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Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg

A technology of hydrophobic chromatography and agarose, applied in the biological field, can solve problems such as uncontrollable ligand density, weakened adsorption capacity of biological macromolecular media, and failure of chromatography media to meet application requirements, etc., to achieve easy maintenance of biological activity and excellent biological Compatibility, the effect of simple and easy purification method

Inactive Publication Date: 2009-05-13
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to overcome the defect that the sulfur-containing agarose chromatography medium prepared by the prior art cannot control the ligand density, so that the biomacromolecule is inactivated in the purification process or the adsorption capacity of the medium is weakened; Inappropriate agarose matrix, resulting in the problem that the final chromatographic medium cannot meet the application requirements, so as to provide a kind of agarose hydrophobic chromatography with controllable ligand density, safe preparation process, good mechanical strength and hydraulic properties Medium, and method for purifying yeast expressing HBsAg using the medium

Method used

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  • Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg
  • Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg
  • Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg

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Embodiment 1

[0040] Take 100.0 g of washed and drained agarose 6FF gel (manufactured by Hangzhou Zhengguang Resin Co., Ltd.), and pour it into a 1-liter three-necked flask. To this was added 150 mL of acetone, 75 mL of epichlorohydrin and 75 mL of 0.5M NaOH solution. The reaction was stirred at 60°C for 4 hours. After the reaction, the gel was washed with water and vacuum-dried, and the washing process was repeated three times. Take 100.0 g of agarose epoxy-activated gel that has been washed and drained, and pour it into a 1-liter three-necked flask. 100 μL of butanethiol, 10 mL of ethanol, and 90 ml of 0.1 M NaOH solution were added thereto. Stir at 60°C for 6 hours. After the reaction, the gel was washed with water and dried, and the washing process was repeated three times to obtain the agarose hydrophobic chromatography medium of the present invention, with a ligand density of 100 μmol / g dry gel, stored in 20% ethanol solution.

Embodiment 2

[0042] Take 100.0 g of washed and drained agarose 6FF gel and pour it into a 1-liter three-necked flask. To this was added 150 mL of epichlorohydrin and 75 mL of 4M NaOH solution. The reaction was stirred at 25°C for 10 hours. After the reaction, the gel was washed with water and dried, and the washing process was repeated three times. Take 100.0 g of agarose epoxy-activated gel that has been washed and drained, and pour it into a 1-liter three-necked flask. 2000 μL of butanethiol, 10 mL of ethanol and 90 ml of 4M NaOH solution were added thereto. Stir at 25°C for 10 hours. After the reaction, the gel was washed with water and dried, and the washing process was repeated three times to obtain the agarose hydrophobic chromatography medium of the present invention with a ligand density of 120 μmol / g dry gel, which was stored in 20% ethanol solution.

Embodiment 3

[0044] Take 100.0 g of washed and drained agarose 6FF gel and pour it into a 1-liter three-necked flask. Thereto were added 150 mL of acetone, 75 mL of 1,4-butanediol diglycidyl ether and 75 mL of 0.5M NaOH solution. The reaction was stirred at 60°C for 4 hours. After the reaction, the gel was washed and dried, and the washing process was repeated three times. Take 100.0 g of agarose epoxy-activated gel that has been washed and drained, and pour it into a 1-liter three-necked flask. 100 μL of butanethiol, 10 mL of ethanol and 90 ml of 0.1 M NaOH solution were added thereto. Stir at 60°C for 6 hours. After the reaction, the gel was washed and dried, and the washing process was repeated three times to obtain the agarose hydrophobic chromatography medium of the present invention with a ligand density of 61 μmol / g dry gel, which was stored in 20% ethanol solution.

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Abstract

A hydrophobic agarose used as the chromatographic medium is prepared from agarose gel through epoxy activating and coupling with butyl sulfhydrate. It can be used in purifying the yeast expression HbsAg. It has high strength, throughput and controllable density.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an agarose hydrophobic chromatography medium and its application in purifying yeast to express HBsAg. Background technique [0002] Hepatitis B is one of the most common infectious diseases. It has the characteristics of strong infectivity, high carrier rate, wide prevalence and serious tendency of chronicity. According to statistics, more than 2 billion people in the world have experienced HBV (Hepatitis B Virus) infection, of which 350 million are chronic HBV carriers, and my country accounts for 120 million. Every year, 1 million to 1.5 million patients worldwide die from liver cirrhosis, liver failure and liver cancer caused by acute or chronic HBV infection. Vaccination against hepatitis B is a safe and effective method of prevention. HBV vaccine can not only effectively block the transmission of HBV, but also effectively control the carrier rate of the population,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/291C12N1/02C12N15/10
Inventor 赵岚苏志国闭静秀周卫斌王阳木李岩黄永东
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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