Purification method of streptococcus pneumoniae capsular polysaccharide

A technology of Streptococcus pneumoniae and capsular polysaccharide, applied in the field of simplified purification, can solve the problems of potential safety hazards, many steps, and high requirements for operators, and achieve large-scale amplification, avoid the use of phenol, and shorten the process time. Effect

Active Publication Date: 2015-12-09
LANZHOU INST OF BIOLOGICAL PROD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

US20100129881 and NormaSuarez et al. (NORMASUA′REZ, LAURAFRANCO, et al., FRAGUAS APPLIEDANDENVIRONMENTALMICROBIOLOGY, 2001, p.969–971Vol.67, No.2), introduced the method of column chromatography in the purification process of Streptococcus pneumoniae capsular polysaccharide, However, this method has high requirements for operators, takes a long time for sample processing, and the operation and maintenance costs of equipment are high, so it is difficult to meet the needs of large-scale production
CA1206905 proposed enzymatic hydrolysis method to purify capsular polysaccharide of Streptococcus pneumoniae, VivianeMaimoni (Viviane Maimoni etal., Simple and effi

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  • Purification method of streptococcus pneumoniae capsular polysaccharide

Examples

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Embodiment 1

[0031] Example 1: Purification of Type 1 Streptococcus pneumoniae capsular polysaccharide

[0032] Add sodium deoxycholate to 5 L of type 1 pneumococcal fermentation culture medium to make the final concentration 0.15%, and place it at room temperature for 12 hours to lyse. Then the lysate was centrifuged (centrifugal force: 6000 g, centrifugation time: 35 minutes), and the supernatant was collected. The supernatant was then concentrated to 1000 ml by ultrafiltration in a 100 Kd membrane bag. Add sodium deoxycholate to make the final concentration 0.5%, adjust the pH to 4.4, let stand at room temperature for 10 minutes, perform centrifugation (centrifugal force: 6000g, centrifugation time: 15 minutes), and collect the supernatant. Then repeat the above steps, then add 0.5% sodium deoxycholate, adjust the pH to 4.4, let stand at room temperature for 10 minutes, perform centrifugation (centrifugal force: 6000g, centrifugation time: 15 minutes), and collect the supernatant. The...

Embodiment 2

[0035] Example 2: Purification of Type 3 Streptococcus pneumoniae capsular polysaccharide

[0036]Type 3 Streptococcus pneumoniae fermentation culture medium 1L was added with sodium deoxycholate to make the final concentration 0.05%, and placed at room temperature for 15 hours to lyse. Then the lysate was centrifuged (centrifugal force: 6000g, centrifugation time: 35 minutes), and the supernatant was collected. The supernatant was then concentrated to 1000 ml by ultrafiltration in a 100 Kd membrane bag. Add sodium deoxycholate to make the final concentration 1%, adjust the pH to 4.0, let stand at room temperature for 10 minutes, perform centrifugation (centrifugal force: 6000g, centrifugation time: 15 minutes), and collect the supernatant. Then repeat the above steps, then add 1% sodium deoxycholate, adjust the pH to 4.0, let stand at room temperature for 10 minutes, centrifuge (centrifugal force: 6000g, centrifugation time: 15 minutes), and collect the supernatant. Then ad...

Embodiment 3

[0039] Example 3: Purification of Type 5 Streptococcus pneumoniae capsular polysaccharide

[0040] Add sodium deoxycholate to 6 L of type 5 Streptococcus pneumoniae fermentation broth to make the final concentration 0.12%, and place it at room temperature for 15 hours to lyse. Then the lysate was centrifuged (centrifugal force: 6000g, centrifugation time: 35 minutes), and the supernatant was collected. The supernatant was then concentrated to 1000 ml by ultrafiltration in a 100 Kd membrane bag. Add sodium deoxycholate to make the final concentration 0.5%, adjust the pH to 4.5, let stand at room temperature for 10 minutes, perform centrifugation (centrifugal force: 6000g, centrifugation time: 15 minutes), and collect the supernatant. Then repeat the above steps, then add 0.5% sodium deoxycholate, adjust the pH to 4.5, let stand at room temperature for 10 minutes, perform centrifugation (centrifugal force: 6000g, centrifugation time: 15 minutes), and collect the supernatant. T...

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Abstract

The invention relates to a purification method of streptococcus pneumoniae capsular polysaccharide. The purification method comprises the following steps: subjecting streptococcus pneumoniae lysate to ultrafiltration and condensation, adding sodium deoxycholate, adjusting the pH to 4-5, carrying out a centrifugal treatment, collecting the supernate, adding nuclease into the supernate, carrying out reactions for 3-8 hours to obtain acidified lysate, adding sodium deoxycholate into the acidified lysate, subjecting acidified lysate to ultrafiltration, performing acidification and centrifugation, collecting the supernate, and finally subjecting the supernate to ultrafiltration and condensation to obtain a purified polysaccharide solution. In the provided purification method, phenol is not used; after ethanol drying, the obtained purified streptococcus pneumoniae capsular polysaccharide is the cell lysate after one nuclease treatment, the product is more environment-friendly and safer, the process time is short, the production cost is low, and massive production can be achieved.

Description

technical field [0001] The field of the present invention relates to a purification method of capsular polysaccharide of Streptococcus pneumoniae, especially a simplified purification method. Background technique [0002] Streptococcus pneumoniae is the primary pathogen of respiratory tract infection, not only the most common pathogen causing community-acquired pneumonia in children, but also the main pathogen causing fatal pneumonia infection in the elderly. At present, more than 90 serotypes have been found, of which about 30 serotype strains are pathogenic to humans. Capsular polysaccharide is the main virulence factor of Streptococcus pneumoniae. The research results show that the capsular polysaccharide vaccine of Streptococcus pneumoniae has good immunogenicity and safety. [0003] The general process of preparing the Streptococcus pneumoniae capsular polysaccharide vaccine is as follows: the bacteria are grown in a fermenter, and at the end of the fermentation, sodiu...

Claims

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Application Information

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IPC IPC(8): C08B37/00
Inventor 任克明王玺王剑虹白贵杰沈荣
Owner LANZHOU INST OF BIOLOGICAL PROD
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