CN104945527A - Galactomannan antigen and preparation method thereof - Google Patents
Galactomannan antigen and preparation method thereof Download PDFInfo
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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
The present invention relates to antigen preparation field, particularly a kind of polygalactomannan antigen and preparation method thereof.
Background technology
Polygalactomannan (Galactomannan, be called for short GM) be present in fungi (as Eurotium, Penicillium etc.) and endosperm bean (as trigonella, Gua Erdou, angle beans etc.) cell walls, be widely used in food and medical science, clinical field.
At present, due to a large amount of uses of microbiotic and immunological reagent, fungi infestation is in ascendant trend year by year, and wherein, aspergillus particularly Aspergillus fumigatus has become a kind of important pathomycete clinically gradually.Polygalactomannan is as the skeleton of aspergillus cell wall, and be also aspergillus Exoantigen, polygalactomannan antigen is considered to the target of Aspergillosis early diagnosis simultaneously.Therefore, obtain the antibody of specific anti-polygalactomannan, diagnosis for aspergillosis has great importance, but, adopt the antibody that restructuring polygalactomannan antigen prepares, only part can, with the polygalactomannan antigen site specific combination in aspergillus fumigatus filament, adopt natural galacto mannan antigen may solve this problem, but current natural galacto mannan antigen is purified and be there is certain difficulty.
In prior art, natural galacto mannan antigen is mainly separated from aspergillus fumigatus nutrient solution, and the polygalactomannan antigen of extraction is mainly crude extract, uses alcohol settling nutrient solution supernatant liquor, then uses the component after ethanol purge alcohol precipitation, obtain antigen crude extract.But, when polygalactomannan uses as the antigen for the preparation of antibody, owing to containing the impurity such as osamine, albumen in crude extract, requirement prepared by antibody cannot be met, also the research that crude extract is further purified is had in prior art, such as application number is that the Chinese invention patent application of CN201110289099.8 discloses polygalactomannan crude extract is carried out uf processing, to the protein of certain molecular weight, assorted sugar and glycoprotein filtering be less than, thus obtain the polygalactomannan antigen of purity more than 90%.But, the method is used to carry out purifying to polygalactomannan, only can remove the impurity that molecular weight is significantly less than galactomannan molecule amount, when the content of assorted sugar or protein is close with galactomannan molecule or when being greater than galactomannan molecule, then cannot filtering.In addition, also have in prior art logical adopt hydrazinolysis, the mode of hydrolysis or chromatography purifies to crude extract, but this purifying is mainly used in the sign of galactomannan structure, the purity of polygalactomannan and suitable molecular weight distribution cannot be ensured, be unfavorable for that industry is produced in batches.
Summary of the invention
The object of this invention is to provide a kind of purity higher and be suitable for polygalactomannan antigen and preparation method thereof of producing in batches.
In order to realize object of the present invention, the present invention adopts following technical scheme:
A preparation method for polygalactomannan antigen, comprises the steps:
A () is rich in the organism of polygalactomannan through broken, centrifugal, alcohol precipitation, washing, drying treatment, be separated and obtain polygalactomannan crude extract;
B polygalactomannan crude extract obtained for step (a) is carried out hydrazinolysis, hydrolysis by () successively;
C () carries out enzymolysis by adding enzyme in the hydrolysate in step (b), and the enzyme removed in enzymolysis product and small-molecule substance;
D product that step (c) obtains by (), through ion exchange column, affinity column and gel chromatography purifying, obtains polygalactomannan antigen sterling.To the polygalactomannan antigen sterling obtained, identify.
Preferably, described organism comprises biotic population, individuality or in vitro tissue, and concrete comprises fungi or endosperm bean; Described fungi comprises aspergillus tubigensis, mucormycosis, Penicillium notatum, pearl bacterium, candidiasis etc., and described endosperm bean comprises trigonella, Gua Erdou, angle beans, locust bean, pass China beans, Chinese honey locust, Semen adenantherae pavoninae, Leucaena leucocephala (L.) etc.Those skilled in the art know, as long as described organism is rich in polygalactomannan, are not limited to above-mentioned concrete biology.
In a concrete embodiment, preferably, described step (a) specifically comprises: the organism of being rich in polygalactomannan is cultivated polygalactomannan content the highest time, inactivation treatment; Filter, be separated by the organism after deactivation with nutrient solution and/or tissue juice, organism carries out cytoclasis, centrifugal, is separated by tenuigenin, obtains active principle with cell walls; Described active principle is through alcohol precipitation, and washing, the art breading such as drying, obtain polygalactomannan crude extract.Wherein, described active principle refers to the component being rich in polygalactomannan, as the fermented liquid of aspergillus tubigensis, tenuigenin and cell wall constituent, be rich in the vegetable jelly (as trigonella bean gum, tara gum, Viscogum BE, carob bean gum, guar gum, pass China bean gum etc.) of polygalactomannan.
Preferably, the substratum made in described organism culturing process be selected from the substratum such as PDA substratum, sabouraud culture medium, czapek's solution, malt extract medium one or more.Cultural method comprises the slat chain conveyor and slant culture that use solid medium, the shake flask cultivation of use liquid nutrient medium and fermentor cultivation etc., temperature 20-40 DEG C, time 3-15 days.
Preferably, described ablation method comprises chemical reagent, ray, high temperature, high osmotic pressure etc.
Preferably, described cytoclasis uses one or more in the methods such as mechanical crushing method, swelling method, enzymolysis process.
Preferably, alcohol precipitation uses methyl alcohol or ethanol, and the volume of alcohol precipitation is 2-6 times of active principle liquor capacity, and the alcohol precipitation time is 1-48 hour.Preferred, alcohol precipitation volume is 4 times of active principle liquor capacities, and the alcohol precipitation time is 6-24 hour.
Preferably, washing uses methyl alcohol or ethanol; Drying means is one or more in oven dry, freeze-drying, drying under reduced pressure.
In a concrete embodiment, preferably, described step (a) also comprises activated carbon decolorizing.
Concrete, active principle is soluble in water, and add active carbon powder, room temperature decolouring 6-24 hour, treat that solution brown color is taken off, funnel filters, and filtrate, through 0.22 μm of membrane filtration, namely obtains the polygalactomannan extract removing pigment.Preferably, 0.02-0.05g gac is added in every ml soln.
In a concrete embodiment, preferably, in described step (b) during hydrazinolysis: the anhydrous hydrazine adding 10-100 μ l in every milligram of polygalactomannan crude extract, hydrazinolysis temperature is 100-150 DEG C, and the hydrazinolysis time is 2-12 hour.Preferred, described hydrazinolysis temperature is 110-130 DEG C of hydrazinolysis time is 3-8 hour.
Preferably, in described step (b), hydrazinolysis specifically comprises: in every milligram of polygalactomannan crude extract, add 10-100 μ l enter anhydrous hydrazine, mixing, and sealing, is incubated 2-12 hour at 100-150 DEG C; The hydrazinolysis product of gained is added ethanol carry out centrifugal, washing and lyophilize, obtain the dry powder after hydrazinolysis.
In a concrete embodiment, preferably, in described step (b), hydrolysis adopts nitrous acid hydrolysis, during nitrous acid hydrolysis, nitrous acid concentration is 1.5-5.0mol/L, hydrolysis temperature is 30 DEG C, and hydrolysis time is 2-12 hour, passes into air after hydrolysis terminates.Preferred, described nitrous acid concentration is 1.6-2.4mol/L, and hydrolysis time is 3-8 hour.
Preferably, in described step (b), hydrolysis specifically comprises: the dry powder after hydrazinolysis being added concentration is 1.5-5.0mol/L, and hydrolysis temperature is 30 DEG C, and hydrolysis time is 2-12 hour, passes into air after hydrolysis terminates; Hydrolyzed solution through water dialysed overnight, add in dialyzate ethanol carry out centrifugal, washing and lyophilize, obtain be hydrolyzed after dry powder.
After hydrazinolysis and hydrolysis, the albumen effectively in removing polygalactomannan and amino; And by the optimization to hydrazinolysis, hydrolysising condition, make the loss amount of polygalactomannan in hydrazinolysis and hydrolytic process less, and vestiges of protein quantity not sufficient 4%.
In a concrete embodiment, preferably, in step (c), described enzyme be selected from dextranase, cellulase, polygalacturonase, chitinase one or more, enzymolysis damping fluid is 0.01M PBS, pH is 7.4, temperature is 20-60 DEG C, and enzymolysis time is 6-48 hour.Preferred, described hydrolysis temperature is 30-45 DEG C, and enzymolysis time is 12-24 hour.
Preferably, in described step (c), enzymolysis specifically comprises: the dry powder after hydrolysis is prepared into solution, add the one in the dextranase, cellulase, polygalacturonase, chitinase etc. that concentration is 2-5% or kind, enzymolysis damping fluid is 0.01M PBS, pH is 7.4, enzymolysis 6-48 hour at 20-60 DEG C; Adopt sevage method (adding propyl carbinol and chloroformic solution, vibration, centrifugal) removing enzyme, adopt the method removing small-molecule substances such as dialysis, ultrafiltration or Sephadex G molecular sieve.
By the polysaccharide impurity such as dextran, chitin, Mierocrystalline cellulose, pectin mixed in enzymolysis removing product, further increase the purity of polygalactomannan.
In a concrete embodiment, preferably, in step (d), ion exchange column is anion-exchange column.
Preferably, anion-exchange column is Mono Q anion-exchange column, and damping fluid and solvent are Tris-HCl damping fluid, PH is 7 ~ 8.5, temperature 4 DEG C.
Due to polygalactomannan sterling neutral, electronegative albumen, nucleic acid etc. are adsorbed by anion-exchange column, albumen remaining in further removal process (b) and nucleic acid.
In a concrete embodiment, preferably, in step (d), affinity column is Con A-Sepharose 4B affinity column.Wherein, damping fluid is that (solute is 0.5mol/L NaCl, 1mmol/L CaCl for the Tris-HCl damping fluid of PH 7-8.5 (be preferably PH7.4)
2, 1mmol/L MgCl
2), temperature 4 DEG C, elutriant is the elutriant 0.2-0.3M Alpha-Methyl-D-MANNOSE glycosides of PH 8-8.5 (being preferably PH 8.5).
In a concrete embodiment, preferably, in step (d), gel chromatographic columns comprises the one in dextrane gel (sephadex G, sephadex LH ?20), sepharose (sepharose), cellulose gel (cellufine GCL ?2000), polyacrylamide gel (Bio ?gel P) etc.
Polygalactomannan is separated by gel chromatographic columns, different according to the polysaccharide molecule elution time of different size and shape, thus obtain the polygalactomannan sterling of size uniform.
In addition, the present invention also provide a kind of adopt preparation method of the present invention to prepare polygalactomannan antigen and preparing the application in polygalactomannan polyclonal antibody.Especially can be used for the preparation of aspergillus tubigensis polygalactomannan polyclonal antibody.Use polygalactomannan antigen-immunized animal, measure the serum titer of animals following immunization, in the animal body after immunity, get blood; Then with saturated ammonium sulphate salting-out process and affinity chromatography, purifying is carried out to serum, obtain the antibody of purifying.
Further, the present invention also provides the application of a kind of polygalactomannan antigen adopting preparation method of the present invention to prepare in the test kit for the preparation of detection invasive fungi disease detection, and described invasive fungi is selected from aspergillus tubigensis, mucormycosis, Penicillium notatum, pearl bacterium, candidiasis.Preferably, described test kit is for detecting aspergillus tubigensis.Those skilled in the art know, and test kit can comprise antigen, antibody, enzyme labelled antibody, washings, substrate nitrite ion, stop buffer etc.
Beneficial effect of the present invention:
1. the polygalactomannan antigen purity using the inventive method to obtain is higher, eliminates the interference of GalN, albumen or other impurity.
2. homogeneous, the size uniform of polygalactomannan antigen molecular distribution, eliminates the interference of assorted sugar in crude extract.
3. the present invention adopts vegetable jelly to extract polygalactomannan and the polygalactomannan antigen raw material sources obtained its purification is extensive, cost is low and have excellent immunocompetence.
Accompanying drawing explanation
Fig. 1 determination of polysaccharide typical curve.
Fig. 2 embodiment of the present invention 5 polygalactomannan antigen HPLC differential pulse polarograpll result.
Monosaccharide component liquid chromatographic detection result in Fig. 3 embodiment of the present invention 5 polygalactomannan antigen.
Fig. 4 polysaccharide molecular weight bioassay standard curve.
Fig. 5 embodiment of the present invention 5 polygalactomannan antigen high productivity computing differential pulse polarograpll result.
Embodiment
The preparation method of polygalactomannan antigen of the present invention, comprises the steps:
A () will be rich in the organism of polygalactomannan through broken, centrifugal, alcohol precipitation, washing, drying treatment, be separated and obtain polygalactomannan crude extract;
B polygalactomannan crude extract obtained for step (a) is carried out hydrazinolysis, hydrolysis by () successively;
C () carries out enzymolysis by adding enzyme in the hydrolysate in step (b), and the enzyme removed in enzymolysis product and small-molecule substance;
D product that step (c) obtains by (), through ion exchange column, affinity column and gel chromatography purifying, obtains polygalactomannan antigen sterling.
Wherein, organism can be biotic population, individuality or in vitro tissue, and concrete comprises fungi (such as aspergillus tubigensis, mucormycosis, Penicillium notatum, pearl bacterium, beads etc.) or endosperm bean (such as trigonella, Gua Erdou, angle beans, locust bean, pass China beans, Chinese honey locust, Semen adenantherae pavoninae, Leucaena leucocephala (L.) etc.).Those skilled in the art know, and organism is not limited to above-mentioned listed concrete biological, as long as be rich in polygalactomannan.
In the embodiment that the present invention one is concrete, step (a) specifically comprises: the organism of being rich in polygalactomannan is cultivated polygalactomannan content the highest time, inactivation treatment (such as chemical reagent, ray, high temperature, high osmotic pressure etc.); Filter, be separated by the organism after deactivation with nutrient solution and/or tissue juice, organism carries out cytoclasis (such as using mechanical crushing method, swelling method, enzymolysis process etc.), centrifugal, is separated by tenuigenin, obtains active principle with cell walls; Active principle is through alcohol precipitation, and washing, the art breading such as drying, obtain polygalactomannan crude extract.Wherein, active principle refers to the component being rich in polygalactomannan, as the fermented liquid of aspergillus tubigensis, tenuigenin and cell wall constituent, be rich in the vegetable jelly (as trigonella bean gum, tara gum, Viscogum BE, carob bean gum, guar gum, pass China bean gum etc.) of polygalactomannan.
In the embodiment that the present invention one is concrete, the substratum made in described organism culturing process be selected from the substratum such as PDA substratum, sabouraud culture medium, czapek's solution, malt extract medium one or more.Cultural method comprises the slat chain conveyor and slant culture that use solid medium, the shake flask cultivation of use liquid nutrient medium and fermentor cultivation etc., temperature 20-40 DEG C, time 3-15 days.
Wherein, the proportioning of each substratum is as follows:
Sabouraud culture medium: peptone 10g, glucose 40g, adding distil water, is settled to 1L.
Czapek's solution: SODIUMNITRATE 3g, dipotassium hydrogen phosphate 1g, magnesium sulfate (MgSO
47H
2o) 0.5g, Repone K 0.5g, ferrous sulfate 0.01g, sucrose 30g, adding distil water, is settled to 1L.
Malt extract medium: malt extract 20g, glucose 20g, peptone 1g, adding distil water, is settled to 1L.
PDA substratum: get potato and to be cut into small pieces 300g, add water boil 1h, filters, adds glucose 20g, distilled water, be settled to 1L.
Solid medium: on the basis of aforesaid liquid substratum, adds the agar of 1.5-2%.
The numerical value of concrete proportioning is only for illustration of the ratio between each component of substratum above, is not intended to limit the present invention.Those skilled in the art know, and according to waiting the demand of cultivating organism and concrete culture condition, can select suitable substratum, its proportioning also can be finely tuned.
Biological body weight culture condition is specific as follows:
Solid culture condition: culture temperature is 25-37 DEG C, quiescent culture 3-15 days.
Shake flask culture condition: culture temperature is 25-37 DEG C, shaking speed is 150-200rpm, cultivates 3-15 days, stops cultivating when nutrient solution pH value is down to 4.2-4.5.
Fermentor cultivation condition: produce fermentor tank: cultivate at 25 DEG C at 20L fermentor tank, 500rpm, per minute air flow is 0.5 times of culture volume, 2% glucose and 1% protein culture medium (initial pH value is 6.3), and inoculum size is 8%; Seed fermentation tank: the mycelia growing 60h under adopting the identical condition of 2L fermentor tank.
Sterilising conditions is specific as follows:
Medium sterilization: 121 DEG C of moist heat sterilization 20-30 minute.
Thalline deactivation: 121 DEG C of moist heat sterilization 20-30 minute; 3.7% formaldehyde soaked overnight; Radiation exposure etc.
The design parameter of concrete culture condition and sterilising conditions is only for illustrating above, is not intended to limit the present invention.Those skilled in the art know, and can select suitable culture condition and sterilising conditions according to the actual requirements.
In the embodiment that the present invention one is concrete, alcohol precipitation uses methyl alcohol or ethanol, and the volume of alcohol precipitation is 2-6 times of active principle liquor capacity, and the alcohol precipitation time is 1-48 hour; Washing uses methyl alcohol or ethanol; Drying means is one or more in oven dry, freeze-drying, drying under reduced pressure.Those skilled in the art know, and alcohol precipitation, washing and drying are this area separating polyose routine techniques means, and therefore not to repeat here.
Polygalactomannan is brown color material, and pigment content is higher, in the embodiment that the present invention one is concrete, also comprises activated carbon decolorizing step before step (a) alcohol precipitation.Concrete, active principle is soluble in water, add active carbon powder (adding 0.02-0.05g gac in every ml soln), room temperature decolouring 6-24 hour, treat that solution brown color is taken off, funnel filters, and filtrate, through 0.22 μm of membrane filtration, namely obtains the polygalactomannan extract removing pigment.
In the embodiment that the present invention one is concrete, in step (b) during hydrazinolysis: the anhydrous hydrazine adding 10-100 μ L in every milligram of polygalactomannan crude extract, hydrazinolysis temperature is 100-150 DEG C, and the hydrazinolysis time is 2-12 hour.Concrete, in polygalactomannan crude extract, add 10-100 μ L enter anhydrous hydrazine, mixing, sealing, is incubated 3-8 hour at 110-130 DEG C; The hydrazinolysis product of gained is added ethanol carry out centrifugal, washing and lyophilize, obtain the dry powder after hydrazinolysis.
In the embodiment that the present invention one is concrete, in step (b), hydrolysis adopts nitrous acid hydrolysis, and during nitrous acid hydrolysis, nitrous acid concentration is 1.5-5.0mol/L, and hydrolysis temperature is 30 DEG C, and hydrolysis time is 2-12 hour, passes into air after hydrolysis terminates.Concrete, the dry powder after hydrazinolysis is added the nitrous acid solution that concentration is 1.6-2.4mol/L, and hydrolysis temperature is 30 DEG C, and hydrolysis time is 3-8 hour, passes into air after hydrolysis terminates; Hydrolyzed solution through water dialysed overnight, add in dialyzate ethanol carry out centrifugal, washing and lyophilize, obtain be hydrolyzed after dry powder.
After hydrazinolysis and hydrolysis, the albumen effectively in removing polygalactomannan and amino; And by the optimization to hydrazinolysis, hydrolysising condition, make the loss amount of polygalactomannan in hydrazinolysis and hydrolytic process less, and vestiges of protein quantity not sufficient 3%.
In the embodiment that the present invention one is concrete, in step (c), enzyme be selected from dextranase, cellulase, polygalacturonase, chitinase one or more, enzymolysis damping fluid is 0.01M PBS, pH is 7.4, and hydrolysis temperature is 20-60 DEG C, and enzymolysis time is 6-48 hour.Concrete, the dry powder after hydrolysis is prepared into solution, adds the one in the dextranase, cellulase, polygalacturonase, chitinase etc. that concentration is 2-5% or kind, enzymolysis 12-24 hour at 30-45 DEG C; Adopt sevage method (adding propyl carbinol and chloroformic solution, vibration, centrifugal) removing enzyme, adopt the method removing small-molecule substances such as dialysis, ultrafiltration or Sephadex G molecular sieve.
By the polysaccharide impurity such as dextran, chitin, Mierocrystalline cellulose, pectin mixed in enzymolysis removing product, further increase the purity of polygalactomannan.
In the embodiment that the present invention one is concrete, in step (d), ion exchange column is anion-exchange column, such as Mono Q anion-exchange column, and wherein, it is 7 ~ 8.5Tris-HCl damping fluid, temperature 4 DEG C that damping fluid and solvent are PH.
Due to polygalactomannan antigen sterling neutral, electronegative albumen, nucleic acid etc. are adsorbed by anion-exchange column, albumen remaining in further removal process (b) and nucleic acid.
In the embodiment that the present invention one is concrete, in step (d), affinity column is Con A-Sepharose 4B affinity column.Wherein, damping fluid is that (solute is 0.5mol/L NaCl, 1mmol/L CaCl for the Tris-HCl damping fluid of PH7 ~ 8.5 (such as 7.4)
2, 1mmol/L MgCl
2), temperature 4 DEG C, elutriant is the elutriant 0.2-0.3M Alpha-Methyl-D-MANNOSE glycosides of PH 8-8.5.
In the embodiment that the present invention one is concrete, in step (d), gel chromatographic columns comprises the one in dextrane gel (sephadex G, sephadex LH ?20), sepharose (sepharose), cellulose gel (cellufine GCL ?2000), polyacrylamide gel (Bio ?gel P) etc.
Polygalactomannan antigen is separated by gel chromatographic columns, different according to the polysaccharide molecule elution time of different size and shape, thus obtain the polygalactomannan sterling of size uniform.
The polygalactomannan antigen adopting preparation method of the present invention to prepare can be used for the preparation of polygalactomannan polyclonal antibody, especially can be used for the preparation of aspergillus fumigatus polygalactomannan polyclonal antibody.Use polygalactomannan antigen-immunized animal, measure the serum titer of animals following immunization, in the animal body after immunity, get blood; Then with saturated ammonium sulphate salting-out process and affinity chromatography, purifying is carried out to serum, obtain the antibody of purifying.
Further, the present invention also provides the application of a kind of polygalactomannan adopting preparation method of the present invention to prepare in the test kit for the preparation of detection invasive fungi disease detection, and described invasive fungi is selected from aspergillus tubigensis, mucormycosis, Penicillium notatum, pearl bacterium, beads.Be particularly useful for detecting aspergillus tubigensis.
Those skilled in the art know, and test kit can comprise antigen, antibody, enzyme labelled antibody, washings, substrate nitrite ion, stop buffer etc., and therefore not to repeat here.
Below in conjunction with specific embodiment, the present invention is further elaborated.
Embodiment 1: the separation taking aspergillus tubigensis as the microbial components of representative
Aspergillus strain of the present invention is bought from Chinese medicine Microbiological Culture Collection administrative center (CMCC) and the biological product collecting center (ATCC) of USS.
Preparation Sharpe liquid nutrient medium 1.0L, adopt shaking flask concussion training method to cultivate, culture temperature is 35 DEG C, and shaking speed is 150rpm, cultivates after 3 days, 121 DEG C of moist heat sterilizations 30 minutes.The nutrient solution of deactivation, by filtered through gauze, filter paper suction filtration, then use 0.22 μm of membrane filtration, nutrient solution (comprising tissue juice etc.) is separated with thalline.Thalline physiological saline repetitive scrubbing, lyophilize.Adopt ultrasonic wave ice broken, 10000g ultracentrifugation, is separated tenuigenin with cell walls.Drying can obtain in aspergillus tubigensis the fermented liquid, tenuigenin and the cell wall constituent that are rich in polygalactomannan.
Embodiment 2: the separation taking guar seed as the vegetable jelly of representative
Guar seed is broken, and add flooding, filter, add organic solvent (ethanol or methyl alcohol) sedimentation, oven dry, abrasive dust, can obtain the vegetable jelly being rich in polygalactomannan.
Those skilled in the art know, and melon beans seed can be the melon beans of field growth natural maturity, also can be the in vitro tissue obtained by external sterile culture.When it is the in vitro tissue obtained by external sterile culture, described in its separation method and embodiment 1, the separation method of aspergillus tubigensis is similar, and therefore not to repeat here.
Embodiment 3: the preparation of polygalactomannan crude extract
Be dissolved in water by the active principle being rich in polygalactomannan that embodiment 1 or 2 obtains, add 3 times of volume dehydrated alcohols, in 4 DEG C of refrigerators, alcohol precipitation spends the night, centrifugal 15 minutes of 10000g, absolute ethanol washing three times of gained precipitation, lyophilize.By soluble in water for polygalactomannan dry powder, under agitation add gac, room temperature is decoloured 8 hours, and funnel filters, and filtrate, through 0.22 μm of membrane filtration, namely obtains the polygalactomannan crude extract removing pigment.
Embodiment 4: the preparation of polygalactomannan antigen sterling
(1) hydrazinolysis
Anhydrous hydrazine (adding 10-100 μ L anhydrous hydrazine in every milligram of alcohol hypostasis) is added, mixing, sealing in the galactomannan crude extract that embodiment 3 is obtained.With insulation at oil bath pan 110 DEG C 6 hours in stink cupboard.The ethanol of 4 times of volumes will be added in the hydrazinolysis thing of gained, the centrifugal 15min of 3000g, then precipitate 3 times with absolute ethanol washing, finally that pellet frozen is dry.
(2) nitrous acid hydrolysis
The nitrous acid solution adding the 2mol/L of 5ml in the dry powder after hydrazinolysis redissolves, and 30 DEG C are hydrolyzed 6 hours.Hydrolysis terminates to pass into air, hydrolyzed solution water dialysed overnight in backward solution, and dialyzate adds the ethanol of 4 times of volumes, the centrifugal 15min of 6000rpm, collecting precipitation, lyophilize; Repeat above-mentioned hydrazinolysis, hydrolytic process.
(3) enzymolysis
After hydrolysis, add the dextranase of 2-5% in gala mannan solution, at 37 DEG C, enzymolysis spends the night, and adopts sevage method, adds propyl carbinol and chloroformic solution in the solution, vibration, the centrifugal 15min of 6000rpm, removing enzyme; Again by solution by removing small-molecule substance in centrifugal ultrafiltration pipe.
(4) Mono Q anion-exchange column
By the polygalactomannan dry powder after enzymolysis in Tris-HCl (pH 7.4) buffer system of 25mM, cross Mono Q post, reclaim uncharged material.
(5) ConA-Sepharose 4B affinity column
First with PH be 7.4 Tris-HCl damping fluid (solute is 0.5mol/L NaCl, 1mmol/L CaCl
2, 1mmol/L MgCl
2) pre-equilibration affinity column, be then splined in ConA-Sepharose 4B affinity column by the polysaccharide soln after being separated through anion-exchange column, loading flow velocity is 1mL/min, and the time is 1h.Be the elutriant 0.2M Alpha-Methyl-polysaccharide of D-MANNOSE glycosides elution of bound on affinity column of 8.5 again with PH.Collect elutriant, alcohol precipitation, washing, drying, obtain polysaccharide dry powder.
(6) gel chromatographic columns
Polysaccharide dry powder is dissolved in 0.7% sodium sulfate, be splined in sephadex chromatography post sephadex G, after completion of the sample at 35 DEG C, 0.7% sodium sulfate is adopted to flow through gel chromatographic columns as moving phase with the speed of 0.5ml/min, the first outflow chromatographic column that molecular weight is large, the rear outflow chromatographic column that molecular weight is little, successively collects elutriant according to the different of molecular weight, thus obtains the homogeneous polygalactomannan antigen sterling of size uniform, molecular weight distribution.
Embodiment 5: by the method preparing polygalactomannan antigen of the present invention, for aspergillus tubigensis polygalactomannan, identify:
1. conventional biochemical method
(1) polysaccharide concentration measures-Dubois-Phenol-sulphate acid method
Adopt Dubois-Phenol-sulphate acid method to carry out determination of polysaccharide to the polygalactomannan purification of samples that preparation method of the present invention obtains, detected result as shown in following table 1 and 2, polygalactomannan bioassay standard curve as shown in Figure 1:
Table 1: polygalactomannan absorbance measurement
Table 2: polygalactomannan sugar degree
| Volume (mL) | Sugar content (mg) | |
| Detect sample | 0.200 | 0.310 |
| Whole sample | 100 | 155 |
From above-mentioned data, 155mg polygalactomannan sterling finally can be obtained by preparation method of the present invention from 1L Aspergillus fumigatus nutrient solution.
(2) hexosamine concentration determination-p-dimethylin phenyl aldehyde spectrophotometry
Adopt p-dimethylin phenyl aldehyde spectrophotometry hexosamine concentration.Polygalactomannan antigen purification sample the present invention prepared, is enclosed in ampoul tube, and adopt 8mol/LHCl at 100 DEG C of hydrolysis 2h, lyophilize, opens ampoul tube after hydrolysis, and by sample vacuum-drying, add water constant volume.Get 2mL and add 1mL methyl ethyl diketone reagent, shake up, test tube mouth glass bubbles makes cap seal, escapes to overflow, heat 20min, be cooled to room temperature with frozen water in boiling water bath to prevent methyl ethyl diketone.Add the p-dimethylaminobenzaldehyde of 1mL after adding dehydrated alcohol 5mL, then supply reaction solution cumulative volume to 10mL with anhydrous ethyl ketone.After Tube contents is fully mixed, 70 DEG C of heating in water bath 10min, are then cooled to room temperature.Absorbancy is measured at 530nm place.Using 100mg/L glucosamine as standard substance, water replaces sample, and the blank measured is used as in the same process, and 530nm place measures absorbancy.Detected result is as following table 3:
Table 3: hexosamine concentration determination
| OD530 | |
| Standard substance | 1.826 |
| Detect sample | 0.054 |
There is not hexosamine composition in hydrolyzed solution after measured, as can be seen here, the inventive method is extracted in sterling does not exist glycoprotein.
2. stratographic analysis
(1) ultraviolet absorption spectroscopy polysaccharide fraction
UV absorbance detection is carried out to the polygalactomannan antigen purification sample that preparation method of the present invention obtains.Under 260nm, 280nm and 320nm wavelength, detect sample respectively, result is as following table 4.
Table 4: polygalactomannan DNA, protein and polysaccharide determination result
| Test item | Concentration (μ g/mL) | Content (%) |
| DNA | 3.259 | 0.20 |
| Protein | 63.168 | 3.91 |
| Polysaccharide | 1550 | 95.89 |
From table 4 detected result, the total content of nucleic acid and protein and the ratio of polysaccharide content are less than 5%, that is nucleic acid and protein content are less than 5% of total substances content, are no more than 5% of sample total mass.
(2) differential refraction method analyzes polysaccharide composition
Carry out HPLC detection to the polygalactomannan antigen purification sample that preparation method of the present invention obtains, detector is differential refraction detector, and result is as accompanying drawing 2.As can be seen from Figure 2, it is single that sample goes out peak, and point is narrow, and without peak of obviously mixing, illustrate that the material size that the present invention extracts is homogeneous, purity is higher.
(3) monosaccharide component in liquid-phase chromatographic analysis polysaccharide
To the polygalactomannan antigen purification sample that preparation method of the present invention obtains, trifluoroacetic acid hydrolysis, then with 1-phenyl-3-methyl-5-pyrazolones ketone, derivatize is carried out to monose after hydrolysis.Determined wavelength is 250nm, and result as shown in Figure 3.As can be seen from Figure 3, except derivative peak, only containing a small amount of glucose, to mix peak without other monose.Prove thus, the polysaccharide main component that the inventive method obtains is polygalactomannan, and other polysaccharide content is extremely low.
(4) immunologic competence qualification
Adopt the Aspergillus fumigatus antigen detection kit of Bio-Rad company of the U.S. to detect polygalactomannan antigen samples prepared by method of the present invention, concentration of specimens is 1ng/ml.Detected result is positive, and OD value is greater than positive quality control.The polygalactomannan that method in explanation the present invention can obtain Aspergillus fumigatus has immunologic competence.The results are shown in following table 5:
Table 5: polygalactomannan immunocompetence measures
Data presentation, the high and even molecular weight distribution by the Aspergillus fumigatus polygalactomannan antigen purity of purifying of the present invention, possesses immunocompetence.
(5) polysaccharide molecule flow measurement
Adopt high productivity computing method to measure the dextran standards retention time tR of different molecular weight, detector is differential refraction detector, and data are in table 6.With the logarithm lgMw of dextran standards weight-average molecular weight, regression treatment is carried out to retention time tR, draw weight-average molecular weight typical curve, the results are shown in Figure 4, typical curve equation: tR=33.95188-3.74663*logMw, R
2=0.99957.By high productivity computing method, the polygalactomannan purification of samples that preparation method of the present invention obtains is measured, the results are shown in Figure 5, obtain retention time tR
0=17.815, substituted into typical curve, obtaining galactomannan molecule amount Mw is 20279Da.
The tR table of table 6 dextran standards
| Dextran standards | Molecular weight Mw (D) | logMw | Retention time tR (min) |
| T7 | 7100 | 3.851 | 19.500 |
| T10 | 10000 | 4.000 | 19.015 |
| T20 | 21400 | 4.382 | 17.510 |
| T40 | 41100 | 4.614 | 16.641 |
| T80 | 84400 | 4.926 | 15.518 |
Data presentation, the high and even molecular weight distribution by the Aspergillus fumigatus polygalactomannan antigen purity of purifying of the present invention.
Above-described embodiment is only for illustrating technical conceive of the present invention and feature, and not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement etc., all should be included within protection scope of the present invention.
Claims (11)
1. a preparation method for polygalactomannan antigen, comprises the steps:
A () is rich in the organism of polygalactomannan through broken, centrifugal, alcohol precipitation, washing, drying treatment, be separated and obtain polygalactomannan crude extract;
B polygalactomannan crude extract obtained for step (a) is carried out hydrazinolysis, hydrolysis by () successively;
C () carries out enzymolysis by adding enzyme in the hydrolysate in step (b), and the enzyme removed in enzymolysis product and small-molecule substance;
D product that step (c) obtains by (), through ion exchange column, affinity column and gel chromatography purifying, obtains polygalactomannan antigen sterling.
2. the preparation method of polygalactomannan antigen as claimed in claim 1, is characterized in that: described organism comprises fungi or endosperm bean; Preferably, described fungi comprises aspergillus tubigensis, mucormycosis, Penicillium notatum, pearl bacterium or candidiasis, and described endosperm bean comprises trigonella, Gua Erdou, angle beans, locust bean, pass China beans, Chinese honey locust, Semen adenantherae pavoninae or Leucaena leucocephala (L.).
3. the preparation method of polygalactomannan antigen as claimed in claim 1 or 2, is characterized in that: described step (a) comprising: the organism of being rich in polygalactomannan is cultivated polygalactomannan content the highest time, inactivation treatment; Filter, be separated by the organism after deactivation with nutrient solution and/or tissue juice, organism carries out cytoclasis, centrifugal, is separated by tenuigenin, obtains active principle with cell walls; Described active principle is through alcohol precipitation, and wash, drying process process, obtains polygalactomannan crude extract;
Or described step (a) comprising: organism is carried out fragmentation, adds flooding, filter, active principle is through alcohol precipitation, and washing, drying process process, obtain polygalactomannan crude extract.
4. the preparation method of polygalactomannan antigen as claimed in claim 3, it is characterized in that: in described step (a), the substratum used in organism culturing process be selected from PDA substratum, sabouraud culture medium, czapek's solution, malt extract medium one or more; Cultural method is selected from the slat chain conveyor or slant culture that use solid medium, or uses the shake flask of liquid nutrient medium to cultivate or fermentor cultivation.
5. the preparation method of polygalactomannan antigen as claimed in claim 3, it is characterized in that: in described step (a), ablation method comprises chemical reagent, ray, high temperature, high osmotic pressure;
And/or, in described step (a), described cytoclasis use in mechanical crushing method, swelling method, enzymolysis process one or more;
And/or in described step (a), described alcohol precipitation uses methyl alcohol or ethanol, the volume of alcohol precipitation is 2-6 times of active principle liquor capacity, and the alcohol precipitation time is 1-48 hour;
And/or in described step (a), described washing uses methyl alcohol or ethanol; Drying means is one or more in oven dry, freeze-drying, drying under reduced pressure.
6. the preparation method of polygalactomannan antigen as claimed in claim 1 or 2, is characterized in that: described step (a) also comprises activated carbon decolorizing.
7. the preparation method of polygalactomannan antigen as claimed in claim 1 or 2, it is characterized in that: in described step (b) during hydrazinolysis: the anhydrous hydrazine adding 10-100 μ L in every milligram of polygalactomannan crude extract, hydrazinolysis temperature is 100-150 DEG C, and the hydrazinolysis time is 2-12 hour;
And/or in described step (b) during hydrolysis, adopt nitrous acid hydrolysis, during nitrous acid hydrolysis, nitrous acid concentration is 1.5-3.0mol/L, and hydrolysis temperature is 30 DEG C, and hydrolysis time is 2-12 hour, passes into air after hydrolysis terminates.
8. the preparation method of polygalactomannan antigen as claimed in claim 1 or 2, it is characterized in that: in described step (c), described enzyme be selected from dextranase, cellulase, polygalacturonase, chitinase one or more, hydrolysis temperature is 20-60 DEG C, and enzymolysis time is 6-48 hour;
And/or in described step (c), add propyl carbinol and chloroformic solution, vibration, centrifugal, removing enzyme, adopts dialysis, ultrafiltration or molecular sieve removing small-molecule substance.
9. the preparation method of polygalactomannan antigen as claimed in claim 1 or 2, it is characterized in that: in described step (d), ion exchange column is anion-exchange column, damping fluid to be PH be 7 ~ 8.5 Tris-HCl damping fluid, temperature 4 DEG C;
And/or, in described step (d), affinity column is Con A-Sepharose 4B affinity column, and damping fluid is the Tris-HCl damping fluid of PH 7 ~ 8.5, temperature 4 DEG C, the Alpha-Methyl-D-MANNOSE glycosides of elutriant to be the concentration of PH 8 ~ 8.5 be 0.2 ~ 0.3mol/L;
And/or in described step (d), gel chromatographic columns is selected from dextrane gel, sepharose, cellulose gel, polyacrylamide gel.
10. a polygalactomannan antigen, is characterized in that: adopt the preparation method of the polygalactomannan antigen as described in any one of claim 1-9 to prepare.
11. polygalactomannan antigens as claimed in claim 10 are preparing polygalactomannan polyclonal antibody or for the preparation of the application detected in invasive fungi disease detection test kit.
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| CN111171119A (en) * | 2019-09-18 | 2020-05-19 | 吉林大学第一医院 | Method for extracting spore wall antigen |
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| CN112759666A (en) * | 2020-12-24 | 2021-05-07 | 承德康尔润食品有限公司 | Preparation method of semen cassiae-derived galactomannan |
| CN112759666B (en) * | 2020-12-24 | 2022-09-06 | 承德康尔润食品有限公司 | Preparation method of semen cassiae-derived galactomannan |
| CN113373190A (en) * | 2021-05-27 | 2021-09-10 | 承德康尔润食品有限公司 | Method for preparing galactomannan from semen Trigonellae |
| CN113373190B (en) * | 2021-05-27 | 2022-11-11 | 承德康尔润食品有限公司 | Method for preparing galactomannan from semen Trigonellae |
| CN114409824A (en) * | 2022-02-23 | 2022-04-29 | 皖南医学院 | Mucor exopolysaccharide and preparation method and application thereof |
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