Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method

A technology for hemagglutinin and scutellaria, which is applied in the field of separation and purification to obtain hemagglutinin and its preparation, and achieves the effects of stable preparation process, shortened coagulation time and low toxicity

Inactive Publication Date: 2007-04-11
沈居仁 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the technical difficulties in the separation and purification of the single-chain hemagglutinase from Agkistrodon akistrodon venom, there are very few relevant literatures with practical reference significance

Method used

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  • Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method
  • Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method
  • Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, 20060418 batches of Agkistrodon acutus venom 3.6KD hemagglutinase production example

[0032] Weigh 10g of Agkistrodon venom freeze-dried product, add 50ml of sterile water for injection, dialyze with deionized water at 4°C overnight, centrifuge at 8000rpm, 15min, 4°C, and adjust the conductivity of the supernatant to 3.5 with 1M Tris-HCl (PH8.0) After ms / cm, put on the affinity chromatography column Metal Sepharose F.F column which was pre-equilibrated with 0.05M Tris-HCl (PH8.0), 1M NH 4 Cl was eluted in stages, and the breakthrough peak was collected; the DEAE-Sepharose column on the breakthrough peak was equilibrated, and after zero washing with 0.05M Tris-HCl (PH8.0) for 1 column bed volume, wash with 0.05M Tris-HCl+1.0 M NaCl (PH8.0) was used for gradient elution, and the eluted 5 peaks (that is, the crude fraction of hemocoagulase) were collected under the detection of ultraviolet 280nm, and the active peak was desalted and freeze-dried. The lyophi...

Embodiment 2

[0033] Embodiment two, 20060428 batches of Agkistrodon acutus venom 3.6KD hemagglutinase production example

[0034] Weigh 10g of Agkistrodon venom freeze-dried product, add 50ml of sterile water for injection, dialyze with deionized water at 4°C overnight, centrifuge at 8000rpm, 15min, 4°C, and adjust the conductivity of the supernatant to 3.5 with 1M Tris-HCl (PH8.0) After ms / cm, put on the affinity chromatography column Metal Sepharose F.F column which was pre-equilibrated with 0.05M Tris-HCl (PH8.0), 1M NH 4 Cl was eluted in stages, and the breakthrough peak was collected; the DEAE-Sepharose column on the breakthrough peak was equilibrated, and after zero washing with 0.05M Tris-HCl (PH8.0) for 1 column bed volume, wash with 0.05M Tris-HCl+1.0 M NaCl (PH8.0) was used for gradient elution, and the eluted 5 peaks (that is, the crude fraction of hemocoagulase) were collected under the detection of ultraviolet 280nm, and the active peak was desalted and freeze-dried. The lyop...

Embodiment 3

[0035]Embodiment three, 20060523 batches of Agkistrodon acutus venom 3.6KD hemagglutinase production example

[0036] Weigh 10g head of Agkistrodon venom freeze-dried product, add 50ml sterile water for injection, dialyze with deionized water overnight at 4°C, centrifuge at 8000rpm, 15min, 4°C, and use 1M Tris-HCl (PH8.0) to adjust the conductivity of the supernatant to After 3.3ms / cm, put on the Metal sepharose F.F XK50 / 30 column (product of GE Healthcare) equilibrated with 0.05MTris-HCl (PH8.0) in advance, 1M NH 4 Cl was eluted in stages, and the breakthrough peak was collected; the DEAE-sepharose column on the breakthrough peak was equilibrated, and after zero washing with 0.05M Tris-HCl (PH8.0) for 1 column bed volume, wash with 0.05M Tris-HCl+1.0 M NaCl (PH8.0) was used for gradient elution, and the eluted 5 peaks (that is, the crude fraction of hemocoagulase) were collected under the detection of ultraviolet 280nm, and the active peak was desalted and freeze-dried. Diss...

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Abstract

The present invention is Agkistrodon acutus venon 36KD single-stranded hemocoagulase and its preparation process, and aims at providing single-stranded Agkistrodon acutus venon hemocoagulase with high potency and low toxicity. SDS-PAGE electrophoresis shows that the hemocoagulase has molecular weight of 36KD+/-2KD, single color zone, and at least 90 % homogeneity with the N end sequence of the single-stranded amino acid shown in SEQ ID No. 1. The preparation process includes dissolving Agkistrodon acutus venon in Tris-HCl buffering solution (pH8.0) and dialysis; adding the supernatant to Metal Sepharose F.F affinity chromatographic column and collecting the penetrating peak component; adding the penetrating peak component to DEAE-Sepharose F.F ion exchange column and collecting active peak component; separating in Superdex 75 column and Sephacryl S-100 column; desalting in Sephadex G-25 column; sterilizing and freeze drying.

Description

Technical field [0001] The invention relates to an enzyme and a method for separating and purifying the enzyme, in particular to separating and purifying hemagglutinase from Agkistrodon acutus and a method for preparing the same. Background technique [0002] In 1936, K1obusizky and Konig isolated an enzyme protein that can shorten the coagulation time from the venom of the willow-leaf viper, named Coagulase, and many scholars successively confirmed the existence of this enzyme in different Vipers of the Viper family. Since the 1960s, with the improvement of protein purification and separation technology and the in-depth research of biochemistry and pharmacology, the great value of hemocoagulase in clinical hemostasis application has been determined. Among them, Reptilase is a product of Solco Basle Ltd in Switzerland and Brazil The hemostatic agent prepared from the single-chain hemocoagulase with a molecular weight of about 31KD extracted fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74
Inventor 沈居仁郑颖范泉水
Owner 沈居仁
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