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Process for purifying polysaccharide of bacteria by using Sepharose 4FF gel

A bacterial polysaccharide and gel technology, which is applied in the field of bacterial polysaccharide purification, can solve problems such as unfavorable product quality control, no literature reports on bacterial polysaccharide purification, and large process fluctuations, so as to improve safety and effectiveness, and be easy to control. The effect of easy operation

Active Publication Date: 2007-05-30
YUXI WALVAX BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many steps in this process, and each process involves many process parameters, so the process fluctuates greatly, which brings unfavorable factors to the quality control of products. At the same time, the large-scale use of phenol has brought great pressure to the environmental protection of enterprises.
[0005] Sepharose 4FF gel has been applied in the purification of viruses and nucleic acids, but there is no literature report on the use of Sepharose 4FF gel to purify bacterial polysaccharides

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Haemophilus influenzae type b was fermented and cultured in a bioreactor; after sterilization in the late logarithmic growth period, the supernatant was collected by centrifugation; cetyltrimethylammonium bromide was added to the supernatant to a final concentration of 0.1% After standing at room temperature for 12 hours, centrifuge at 4000rpm for 15 minutes to collect the precipitate; add 1mol / LCaCl to the precipitate 2 solution, after stirring for 3 hours, add ethanol to a final concentration of 25%, and centrifuge to collect the supernatant; add ethanol to the supernatant to a final concentration of 80%, and centrifuge to collect the polysaccharide precipitate; the polysaccharide precipitate is washed twice with absolute ethanol and acetone respectively ; The washed polysaccharide is freeze-dried to obtain the crude polysaccharide of Haemophilus influenzae type b.

[0020] Dissolve 10 grams of crude sugar at 50 mg / ml in water for injection, add chloroform-n-butanol m...

Embodiment 2

[0030] Use a bioreactor to ferment and cultivate group A meningococci; after sterilization in the late logarithmic growth period, centrifuge to collect the supernatant; add cetyltrimethylammonium bromide to the supernatant to a final concentration of 0.1%, After standing at room temperature for 12 hours, the precipitate was collected by centrifugation; 1mol / L CaCl was added to the precipitate 2 solution, after stirring for 3 hours, add ethanol to a final concentration of 25%, and centrifuge to collect the supernatant; add ethanol to the supernatant to a final concentration of 75%, and centrifuge to collect the polysaccharide precipitate; the polysaccharide precipitate is washed twice with absolute ethanol and acetone respectively ; The washed polysaccharide is freeze-dried to obtain the crude polysaccharide of group A meningococcus.

[0031] Dissolve 40 grams of crude sugar at 10 mg / ml in water for injection, add chloroform-n-butanol mixture (chloroform: n-butanol volume ratio...

Embodiment 3

[0041] Use a bioreactor to ferment and cultivate group C meningococci; after sterilization in the late logarithmic growth period, centrifuge to collect the supernatant; add cetyltrimethylammonium bromide to the supernatant to a final concentration of 0.1%, After standing at room temperature for 12 hours, centrifuge to collect the precipitate; add 1mol / L CaCl to the precipitate 2 solution, after stirring for 3 hours, add ethanol to a final concentration of 25%, and centrifuge to collect the supernatant; add ethanol to the supernatant to a final concentration of 75%, and centrifuge to collect the polysaccharide precipitate; the polysaccharide precipitate is washed twice with absolute ethanol and acetone respectively ; The washed polysaccharide is freeze-dried to obtain the crude polysaccharide of group C meningococcus.

[0042] Dissolve 20 grams of crude sugar at 10 mg / ml in water for injection, add chloroform-n-butanol mixture (chloroform: n-butanol volume ratio 5:1) according ...

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Abstract

The invention discloses a purifying method of bacterial polysaccharide through Sepharose 4FF gel in the biological technical domain, which comprises the following steps: predisposing bacterial polysaccharide; purifying; collecting component before Kd0.5; obtaining high-purity and low-endotoxin product.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and more specifically relates to a method for purifying bacterial polysaccharides. Background technique: [0002] Bacterial capsular polysaccharides are important protective antigens of bacteria. Inoculation of these polysaccharides can provide immune protection for groups over 2 years old. [0003] In the process of bacterial fermentation and cultivation, in addition to producing capsular polysaccharides, a large amount of protein, nucleic acid, endotoxin and other components are also produced. When the content of these components in the vaccine exceeds a certain standard, it may cause serious side effects after vaccination. The bacterial polysaccharide vaccine regulations issued by the World Health Organization (WHO) and the polysaccharide vaccine standards of the "Chinese Pharmacopoeia" all have strict requirements on the protein content, nucleic acid content and endotoxin content in the capsular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04C08B37/00C12R1/46C12R1/42C12R1/21C12R1/01
Inventor 黄镇向左云
Owner YUXI WALVAX BIOTECH CO LTD
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