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Method for purifying streptococcus pneumoniae capsular polysaccharide

A technology of Streptococcus pneumoniae and capsular polysaccharide, applied in the field of medicine, can solve the problems of restricting large-scale use, expensive nuclease, etc., and achieves the effects of mild reagents, no damage to the environment and human body, and simple preparation process

Inactive Publication Date: 2017-08-22
HUALAN BIOLOGICAL ENG INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nuclease enzymatic hydrolysis is commonly used to remove nucleic acid with deoxyribonuclease and ribonuclease. This method is generally more thorough in removing nucleic acid, but the price of nuclease is relatively expensive, which limits the large-scale use of this method

Method used

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  • Method for purifying streptococcus pneumoniae capsular polysaccharide
  • Method for purifying streptococcus pneumoniae capsular polysaccharide
  • Method for purifying streptococcus pneumoniae capsular polysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1) Take Streptococcus pneumoniae culture solution with serotype 7F, add sodium deoxycholate (DOC) with a final concentration of 0.12%, stir overnight, centrifuge to remove large particles of impurities, and collect the supernatant;

[0044] 2) Use an ultrafiltration membrane with a molecular weight cut-off of 100KDa for ultrafiltration and liquid replacement. Before ultrafiltration, use a 0.45-0.8 μm filter for pre-filtration, use 0.1M phosphate buffer as the ultrafiltration buffer, and the pH value is 7.0 ;

[0045] 3) Adjust the pH of the filtrate obtained after ultrafiltration to 4.0, place it in a cold storage and stir for 30 minutes, and retain the supernatant by high-speed refrigerated centrifugation;

[0046] 4) Adding mass percent concentration to the supernatant is 20% CaCl 2 Mix the aqueous solution to a final concentration of 3% in the system, stir for 10-30 minutes, adjust the pH to about 9.0, place in a cold storage and stir for 30 minutes, and centrifuge at...

Embodiment 2

[0049] Take the Streptococcus pneumoniae culture fluid with serotype 12F, add sodium deoxycholate (DOC) with a final concentration of 0.12%, stir overnight, centrifuge to remove large particles of impurities, and collect the supernatant; and use ultrafiltration with a molecular weight cut-off of 100KDa The membrane is used for ultrafiltration and liquid replacement. Before ultrafiltration, use a 0.45-0.8μm filter for pre-filtration. The ultrafiltration buffer uses 0.1M phosphate buffer, and the pH value is about 7.0; adjust the pH of the sample after ultrafiltration to 4.0, placed in the cold storage and stirred for 30 minutes, high-speed refrigerated centrifugation to retain the supernatant; add CaCl with a final concentration of 3% to the supernatant 2 solution, stirred for 10 to 30 minutes, adjusted to a pH of about 9.0, placed in a cold storage and stirred for 30 minutes, high-speed refrigerated and centrifuged to retain the supernatant; then added ethanol to the supernatan...

Embodiment 3

[0051] Take the Streptococcus pneumoniae culture fluid with serotype 14, add sodium deoxycholate (DOC) with a final concentration of 0.12%, stir overnight, centrifuge to remove large particles of impurities, and collect the supernatant; and use ultrafiltration with a molecular weight cut-off of 100KDa The membrane is used for ultrafiltration and liquid replacement. Before ultrafiltration, use a 0.45-0.8μm filter for pre-filtration. The ultrafiltration buffer uses 0.1M phosphate buffer, and the pH value is about 7.0; adjust the pH of the sample after ultrafiltration to 4.0, placed at room temperature and stirred for 30 minutes, high-speed refrigerated centrifugation to retain the supernatant; add a final concentration of 3% CaCl to the supernatant 2 Solution, stirred for 10-30 minutes, adjusted to pH 9.0, placed at room temperature and stirred for 30 minutes, high-speed refrigerated and centrifuged to retain the supernatant; The buffer solution is 0.5M sodium chloride, and then...

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Abstract

The invention discloses a method for purifying streptococcus pneumoniae capsular polysaccharide. The method comprises 1) inactivating streptococcus pneumoniae, centrifuging the streptococcus pneumoniae and collecting the supernatant, 2) carrying out ultrafiltration and collecting the filtrate, 3) adjusting the pH of the filtrate obtained in step 2) to 3-5, centrifuging the filtrate and collecting the supernatant, (4) adding a calcium salt obtained by the step 3) into the supernatant, adjusting the pH to more than 7, carrying out centrifugation and collecting the supernatant, and 5) adding a precipitant into the supernatant obtained in the step 4), carrying out precipitation, collecting the precipitates, and washing and drying the precipitates so that the purification of the streptococcus pneumoniae capsular polysaccharide is realized. Through the optimization of the purification process, the capsular polysaccharides having protein content, nucleic acid content, sugar content, phosphorus content and nitrogen content satisfying indexes. The method has high content and high yield of streptococcus pneumoniae, simple processes and enlargement easiness and is suitable for the production of a streptococcus pneumoniae polysaccharide vaccine.

Description

technical field [0001] The invention belongs to the field of medicine, and relates to a purification method of Streptococcus pneumoniae capsular polysaccharide. Background technique [0002] Streptococcus pneumoniae is a Gram-positive coccus that is arranged in pairs and arranged in spearheads. More than 90 serotypes have been found, of which about 30 serotypes are pathogenic to humans and often cause infections in children and the elderly. Human infection with this pathogen can lead to a series of diseases, including mild otitis media and severe invasive diseases such as pneumonia, blood disease, meningitis, and sepsis. Infection with Streptococcus pneumoniae is the leading cause of death for children under 5 years of age. The World Health Organization estimates that 1.6 million people worldwide die from Streptococcus pneumoniae infection each year, and the vast majority of deaths occur in low-income countries. In addition, the problem of drug resistance of Streptococcus p...

Claims

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Application Information

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IPC IPC(8): C08B37/00
CPCC08B37/0003
Inventor 王立波吴贺高瑞芳李涛
Owner HUALAN BIOLOGICAL ENG INC
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