Specific polysaccharide preparation process

A specific and species-specific technology, applied in the field of preparation of conjugated vaccines, can solve problems such as low yield and complicated preparation process, and achieve the effects of simplifying the process, reducing operation links and saving time

Active Publication Date: 2011-09-21
BEIJING ZHIFEI LVZHU BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after the extraction of lipopolysaccharide and then the purification of specific polysaccharides, there are many unfavorable factors such as complex preparation process, use of phenol and other organic reagents that are harmful to the human body and the environment, and low yield. More concise and effective preparation methods are needed.

Method used

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Examples

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Embodiment 1

[0026] Embodiment one bacterial fermentation culture

[0027] After the Escherichia coli strain is turned on, inoculate it into 5ml LB medium, culture it with shaking at 35°C for 6 hours, transfer it to 500ml LB medium, cultivate it with shaking at 35°C for 6-8 hours, and transfer it to a 10L strain tank. After culturing for 6-8 hours, inoculate a 100L fermenter, cultivate to the end of logarithmic growth, and collect the bacteria by centrifugation after formaldehyde inactivation. About 1.2Kg of wet bacteria can be obtained from 50L of culture solution.

Embodiment 2

[0028] Example two specific polysaccharide extraction

[0029] Dissolve 200g of bacteria into 4000ml of water for injection, stir well, raise the temperature to 100°C, add glacial acetic acid to a final concentration of 1%, stir for 90 minutes, cool down to 4°C, adjust the pH value to neutral, and remove the bacteria by centrifugation. Add ethanol to a concentration of 25%, stand for more than 3 hours, centrifuge to collect the supernatant, add ethanol to a final concentration of 70-80%, stand overnight, and centrifuge to collect the precipitate. The precipitate was washed twice with absolute ethanol and acetone respectively, and dried to obtain the crude product of specific polysaccharide, which can be used in fields with lower purity requirements. About 4g of crude specific polysaccharide can be obtained from 200g of bacteria.

Embodiment 3

[0030] Example three specific polysaccharide purification

[0031] The specific polysaccharide prepared in Example 2 can be controlled within 3% of protein and within 5% of nucleic acid. If higher purity is required, it can be further purified according to this example.

[0032] Dissolve the specific polysaccharide to 5-20 mg / ml, centrifuge to collect the supernatant, wash the 10K ultrafiltration membrane circularly, collect the filtrate, concentrate and wash the 3K ultrafiltration membrane, collect the retentate, ethanol precipitation or lyophilize to collect the purified polysaccharide. It can control within 1% of protein and within 1% of nucleic acid.

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Abstract

Specific polysaccharide (also named O-specific polysaccharide) is one component of lipopolysaccharide. The invention relates to a preparation process of specific polysaccharide. The process adopts perfect thallus as starting material through the measure of hydrolysis, and can directly hydrolyse thallus to prepare specific polysaccharide, and the prepared specific polysaccharide can be used as rawmaterial or final products for disease diagnosis, prevention, treatment and the like, also can be used for preparing standard products or reference products of specific polysaccharide, in particular to preparing coupling vaccine.

Description

Technical field: [0001] The invention relates to a preparation method of Gram-negative bacteria specific polysaccharide (also known as O-specific polysaccharide). The specific polysaccharides specifically involved in the present invention can be used as raw materials or final products for disease diagnosis, prevention, treatment, etc., and can also be used to prepare standard or reference products of specific polysaccharides, especially for the preparation of conjugated vaccines. Background technique: [0002] The specific polysaccharides of Gram-negative bacteria are part of lipopolysaccharides. Lipopolysaccharide is the most important component of the outer membrane of Gram-negative bacteria and consists of three parts: specific polysaccharide, core polysaccharide and lipid A. Among them, the specific polysaccharide is the outermost structure of the bacterium, which is the protective layer for the bacterium to escape the immune attack of the host, and is also the target f...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C08B37/00A61K39/02A61P31/04
Inventor杜琳朱卫华
OwnerBEIJING ZHIFEI LVZHU BIOPHARM