%.
Embodiment three:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in the Tris-HCL damping fluid that 80ml pH8.0 contains 0.6M NaCL, soak in 3 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 0.6M NaCL with pH8.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 0.6M NaCL with pH8.0 behind the end of the sample carries out wash-out, reaches baseline to Protein Detection instrument reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.3M NaCL and 0.15M NSC 2020 (BenzamidineHCL) with pH8.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.015M phosphate buffered saline buffer with the method displacement pH5.8 concentration of dialysis;
6, metilsulfate sepharose (S Sepharose F.F.) chromatography column is at least 10 column volumes of 0.015M phosphate buffered saline buffer balance through pH5.8 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the phosphate buffered saline buffer that contains 0.2-1.0M NaCL respectively, collect the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S200 (SephacrylS200) molecular sieve chromatography (1.6cm * 100cm), the phosphate buffered saline buffer wash-out that contains 0.3M NaCL with pH6.8, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 97%, is 1380BU/mg albumen than vigor, and activity yield is 35%.
Embodiment four:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in glycine-NaOH damping fluid that 80ml pH9.0 contains 1.5M NaCL, soak in 6 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (glycine-NaOH damping fluid that 2.6cm * 30cm) contains 1.5M NaCL with pH9.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the glycine-NaOH damping fluid that contains 1.5M NaCL with pH9.0 behind the end of the sample carries out wash-out, reaches baseline to Protein Detection instrument reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, glycine-NaOH the buffer solution elution that contains 0.3M NaCL and 0.2M NSC 2020 (BenzamidineHCL) with pH8.8, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.015M acetate buffer with the method displacement pH6.2 concentration of dialysis;
6, metilsulfate sepharose (S Sepharose F.F.) chromatography column is at least 10 column volumes of 0.015M acetate buffer balance through pH6.2 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the acetate buffer that contains 0-0.8M NaCL respectively, collect the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last Superdex-75 molecular sieve chromatography (1.6cm * 100cm), the acetate buffer wash-out that contains 0.2M NaCL with pH6.8, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 99%, is 1580BU/mg albumen than vigor, and activity yield is 30%.
Embodiment five:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 3g, be dissolved in the Tris-HCL damping fluid that 60ml pH7.0 contains 0.1M NaCL, soak in 3.5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 0.1M NaCL with pH7.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 0.1M NaCL with pH7.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, glycine-NaOH the buffer solution elution that contains 0.2M NaCL and 0.25M NSC 2020 (BenzamidineHCL) with pH10.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.04M phosphate buffered saline buffer with the method displacement pH5.5 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.04M phosphate buffered saline buffer balance through pH5.5 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the phosphate buffered saline buffer that contains 0.1-0.8MNaCL respectively, collect the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the Tris-HCL buffer solution elution that contains 0.2M NaCL with pH7.4, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1352BU/mg albumen than vigor, and activity yield is 29%.
Embodiment six:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 4g, be dissolved in the phosphate buffered saline buffer that 65ml pH7.5 contains 1.2M NaCL, soak in 4 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (phosphate buffered saline buffer that 2.6cm * 30cm) contains 1.2M NaCL with pH7.5 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the phosphate buffered saline buffer that contains 1.2M NaCL with pH7.5 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the phosphate buffered saline buffer wash-out that contains 0.2M NaCL and 0.25M NSC 2020 (BenzamidineHCL) with pH8.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.02M citrate buffer with the method displacement pH5.9 concentration of dialysis;
6, carboxymethyl sepharose (CM Sepharose F.F.) chromatography column is at least 10 column volumes of 0.02M citrate buffer balance through pH5.9 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the citrate buffer that contains 0.05-1.0M NaCL respectively, collect the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last propylene dextrane gel S100 (Sephacryl S100) molecular sieve chromatography (1.6cm * 100cm), contain the phosphate buffered saline buffer wash-out of 0.3M NaCL with pH6.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 97%, is 1456BU/mg albumen than vigor, and activity yield is 31%.
Embodiment seven:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in glycine-NaOH damping fluid that 70ml pH6.8 contains 1.8M NaCL, soak in 8 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (glycine-NaOH damping fluid that 2.6cm * 30cm) contains 1.8M NaCL with pH6.8 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the glycine-NaOH damping fluid that contains 1.8M NaCL with pH6.8 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, glycine-NaOH the buffer solution elution that contains 0.3M NaCL and 0.3M NSC 2020 (BenzamidineHCL) with pH8.5, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.02M acetate buffer with the method displacement pH6.0 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.02M acetate buffer balance through pH6.5 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the acetate buffer that contains 0.1-0.9M NaCL respectively, collect the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the citrate buffer wash-out that contains 0.25M NaCL with pH5.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 97%, is 1466BU/mg albumen than vigor, and activity yield is 31%.
Embodiment eight:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in the phosphate buffered saline buffer that 60ml pH8.0 contains 1.0M NaCL, soak in 4.5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (phosphate buffered saline buffer that 2.6cm * 30cm) contains 1.0M NaCL with pH8.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the phosphate buffered saline buffer that contains 1.0M NaCL with pH8.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.2M NaCL and 0.5M NSC 2020 (BenzamidineHCL) with pH8.5, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.03M citrate buffer with the method displacement pH5.8 concentration of dialysis;
6, metilsulfate sepharose (S Sepharose) chromatography column is at least 10 column volumes of 0.03M citrate buffer balance through pH5.8 concentration, batroxobin crude extract upper prop is separated, Tris-HCL damping fluid with pH value 6.5-9.0 carries out stage gradient wash-out or straight line gradient elution respectively, collects the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S100 (Sephacryl S100) molecular sieve chromatography (1.6cm * 100cm), the Tris-HCL buffer solution elution that contains 0.3M NaCL with pH7.2, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 99%, is 1516BU/mg albumen than vigor, and activity yield is 27%.
Embodiment nine:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 3g, be dissolved in the Tris-HCL damping fluid that 60ml pH9.5 contains 2.0M NaCL, soak in 6 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 2.0M NaCL with pH9.5 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 2.0M NaCL with pH9.5 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, glycine-NaOH the buffer solution elution that contains 0.1M NaCL and 0.3M NSC 2020 (BenzamidineHCL) with pH8.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.015M phosphate buffered saline buffer with the method displacement pH5.8 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.015M phosphate buffered saline buffer balance through pH5.8 concentration, batroxobin crude extract upper prop is separated, phosphate buffered saline buffer with pH value 6.0-8.5 carries out stage gradient wash-out or straight line gradient elution respectively, collects the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last Superdex-75 molecular sieve chromatography (1.6cm * 100cm), the citrate buffer wash-out that contains 0.3M NaCL with pH6.4, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 97%, is 1425BU/mg albumen than vigor, and activity yield is 28%.
Embodiment ten:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 4g, be dissolved in the phosphate buffered saline buffer that 70ml pH9.0 contains 1.6M NaCL, soak in 5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (phosphate buffered saline buffer that 2.6cm * 30cm) contains 1.6M NaCL with pH9.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the phosphate buffered saline buffer that contains 1.6M NaCL with pH9.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the phosphate buffered saline buffer wash-out that contains 0.3M NaCL and 0.18M NSC 2020 (BenzamidineHCL) with pH8.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.015M citrate buffer with the method displacement pH5.6 concentration of dialysis;
6, metilsulfate sepharose (S Sepharose) chromatography column is at least 10 column volumes of 0.015M citrate buffer balance through pH5.6 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the citrate buffer that contains 0.15-0.7M NaCL respectively, collect the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the Tris-HCL buffer solution elution that contains 0.4M NaCL with pH7.5, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1280BU/mg albumen than vigor, and activity yield is 26%.
Embodiment 11:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 3g, be dissolved in glycine-NaOH damping fluid that 80ml pH8.5 contains 1.2M NaCL, soak in 3.5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (glycine-NaOH damping fluid that 2.6cm * 30cm) contains 1.2M NaCL with pH8.5 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the glycine-NaOH damping fluid that contains 1.2M NaCL with pH8.5 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.5M NaCL and 0.1M NSC 2020 (BenzamidineHCL) with pH10.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.008M phosphate buffered saline buffer with the method displacement pH5.4 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.008M phosphate buffered saline buffer balance through pH5.4 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the Tris-HCL damping fluid that contains 0.1-0.6M NaCL respectively, collect the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last Superdex-75 molecular sieve chromatography (1.6cm * 100cm), the citrate buffer wash-out that contains 0.01M NaCL with pH5.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1444BU/mg albumen than vigor, and activity yield is 32%.
Embodiment 12:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in the phosphate buffered saline buffer that 65ml pH10.0 contains 1.8M NaCL, soak in 3 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (phosphate buffered saline buffer that 2.6cm * 30cm) contains 1.8M NaCL with pH10.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the phosphate buffered saline buffer that contains 1.8M NaCL with pH10.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the phosphate buffered saline buffer wash-out that contains 0.5M NaCL and 0.2M NSC 2020 (BenzamidineHCL) with pH7.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.001M phosphate buffered saline buffer with the method displacement pH5.3 concentration of dialysis;
6, metilsulfate sepharose (S Sepharose) chromatography column is at least 10 column volumes of 0.001M phosphate buffered saline buffer balance through pH5.3 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the phosphate buffered saline buffer that contains 0.1-0.7M NaCL respectively, collect the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last propylene dextrane gel S100 (Sephacryl S100) molecular sieve chromatography (1.6cm * 100cm), the Tris-HCL buffer solution elution that contains 0.01M NaCL with pH8.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 99%, is 1278BU/mg albumen than vigor, and activity yield is 29%.
Embodiment 13:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 4g, be dissolved in glycine-NaOH damping fluid that 70ml pH6.5 contains 1.0M NaCL, soak in 4 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (glycine-NaOH damping fluid that 2.6cm * 30cm) contains 1.0M NaCL with pH6.5 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the glycine-NaOH damping fluid that contains 1.0M NaCL with pH6.5 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.1M NaCL and 0.4M NSC 2020 (BenzamidineHCL) with pH7.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.04M citrate buffer with the method displacement pH5.7 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column pH5.7 concentration is at least 10 column volumes of 0.04M citrate buffer balance, batroxobin crude extract upper prop is separated, citrate buffer with pH value 5.8-9.0 carries out stage gradient wash-out or straight line gradient elution respectively, collects the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last Superdex-7 molecular sieve chromatography (1.6cm * 100cm) contains the phosphate buffered saline buffer wash-out of 0.4M NaCL with pH7.0, nucleic acid-protein detector test sample peak, collection obtains the batroxobin of single composition.Detecting purity is 99%, is 1312BU/mg albumen than vigor, and activity yield is 33%.
Embodiment 14:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 3g, be dissolved in the Tris-HCL damping fluid that 60ml pH7.5 contains 0.4M NaCL, soak in 4.5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 0.4M NaCL with pH7.5 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 0.4M NaCL with pH7.5 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, glycine-NaOH the buffer solution elution that contains 0.4M NaCL and 0.5M NSC 2020 (BenzamidineHCL) with pH8.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.03M phosphate buffered saline buffer with the method displacement pH6.0 concentration of dialysis;
6, carboxymethyl sepharose (CM Sepharose) chromatography column is at least 10 column volumes of 0.03M phosphate buffered saline buffer balance through pH6.0 concentration, batroxobin crude extract upper prop is separated, phosphate buffered saline buffer with pH value 6.0-9.0 carries out stage gradient wash-out or straight line gradient elution respectively, collects the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the citrate buffer wash-out that contains 0.1M NaCL with pH7.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 99%, is 1368BU/mg albumen than vigor, and activity yield is 28%.
Embodiment 15:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 3g, be dissolved in glycine-NaOH damping fluid that 80ml pH8.0 contains 1.8M NaCL, soak in 5.5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (glycine-NaOH damping fluid that 2.6cm * 30cm) contains 1.8M NaCL with pH8.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the glycine-NaOH damping fluid that contains 1.8M NaCL with pH8.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the phosphate buffered saline buffer wash-out that contains 0.1M NaCL and 0.3M NSC 2020 (BenzamidineHCL) with pH6.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.001M citrate buffer with the method displacement pH5.0 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.001M citrate buffer balance through pH5.0 concentration, batroxobin crude extract upper prop is separated, citrate buffer with pH value 5.0-8.0 carries out stage gradient wash-out or straight line gradient elution respectively, collects the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S100 (Sephacryl S100) molecular sieve chromatography (1.6cm * 100cm), the acetate buffer wash-out that contains 0.2M NaCL with pH4.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1186BU/mg albumen than vigor, and activity yield is 25%.
Embodiment 16:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in the phosphate buffered saline buffer that 70ml pH7.0 contains 1.4M NaCL, soak in 4 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (phosphate buffered saline buffer that 2.6cm * 30cm) contains 1.4M NaCL with pH7.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the phosphate buffered saline buffer that contains 1.4M NaCL with pH7.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.3M NaCL and 0.3M NSC 2020 (BenzamidineHCL) with pH6.5, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.02M acetate buffer with the method displacement pH5.7 concentration of dialysis;
6, carboxymethyl sepharose (CM Sepharose) chromatography column is at least 10 column volumes of 0.02M acetate buffer balance through pH5.7 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the acetate buffer that contains 0.05-0.8M NaCL respectively, collect the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last Superdex-75 molecular sieve chromatography (1.6cm * 100cm), the Tris-HCL buffer solution elution that contains 0.001M NaCL with pH4.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 96%, is 1255BU/mg albumen than vigor, and activity yield is 29%.
Embodiment 17:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 5g, be dissolved in the Tris-HCL damping fluid that 70ml pH9.0 contains 0.6M NaCL, soak in 6.5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 0.6MNaCL with pH9.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 0.6M NaCL with pH9.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.4M NaCL and 0.3M NSC 2020 (BenzamidineHCL) with pH9.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is a 0.01M Tris-HCL damping fluid with the method displacement pH6.2 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.01M Tris-HCL damping fluid balance through pH6.2 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the Tris-HCL damping fluid that contains 0-0.9M NaCL respectively, collect the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the citrate buffer wash-out that contains 0.2M NaCL with pH5.8, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1400BU/mg albumen than vigor, and activity yield is 27%.
Embodiment 18:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 4g, be dissolved in glycine-NaOH damping fluid that 75ml pH7.0 contains 1.0M NaCL, soak in 3 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (glycine-NaOH damping fluid that 2.6cm * 30cm) contains 1.0M NaCL with pH7.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose 4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the glycine-NaOH damping fluid that contains 1.0M NaCL with pH7.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the phosphate buffered saline buffer wash-out that contains 0.4M NaCL and 0.5M NSC 2020 (BenzamidineHCL) with pH9.5, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.01M citrate buffer with the method displacement pH6.4 concentration of dialysis;
6, metilsulfate sepharose (S Sepharose) chromatography column is at least 10 column volumes of 0.01M citrate buffer balance through pH6.4 concentration, batroxobin crude extract upper prop is separated, citrate buffer with pH value 6.2-8.6 carries out stage gradient wash-out or straight line gradient elution respectively, collects the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last Superdex-75 molecular sieve chromatography (1.6cm * 100cm) contains the acetate buffer wash-out of 0.01M NaCL with pH5.0, nucleic acid-protein detector test sample peak, collection obtains the batroxobin of single composition.Detecting purity is 97%, is 1280BU/mg albumen than vigor, and activity yield is 31%.
Embodiment 19:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 3g, be dissolved in the Tris-HCL damping fluid that 80ml pH10.0 contains 0.8M NaCL, soak in 7 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 0.8M NaCL with pH10.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 0.8M NaCL with pH10.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, glycine-NaOH the buffer solution elution that contains 0.3M NaCL and 0.1M NSC 2020 (BenzamidineHCL) with pH7.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.025M phosphate buffered saline buffer with the method displacement pH6.1 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.025M phosphate buffered saline buffer balance through pH6.1 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the phosphate buffered saline buffer that contains 0.05-0.9M NaCL respectively, collect the sample peak;
7, sample through ultrafiltration and concentration to about 3ml, last propylene dextrane gel S100 (Sephacryl S100) molecular sieve chromatography (1.6cm * 100cm), the citrate buffer wash-out that contains 0.5M NaCL with pH5.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 97%, is 1088BU/mg albumen than vigor, and activity yield is 27%.
Embodiment 20:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 4g, be dissolved in glycine-NaOH damping fluid that 70ml pH10.0 contains 2.0M NaCL, soak in 5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (glycine-NaOH damping fluid that 2.6cm * 30cm) contains 2.0M NaCL with pH10.0 is 5 column volumes of balance at least for benzenyl amidine sepharose 6B (Benzamidine Sepharose 6B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the glycine-NaOH damping fluid that contains 2.0M NaCL with pH10.0 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, the Tris-HCL buffer solution elution that contains 0.3M NaCL and 0.2M NSC 2020 (BenzamidineHCL) with pH9.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.035M citrate buffer with the method displacement pH6.0 concentration of dialysis;
6, metilsulfate sepharose (S Sepharose) chromatography column is at least 10 column volumes of 0.035M citrate buffer balance through pH6.0 concentration, batroxobin crude extract upper prop is separated, citrate buffer with pH value 6.0-8.5 carries out stage gradient wash-out or straight line gradient elution respectively, collects the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S200 (Sephacryl S200) molecular sieve chromatography (1.6cm * 100cm), the Tris-HCL buffer solution elution that contains 0.5M NaCL with pH7.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1602BU/mg albumen than vigor, and activity yield is 26%.
Embodiment 21:
The concrete steps of present embodiment are as follows:
1, get spearhead agkistrodon halyx pallas venom 4g, be dissolved in the Tris-HCL damping fluid that 65ml pH7.5 contains 1.2M NaCL, soak in 3.5 ℃ of refrigerators it is fully dissolved, the centrifugal 10min of 8000rpm removes precipitation then, collects the snake venom supernatant liquor;
2, (the Tris-HCL damping fluid that 2.6cm * 30cm) contains 1.2M NaCL with pH7.5 is 5 column volumes of balance at least for benzenyl amidine sepharose 4B (Benzamidine Sepharose4B) affinity column, with above-mentioned snake venom supernatant liquor upper prop, control flow velocity 0.3ml/min;
3, the Tris-HCL damping fluid that contains 1.2M NaCL with pH7.5 behind the end of the sample carries out wash-out, reaches baseline to nucleic acid-protein detector reading;
4, with affinity column and sephadex G 25 (Sephadex G25) desalting column (1.6cm * 100cm) be connected, glycine-NaOH the buffer solution elution that contains 0.5M NaCL and 0.2M NSC 2020 (BenzamidineHCL) with pH9.0, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
5, the batroxobin crude extract is the 0.05M acetate buffer with the method displacement pH5.8 concentration of dialysis;
6, sulfopropyl sepharose (SP Sepharose F.F.) chromatography column is at least 10 column volumes of 0.05M acetate buffer balance through pH5.8 concentration, batroxobin crude extract upper prop is separated, carry out stage gradient wash-out or straight line gradient elution with the acetate buffer that contains 0.05-0.6M NaCL respectively, collect the sample peak;
7, sample through Macrogol 2000 0 (PEG20000) embedding, be concentrated into about 3ml, last propylene dextrane gel S100 (Sephacryl S100) molecular sieve chromatography (1.6cm * 100cm), the acetate buffer wash-out that contains 0.5M NaCL with pH8.0, the batroxobin that obtains single composition is collected at nucleic acid-protein detector test sample peak.Detecting purity is 98%, is 1361BU/mg albumen than vigor, and activity yield is 25%.
Gained sample of the present invention detects purity according to SDS-polyacrylamide gel electrophoresis (2005 editions two appendix VF the 5th methods of Chinese Pharmacopoeia).Wherein, marker is the lower molecular weight standard protein, and standard substance are the batroxobin standard substance, and sample only shows an electrophoresis band for the batroxobin sample by embodiment of the invention preparation method gained, and scanning purity is greater than 95%.
(seeing accompanying drawing)
Claims (4)
1, extract the method for batroxobin from two the subspecies Bothrops moojeni of spearhead pallas pit viper or Bothrops atrox snake venom, step comprises:
(1) snake venom soaks, dissolves and spend the night with damping fluid I, and centrifugal then removal precipitation gets supernatant liquor;
(2) affinity column on the snake venom supernatant liquor carries out wash-out with damping fluid I, reaches baseline to Protein Detection instrument reading, uses damping fluid II wash-out again, nucleic acid-protein detector test sample peak, collect the batroxobin crude extract;
(3) batroxobin crude extract sephadex G 25 desalting columns of directly flowing through are removed NSC 2020;
(4) the batroxobin crude extract of removing NSC 2020 is with dialysis membrane or ultra-filtration equipment or desalting column exchange buffering liquid III;
(5) on the batroxobin crude extract through damping fluid III equilibrated cation-exchange chromatography post, use NaCl ionic strength gradient buffering liquid or pH gradient buffering liquid wash-out, sample solution is collected at nucleic acid-protein detector test sample peak;
(6) sample solution concentrates through Macrogol 2000 0 embedding, or through ultrafiltration and concentration, through molecular sieve chromatography, with damping fluid IV wash-out, promptly gets batroxobin;
In above-mentioned steps, damping fluid I is that pH is 6.0～10.0 and all contain Tris-HCL damping fluid or glycine-NaOH damping fluid or the phosphate buffered saline buffer of 0.1～2.0M NaCL;
Damping fluid II is that pH is 6.0～10.0 and all contain the Tris-HCL damping fluid or the glycine-NaOH damping fluid or the phosphate buffered saline buffer of 0.1～0.5M NaCL and 0.1～0.5M NSC 2020;
Damping fluid III is phosphate buffered saline buffer or acetate buffer or citrate buffer or the Tris-HCL damping fluid that pH is 5.0～6.5, concentration is 0.001～0.05M;
Damping fluid IV is that pH is 4.0～8.0 and all contain citrate buffer or Tris-HCL damping fluid or phosphate buffered saline buffer or the acetate buffer of 0.01～0.5M NaCL;
Affinity column described in the step (2) is meant benzenyl amidine sepharose 6B or benzenyl amidine sepharose 4B chromatography column;
Cation-exchange chromatography post described in the step (5) is meant sulfopropyl sepharose, metilsulfate sepharose or carboxymethyl sepharose chromatography column;
Molecular sieve chromatography described in the step (6) is propylene dextrane gel S200 or propylene dextrane gel S100 or Superdex-75 chromatography column.
2, the method for extracting batroxobin from snake venom according to claim 1 is characterized in that the affinity column in the described step (2) and the desalting column in the step (3) can be cascaded in the sample elution process or separately.
3, the method for extracting batroxobin from snake venom according to claim 1 and 2 is characterized in that the ionic strength gradient in the described step (5) or the gradient of pH gradient elution are: 0～1.0M NaCL or pH value 4.0～9.0.
4, the method for extracting batroxobin from snake venom according to claim 1 and 2, it is characterized in that: each step is carried out under 3～8 ℃ environment.
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| MX2015006149A (en) * | 2012-11-15 | 2015-08-05 | Genentech Inc | IONIC STRENGTH-MEDIATED pH GRADIENT ION EXCHANGE CHROMATOGRAPHY. |
| CN104013648B (en) * | 2014-04-14 | 2017-06-16 | 浙江中医药大学 | A kind of Agkistrodon extract and its application |
| CN108611341A (en) * | 2016-12-09 | 2018-10-02 | 辽宁远大诺康生物制药有限公司 | The hemagglutinase and its preparation method and application extracted from spearhead viper venom |
| CN109929020A (en) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | A kind of purification process of cobra venom and products thereof |
| CN114686463A (en) * | 2020-12-30 | 2022-07-01 | 远大生命科学(辽宁)有限公司 | Purification method of haemocoagulase atrox |
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2006
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Non-Patent Citations (2)
| Title |
|---|
| 长白山白眉蝮蛇毒类凝血酶的分离纯化. 牟萍等.锦州医学院学报，第26卷第5期. 2005 |
| 长白山白眉蝮蛇毒类凝血酶的分离纯化. 牟萍等.锦州医学院学报，第26卷第5期. 2005 * |
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