Preparation method of human derived thrombomodulin capable of being industrially produced

A technology for regulating protein and urinary protein, which is applied in the field of preparation of human-derived hemagglutination regulatory protein, can solve the problems of low yield and unsuitability for large-scale industrial production, and achieve high biological activity, industrialization basis, and high yield. Effect

Inactive Publication Date: 2018-05-04
YANGZHOU AIDEA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In summary, it can be seen that the various purification methods currently used to extract TM from human urine all exhibit low yields and are not suitable for large-scale industrial production.

Method used

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  • Preparation method of human derived thrombomodulin capable of being industrially produced
  • Preparation method of human derived thrombomodulin capable of being industrially produced
  • Preparation method of human derived thrombomodulin capable of being industrially produced

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Collect 100 kilograms of resins that absorb urine protein, wash with water, pack column; Carry out elution with the NaCl aqueous solution of 0.5mol / L, collect elution peak, record the content of hemagglutinin wherein is 11mg.

[0030] a. Metal chelate affinity chromatography

[0031] Use an ultrafiltration membrane with a molecular weight cutoff of 30K for ultrafiltration concentration, adjust the pH of the ultrafiltration concentrate to 5.0, and the conductivity to 0.5mS / cm, and the metal ion that has been balanced on the surface is Cu 2+ The metal chelating affinity chromatography column (Chelating sepharoseFF); then balance liquid (equilibrium liquid formula: 0.2mol / L acetate buffer solution, containing 0.05mol / LNH 4 Ac, pH is 5.0) to wash the metal chelate affinity chromatography column, collect the sample loading and wash the breakthrough, wash with the washing solution (0.2mol / L acetate buffer, containing 0.1mol / LNH 4 Ac, pH is 5.0) washing, with eluent (0.2mol / L...

Embodiment 2

[0042] Collect 100 kilograms of resins that absorb urine protein, rinse with water, pack into columns; carry out elution with the NaCl aqueous solution of 1.5mol / L, collect elution peak, measure the content of hemagglutinin wherein is 10mg.

[0043] a. Metal chelate affinity chromatography

[0044] Use an ultrafiltration membrane with a molecular weight cut-off of 30K for ultrafiltration concentration, adjust the pH of the ultrafiltration concentrate to 8.0, and the conductivity to 10mS / cm, and the balanced metal ion on it is Zn 2+ The metal chelate affinity chromatography column (Chelating sepharoseFF), and then balance liquid (equilibrium liquid formula: 0.02mol / L phosphate buffer, containing 0.5mol / LNH 4 Ac, the pH is 8.0) to wash the metal chelate affinity chromatography column, collect the sample loading and washing breakthrough, and use the washing solution (0.02mol / L phosphate buffer, containing 0.5mol / LNH 4 Ac, pH is 8.0) washing, with eluent (0.02mol / L phosphate aque...

Embodiment 3

[0054] Collect 5 tons of resin that absorbs urinary protein, rinse with water, pack column; Carry out elution with the NaCl aqueous solution of 0.8mol / L, collect elution peak, record the hemagglutinin content wherein is 500mg.

[0055] a. Metal chelate affinity chromatography

[0056] Use an ultrafiltration membrane with a molecular weight cutoff of 30K for ultrafiltration concentration, adjust the pH of the ultrafiltration concentrate to 6.0, and the conductivity to 5.0mS / cm, and the metal ion that has been balanced on the surface is Cu 2+ The metal chelating affinity chromatography column (Chelating sepharoseFF); then use the equilibrium solution (equilibrium solution formula: 0.1mol / L phosphate buffer, containing 0.1mol / LNH 4 Ac, pH is 6.0) to wash the metal chelate affinity chromatography column, collect the sample loading and wash the breakthrough, wash with the flushing solution (0.1mol / L phosphate buffer, containing 0.2mol / L NH 4 Ac, pH 6.0) wash, with eluent (0.1mol / L...

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Abstract

The invention discloses a preparation method of human derived thrombomodulin capable of being industrially produced. According to the method, urine protein concentrate extracted from human urine is made to go through a metal chelate affinity chromatography column, an anion exchange column, a thrombin affinity chromatography column and a hydrophobic chromatography column so as to obtain thrombomodulin. The provided method has the advantages of simple and convenient operation, low cost, and high yield. The prepared thrombomodulin is purely human-derived, has the advantages of no foreign proteinpollution, more safety, more reliability, high bioactivity, no immunogenicity and stable quality, and is suitable for large scale production.

Description

technical field [0001] The present invention relates to a method for preparing human-derived hemagglutination regulatory protein, in particular to a new method for large-scale preparation of human-derived hemagglutination regulatory protein that can be industrially produced. Background technique [0002] Thrombomodulin (TM), also known as thrombin modulin, is a single-chain transmembrane glycoprotein present on the membrane of vascular endothelial cells. The relative molecular mass of the complete molecule is 75,000 Da, and the relative molecular mass after degrading the disulfide bond is 105,000 Da, composed of 575 amino acids. TM was originally discovered by N.L.Esmon et al. in 1981. Its structure includes an amino-terminal plant agglutination-like domain, 6 repeated endothelial growth factor (endothelial growth factor, EGF)-like domains, and a serine / threonine-rich region (hydrophobic region), a transmembrane region and a short intracellular tail. The structure of throm...

Claims

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Application Information

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IPC IPC(8): C07K14/745C07K1/22C07K1/20C07K1/18
CPCC07K14/7455
Inventor 侯晓彦傅和亮李文全袁玉王筱蒙居维艳
Owner YANGZHOU AIDEA BIOTECH
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