Method for preparing activated pig plasma blood coagulation factor X

A blood coagulation factor and porcine plasma technology, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve the problems of low purity of porcine FXa, low purity of porcine FX, and difficulty in scaling up, and solve the problem of limited plasma sources , Improve the separation efficiency and the effect of improving the yield

Active Publication Date: 2017-12-12
SHANGHAI SUNBIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few reports about the preparation of FX and FXa from pig plasma at home and abroad.
Zhang Qiang et al. and He Huan prepared porcine FX by extracting and purifying porcine FX according to the preparation method of bovine FX, but the purity of porcine FX in the report was not high and the loss was large (He Huan. Extraction of three coagulation factors in porcine blood and coagulation factors Protein analysis of X[D]. Hunan Agricultural University, 2009; Zhang Qiang, Gao Xuejun, Qian Shanshan, et al. Study on the preparation technology of porcine coagulation factor Ⅹ and coagulation factor Ⅹa[J]. Chinese Journal of Biochemical Medicine, 2005, 26( 1):24-26), difficult to enlarge
Purify porcine plasma FX and FXa through traditional ion-exchange chromatography packing, hydrophobic chromatography packing, and heparin affinity chromatography using the currently generally recognized and effective methods for FX purification. The purity of porcine FXa is also not high and the loss is large

Method used

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  • Method for preparing activated pig plasma blood coagulation factor X
  • Method for preparing activated pig plasma blood coagulation factor X
  • Method for preparing activated pig plasma blood coagulation factor X

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Extracting and Purifying FX from Pig Plasma

[0046] Add 80mL of 1M barium chloride solution to each liter of pig plasma, stir slowly for 30 minutes, and centrifuge to obtain the precipitate of FX crude extract. The obtained precipitate was dissolved in 0.2M EDTA pH7.4 solution, and the pH was adjusted to 7.4 before loading. Samples were loaded onto the Capto Adhere column equilibrated with 0.02M Tris-HCl buffer pH 7.4 buffer, and then unbound impurities were washed with 0.02M Tris-HCl buffer pH 7.4 buffer until the baseline was stable. 0.025M citric acid-sodium citrate buffer (pH6.5) containing 0.1M NaCl, 0.05M sodium citrate buffer (pH5.8) containing 0.15M NaCl, 0.05M citric acid-sodium citrate buffer ( pH3.0) stage elution, elution speed 1.0mL / min, 5min / tube, automatically collect eluate. Monitor with a nucleic acid and protein detector, according to A 280nm Values ​​plot the elution line, such as figure 1 shown. Use the chromogenic substrate method to...

Embodiment 2

[0047] Example 2 Plasma directly prepares FX through the chromatographic column

[0048] Add an appropriate amount of PPA HyperCel filler that has been equilibrated with an equilibration buffer (0.02M Tris-HCl buffer pH 7.4) into the porcine plasma, and stir gently for 1 hour. Pack the protein-adsorbed filler into the column, and then fully wash the unbound foreign protein with the equilibration buffer, about 10 times the column bed volume of the equilibration buffer is required. 0.025M citric acid-sodium citrate buffer (pH6.5) containing 0.1M NaCl, 0.05M citric acid-sodium citrate buffer (pH5.8) containing 0.15M NaCl, 0.05M citric acid-sodium citrate buffer Buffer (pH3.0) stage elution, each elution stage requires about 3 times the column bed volume of elution buffer, and collect the eluate. The chromogenic substrate method was used to detect the activity of each collected part, and the eluate with FX activity was determined, and the collected part was dialyzed and concentra...

Embodiment 3

[0049] Example 3 activates FX

[0050] Collect the eluate with FX activity in Example 1 or Example 2 and add sodium citrate solid to make the final concentration 0.35g / ml, and activate at room temperature for 23 hours. Then add purified RVV-X freeze-dried powder to it to make the concentration 0.5mg / ml, then add 100mL 0.2M calcium chloride solution and 5mL rabbit brain phospholipid to each liter of eluent, activate at 37°C for 3 hours . After activation is complete, the concentrated sample is dialyzed and immediately isolated and purified.

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Abstract

The invention belongs to the technical field of biology, and specifically discloses a method for preparing an activated pig plasma blood coagulation factor X. The method comprises the steps of performing column chromatography on pig plasma through compound chromatography filler, stepwise eluting a buffer solution, and collecting an eluant of the buffer solution with pH of 3.2 to 2.8; adding an activating agent to activate the blood coagulation factor X; performing benzamidine affinity column chromatographic purification on the activated blood coagulation factor X. Compared with the prior art, the preparation method disclosed by the invention can be used for purifying the pig plasma to prepare FXa through the compound chromatography filler, has the advantages of convenience in preparation, reducion of a separation step, improvement of separation efficiency, reduction of cost, low consumption and high yield and purity, and solving of the problem that a plasma source is limited to a certain extent, and is suitable for large-scale industrialization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing activated porcine blood coagulation factor X. Background technique [0002] Coagulation factor X (FX) is a vitamin K-dependent serine protease and a plasma protein, which consists of two peptide chains connected by disulfide bonds. In the blood coagulation cascade reaction, the activated coagulation factor X (FXa) is at the junction of endogenous, exogenous and common pathways, so it plays a key role in the chain reaction of blood coagulation. In the plasma of normal people, the content of coagulation factor X is usually 10 μg / ml. [0003] There are many suitable ways to activate blood coagulation factor X. In addition to blood components in the endogenous and exogenous blood coagulation pathways that can activate blood coagulation factor X, RVV-X, trypsin, and 25% sodium citrate in viper venom etc. can also activate coagulation factor X. There a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
CPCC12N9/6432C12Y304/21006
Inventor 谢永华侯冬梅
Owner SHANGHAI SUNBIO TECH
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