Method for separating and purifying functional protein in plasma

A technology for separation and purification of plasma proteins, applied in the field of bioengineering, which can solve the problems of huge differences in protein types and contents, failure to overcome the Cohn method, high cost of affinity media, etc., and achieve easy industrial scale-up, low cost, and convenient solvent recovery Effect

Active Publication Date: 2013-09-11
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent authorization announcement number CN1259338C discloses a plasma protein separation method that can be carried out in a sealed pipeline. This method improves the Cohn method, and uses pressure filtration to replace the centrifugation of the solid-liquid separation part in the Cohn method, and the production cycle can be shortened. half, but still does not overcome the problems of Cohn's law
Chinese patent authorization announcement number CN1089609C discloses a method for obtaining high-purity IgG by affinity chromatography, but the high cost of the affinity medium limits the application of this method
However, compared with yeast fermentation liquid, plasma not only has a large difference in shape, but also has a huge difference in the type and content of protein in it.

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  • Method for separating and purifying functional protein in plasma
  • Method for separating and purifying functional protein in plasma
  • Method for separating and purifying functional protein in plasma

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1 Purification of Human Plasma Functional Proteins

[0052] (1) Two-phase solid-liquid separation treatment

[0053] Prepare 40%K 2 HPO 4 solution, adjusted to pH 7.0 with phosphoric acid, at room temperature (24°C), weigh 2.5 g of 40% K with pH 7.0 2 HPO 4 Solution, 0.8g water, 1.0g human plasma, 0.7g ethanol, stir and mix well, stand at 4°C for 2 hours to form a two-phase aqueous phase, collect the upper phase extract, protein concentration analysis shows that the upper phase extract is rich in IgG and albumin, the yields were 81.18% and 87.61%.

[0054] (2) Hydrophobic chromatography purification

[0055] After adjusting the pH of the upper layer extract in step (1) to 6.0, carry out vacuum distillation to recover ethanol, the distillation temperature is 37°C, the distillation time is 30min, and the ethanol content in the upper layer extract is less than 10%.

[0056] Phenyl Sepharose 6FF TM Pack the hydrophobic medium into the XK16 / 20 column, the colu...

Embodiment 2

[0066] Example 2 Separation and Purification of Porcine Plasma Functional Proteins

[0067] (1) Two-phase solid-liquid separation treatment

[0068] At room temperature (24°C), take 1,000ml of fresh pig blood, centrifuge twice at 3000r / min for 15min, remove blood cells, take the supernatant, and dilute it with 0.9% normal saline to obtain a pig plasma solution. Weigh 20gK 2 HPO 4Dissolve in 46g of water, slowly add 20g of plasma and 14g of ethanol, stir well, and measure the pH of the system to be 9.6. After standing at room temperature for 2 hours, an aqueous two-phase phase was formed, and the upper phase extract was collected. Protein concentration analysis showed that the upper phase extract was rich in IgG and albumin, and the yields were 95.7% and 93.0%, respectively.

[0069] (2) Hydrophobic chromatography purification

[0070] Adjust the pH of the upper layer extract in step (1) to 7.0, then carry out vacuum distillation to recover ethanol, the distillation tempera...

Embodiment 3

[0078] Example 3 Effect of Organic Solvent and Salt Concentration on the Yield of IgG in Two-phase Extraction

[0079] Select different concentrations of dipotassium hydrogen phosphate, sodium citrate and sodium carbonate with different concentrations of ethanol and appropriate amount of water to form a two-phase system, the pH of the system is 7.0, and investigate the organic solvent in different two-phase systems. , The effect of salt concentration on the extraction yield of human IgG. In all systems, the amount of IgG added was 1 mg, and the standing time was 8 hours. The upper phase extract was collected, and the protein content in it was determined by Bradford method, and the IgG recovery (%) was calculated.

[0080] In the dipotassium hydrogen phosphate-ethanol system, the final concentration of ethanol is 14-16% (w / w), the concentration of dipotassium hydrogen phosphate is 18-24% (w / w), and the temperature is 15°C; IgG recovery rate results such as image 3 shown.

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Abstract

The invention provides a method for separating and purifying functional protein in plasma, which comprises the following steps of: adding soluble inorganic salt and a hydrophilic organic solvent into a plasma solution to form a two-aqueous phase extraction system, and obtaining the upper-phase extraction liquid rich in plasma functional protein; and performing further separation and purification of the extraction liquid through hydrophobic chromatography and ion exchange chromatography to obtain the plasma functional protein of which the electrophoresis purity is higher than 95%. The method provided by the invention simplifies the purifying steps of the plasma functional protein; the organic solvent/salt two-aqueous phase extraction has the advantages of convenience in solvent recovery, low cost and short phase separating time, does not need back-extraction operation and can selectively remove glucose and partial protein in the plasma; and the extraction liquid is directly separated and purified by hydrophobic chromatography and ion exchange chromatography, the complicated steps of desalination are avoided, and the separation and purification effects of the plasma functional protein are remarkably improved. The method provided by the invention solves the problems of complicated separation steps, high cost, low purity and the like in the separation and purification of functional protein in the plasma protein.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for separating and purifying proteins, in particular to a method for separating and purifying functional proteins in blood plasma. Background technique [0002] Plasma is composed of 90% water, 7% protein and 3% sugar organic matter. The 7% protein contains hundreds of proteins with a wide range of physiological functions, making plasma a fractionated product of most therapeutic products or biological drugs The raw materials, functional proteins separated and extracted from plasma include albumin, immunoglobulin, coagulation factors (fibrin, procoagulation factor VIII), protein inhibitors and other varieties. Among them, albumin has the highest content, accounting for more than half of the total. It is a single-chain, non-glycosylated protein, and is mainly involved in maintaining osmotic pressure, transporting fatty acids, and assisting human detoxification. Album...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K16/06C07K1/20C07K1/18C07K1/14
Inventor 董悦生李玲玲于芳修志龙
Owner DALIAN UNIV OF TECH
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