Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Separation and purification method of recombinant hepatitis B core antigen

A 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 quality standards of medicine, and achieve the effects of low host protein and host nucleic acid residue, simple production process, and low host residue

Active Publication Date: 2021-06-15
JIANGSU THERAVAC BIO PHARMA CO LTD
View PDF8 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Separation and purification method of recombinant hepatitis B core antigen
  • Separation and purification method of recombinant hepatitis B core antigen
  • Separation and purification method of recombinant hepatitis B core antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0072] 1) Bacteria 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 concentrati...

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) Bacteria 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 of...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a separation and purification method of recombinant hepatitis B core antigen, which comprises heat denaturation and clarification, ammonium sulfate precipitation, ultrafiltration and concentration, diafiltration and liquid replacement, depolymerization, the first step of molecular sieve chromatography, ultrafiltration and concentration, diafiltration And the steps of changing liquid, repolymerization and second step molecular sieve chromatography. The method can obtain recombinant hepatitis B core antigen with high purity, low host residue, uniform particle structure, and high stability, that is, the method has the characteristics of high antigen purity, low host protein and host nucleic acid residue, uniform antigen particles, and easy industrial scale-up.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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 CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com