Preparation method of blood coagulation factor X activator RVV-X and prepared RVV-X

A technology of coagulation factor, RVV-X, applied in the direction of biochemical equipment and methods, enzymes, peptidases, etc., can solve the conventional research that limits the scope of RVV-X market use, RVV-X is difficult to mass-produce, and the filler is physically stable problems such as poor performance, to achieve the effect of maintaining the volume and structural stability of the column bed, which is conducive to scale-up production and has fewer preparation steps.

Active Publication Date: 2019-04-19
SHANGHAI SUNBIO TECH
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  • Application Information

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Problems solved by technology

[0004] In the current literature reports, the purification of RVV-X is mainly through the mixed use of molecular sieve filtration, anion exchange column and cation exchange, among which the molecular sieve is mainly Sephadex G-150, Sephadex G-100, Sepherdex G-75 or Superdex 75, but these fillers have poor physical stability and slow speed, which is not conducive to scale-up production. At the same time, because the entire separation and purification process is complicated, the yield is difficult to meet the actual demand of the market, making it difficult for RVV-X to be produced on a large scale, thus limiting The scope of market use of RVV-X and its routine research

Method used

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  • Preparation method of blood coagulation factor X activator RVV-X and prepared RVV-X
  • Preparation method of blood coagulation factor X activator RVV-X and prepared RVV-X
  • Preparation method of blood coagulation factor X activator RVV-X and prepared RVV-X

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preparation example Construction

[0026] The preparation method of blood coagulation factor X activator RVV-X comprises the following steps:

[0027] 1) Viper venom is dissolved in buffer, separated by molecular sieve chromatography column, eluted with buffer, monitored with a nucleic acid protein detector, and the elution line is drawn according to the A280nm value, and the chromogenic substrate method is used to detect RVV- The elution peak of Ⅹ activity is collected and concentrated;

[0028] 2) The active peak concentrate in step 1) is separated by a weak cationic column, and gradient eluted with a buffer solution containing 0-0.5mol / L NaCl, monitored by a nucleic acid protein detector, and the elution line is drawn according to the A280nm value, The elution peaks with RVV-X activity were collected respectively by chromogenic substrate detection.

[0029] Step 1) of the preparation method of the present invention uses viper venom as a raw material, dissolves it in a buffer, conducts rough separation throu...

Embodiment 1

[0056] Dissolve 100 mg of Viper venom in 1.5 mL of 0.05 mol / L TRIS-HCl buffer (pH 7.4), centrifuge at 12,000 rpm for 10 min, take the supernatant and load it onto a Sephacryl S-100HR column that has been equilibrated with the above buffer, and continue to add buffer Liquid elution, flow rate 0.2ml / min, automatic collection of eluent. Monitor with a nucleic acid protein detector, draw the elution line according to the A280nm value (see figure 1 ), while using the chromogenic substrate method to detect and determine the elution peak with RVV-X activity. The results showed that three peaks were mainly obtained in this step of separation, and the peaks with RVV-X activity were collected and concentrated for electropherogram analysis, which showed that most of the small molecular substances were removed in this step.

[0057] The peak with RVV-X activity was concentrated and loaded onto a weak cationic CM Sepharose Fast Flow column equilibrated with 0.05mol / L ammonium acetate buff...

Embodiment 2

[0059] Dissolve 100mg of Viper venom in 1.5mL 0.05mol / L TRIS-HCl buffer solution (pH 7.4), centrifuge at 12000rpm for 10min, take the supernatant and load it onto a Superdex 200 column equilibrated with the above buffer solution, and continue to add buffer solution for elution , the flow rate is 0.2mL / min, and the eluate is collected automatically. Monitor with a nucleic acid protein detector, draw the elution line according to the A280nm value, and use the chromogenic substrate method to detect and determine the elution peak with RVV-X activity. result with figure 1 Similarly, three peaks were mainly obtained in this step of separation, and the peaks with RVV-X activity were collected and concentrated for electropherogram analysis. It can be seen that most of the small molecular substances were removed in this step.

[0060] The peak with RVV-X activity was concentrated and loaded onto a weak cationic CM Sepharose Fast Flow column equilibrated with 0.05mol / L ammonium acetate...

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Abstract

The invention belongs to the technical field of biology and discloses a preparation method of a coagulation factor X activator RVV-X and a prepared RVV-X. The preparation method comprises the following steps of: dissolving viper venom, as a raw material, in a buffer solution, carrying out coarse separation by using a molecular sieve chromatographic column, eluting the buffer solution, collecting an elution peak with RVV-X activity, concentrating, separating and purifying by using a weak cation column, carrying out gradient elution by using a buffer solution containing 0-0.5mol / L NaCl, collecting elution peaks with RVV-X activity. According to the preparation method, the raw material, viper venom, is easy to obtain; the coarse separation is carried out by adopting the molecular sieve chromatographic column; the physical properties of the molecular sieve filler are stable; the process of reloading the column each time is avoided; and the method is more beneficial to scale-up production.According to the preparation method, two kinds of RVV-X of 79KD and 98KD can be obtained through two-step column-passing separation, the preparation steps are few, the yield is high, the prepared twokinds of RVV-X have high purity and good stability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of blood coagulation factor X activator RVV-X and the prepared RVV-X. Background technique [0002] Coagulation factor X activator (FX activator) is a kind of procoagulant component that can activate blood coagulation factor X existing in snake venoms such as Viperidae, Viperidae, and Cobra family. It can activate blood coagulation factor X and accelerate the coagulation process. Promotes blood clotting. Studies have found that the FX activators present in a variety of snake venoms can be divided into metalloproteinases and serine proteases. The FX activator (RVV- X). FX activators are widely used, and the market demand potential is huge. As the main component of an in vitro diagnostic kit, it can be used for FX itself, the difference between FVII and FX deficiency, lupus anticoagulant diagnosis and platelet disease diagnosis. For example, using v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
CPCC12N9/6416C12N9/6418C12Y304/21006C12Y304/24001
Inventor 谢永华李存存
Owner SHANGHAI SUNBIO TECH
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