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Separation and purification method for recombinant hepatitis B core antigen

A kind of hepatitis B core antigen, separation and purification technology, applied in the field of protein separation and purification, can solve the problem that HBcAg is difficult to meet the pharmaceutical quality standards and other problems

Active Publication Date: 2018-05-18
JIANGSU THERAVAC BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of the core protein in the research of hepatitis B diagnostic reagents or therapeutic vaccines has been paid more and more attention. At present, full-length or truncated HBcAg has been overexpressed in Escherichia coli, Pichia pastoris, Hansenula and insect cells, but Due to its preparation process, the obtained HBcAg is difficult to meet the pharmaceutical quality standard in terms of residual substances

Method used

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  • Separation and purification method for recombinant hepatitis B core antigen
  • Separation and purification method for recombinant hepatitis B core antigen
  • Separation and purification method for recombinant hepatitis B core antigen

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0071] The purification of embodiment 1 recombinant hepatitis B core antigen

[0072] 1) Bacterial fragmentation

[0073] Take the fermented bacteria, resuspend with 50mM Tris-HCl containing 150mM NaCl, pH 8.0 buffer, maintain the solid content at 10%-30%, obtain the initial bacterial suspension, use a high-pressure homogenizer 1500bar to repeatedly break up The initial bacterial suspension was taken 6 times until the cell disruption rate reached 80-90%; centrifuged at 12000g at 4°C for 30min to remove the precipitate and obtain the supernatant.

[0074] 2) thermal denaturation clarification

[0075] The supernatant obtained in step 1) was placed in a water bath at 60° C. and kept shaking at 50-200 rpm for 0.5-2 hours. Then centrifuge at 12000g, 4°C for 30min, collect the supernatant, and obtain the supernatant;

[0076] 3) Ammonium sulfate precipitation treatment

[0077] Add ammonium sulfate solution dropwise to the supernatant obtained in step 2), the final concentrat...

Embodiment 2

[0090] Example 2 Purification of Recombinant Hepatitis B Core Antigen

[0091] 1) Bacteria fragmentation

[0092] Take the fermented bacteria, resuspend with 50mM Tris-HCl containing 150mM NaCl, pH 8.0 buffer, maintain the solid content at 10%-30%, obtain the initial bacterial suspension, use a high-pressure homogenizer 1500bar to repeatedly break up The initial bacterial suspension was taken 6 times until the cell disruption rate reached 80-90%; centrifuged at 12000g at 4°C for 30min to remove the precipitate and obtain the supernatant.

[0093] 2) thermal denaturation clarification

[0094] The supernatant obtained in step 1) was placed in a water bath at 60° C. and kept shaking at 50-200 rpm for 0.5-2 hours. Then centrifuge at 12000g, 4°C for 30min, collect the supernatant, and obtain the supernatant;

[0095] 3) Ammonium sulfate precipitation treatment

[0096] Add ammonium sulfate solution dropwise to the supernatant obtained in step 2), the final concentration of a...

Embodiment 3

[0109] Example 3 Purification of Recombinant Hepatitis B Core Antigen

[0110] 1) Bacterial fragmentation

[0111] Take the fermented bacteria, resuspend with 50mM Tris-HCl containing 150mM NaCl, pH 8.0 buffer, keep the solid content at 10%-30%, obtain the initial bacterial suspension, use a high-pressure homogenizer 1800bar to repeatedly crush and obtain The initial bacterial suspension was taken 4 times until the cell disruption rate reached 80-90%; centrifuged at 12000g at 4°C for 30min to remove the precipitate to obtain the supernatant.

[0112] 2) thermal denaturation clarification

[0113] The supernatant obtained in step 1) was placed in a water bath at 50° C. and kept shaking at 50-200 rpm for 0.5-2 hours. Then centrifuge at 12000g, 4°C for 30min, collect the supernatant, and obtain the supernatant;

[0114] 3) Ammonium sulfate precipitation treatment

[0115]Add ammonium sulfate solution dropwise to the supernatant obtained in step 2), the final concentration o...

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Abstract

The invention relates to a separation and purification method for a recombinant hepatitis B core antigen. The method includes the steps of thermal denaturation and clarification; ammonium sulfate precipitation; ultrafiltration and concentration, washing filtering and liquid exchange; depolymerization; first-step molecular sieve chromatography; ultrafiltration and concentration, washing filtering and liquid exchange; repolymerization; second-step chromatography. By means of the method, the high-purity, low-host-residue and high-stability recombinant hepatitis B core antigen with a uniform granular structure can be obtained, that is to say, the method has the advantages that the antigen purity is high, the residue of host protein and host nucleic acid is low, antigen particles are uniform, and industrial enlargement is easy.

Description

technical field [0001] The invention belongs to the field of protein separation and purification, in particular to a method for separation and purification of recombinant hepatitis B core antigen. Background technique [0002] Hepatitis B virus (HBV) is a double-stranded DNA enveloped virus that can cause various acute and chronic hepatitis and even liver cancer. According to the report of the World Health Organization, about 2 billion people in the world have been infected with HBV, and about 650,000 people die of liver failure, cirrhosis and liver cancer caused by HBV infection every year (Rehermann et al., Nat Rev Immunol, 2005; Michel et al ., Vaccine, 2002; Jung et al., Lancet Infect Dis, 2002). [0003] HBV virus particles are composed of two parts, the nucleocapsid and the outer envelope (envelop), which wrap the nucleic acid in the center. The nucleocapsid is composed of core protein (HBcAg). The core protein has a total of 183 amino acids and can be divided into t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/02C07K1/36C07K1/30C07K1/16
CPCC07K14/005C12N2730/10151
Inventor 李建强任苏林顾月周童孙洪林葛君戚凤春鲍梦汝韩杰陈晓晓
Owner JIANGSU THERAVAC BIO PHARMA
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