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Galactomannan antigen and preparation method thereof

A galactomannan and antigen technology, which is applied in the field of galactomannan antigen and its preparation, can solve the problems of unfavorable industrial mass production, inability to guarantee the purity and molecular weight distribution of galactomannan, and inability to filter out the galactomannan. Eliminate the interference of heterosaccharides, uniform size and uniform molecular weight distribution

Inactive Publication Date: 2015-09-30
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when galactomannan is used as an antigen for preparing antibodies, the crude extract contains impurities such as glycosamines and proteins, which cannot meet the requirements for antibody preparation. In the prior art, the crude extract is also further purified For example, the Chinese invention patent application with the application number CN201110289099.8 discloses that the crude extract of galactomannan is subjected to ultrafiltration treatment, and proteins, miscellaneous sugars and glycoproteins less than a certain molecular weight are filtered out, thereby obtaining a purity of 90 % above galactomannan antigen
However, using this method to purify galactomannan can only remove impurities with a molecular weight significantly smaller than that of galactomannan. Sugar molecules cannot be filtered out
In addition, in the prior art, the crude extract is also purified by hydrazinolysis, hydrolysis or chromatography. However, this purification is mainly used for the characterization of the structure of galactomannan, and the purity of galactomannan cannot be guaranteed. And suitable molecular weight distribution, not conducive to industrial mass production

Method used

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  • Galactomannan antigen and preparation method thereof
  • Galactomannan antigen and preparation method thereof
  • Galactomannan antigen and preparation method thereof

Examples

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preparation example Construction

[0046] The preparation method of the galactomannan antigen of the present invention comprises the following steps:

[0047] (a) subjecting organisms rich in galactomannan to crushing, centrifuging, ethanol precipitation, washing, and drying to obtain a crude galactomannan extract;

[0048] (b) carrying out hydrazinolysis and hydrolysis of the galactomannan crude extract prepared in step (a) successively;

[0049] (c) adding enzymes to the hydrolyzate in step (b) for enzymolysis, and removing enzymes and small molecular substances in the enzymolyzate;

[0050] (d) purifying the product obtained in step (c) through ion exchange column, affinity chromatography column and gel chromatography to obtain pure galactomannan antigen.

[0051] Wherein, the organism can be a biological population, an individual or an isolated tissue, specifically including fungi (such as Aspergillus, Mucor, Penicillium, Candida, Candida, etc.) or endosperm legumes (such as Fenugreek, Guar, Carob, Locust...

Embodiment 1

[0085] Embodiment 1: the separation of the microbial components represented by Aspergillus

[0086] The Aspergillus strains used in the present invention were purchased from the China Medical Microbiology Collection (CMCC) and the American Standard Biological Collection (ATCC).

[0087] Prepare 1.0 L of Sabouraud liquid culture medium, and cultivate it by shaking flask shaking culture method, the culture temperature is 35°C, the shaker speed is 150rpm, after culturing for 3 days, it is sterilized by moist heat at 121°C for 30 minutes. The inactivated culture solution was filtered with gauze, filtered with filter paper, and then filtered with a 0.22 μm filter membrane to separate the culture solution (including tissue fluid, etc.) from the bacteria. The cells were washed repeatedly with saline and freeze-dried. Ultrasonic ice breaking and 10000g ultra-high speed centrifugation were used to separate the cytoplasm and cell wall. The galactomannan-rich fermentation broth, cytopl...

Embodiment 2

[0088] Embodiment 2: the separation of the vegetable gum represented by guar seed

[0089] The guar seeds are crushed, extracted with water, filtered, added with an organic solvent (ethanol or methanol) for sedimentation, dried and ground to obtain vegetable gum rich in galactomannan.

[0090] Those skilled in the art know that the guar bean seeds can be naturally matured guar beans grown in the field, and can also be isolated tissues obtained by aseptic culture in vitro. When it is an isolated tissue obtained by aseptic culture in vitro, its isolation method is similar to that of Aspergillus described in Example 1, and will not be repeated here.

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Abstract

The invention relates to a preparation method of a galactomannan antigen. The method comprises the steps that breaking, centrifugation, alcohol precipitation, washing and drying are carried out on a living body rich in galactomannan, and galactomannan crude extract is obtained through separation; hydrazinolysis and hydrolysis are carried out on the obtained galactomannan crude extract in sequence; an enzyme is added to a hydrolysis product for enzymolysis, and the enzyme and small molecular substances in an enzymolysis product are removed; a product is purified through an ion exchange column, an affinity column and gel chromatography, and the pure galactomannan antigen is obtained; the obtained pure galactomannan antigen is identified. The galactomannan antigen obtained through the preparation method is high in purity, the inference of galactosamine, protein, miscellaneous sugar possibly included in the galactomannan crude extract is eliminated, and the preparation method can be used for preparing aspergillus fumigatus antibodies.

Description

technical field [0001] The invention relates to the field of antigen preparation, in particular to a galactomannan antigen and a preparation method thereof. Background technique [0002] Galactomannan (GM for short) exists in the cell walls of fungi (such as Aspergillus, Penicillium, etc.) and endosperm legumes (such as fenugreek, guar bean, carob, etc.), and is widely used in food and medical and clinical fields. [0003] At present, due to the extensive use of antibiotics and immune preparations, fungal infections are on the rise year by year, and Aspergillus, especially Aspergillus fumigatus, has gradually become an important pathogenic fungus clinically. Galactomannan, as the skeleton of Aspergillus cell wall, is also the secreted antigen of Aspergillus. Galactomannan antigen is considered to be the target of early diagnosis of invasive aspergillosis. Therefore, obtaining specific anti-galactomannan antibodies is of great significance for the diagnosis of aspergillosis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00
Inventor 周泽奇刘春龙翟栓柱张舟李宁粟艳
Owner DYNAMIKER BIOTECH TIANJIN
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