CN103030702A - Aspergillus fumigatus galactomannan antigen extracting and purifying method - Google Patents
Aspergillus fumigatus galactomannan antigen extracting and purifying method Download PDFInfo
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- CN103030702A CN103030702A CN 201110289099 CN201110289099A CN103030702A CN 103030702 A CN103030702 A CN 103030702A CN 201110289099 CN201110289099 CN 201110289099 CN 201110289099 A CN201110289099 A CN 201110289099A CN 103030702 A CN103030702 A CN 103030702A
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Abstract
The invention relates to the fungal polysaccharide preparation field, concretely relates to a preparation method of Aspergillus fumigatus excretion galactomannan, and provides a method for preparing the Aspergillus fumigatus excretion galactomannan through the combination of alcohol precipitation, decoloring and ultrafiltration. The method comprises the following steps: processing extracellular galactomannan secreted by Aspergillus fumigatus through alcohol to obtain a precipitate which is a crude galactomannan extract; decoloring the crude extract by active carbon; and purifying the decolored polysaccharide extract through using an ultrafiltration process to finally obtain pure galactomannan. The method for preparing the Aspergillus fumigatus excretion galactomannan has the advantages of no need of the processes comprising acid hydrolysis or column chromatography, simple preparation technology, and easy quality control, and is in favor of the technological production of the galactomannan, and the preparation of antibodies.
Description
Technical field
The present invention relates to diagnosis and use the antibody preparation field, especially for extraction and the purifying of the required antigen of immune animal in the antibody preparation process of polygalactomannan (Galactomannan).
Background technology
In recent years, along with antibiotic a large amount of abuses and immunosuppressor in clinical being widely used, sickness rate and the case fatality rate of aggressive deep fungal infection are more and more higher.Wherein, aspergillus particularly Aspergillus fumigatus (Aspergillus fumigatus) become gradually clinically a kind of important pathomycete.The gold standard of at present aggressive aspergillin infection detection is that aseptic body fluid is cultivated and biopsy.Yet aggressive aspergillus infection disease progression is very fast, and the method positive rate of traditional separation and Culture is low, the cycle is long, often cause fail to pinpoint a disease in diagnosis, mistaken diagnosis, cause and affect the state of an illness adversely.Therefore, detect the effective ways that Aspergillus fumigatus antigen is at present clinical generally acknowledged quick early diagnosis aspergillus fumigatus infection.The Aspergillus fumigatus Exoantigen is polygalactomannan antigen, can find from blood samples of patients in early days in infection.Based on this, its polygalactomannan antigen of separation and Extraction and to prepare monoclonal antibody be to realize that the infection by Aspergillus fumigatus early immune learns the Critical policies of diagnosis from aspergillus fumigatus.The present invention has set up a kind of polygalactomannan antigen extraction and purification method that immune animal is realized the antibody preparation that can be used for.
It is a kind of polysaccharide that Aspergillus fumigatus is secreted into the outer polygalactomannan antigen of born of the same parents, generally extracts to obtain from the nutrient solution of this bacterium.Domestic research to polygalactomannan antigen extraction purifying is few, separating-purifying polygalactomannan antigen is mainly crude extract from nutrient solution at present, precipitate Aspergillus fumigatus nutrient solution supernatants with 4 times of volume dehydrated alcohols in the document 1, wash 3 times with 75% ethanol again, namely obtain Aspergillus fumigatus secretor type antigen crude extract.Mention in the document 2 with 4 ℃ of the ethanol of 3 times of volumes precipitation nutrient solution filtrates and spending the night, in centrifugal 10 minutes of 4 ℃ of lower 3000g, namely obtain crude extract, then in 1.5 moles nitrous acid, processing 2 hours under 100 ℃ again.Gained filtrate is polygalactomannan antigen sterling behind the 0.45um membrane filtration.After the ethanol precipitation Aspergillus fumigatus nutrient solution filtrate of 4 times of volumes of document 3 usefulness, will precipitate with dehydrated alcohol and wash 3 times, freeze-drying, every 15mg throw out adds in the 1ml anhydrous hydrazine, and 105 ℃ are spent the night.Afterwards with 4 times of volume dehydrated alcohol precipitations, absolute ethanol washing, tap water dialysis.Process in 1.5 ambient temperature overnight of rubbing in the nitrous acid solution of every liter of final concentration again, then in solution, blast air.After dialysis, with 4 times of volume dehydrated alcohol precipitations, use again absolute ethanol washing, freeze-drying.Repeating above-mentioned anhydrous hydrazine and nitrous acid treatment step once.To through the material that above-mentioned processing obtains MonoQ chromatography column purifying, finally obtain the polygalactomannan sterling.
The polysaccharide fraction that is secreted in the nutrient solution in view of Aspergillus fumigatus is mainly polygalactomannan, therefore the polysaccharide secretory volume of other kinds seldom considers that the crude extract that ethanol precipitation Aspergillus fumigatus nutrient solution filtrate obtains just can reach very high purity through the simple suitable purifying of method again.Ultra-filtration technique is a kind of membrane sepn process take screening as separation principle, take pressure as impellent, utilizes ultra-filtration technique can effectively remove the following macromolecule organic of specified molecular weight in the solution system.This research namely is based on ultra-filtration technique Aspergillus fumigatus antigen crude extract is carried out purifying, thereby has obtained the polygalactomannan antigen of purity more than 90%.Present method principle is simple, and the operating time is short, and condition is easily controlled, and is convenient to batch production, is very beneficial for the immunogen preparation as the antibody producing process.
The present invention describes in detail
The object of the present invention is to provide a kind of method of utilizing ultra-filtration technique to come purifying Aspergillus fumigatus secretor type antigen.
Aspergillus fumigatus bacterial strain of the present invention is bought from Chinese medicine microbial strains preservation administrative center (CMCC).
Technical solution of the present invention is as follows:
Step:
1) the Aspergillus fumigatus liquid culture adopts general Sha Shi liquid nutrient medium, and composition is that every liter of substratum contains 1% peptone, 4%D-glucose.Culture temperature is 30 ℃, and shaking speed speed is 200 to turn per minute, about 46 hours of incubation time.When being down to 4.2-4.5, medium pH value stops to cultivate.
2) above-mentioned nutrient solution is through 121 ℃ of moist heat sterilizations 40 minutes, the gemma that kills thalline and may exist.Filter through qualitative filter paper under the gained bacterium liquid chamber temperature, filtrate through 0.22 μ m membrane filtration to remove thalline.
3) gained filtrate is transferred in the clean beaker, adds 4 times of volume dehydrated alcohols.4 ℃ of hold over night.
4) 4 ℃, centrifugal 15 minutes of 16,000g is dissolved in precipitation in the deionized water.
5) add 4 times of volume dehydrated alcohols, left standstill 1 hour.
6) centrifugation precipitation, precipitation absolute ethanol washing three times.
7) 4 ℃, centrifugal 15 minutes of 16,000g abandons supernatant.
8) with the dissolving of gained precipitate with deionized water, add again active carbon powder, stirring at room 2 hours.
9) the room temperature suction filtration is removed activated carbon granule.
10) gained filtrate is through 0.22 μ m membrane filtration.
11) filtrate is transferred to 10KD centrifugal ultrafiltration pipe, centrifugal 10 minutes of 5000g.Namely obtain the polygalactomannan sterling.
Among the present invention, because polygalactomannan antigen crude extract is the brown material, pigment content is very high.Gac is usually used in the Sugarcane juice decolorization of sugar-refining industry, so this research is decoloured to antigenic solution with gac, the antigenic solution color homogeneous clarification that obtains slightly has light brown.Because polygalactomannan antigen is the molecule about 30KD, so do uf processing with the 10KD super filter tube, can effectively remove the following protein of 100KD, assorted sugar and glycoprotein.And polygalactomannan can be held back by ultra-filtration membrane greatly because of molecular volume, thereby realizes the purifying of polygalactomannan.
Accompanying drawing is described:
Fig. 1 has shown the result that HPLC detects.
Fig. 2 has shown method flow of the present invention.
Fig. 3 has shown the typical curve of determination of polysaccharide experiment in the exemplifying embodiment 4.
Embodiment 1, extracts aspergillus fumigatus polygalactomannan antigen with preparation method of the present invention, and its step is as follows:
Preparation Sha Shi liquid nutrient medium 2.0L, its composition is peptone 20.0g, D-Glucose 80.0g.Culture temperature is 30 ℃, and shaking speed is 200 to turn per minute.Cultivation 46 is 121 ℃ of moist heat sterilizations as a child.0.22um membrane filtration bacterium liquid is collected filtrate.The dehydrated alcohol that adds 3 times of volumes in the filtrate, 4 ℃ of hold over night.Centrifugal 30 minutes of all liquid 16000g is precipitated.To precipitate and be dissolved in deionized water after washing 3 times with 75% ethanol, 60 ℃ of oven dry obtain polygalactomannan.
Embodiment 2, use preparation method of the present invention, with the method for charcoal absorption polygalactomannan carried out purifying, and concrete steps are as follows:
Polygalactomannan is dissolved in the 200ml deionized water, and the limit is stirred and just slowly to be added active carbon powder 5.0g, and room temperature was decoloured more than 6 hours, treats that the solution brown color takes off, and filters with Büchner funnel.Repeatedly be filtered to the solution clarification.Namely obtain removing the polygalactomannan extract of pigment.
Embodiment 3, use preparation method of the present invention, with the method for charcoal absorption polygalactomannan carried out purifying, and concrete steps are as follows:
With removing centrifugal 15 minutes of the polygalactomannan extract 16000g of pigment, keep supernatant, be transferred in the centrifugal ultrafiltration pipe of molecular weight cut-off 3KD centrifugal 15 minutes of 5000g.With the concentrated solution freeze-drying that obtains, namely obtain polygalactomannan antigen sterling.
Embodiment 4, use preparation method of the present invention, and polygalactomannan is carried out purifying, and the polygalactomannan sample that obtains is carried out polysaccharide, protein and nucleic acid content detection:
1) with Dubois-phenol sulfuric acid method the polygalactomannan antigen purification sample that preparation method of the present invention obtains is carried out the desugar assay, the result is as follows:
| Volume (ml) | Sugar content (mg) | |
| Test sample | 0.02 | 0.027 |
| Whole samples | 100.00 | 135.00 |
As seen, the preparation method with this patent finally can obtain 150mg polygalactomannan antigen sterling from 2L Aspergillus fumigatus nutrient solution.
2) the polygalactomannan antigen purification sample that preparation method of the present invention is obtained carries out uv-absorbing and detects.Under UV260 nanometer, UV280 nanometer, UV320 nano wave length, detect sample respectively, result such as following table.As seen the ratio of the total content of nucleic acid and protein and polysaccharide content is less than 10%, that is nucleic acid and protein content are no more than 7% of sample total mass less than 10% of total substances content.
| Test item | Concentration | Content (%) |
| DNA | 4.499ug/mL | 0.33 |
| Protein | 87.643ug/mL | 6.49 |
| Polysaccharide | 1.35mg/mL | 93.18 |
Embodiment 5, use preparation method of the present invention, and polygalactomannan is carried out purifying, the polygalactomannan sample that obtains is carried out HPLC detect:
The polygalactomannan antigen purification sample that preparation method of the present invention is obtained carries out the HPLC detection.Detector is differential refraction detector.Result such as accompanying drawing 1.As can be seen from Figure, it is single that sample goes out the peak, and point is narrow, there is no obviously assorted peak and occur, and contained material size homogeneous is described, purity is higher.
Only have in the world at present a kind of test kit that detects Aspergillus fumigatus antigen, i.e. the Aspergillus fumigatus antigen detection kit of U.S. Bio-Raid company.With this test kit the polygalactomannan antigen samples with method preparation of the present invention is detected, concentration of specimens is 1ng/ml.Detected result is positive, and the OD value is greater than positive quality control.The polygalactomannan antigen that can obtain Aspergillus fumigatus with the method among the present invention is described.The results are shown in following table:
Data presentation, the present invention compares with additive method, and the time spent is short, and easy to drawing materials is easy and simple to handle.Aspergillus fumigatus polygalactomannan antigen with purifying of the present invention can reach higher degree.
Should know that just invention has been described with exemplifying embodiment, the improvement of making on basis of the present invention still belongs to category of the present invention.
Reference (it is for referencial use to fit into this paper in it)
[1] Che Xiaoyan etc., the preparation of (2008) the 1 mould monoclonal antibodies of suite and evaluation. Chinese mycology magazine .3 (3): 129-133
[2] DIRK STYNEN etc., (1992) INFECION AND IMMUNITY.60 (6): 2237-2245
[3] JEAN-PAUL LATGE etc., (1994) INFECTION AND IMMUNITY.60 (12): 5424-5433
Claims (9)
1. the preparation and purification method of an Aspergillus fumigatus secretor type polygalactomannan may further comprise the steps:
(a) after the aspergillus fumigatus liquid medium was processed through autoclaving, the method for filtration was removed thalline;
(b) obtain in the bacterium liquid supernatant to (a) step, add methyl alcohol or the ethanolic soln of dehydrated alcohol or other concentration, produce precipitation, centrifugal, precipitation is preserved with methods such as oven dry or freeze-drying with the ethanolic soln washing again;
(c) step (b) gained precipitation water or physiological saline, PBS etc. can dissolve the solution dissolving of polysaccharide;
(d) in the solution of step (c) gained, add the decolouring materials such as gac, or make decolorizing column with the decolouring material, GM crude extract solution is decoloured;
(e) step (d) gained solution is used the super filter tube ultrafiltration behind membrane filtration, and freeze-drying is the purifying mannosans.
2. method as claimed in claim 1 is characterized in that, removes thalline with membrane filtration, obtains the nutrient solution supernatant without thalline.
3. the method for claim 1 is characterized in that, in the step (b), processes Aspergillus fumigatus nutrient solution supernatant with ethanol or methanol solution, obtains the polygalactomannan crude extract.
4. method as claimed in claim 1 is characterized in that in the step (b), the volume of used dehydrated alcohol is 4 times of nutrient solution supernatant volume.
5. method as claimed in claim 1 is characterized in that in the step (c), the solvent that dissolution precipitation is used is water or PBS, or the solution of the solubilized polysaccharide such as physiological saline.
6. method as claimed in claim 1 is characterized in that step (d) decoloring medium of using is gac, and its shape can be the sources such as charcoal, coal, bone black, can be the different shapeies such as Powdered, particulate state, column.Decoloring medium can be other decoloring medium except gac, such as resin, atlapulgite etc.
7. method as claimed in claim 1 is characterized in that in the step (e), the purification process that uses is ultrafiltration.
8. method as claimed in claim 1 is characterized in that, in the step (e), the ultra-filtration equipment that uses be the centrifugal ultrafiltration pipe, molecular weight cut-off can be the following any molecular weight of 100KD.
9. method as claimed in claim 1 is characterized in that, step (a) and (e) in, solution all uses 0.22 μ m filter membrane to filter.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104945527A (en) * | 2015-07-03 | 2015-09-30 | 丹娜（天津）生物科技有限公司 | Galactomannan antigen and preparation method thereof |
| CN105866407A (en) * | 2016-04-22 | 2016-08-17 | 丹娜（天津）生物科技有限公司 | Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof |
| CN106093414A (en) * | 2016-08-03 | 2016-11-09 | 天津喜诺生物医药有限公司 | One detects aspergillosis and cryptococcal immune colloid gold test paper simultaneously |
| CN110894236A (en) * | 2019-12-17 | 2020-03-20 | 丹娜（天津）生物科技有限公司 | Anti-aspergillus galactomannan monoclonal antibody and application thereof |
| CN114835828A (en) * | 2022-04-20 | 2022-08-02 | 武汉轻工大学 | Preparation method of auricularia auricula crude polysaccharide |
| CN115043953A (en) * | 2022-06-27 | 2022-09-13 | 江西赛基生物技术有限公司 | Method for producing galactomannan antigen and galactomannan antigen |
-
2011
- 2011-09-29 CN CN 201110289099 patent/CN103030702A/en not_active Withdrawn
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104945527A (en) * | 2015-07-03 | 2015-09-30 | 丹娜（天津）生物科技有限公司 | Galactomannan antigen and preparation method thereof |
| CN105866407A (en) * | 2016-04-22 | 2016-08-17 | 丹娜（天津）生物科技有限公司 | Aspergillus galactomannan (GM) antigen immunodetection kit as well as preparation method and application thereof |
| CN106093414A (en) * | 2016-08-03 | 2016-11-09 | 天津喜诺生物医药有限公司 | One detects aspergillosis and cryptococcal immune colloid gold test paper simultaneously |
| CN110894236A (en) * | 2019-12-17 | 2020-03-20 | 丹娜（天津）生物科技有限公司 | Anti-aspergillus galactomannan monoclonal antibody and application thereof |
| CN110894236B (en) * | 2019-12-17 | 2021-01-01 | 丹娜(天津)生物科技股份有限公司 | Anti-aspergillus galactomannan monoclonal antibody and application thereof |
| CN114835828A (en) * | 2022-04-20 | 2022-08-02 | 武汉轻工大学 | Preparation method of auricularia auricula crude polysaccharide |
| CN114835828B (en) * | 2022-04-20 | 2024-01-09 | 武汉轻工大学 | Preparation method of black fungus crude polysaccharide |
| CN115043953A (en) * | 2022-06-27 | 2022-09-13 | 江西赛基生物技术有限公司 | Method for producing galactomannan antigen and galactomannan antigen |
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Application publication date: 20130410 |