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

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

Inactive Publication Date: 2007-07-25
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

[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,

Method used

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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

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Embodiment 1

[0035] Embodiment 1, laboratory scale separation and purification experiment 1

[0036] Take 200ml of the culture supernatant of recombinant HBsAg expressed by Hansenula spp. obtained by conventional process fermentation culture, first centrifuge and wash the Hansenula saccharomyces cells used to express HBsAg, then place it in a high-pressure homogenizer, add The weight ratio of the crushing liquid is 0.05% Triton X-100 and 1 mM PMSF, and the cell crushing rate reaches 90% after repeated crushing for 6 times.

[0037]The broken cells were centrifuged at 20000g for 20min to remove cell debris, the supernatant was adjusted to pH 5.5 with 1.0M NaOH and 1.0M HCl, and the conductivity was less than 5.0mS / cm, and then fed to Q SepharoseFF ion exchange In a chromatography column (GE Healthcare, 30cm×15cm I.D.), the column volume (CV) is 2000ml, and the flow rate is 1.0ml / (h·ml gel). The chromatography process is followed by buffer I (20mM phosphate buffer (PB), pH5.5) equilibration...

Embodiment 2

[0041] Embodiment 2, laboratory scale separation and purification experiment 2

[0042] Take 200ml of the culture supernatant of recombinant HBsAg expressed by Hansenula spp. obtained by conventional fermentation and culture, first centrifuge and wash the Hansenula saccharomyces cells used to express HBsAg, then place it on a ball mill, and add the broken solution The weight ratio of 0.3% Triton X-100 and 0.1mM PMSF was repeatedly crushed twice, and the cell crushing rate reached 50%.

[0043] The broken cells were centrifuged at 6000g for 30min to remove cell debris, the supernatant was adjusted to pH 8.5 with 1.0M NaOH and 1.0M HCl, and the conductivity was less than 20mS / cm, and then fed to the Q SepharoseFF ion exchange layer In a column (GE Healthcare, 30cm×15cm I.D.), the column volume (CV) is 2000ml, and the flow rate is 20ml / (h·ml gel). The chromatography process is followed by buffer I (20mM Tris-HCl buffer, pH8.5) equilibration, feeding, buffer I rebalance, 0-100% b...

Embodiment 3

[0047] Embodiment 3, laboratory scale separation and purification experiment 3

[0048] Take 50ml of the culture supernatant of recombinant HBsAg expressed by Hansenula spp. obtained by conventional fermentation and culture, first centrifuge and wash the Hansenula saccharomyces cells used to express HBsAg, then place it on a high-pressure homogenizer, and add The weight ratio of the disrupting fluid is 0.1% Triton X-100 and 10mM PMSF, and the disrupting solution is repeated 3 times, and the cell disrupting rate reaches 70%.

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

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Abstract

This invention relates to a method for separating and purifying recombinant hepatitis B virus surface antigen expressed in Hansenula polymorpha. The method comprises: (1) crushing culture solution of recombinant B virus surface antigen expressed in Hansenula polymorpha till 50-90% cells are crushed; (2) centrifuging to remove cell debris, adjusting pH value and electrical conductivity, performing anion exchange chromatography, and collecting the eluates with activity higher than 20%; (3) incorporating the eluates, adjusting the pH value and electrical conductivity, and performing hydrophobic chromatography; (4) concentrating the eluate with ultrafiltration membrane; (5) separating by a gel filtration column to obtain recombinant B virus surface antigen expressed in Hansenula polymorpha with purity higher than 99%. The method has such advantages as high recovery rate, high product purity, few steps, and short time.

Description

technical field [0001] The invention belongs to the field of protein separation and purification, and in particular relates to a method for separation and purification of recombinant hepatitis B surface antigen expressed by Hansenula yeast. Background technique [0002] HBV is a serious threat to human health. At present, hepatitis B vaccination is still the main means of preventing and controlling the spread of hepatitis B virus. Due to the problems of carrying 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 (Hepatitis B Surface Antigen, hereinafter referred to as HBsAg) - has been banned by the World Health Organization and replaced by Recombinant Hepatitis B Vaccine. Conventional systems for expressing HBsAg mainly include mammalian cells, yeast cells, and vaccinia systems. Among them, yeast cells are favored by manufacturers because of their expression level muc...

Claims

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Application Information

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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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