Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast

A technology of hepatitis B surface antigen and Hansenula yeast, applied in the field of protein separation and purification, can solve the problems of low activity recovery rate, many HBsAg steps, long cycle, etc., and achieve the effects of simple production process, great practical application value and short operation cycle.

Inactive Publication Date: 2009-09-23
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects of many steps, long cycle and low activity recovery rate in the existing separation and purification process of HBsAg expressed by Hansenula, thereby providing a simple process, short cycle, low cost and high purity. Method for Separating and Purifying Recombinant Hepatitis B Surface Antigen Expressed by Hansenula Yeast with High Activity Recovery Rate

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  • Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast
  • Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast
  • Method for separating and purifying recombined hepatitis b surface antigen expressed by Hansenula yeast

Examples

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Embodiment 1

[0035] Example 1. Laboratory-scale separation and purification experiment 1

[0036] Take 200ml of the culture supernatant of the recombinant hepatitis B surface antigen expressed by Hansenula yeast cultured by conventional fermentation, centrifuge and wash the Hansenula yeast cells expressing the hepatitis B surface antigen, then place it in a high-pressure homogenizer and add The weight ratio of the liquid to be disrupted is 0.05% Triton X-100 and 1 mM PMSF, and the disruption is repeated 6 times, and the cell disruption rate reaches 90%.

[0037] Centrifuge the broken cells at 20000g for 20 minutes to remove cell debris. Adjust the pH of the supernatant to 5.5 with 1.0M NaOH and 1.0M HCl, and the conductivity is less than 5.0mS / cm, and then feed it to Q SepharoseFF ion exchange In the chromatography column (GE Healthcare, 30cm×15cm ID), the column volume (CV) is 2000ml, and the flow rate is 1.0ml / (h·ml gel). The chromatographic process is followed by buffer I (20mM phosphate buff...

Embodiment 2

[0041] Example 2: Laboratory-scale separation and purification experiment 2

[0042] Take 200ml of the culture supernatant of the recombinant hepatitis B surface antigen expressed by Hansenula yeast cultured by conventional fermentation, centrifuge and wash the Hansenula yeast cells expressing the hepatitis B surface antigen, then place it on a ball mill, and add the crushing liquid The weight ratio of 0.3% Triton X-100 and 0.1 mM PMSF was repeated twice, and the cell disruption rate reached 50%.

[0043] Centrifuge the broken cells at 6000g for 30min to remove cell debris. Adjust the pH of the supernatant to 8.5 with 1.0M NaOH and 1.0M HCl, and the conductivity is less than 20mS / cm, and then feed it to the Q SepharoseFF ion exchange layer In the analysis column (GE Healthcare, 30cm×15cm ID), the column volume (CV) is 2000ml, and the flow rate is 20ml / (h·ml gel). The chromatographic process is followed by buffer I (20mM Tris-HCl buffer, pH 8.5) balance, feed, buffer I rebalance, 0...

Embodiment 3

[0047] Example 3. Laboratory-scale separation and purification experiment 3

[0048] Take 50ml of the culture supernatant of the recombinant hepatitis B surface antigen expressed by Hansenula yeast cultured by conventional fermentation, centrifuge and wash the Hansenula yeast cells expressing the hepatitis B surface antigen, then place it on a high-pressure homogenizer, and add the The weight ratio of the disrupted liquid was 0.1% Triton X-100 and 10 mM PMSF, and the disruption was repeated 3 times, and the cell disruption rate reached 70%.

[0049] Centrifuge the broken cells at 10000g for 20min to remove cell debris. Adjust the pH of the supernatant to 7.0 with 1.0M NaOH and 1.0M HCl, and the conductivity is less than 10mS / cm, and then feed it to the DEAE QZTFF ion exchange layer In the analysis column (National Biochemical Engineering Center, 30cm×5.5cm ID), the column volume (CV) is 500ml, and the flow rate is 10ml / (h·ml gel). The chromatographic process is followed by buffer ...

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Abstract

The invention relates to a method for separating and purifying recombinant hepatitis B surface antigen expressed by Hansenula, which integrates technologies such as cell crushing, chromatography and membrane separation, and specifically includes: recombinant Hepatitis B expressed by Hansenula obtained by fermentation and culture in a conventional process The surface antigen culture medium is broken until the cell breakage rate reaches 50-90%; the cell debris is removed by centrifugation, the pH value and conductivity are adjusted, and anion exchange chromatography is performed to collect the eluate with an activity recovery rate greater than 20%; combine the eluate , then adjust the conductivity and pH value respectively, then carry out hydrophobic chromatography; use ultrafiltration membrane to concentrate the sample; finally, use gel filtration column for gel filtration to obtain the recombinant HBsAg expressed by Hansenula with a purity of more than 99% . The method has a high recovery rate of purification activity, and the purity can reach more than 99%. It has few operation steps, short operation cycle, relatively low cost, and is easy to scale up to industrial scale production, which has great practical application value.

Description

Technical field [0001] The invention belongs to the field of protein separation and purification, and specifically relates to a method for separating and purifying recombinant hepatitis B surface antigen expressed by Hansenula yeast. Background technique [0002] Hepatitis B virus seriously threatens human health. At present, inoculation of hepatitis B vaccine is still the main means to prevent the spread of hepatitis B virus. Due to the existence of HIV and other viruses and limited sources, the blood-derived hepatitis B vaccine from the blood of hepatitis B virus carriers-Hepatitis B Surface Antigen (hereinafter referred to as HBsAg)-has been banned by the World Health Organization and replaced by it Is a recombinant hepatitis B vaccine. The systems routinely used to express HBsAg mainly include mammalian cells, yeast cells and vaccinia systems. Among them, yeast cells are favored by manufacturers because they have much higher expression levels than animal cells, and hepatitis ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14C07K14/02
Inventor 闭静秀周卫斌苏志国李岩黄永东赵岚张焱
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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