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Method for separating and purifying osteopontin OPN from human milk

An osteopontin and human milk technology, applied in the field of biochemistry, can solve the problems of long time consumption, complicated procedures and high cost, and achieve the effects of simple steps, shortened time and rapid separation

Inactive Publication Date: 2010-02-03
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the 2D-Gel protein separation method consumes a lot of reagents and complicated procedures (Anal.Chem.1996, 68, 850-858); AC method (Protein Expression and Purification.2003, 30: 238-245; Protein Expression and Purification.1997 , 9:309-314), although there are certain advantages in the separation and purification of OPN, the procedures are complex, time-consuming, and the types of reagents used are high, and the cost is high. Chromatography performed on affinity chromatography reagents, not all proteins can find suitable affinity chromatography reagents, there are certain limitations in protein purification

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  • Method for separating and purifying osteopontin OPN from human milk
  • Method for separating and purifying osteopontin OPN from human milk
  • Method for separating and purifying osteopontin OPN from human milk

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Embodiment 1

[0025] Isolate and purify osteopontin (OPN) from human milk according to the following steps:

[0026] (A) Centrifuge 1L of human milk sample at a high speed, the rotation speed must be greater than 12000 rpm, the temperature should not exceed 0°C, and the centrifugation time should be 30-45 minutes; milk fat (60mL), whey (920mL) and milk Granules (20mL), wherein milk fat is in the upper layer, milk grains are in the lower layer, and OPN is in the whey part of the middle layer;

[0027] (B) Preliminary separation: the whey part is adjusted to pH 4.6 with 1mol / L sulfuric acid, precipitated, and after casein is removed, it is separated by high performance liquid chromatography with SCX chromatographic column, mobile phase A (20mmol / L citric acid, 20mmol / L ammonium citrate, pH 3.0) and mobile phase B (20mmol / L citric acid, 20mmol / L ammonium citrate, 0.5mol / L NaCl, pH 3.0) for gradient elution, first eluted with 100% A for 20min , and then the content of mobile phase B was eluted...

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Abstract

The invention provides a method for separating and purifying osteopontin (OPN) from human milk. The method comprises: separating OPN-containing whey from human milk samples through high-speed centrifugation; adopting high-performance liquid chromatography together with SCX chromatographic column to perform preliminary gradient separation; obtaining SCX5 and SCX6 components containing OPN; furtherpurifying the two components on C4 chromatographic column; and obtaining high-purity osteopontin OPN. The method has the advantages of simple steps, high speed, time conservation, few types of used reagents and low cost.

Description

technical field [0001] The invention belongs to the field of biochemistry. Specifically, the present invention relates to a method for isolating and purifying osteopontin OPN from human milk. Background technique [0002] Osteopontin (OPN) is an important multifunctional extracellular matrix protein and a potential pro-inflammatory cytokine related to inflammatory processes, mainly derived from humans, mice, rats, cattle, pigs and rabbits , expressed on bone tissue cells, macrophages, smooth muscle cells, epithelial cells and other cells, mediates chemotaxis, aggregation, adhesion, proliferation and migration of cells, mineralization and reconstruction of bone tissue, immune regulation, important role in signal transduction. Because this protein is a product of cells in the bone matrix and can form a bridge between the cells and the minerals in the matrix, it is named osteopontin. OPN is a negatively charged non-collagenous bone matrix secreted glycosylated phosphoprotein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/20C07K1/14
Inventor 石磊刘晓梅程舸王韶于雷蒋鑫萍
Owner JILIN UNIV