CN1102173C

CN1102173C - Long-noded pit viper enzyme and producing technology thereof

Info

Publication number: CN1102173C
Application number: CN99116406A
Authority: CN
Inventors: 王晴川; 刘广芬
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 1999-03-26
Filing date: 1999-03-26
Publication date: 2003-02-26
Anticipated expiration: 2019-03-26
Also published as: CN1229136A

Abstract

The present invention discloses an agkistrodon enzyme and a production process thereof, which has the technical scheme that freeze-drying venin is dissolved by a known buffer solution and is filtered by dextrane gel G-75 columns, peaks A and B having defibrinogenase vitality are collected, and chromatography is carried out by DEAE-dextrane gel A-50 columns; peaks V, VI and VII having defibrinogenase vitality are collected, and the chromatography is carried out b y CM-dextrane gel C-50 columns; peaks CM1, CM2 and CM3 having defibrinogenase vitality are collected and are filtered by superdex75PG columns, and two peaks (a) and (b) are respectively obtained; peak (b)having high defibrinogenase vitality is collected, and the agkistrodon enzyme with the molecular weight of 24000 to 30000 dalton and the isoelectric point of 4.5 to 5.0 is prepared through mixing.

Description

Effect of Agkistrodon acutus Enzyme and production technique thereof

The present invention relates to a kind of medicine and production technique thereof, relate in particular to a kind of Effect of Agkistrodon acutus Enzyme and production technique thereof.

Document patent application publication number CN1106699A discloses a kind of Acutobin Injection and preparation technology thereof, adopt the agkistrodon acutus poisons sephadex G-75 column chromatography wash-out in Fujian to go out protein peak, get DEAE-dextrane gel A-50 wash-out on the active protein peak of tool, collect V, VI, the VII peak, go up sephadex G-75 again and carry out purifying, examine and determine through SDS-polyacrylamide gel electrophoresis (SDS-PAGE), promptly obtain molecular weight and be respectively 14000,31000 and 66000 daltonian three kinds of isozyme, be mixed with Acutobin Injection again after becoming Effect of Agkistrodon acutus Enzyme by 4: 5: 1 mixed, but the Acutobin Injection that this kind Effect of Agkistrodon acutus Enzyme is mixed with, there are some untoward reactions clinically, it is more to show as platelet count decline, slight mucous membrane occurs, dermatorrhagia and fash.

The object of the present invention is to provide a kind of purity is higher, untoward reaction is less, clinical effectiveness is good Effect of Agkistrodon acutus Enzyme and production technique thereof.

The object of the present invention is achieved like this, described Effect of Agkistrodon acutus Enzyme, its characteristics for get by the ahylysantinfarctase separation and purification have the thrombin-like enzyme activity and arginine ester enzyme activity, molecular weight are that 24000～30000 dalton and iso-electric point are 4.5～5.0 serine-type proteolytic enzyme mixt.

The objective of the invention is to be achieved through the following technical solutions, described Effect of Agkistrodon acutus Enzyme production technique, be 1 in regular turn) point of decon kiss Pallas pit viper freeze-drying snake venom powder sephadex G-75 post on the solution after Tutofusin tris-HCl damping fluid that an amount of pH value is 8.3～8.7, concentration is 0.01～0.05mol/L dissolves, filter A, B, three protein peaks of C are collected A peak and B peak with thrombin-like enzyme activity; 2) A peak of collecting with thrombin-like enzyme activity and B peak get 12 protein peaks with DEAE-dextrane gel A-50 column chromatography, collect V, VI, the VII peak with thrombin-like enzyme activity respectively; 3) CM-dextrane gel C-50 column chromatography is used at the V with thrombin-like enzyme activity, the VI that collects respectively, VII peak respectively, each gets 3 protein peaks, collects CM1, CM2, the CM3 protein peak with thrombin-like enzyme activity respectively; 4) CM1 with thrombin-like enzyme activity, the CM2 that collects respectively, CM3 protein peak filter with the superdex75PG post respectively, the corresponding a that respectively obtains, two protein peaks of b are collected highly purified b protein peak with high thrombin-like enzyme activity, and its mixing is Effect of Agkistrodon acutus Enzyme.

Production technique the 1st of the present invention), 2) step is identical with documents, step and documents be inequality, thereby can collect that to have high purity and high thrombin-like enzyme activity and arginine ester enzyme activity, molecular weight be that 24000～30000 dalton and iso-electric point are 4.5～5.0 serine-type proteolytic enzyme mixt but 3), 4).And the documents production technique is collected molecular weight and is respectively 14000,31000 and 66000 daltonian three kinds of isozyme mixtures.HPLC to the detection of product purity on this technology be single protein peak, purity reaches 95～100%, and documents is 3 protein peaks, and this handicraft product is single band in the SDS-PAGE detection, documents is 3 bands, and the enzyme activity of this handicraft product is also compared than document height.The Effect of Agkistrodon acutus Enzyme that Effect of Agkistrodon acutus Enzyme that production technique of the present invention is produced and documents production technique are produced all is a kind of serine-type proteolytic ferment, can make the Fibrinogen in the blood be cracked into soluble fibrin, and other has the arginine ester enzyme activity.Injection liquid is measured by the method for Ministry of Health regulation all has following characteristics 1) fibrin clot appears in 6 minutes; 2) the arginine ester enzyme activity reaches more than the 1000 μ molTAME/mgmin, and containing Effect of Agkistrodon acutus Enzyme protein is 100 ± 15% of labelled amount; 3) aspect pharmacology, show as the Fibrinogen that can make in the blood and be cracked into soluble fibrin, and eliminate in vivo rapidly, thereby reduce blood viscosity, and the energy anticoagulant, thereby prevent thrombosis; 3) can impel vascular endothelial cell to discharge tissue plasminogen activator, thereby impel thrombolysis; 4) be used to prevent and treat cerebral thrombosis (cerebral infarction) and other peripheral artery vein thrombotic diseases.Acute toxicity and long term toxicity and specific toxicity mensuration are carried out in the toxicology aspect, all meet every requirement of new drug approval.The Effect of Agkistrodon acutus Enzyme that the Effect of Agkistrodon acutus Enzyme that production technique of the present invention is produced and documents production technique are produced is owing to having different molecular weight, and explanation is the different components in the snake venom, and the purity height.Its injection liquid of Effect of Agkistrodon acutus Enzyme untoward reaction clinically that production technique of the present invention is produced is less, 1) showing as platelet count does not have obvious decline, can go up voluntarily very soon after the drug withdrawal; 2) more slight mucous membrane, dermatorrhagia and fash, symptom Lock-out after the drug withdrawal appear in extremely indivedual cases.

The present invention is described in detail below in conjunction with embodiment and accompanying drawing:

Fig. 1 is that snake venom is through sephadex G-75 post filtering A peak and B peak DEAE-dextrane gel A-50 column chromatography figure.

Fig. 2 collects three peaks of V, VI, VII with thrombin-like enzyme activity, filter with sephadex G 75 posts respectively, remove the C peak of non-activity, with activated A, the B peak mixes, and collects CM1, the CM2 that has the thrombin-like enzyme activity separately, the column chromatography figure of CM3 protein peak respectively with CM-dextrane gel C-50 column chromatography.

Fig. 3 is CM1, CM2, the CM3 protein peak column chromatography figure through the superdex75PG post.

Thrombin-like enzyme activity of the present invention and the same document of arginine ester hydrolysing enzyme vigour-testing method " Ahylysantinfarctase thrombase sample The separation of enzyme, purifying and biochemical pharmacology research thereof " " Fujian Medical College's journal " VoL.23 (4): 303～308,1989 public affairs The method of opening. Molecular assay is pressed SDS-PAGE method (Weber K, Osbom M.J Biol Chemistry 1969 244:4406)

Embodiment

After ahylysantinfarctase lyophilized powder 10.0 gram of removing impurity dissolves with Tutofusin tris-HCl damping fluid that an amount of pH value is 8.3～8.7, concentration is 0.01～0.05mol/L, the centrifugation decon, centrifugation solution is below 25 ℃ in temperature, last sephadex G-75 post filters, elutriant with the pH value be 8.3～8.7, concentration is Tutofusin tris-HCl of 0.01～0.05mol/L, with ultraviolet spectrophotometer photometry absorption value, get A, B, three protein peaks of C are measured the thrombin-like enzyme activity.Collection has the A peak and the B peak of thrombin-like enzyme activity, A peak of collecting and B peak DEAE-dextrane gel A-50 column chromatography, elutriant with the pH value be 8.3～8.7, concentration is Tutofusin tris-HCl of 0.01～0.05mol/L, 12 protein peaks, measure the thrombin-like enzyme activity.Collect three peaks of V, VI, VII respectively with thrombin-like enzyme activity, the V that collects, VI, three peaks of VII, filter with sephadex G-75 post respectively, remove the C peak of non-activity, with activated A, the B peak mixes, with CM-dextrane gel C-50 column chromatography, elutriant with the pH value be 8.3～8.7, concentration is Tutofusin tris-HCl of 0.01～0.05mol/L, 3 protein peaks are each got through column chromatography in each peak, measure the thrombin-like enzyme activity.CM1, CM2, the CM3 protein peak that has the thrombin-like enzyme activity separately collected at V, VI, three peaks of VII respectively behind column chromatography, the CM1 that collects, CM2, CM3 protein peak filter with the superdex75PG post respectively again, elutriant contains 0.15mol/LNaCl with Tutofusin tris-HCl that the pH value is 8.3～8.7, concentration is 0.01～0.05mol/L, the corresponding a that respectively obtains, two protein peaks of b are measured the thrombin-like enzyme activity.Collect the highly purified b protein peak of SDS-PAGE calibrating with thrombin-like enzyme height vigor.Above-mentioned three the b protein peak mixing with thrombin-like enzyme activity are the Effect of Agkistrodon acutus Enzyme mother liquor.Its molecular weight is that 24000～30000 dalton and iso-electric point are 4.5～5.0 serine-type proteolytic enzyme mixt after measured.The Effect of Agkistrodon acutus Enzyme mother liquor is through HPLC post TSK G3000-SW (7.5 * 300 millimeters), (eastern ソ one Co., Ltd.'s Nanyang cause is produced) detected, stage number: 10000N/30 centimetre, moving phase: 0.02mol/L sodium phosphate-0.1mol/L sodium sulfate damping fluid, the pH value is 7.0, flow velocity: 1.5 ml/min, recording its purity is 95～100%.The Effect of Agkistrodon acutus Enzyme mother liquor is after No. 6 sintered filter funnels filter, make determining the protein quantity, remake thrombin-like enzyme activity and arginine ester enzyme activity determination, pyrogen and anaphylaxis test, after all qualified, with aseptic, apyrogenic 0.9%NaCl dilution again after No. 6 sintered filter funnels filter, by every 1ml:0.75 unit, the embedding ampoule.

As shown in Figure 1, be A peak and B peak DEAE-dextrane gel A-50 column chromatography figure, chromatography column 50mm * 100cm, total A280nm=14000～15000OD, elutriant with the pH value be 8.3～8.7, concentration is Tutofusin tris-HCl damping fluid of 0～0.3mol/LNaCl--0.01～0.05mol/L, flow velocity: 8 milliliters/15 minutes/pipe, as we know from the figure behind the column chromatography altogether 12 protein peaks, measure the thrombin-like enzyme activity, wherein V, VI, three peaks of VII have the thrombin-like enzyme activity.As shown in Figure 2, for collecting V with thrombin-like enzyme activity, VI, three peaks of VII, filter with sephadex G-75 post respectively, remove the C peak of non-activity, with activated A, the B peak mixes, collect the CM1 that has the thrombin-like enzyme activity separately respectively with CM-dextrane gel C-50 column chromatography, CM2, the column chromatography figure of CM3 protein peak, used post is a CM-dextrane gel C-50 post, column length is 40mm * 450cm, and elutriant is respectively Tutofusin tris-HCl damping fluid of 0.01mol/L with concentration, 0.2mol/L the Tutofusin tris of NaCl--0.01mol/L-HCl damping fluid, 0.3mol/L the Tutofusin tris of the Tutofusin tris of NaCl--0.01mol/L-HCl damping fluid and 0.4mol/LNaCl--0.01mol/L-HCl buffer solution for gradient elution.Flow velocity: 5 milliliters/15 minutes/pipe, sample: 1000O.D (A280nm)/28ml.As shown in Figure 3, be CM1, CM2, the CM3 protein peak is through the column chromatography figure of superdex75PG post, chromatography column superdex75PG 26mm * 74.5cm, flow velocity: 4 ml/min, CM1, CM2, the CM3 protein peak filters respectively, elutriant is 8.3～8.7 with the pH value, concentration is the damping fluid that Tutofusin tris-HCl of 0.15mol/L NaCl--0.01～0.05mol/L contains 0.15mol/L NaCl, the corresponding a that obtains separately behind the column chromatography as we know from the figure, two protein peaks of b, measure the thrombin-like enzyme activity, collect highly purified b protein peak, above-mentioned three the b protein peak mixing with thrombin-like enzyme height vigor are Effect of Agkistrodon acutus Enzyme with high thrombin-like enzyme activity.Post that the present invention is used and gel are Sweden Pharmacia company product.

Claims

1, Effect of Agkistrodon acutus Enzyme, a kind of by the ahylysantinfarctase separation and purification have the thrombin-like enzyme activity and arginine ester enzyme activity, molecular weight are that 24000～30000 dalton and iso-electric point are 4.5～5.0 serine-type proteolytic enzyme mixt.

2, the described Effect of Agkistrodon acutus Enzyme production technique of claim 1, it is characterized in that comprising the steps: in regular turn 1) the ahylysantinfarctase lyophilized powder of decon sephadex G-75 on solution after Tutofusin tris-HCl damping fluid dissolving that an amount of pH value is 8.3～8.7, concentration is 0.01～0.05mol/L filter A, B, three protein peaks of C are collected A peak and B peak with thrombin-like enzyme activity; 2) A peak of collecting with thrombin-like enzyme activity and B peak get 12 protein peaks with DEAE-dextrane gel A-50 column chromatography, collect V, VI, the VII peak with thrombin-like enzyme activity respectively; 3) V with thrombin-like enzyme activity, the VI that collects respectively, VII peak, filter with sephadex G 75 posts respectively, get A, B, three peaks of C remove non-activity C peak, with A, the B peak mixes the back with CM-dextrane gel C-50 column chromatography, gets 3 protein peaks, collects CM1, CM2, the CM3 protein peak that has the thrombin-like enzyme activity separately respectively; 4) CM1 with thrombin-like enzyme activity, the CM2 that collects, CM3 protein peak filter with the superdex75PG post respectively, the corresponding a that respectively obtains, two protein peaks of b, collect highly purified b protein peak, above-mentioned three the b protein peak mixing with thrombin-like enzyme activity are Effect of Agkistrodon acutus Enzyme with thrombin-like enzyme activity.