Structure and purification of venin-class thrombase for preventing and curing thrombotic diseases
A technology for thrombotic diseases and thrombin-like enzymes, which is applied in blood diseases, extracellular fluid diseases, enzymes, etc., can solve problems such as difficulty in obtaining high purity, difficulty in production and purification, and shortage of snake venom resources
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Embodiment 1
[0035] Materials and equipment (the same as below):
[0036] The snake venom is collected from the tip of Wuyi Mountain.
[0037] HiPrep16 / 10DEAE, MonoQ5 / 5HR, SOURCEQ, RESOURCERPC are all produced by Amphamacia.
[0038] Chromatography adopts AKTA expdorer rapid protein purification system, produced by Ampharmacia.
[0039] Dissolve 5 grams of snake venom in 50ml of 0.01mol / L, pH7.5 Tris-HCl buffer, centrifuge it at 7000rpm for 15 minutes after fully dissolving, take the supernatant at 0.01mol / L, pH7.5 Dilute the dialyzed solution by 5 times and filter it with a 0.22 μm microporous membrane. The filtrate was purified by DEAE column chromatography, and the chromatographic conditions were: mobile phase A was 0.01-0.02mol / L, Tris-HCl buffer solution with pH 7.5, B was A solution containing 1mol / L NaCl, and the flow rate was 3ml / min. Using binary gradient elution, The components purified by DEAE column chromatography were purified by MonoQ column, and the chromatographic co...
Embodiment 2
[0041] Dissolve 5 grams of snake venom in 50ml of 0.01mol / L, pH8.2 Tris-HCl buffer, centrifuge it at 7000rpm for 15 minutes after fully dissolving, take the supernatant at 0.01mol / L, pHg.2 Dilute the dialyzed solution by 5 times and filter it with a 0.22 μm microporous membrane. The filtrate was purified by HiPrep16 / 10DEAE column chromatography, and the chromatographic conditions were: mobile phase A was 0.01-0.02mol / L, Tris-HCl buffer solution with pH 8.2, B was A solution containing 1mol / L NaCl, and the flow rate was 3ml / min, using binary gradient elution, The components purified by DEAE column chromatography are purified by SOURCEQ column. The chromatographic conditions are: mobile phase A is 0.01-0.02mol / L, Tris-HCl buffer solution with pH 8.0, B is A solution containing 0.3mol / L NaCl , flow rate: 3ml / min, using binary gradient elution, The components obtained from SOURCEQ column purification were purified by RESOURCE RPC column chromatography. The chromatographic c...
Embodiment 3
[0043] Dissolve 5 grams of snake venom in 0.01mol / L, pH8.5 Tris-HCl buffer solution, centrifuge it at 7000rpm for 15 minutes after fully dissolving, and dialyze the supernatant at 4°C, the dialysate is 0.01mol / L, Tris-HCl buffer solution of pH 8.5, the dialyzed solution was diluted 5 times and then filtered with a 0.22 μm microporous membrane. Take 20ml of the filtrate and purify it by HiPrep16 / 10DEAE column chromatography. The chromatographic conditions are: mobile phase A is 0.01-0.02mol / L, Tris-HCl buffer solution with pH 8.5, B is A solution containing 1mol / L NaCl, and the flow rate is 3ml / min, using binary gradient elution, The components purified by HiPrep16 / 10DEAE column chromatography were purified by SOURCE Q column. The chromatographic conditions were: mobile phase A was 0.01-0.02mol / L, Tris-HCl buffer solution with pH 8.0, B was containing 0.3mol / L NaCl Solution A, flow rate: 3ml / min, using binary gradient elution, The components obtained from SOURCE Q column...
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