CN106179280A - A kind of Candida mannan Mn and the preparation method of immune affinity chromatographic column thereof - Google Patents
A kind of Candida mannan Mn and the preparation method of immune affinity chromatographic column thereof Download PDFInfo
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Abstract
The invention provides and a kind of utilize cetyl trimethylammonium bromide to the method preparing albicans cell wall mannan.Albicans cell is after high temperature high pressure process in citrate solution, supernatant precipitates to obtain mannan crude product in methanol, cetyl trimethylammonium bromide is used first to precipitate foreign protein and nucleic acid, then precipitation is formed with mannan complexation, after complex compound sediment acetic acid, use ethanol precipitate polysaccharides, obtain purified mannan.Then with having cross-linked the immune affinity chromatographic column of Candida mannan Mn monoclonal antibody to mannan Mn process, obtain the Candida mannan Mn of purification, easy, efficiently, low cost, and can effectively avoid using poisonous and hazardous chemical reagent.The invention still further relates to a kind of Candida mannan Mn immune affinity chromatographic column, repeatable utilization, advantageously reduce preparation cost, improve preparation efficiency, preparation method is simple.
Description
Technical field
The present invention relates to antigen preparation field, be specifically related to the preparation method of a kind of Candida mannan Mn.
Background technology
Candida albicans is a kind of opportunistic fungus, and human body will not be felt by Candida albicans under normal circumstances
When dye, only body's immunity or general phylactic power defensive power decline or the mutual restrictive function of normal flora is lacked of proper care, PI white
Candidiasis, causes deep infection, the even Disseminated infection of whole body.Mannan is that the major antigen of albicans cell wall becomes
Point.For the diagnosis of candida albicans infection, except traditional cultural method, measure Candida albicans mannan in serum and resist
Former, it is increasingly becoming simple, the method efficiently of Candida albicans early diagnosis.
And measure the antigen key as candida albicans infection diagnostic method, it is to obtain preparing exempting from of monoclonal antibody
Epidemic focus mannan.The domestic research prepared for albicans cell wall mannan, mainly woods fly minister in ancient times et al. and are adopted
Hot phenol method and hot Methanamide method (woods flies minister in ancient times etc., China Immunology Journal, 1987, volume 3, the 5th phase, 288-292 page).
Hot phenol method is summarized as follows: 1) removing protein: to reading the phosphorus adding the 1/15M pH7.2 containing 45% phenol in bacterium mycopowder in vain
Phthalate buffer, homogenate dialysed overnight removes phenol, is centrifuged and is precipitated thing, and precipitation dehydrated alcohol washes most phenol, adds a small amount of
Water, 100 DEG C of water-baths extract 1 hour；It is centrifuged and is precipitated thing, add the 3%NaOH of 10 times of volumes, flow down at saturated nitrogen, 100 DEG C
Water-bath extracts 6 hours；It is centrifuged again, separates supernatant, neutralize with HCl, distilled water dialysis is desalted, be evaporated to a small amount of
Volume；2) precipitate polysaccharides: adding dehydrated alcohol to ultimate density in above-mentioned supernatant is 80%, and-20 DEG C overnight, many to precipitate
Sugar；It is centrifuged to obtain precipitate, with acetone drying, i.e. obtains rough mannan；3) polysaccharide purification: use DEAE-Sephadex A-
50 (deae dextran gel A-50) purification, first processes deae dextran gel A-50 with conventional method, then is allowed to 0.1N acetic acid
It is converted into acid type, dress post (1.8 × 30cm)；Rough mannan antigen is dissolved in a small amount of deionization redistilled water, to same liquid
Upper prop after body dialysis；With deionization redistilled water eluting, collect eluent.Monitor with detection of nucleic acids albumen instrument, and do Molish survey
Sugar reaction, merges positive each pipe, is evaporated to a small amount of volume, dialyses deionization redistilled water, and subpackage, lyophilization obtains purification
Mannan.The method can obtain the mannan that purity is higher, but preparation process is more complicated, needs twice 100 DEG C of water-baths
Extracting, gel chromatography is wasted time and energy simultaneously, and each fractional dose is few, and when relative molecular mass is estimated not know,
The selection of gel has certain difficulty.
Hot Methanamide method is summarized as follows: 1) removing protein: acetone drying reads mycopowder in vain in round-bottomed flask, adds Methanamide
(20mL/1g mycopowder), oil bath 150 DEG C heating 30min, make thalline rupture, be cooled to room temperature, at 20 DEG C, 2000g is centrifuged 30min,
Abandon precipitation；2) precipitate polysaccharides: supernatant adds 2.5 times of volume acid ethanol (2N HCl 1 part, dehydrated alcohol 19 parts), is stirred at room temperature
30min, at 20 DEG C, 6000g is centrifuged 30min, abandons supernatant, obtains white precipitate polysaccharide；Precipitation distilled water extracts solubility thing
Matter, then precipitation in the ethanol (cold ethanol) of 50%, at 4 DEG C, 6000g is centrifuged 30min, abandons supernatant；Precipitate is molten
In distilled water, add cold ethanol (final volume concentration is 70%) and 1M sodium acetate (5mL aqueous precipitating solution adds 0.2mL sodium acetate), 4
At DEG C, 6000g is centrifuged 30min, abandons supernatant；Precipitation absolute ethanol washing, with acetone drying under reduced pressure, obtains rough
Mannan antigen；3) polysaccharide purification: Crude antigen is dissolved in distilled water, crosses Sephadex G-75 post (1.5x 26-cm), distilled water
Eluting, flow velocity 1mL/6min；Collect each pipe, make sugar qualitative detection with Molish reagent, and detect albumen with nucleic acid-protein instrument
(280nm), merging peaky curve overlapped tubes, lyophilizing obtains purified mannan.The method process is relatively easy, but obtain
Purity of polysaccharide is the highest, containing more foreign protein, and the shortcoming that the method equally exists gel chromatography.
External disclosed albicans cell wall mannan preparation method, the film of Primary Reference Peat et al.
The method (1961, J.Chem.Soc.1:29-34) of agent precipitate.Method particularly includes: mycopowder is at the citrate buffer of 19mM
In, 140 DEG C of sterilizings 2 hours in high-pressure sterilizing pot, centrifugal, after colloid adds water, the same terms autoclaving again；Mixed liquor
Concentrating under reduced pressure, adds acetic acid, and mixed liquor is centrifuged, and separates brown gum, washs with acetum, collects washing liquid, and supernatant is
Containing mannan solution, neutralize with sodium hydroxide；Add ethanol, precipitate and wash twice with 60% ethanol water, be dissolved in water
In, centrifugal, remove brown gum, muddy supernatant adds sodium hydroxide and is adjusted to alkalescence, uses Fehling Regent precipitation, then sinks
Shallow lake washing with alcohol, finally gives mannan precipitation.Although this method does not exist the shortcoming of gel chromatography, but Fehling Regent sinks
Shallow lake polysaccharide, productivity, purity are the most relatively low.Wanting to improve polysaccharide yield and purity, need Reusability Fehling Regent to precipitate, process is numerous
Trivial, its technique is not appropriate for commercial production.
At present, this area is in the urgent need to the Candida mannan developing a kind of simplicity, efficient, low cost, specificity are high
The preparation method of Mn, and can be effectively prevented from using poisonous and hazardous chemical reagent.The present invention overcomes the deficiencies in the prior art,
Provide the preparation method of a kind of Candida mannan Mn.
Summary of the invention
It is an object of the present invention to provide the preparation method of a kind of Candida mannan Mn immune affinity chromatographic column.
The preparation method of described Candida mannan Mn immune affinity chromatographic column, it is characterised in that include walking as follows
Rapid:
(1) Candida mannan Mn monoclonal antibody is dissolved in solution, and is mixed into thin with affinity chromatography matrices
Homogenate；
(2) homogenate obtained with cross-linking buffer washing step (1), and be centrifuged, obtain antibody and affinity chromatography matrices
Mixture；
(3) antibody obtained with the resuspended step of cross-linking buffer (2) and the mixture of affinity chromatography matrices, mixed to obtain
Suspension adds difunctional bonding agent, incubated at room 20-40 minute, and mixes gently, solid-liquor separation, obtain affinity chromatograph base
Matter-antibody linked complex；
(4) affinity chromatography matrices-antibody linked complex obtained with lock solution washing step (3) closes chromatography base
Active group unnecessary in matter is to terminate cross-linking reaction；
(5) being resuspended in lock solution by affinity chromatography matrices-antibody linked complex that step (4) obtains, room temperature is incubated
Educate 1.5-2.5 hour, mix gently；
(6) dress post: after affinity chromatography matrices-antibody linked complex step (5) obtained cross-links successfully after testing, fill out
It is filled with in chromatographic column, prepares Candida mannan Mn immune affinity chromatographic column；
Preferably, (7) preserve: the affinity chromatography matrices that step (6) obtains-antibody linked complex or step (6) obtain
Populated Candida mannan Mn immune affinity chromatographic column preserve under the conditions of 4 DEG C, stable performance in 1 year；
Preferably, (8) regeneration: the chromatographic column after the chromatographic column preserved under the conditions of 4 DEG C in step (7) or use is using
Front 10-25 is washed column regeneration by the buffer that the solvent phase of the sample to be purified of bed volume is same.
Preferably, refill after affinity chromatography matrices-antibody linked complex that step (5) obtains being carried out preservative treatment
Entering in chromatographic column, described preservative treatment is that affinity chromatography matrices-antibody linked complex PBS step (5) obtained delays
Rush liquid washing, and be resuspended in PBS, add thimerosal and preserve；The concentration of described thimerosal is 0.005-
0.015%.
Solution described in step (1) is selected from carbonate buffer solution, Acetic acid-sodium acetate buffer；Preferably, step (1)
Described in affinity chromatography matrices selected from protein A microballon, Protein G microballon, activity microballon, optimization protein A microballon.
Preferably, the solution described in step (1) is selected from pH8.0-9.0 carbonate buffer solution.
Preferably, in step (1), the ratio of affinity chromatography matrices and solution is: add 0.5-2.0mL in every 10mL solution
Affinity chromatography matrices, affinity chromatography matrices with the ratio of antibody is: every 1mL affinity chromatography matrices is combined 1-4mg monoclonal antibody；
It is further preferred that the ratio of affinity chromatography matrices and solution is in step (1): every 10mL solution adds 1mL affinity chromatograph
Substrate, affinity chromatography matrices with the ratio of antibody is: every 1mL affinity chromatography matrices is combined 2mg monoclonal antibody.
Preferably, the sodium borate of the pH8.0-9.5 that cross-linking buffer is 0.1-0.3mol/L described in step (2) is molten
Liquid, consumption is 5-15 times of affinity chromatography matrices volume, it is further preferred that described cross-linking buffer is concentration is 0.2mol/L's
The dobell's solution of pH9.0, consumption is 10 times of affinity chromatography matrices volumes；Described washing times is 1-3 time, and centrifugal condition is
3000g is centrifuged and is centrifuged 30 seconds in 2-5 minute or 10000g.
Preferably, cross-linking buffer described in step (3) is the dobell's solution of the pH8.3-9.5 of 0.1-0.3mol/L,
Consumption is 5-15 times of affinity chromatography matrices volume；It is further preferred that described cross-linking buffer is concentration is 0.2mol/L's
The dobell's solution of pH9.0, consumption is 10 times of affinity chromatography matrices volumes.
Preferably, the difunctional bonding agent described in step (3) selected from dimethyl pimelate, carbonyl dimidazoles, Bromine cyanide.,
Hydroxysuccinimide, acetyl group iodine, preferably dimethyl pimelate, consumption is to make it in affinity chromatography matrices suspension
Final concentration of 15-25mmol/L, preferably 20mmol/L.
Preferably, step (4) and the lock solution described in (5) selected from ethanolamine, aminoethane etc. containing can be in conjunction with amino
The solution of the small-molecule substance of active group, preferably ethanolamine solutions, more preferably concentration is 0.1-0.25mol/L,
PH value is the ethanolamine solutions of the pH8.0 of the ethanolamine solutions of 7.5-8.5, more preferably 0.2mol/L.
Preferably, the detection process of the cross-linking efficiency of the affinity chromatography matrices described in step (6)-antibody linked complex
For: take affinity chromatography matrices sample and affinity chromatography matrices with antibody linked after sample, be separately added in LaemmLi buffer
Boil, take out the two kinds of samples being equivalent to 1mL and 9mL respectively, 10%SDS-polyacrylamide gel carries out electrophoresis, with examining
Maas light blue dyes, as before crosslinking, sample presents heavy chain zone (55kDa), after cross-linking sample then without, show to cross-link successfully.
Preferably, after the dress post described in step (6) completes, use PBS eluant container, collect the affine layer of residual
Analysis substrate, if it is possible, only use in sample to be purified affinity chromatography matrices-antibody linked complex needed for whole Mn.
It is an object of the present invention to provide a kind of Candida mannan Mn immune affinity chromatographic column, it is characterised in that
Described Candida mannan Mn immune affinity chromatographic column is by the system of above-mentioned Candida mannan Mn immune affinity chromatographic column
Preparation Method prepares.
It is an object of the present invention to provide the preparation method of a kind of Candida mannan Mn, it is characterised in that include
Following steps:
(1) extraction of Candida mannan Mn；
(2) Candida mannan Mn immune affinity chromatographic column is utilized to carry out the purification of Candida mannan Mn.
The extraction of described Candida mannan Mn, comprises the steps:
1) Candida albicans is cultivated and is used general Candida albicans condition of culture, specifically culture medium to use YM meat soup
Culture medium, composition is every liter of culture medium yeast extract Han 3g, 5g peptone, 3g maltose extract, 10g D-Glucose.Training
Foster temperature is 30 DEG C, and concussion speed is 200rpm, incubation time about 28h.
2) above-mentioned culture fluid is centrifuged, precipitation i.e. albicans cell 0.9wt% NaCl twice, 56 DEG C
Heating 2h kills cell, obtains Candida albicans mycopowder with acetone drying.
3) gained Candida albicans mycopowder is at pH=6.8-7.2, and concentration is that the citrate buffer mesohigh of 0.02M goes out
Bacterium 90min, centrifugal, retain supernatant, sterilizing under precipitation jelly the same terms, be centrifuged, merge supernatant.
4) above-mentioned supernatant joins in methanol, precipitation occurs；Being centrifugally separating to obtain precipitation, precipitation is dissolved in deionized water,
Dialyse with deionized water, lyophilizing, obtain rough mannan.
5) rough mannan is dissolved in deionized water, adds the aqueous solution of cetyl trimethylammonium bromide, mixed liquor
There is precipitation in static a period of time.
6) centrifugation precipitation, precipitate with deionized water is washed, and merges cleaning mixture and 5) step supernatant, amalgamation liquid adds
The boric acid solution of 1wt%.
7) above-mentioned mixed liquor adds 2M sodium hydroxide while stirring, regulates pH to 8.6-9.0, generates cetyl trimethyl
Ammonium bromide-mannan complex compound sediment, collects precipitation, with the 0.5% sodium acetate washing of pH=8.8, then molten with 2% acetic acid
Solving, gained solution precipitates in ethanol.
8) centrifugal, abandon supernatant, precipitate and wash with the ethanol acetate solution of 2%, neutralize with 2M sodium hydroxide after being dissolved in water,
Distilled water is dialysed, lyophilizing, obtain Candida albicans mannan product.
Preferably, the condition of culture of the candidiasis in the step (1) of the extraction of described Candida mannan Mn is: training
Supporting base and use YM broth bouillon, cultivation temperature is 30 DEG C, and concussion speed is 200rpm, and incubation time is 26-30 hour, described
YM broth bouillon formula be that every liter of culture medium contains: 3g yeast extract, 5g peptone, 3g maltose extract, 10g D-
Glucose.
Preferably, described in step (3) at pH=7.0, concentration is the citrate buffer of 0.02M.
Preferably, the 2M sodium hydroxide described in step (7), regulate pH to 8.8.
The described Candida mannan Mn immune affinity chromatographic column that utilizes carries out Candida mannan Mn crude extract
Purification, comprises the steps:
(1) with the wash buffer above-mentioned Candida mannan Mn immunoaffinity chromatography identical with sample solution to be purified
Post；
(2) sample solution to be purified is made to flow through the chromatographic column after step (1) processes；
(3) post is washed with combining buffer；
(4) post is washed with pre-elution buffer；
(5) use stepwise elution, flow through chromatographic column with elution buffer continuously, be in charge of each component of collection；
(6) detect the Mn content of often pipe, each pipe high for concentration is merged；
Preferably, (7) flow through chromatographic column with the start buffer of 10-25 times of bed volume makes it regenerate, and adds sulfur willow
Hydrargyrum makes its final concentration of 0.005-0.015%, preserves chromatographic column under the conditions of 4 DEG C.
Preferably, the flow velocity of the sample to be purified described in step (2) is 0.5-1.5mL/h, uses pump coutroi velocity, preferably
Use peristaltic pump.
Preferably, combination buffer described in step (3) is selected from the PBS of pH6.0-8.0, pH6.0-8.0
One in Tris-HCl buffer, pH6.0-8.0 Acetic acid-sodium acetate buffer, the PBS of preferably pH6.0-8.0；Step
Suddenly the consumption of the combination buffer described in (3) is 10-25 times of bed volume, preferably 20 times bed volumes.
Preferably, the pre-elution buffer described in step (4) is pH8.5-9.5 carbonate buffer solution, and consumption is 10-25
Times bed volume, more preferably 20 times of bed volumes.
Preferably, the buffer that elution buffer is pH3.0 described in step (5), the 0.1M glycine of preferably pH3.0
Buffer, the citrate-phosphate salt buffer of pH3.0, the citric acid-sodium citrate buffer of pH3.0, the acetic acid-vinegar of pH3.0
Acid sodium buffer, consumption is 0.4-0.8 bed volume.
Preferably, the Mn eluent that step (6) obtains carries out dialysis and goes salt treatment or regulate its pH value with buffer.
It is an object of the present invention to provide a kind of Candida mannan Mn, it is characterised in that described candidiasis is sweet
Dew polysaccharide Mn is prepared by the preparation method of above-mentioned Candida mannan Mn.
Candidiasis of the present invention is purchased from Chinese medicine Microbiological Culture Collection administrative center.
Mannan monoclonal antibody of the present invention is provided by red Na (Tianjin) bio tech ltd.
It is an object of the present invention to provide a kind of Candida mannan Mn immune affinity chromatographic column at purification candidiasis
Application in the early diagnosis of mannan Mn and monilial infection.
It is an object of the present invention to provide a kind of Candida mannan Mn early diagnosis reagent at monilial infection
In application.
The invention provides the preparation method of a kind of Candida mannan Mn, use immunoaffinity chromatography sweet to candidiasis
Dew polysaccharide Mn is purified, and efficiently avoid the use of toxic chemical, it is ensured that the safety of operator, avoids simultaneously
Environmental pollution, and specificity is high, can prepare highly purified Candida mannan while simplifying preparation technology.This
Bright according to target product to be prepared, selection specificity is for the monoclonal antibody of target product, multiple through experiment screening
Condition, obtains a kind of Candida mannan Mn chromatographic column and preparation method thereof.The Candida mannan Mn that the present invention provides
Chromatographic column high specificity, the target product efficiency height of separation correspondence, convenience ease in use, and repeatable utilization, advantageously reduce
Preparation cost, improves preparation efficiency, and its preparation method is simple.
Accompanying drawing explanation
Fig. 1 show the result of the Candida mannan Mn detection by quantitative that one embodiment of the invention provides.
Fig. 2 show the result of the HPLC purity analysis of the Candida mannan Mn that one embodiment of the invention provides.
Fig. 3 show the SDS-PAGE foreign protein content analysis of the Candida mannan Mn that one embodiment of the invention provides
Result.
Detailed description of the invention
Form of presentations such as " start buffer ", " buffer identical with sample solution to be purified " in the present invention has phase
Same implication, is used interchangeably.
Form of presentations such as heretofore described " Candida mannan Mn ", " Mn " has identical implication, interchangeable
Use.
Embodiment 1:
The extraction of Candida mannan Mn, it specifically comprises the following steps that
1) Candida albicans is cultivated and is used general Candida albicans condition of culture, specifically culture medium to use YM meat soup
Culture medium, composition is every liter of culture medium yeast extract Han 3g, 5g peptone, 3g maltose extract, 10g D-Glucose.Training
Foster temperature is 30 DEG C, and concussion speed is 200rpm, incubation time about 28h.
2) above-mentioned culture fluid is centrifuged, precipitation i.e. albicans cell 0.9wt% NaCl twice, 56 DEG C
Heating 2h kills cell, obtains Candida albicans mycopowder with acetone drying.
3) gained Candida albicans mycopowder is at pH=7.0, and concentration is the citrate buffer mesohigh sterilizing of 0.02M
90min, centrifugal, retain supernatant, sterilizing under precipitation jelly the same terms, be centrifuged, merge supernatant.
4) above-mentioned supernatant joins in methanol, precipitation occurs；Being centrifugally separating to obtain precipitation, precipitation is dissolved in deionized water,
Dialyse with deionized water, lyophilizing, obtain rough mannan.
5) rough mannan is dissolved in deionized water, adds the aqueous solution of cetyl trimethylammonium bromide, mixed liquor
There is precipitation in static a period of time.
6) centrifugation precipitation, precipitate with deionized water is washed, and merges cleaning mixture and 5) step supernatant, amalgamation liquid adds
The boric acid solution of 1wt%.
7) above-mentioned mixed liquor adds 2M sodium hydroxide while stirring, regulates pH to 8.8, generates cetyl trimethyl bromination
Ammonium-mannan complex compound sediment, collects precipitation, with the 0.5% sodium acetate washing of pH=8.8, then uses 2% acetic acid,
Gained solution precipitates in ethanol.
8) centrifugal, abandon supernatant, precipitate and wash with the ethanol acetate solution of 2%, neutralize with 2M sodium hydroxide after being dissolved in water,
Distilled water is dialysed, lyophilizing, obtain Candida albicans mannan product.
Embodiment 2
The preparation of Candida mannan Mn immune affinity chromatographic column, it specifically comprises the following steps that
(1) Candida mannan Mn monoclonal antibody is attached on protein A microballon.By every milliliter of wet microballon about
In conjunction with 2mg monoclonal antibody, antibody and protein A can be mixed into thin homogenate, add in the solution that total amount is 10mL
About 1mL microballon, incubated at room 1h, it is shaken gently for mixing.
(2) wash microballon 2 times with the 0.2mol/L sodium borate (pH9.0) of 10 times of volumes, be centrifuged 2min with 3000g every time,
Or 10000g is centrifuged 30s.
(3) with 0.2mol/L sodium borate (pH9.0) the resuspended microballon of 10 times of volumes, the sample being equivalent to the wet microballon of 10mL is left and taken
Product.Add enough dimethyl pimelates (solid) to microballon be homogenized in, make final concentration of 20mmol/L, incubated at room
30min, makes the antibody being combined on protein A microballon crosslink with microballon substrate, and mixes gently, obtains microballon-antibody and hands over
Connection complex.Leave and take the sample being equivalent to 10mL cross-linked composite.
(4) with 0.2mol/L ethanolamine (pH8.0) washing microballon-antibody linked complex 1 time to terminate cross-linking reaction.
(5) microballon-antibody linked complex that step (4) obtains is resuspended in 0.2mol/L ethanolamine solutions, room temperature
Hatch 2h, mix gently.
(6), after the microballon that step (5) is obtained-antibody linked complex PBS washing, it is resuspended in PBS, adds thimerosal
To its final concentration of 0.01% preservation.
(7) microballon-antibody linked complex that step (6) obtains is filled out after cross-linking successfully after testing, be packed into chromatographic column
In, prepare Candida mannan immune affinity chromatographic column, use PBS eluant container, collect the microballon of residual.If it is possible, only
Use the antibody microballon substrate needed for whole Mn in goods to be purified.
Embodiment 3
The purification of the Candida mannan Mn of embodiment 1 preparation, it specifically comprises the following steps that
(1) the Candida mannan Mn immune affinity chromatographic column of Example 2 preparation, with initiateing of 20 times of bed volumes
Post washed by buffer.
(2) sample solution to be purified is added chromatographic column, by the flow velocity of every milliliter of column volume about 1mL/h, make sample molten
Liquid stream crosses chromatographic column, uses peristaltic pump coutroi velocity.
(3) post is washed with the combination buffer of 20 times of bed volumes.
(4) post is washed with the pre-elution buffer of 20 times of bed volumes.
(5) use stepwise elution method, pass through chromatographic column with the elution buffer of 0.5 times of bed volume continuously, be in charge of collection
Each component.
(6) detect the Mn content of often pipe, each pipe high for concentration is merged.According to the purposes of antigen, the antigen collected is washed
De-liquid dialysis.
(7) flow through substrate with the start buffer of 20 times of bed volumes, make column regeneration, add in buffer
0.01% thimerosal, is then stored in chromatographic column in the environment of 4 DEG C.
Wherein, start buffer, combine buffer, pre-elution buffer, elution buffer be respectively high purity water, pH7
PBS, the carbonate buffer solution of pH9, the glycine buffer of 0.1M of pH3.0.
Embodiment 4
The detection of the Candida mannan Mn of embodiment 3 preparation:
A.Dubois-Phenol-sulphate acid method measures sugar content, and it specifically comprises the following steps that
(1) weigh 1g glucose and be dissolved in 100mL dH2In O, it is made into 1% glucose solution；
(2) taking 7 10mL clean tube, every test tube is separately added into 1mL dH2O and 25 μ L phenol solution；
(3) each test tube is separately added into 0,2.5,5,10,15 μ L glucose solution and 10 μ L liquid to be measured；
(4) often pipe is slowly added to 2.5mL concentrated sulphuric acid, all adds unified mixing again after concentrated sulphuric acid；
(5) test tube water-bath about 20 minutes, are cooled to room temperature；
(6) liquid in each pipe is carefully poured in glass cell；
(7) under 485nm, absorbance is measured with ultraviolet spectrophotometer；
(8) standard curve is drawn；
(9) liquid polysaccharide concentration to be measured is calculated according to standard curve.
Standard curve as it is shown in figure 1, linearly the best, can be used for calculating testing sample concentration.
B.HPLC purity analysis, it specifically comprises the following steps that
Take Candida mannan Mn sample 1mL (3.65mg/mL)
Chromatographic column: Sugar ParkT72011A09P/N85188Waters
Flowing phase: ultra-pure water
Flow velocity: 0.5mL/min
Column temperature: 85 DEG C
Detector: differential refraction detector
Sensitivity set point: 1.0 × 10-6-5.0×10-4RIU
Differential scope: 1.00～1.75RIU
Linear dynamic range: 5 × 10-3～5 × 10-9RIU
Noise: ＜ 2 × 10-8RIUATTN
HPLC sample introduction, detects with differential refraction detector, and result as shown in Figure 2, single absworption peak occurs, shows sample
Purity is preferable.
C.SDS-PAGE foreign protein content analysis, it specifically comprises the following steps that
(1) PAGE gel of compound concentration 8%, glue；
(2) every hole adds the Candida mannan sample that 10uL embodiment 3 prepares；
(3) after 10 minutes 120V constant voltage electrophoresis about 1 hour, to bromophenol blue band away from offset plate lower limb 1 for 60V constant voltage electrophoresis
Centimetre time stop electrophoresis；
(4) coomassie brilliant blue staining 1 hour；
(5) 4 DEG C of decolourings are overnight；
(6) take pictures and observe.
Glue figure is as it is shown on figure 3, from contrast, the Candida mannan sample purity that embodiment 3 prepares is higher, without miscellaneous
Albumen.
Embodiment 5:
The repeatability of Candida mannan Mn immune affinity chromatographic column, the response rate, stability are verified:
A. repeatability checking, specifically comprises the following steps that
(1) prepare 5 Candida mannan Mn immune affinity chromatographic columns by the step of embodiment 2, be numbered respectively
001,002,003,004 and 005.
(2) Candida mannan of Example 1 preparation is sample to be purified, is uniformly divided into 5 parts.
(3) 5 parts of 2mL samples to be purified are separately added in the chromatographic column of 5 parallel tests, enter by the operation of embodiment 3
Row purification.
(4) qualification of the polysaccharide MN after being purified by the operation of embodiment 4, result is as follows:
Phenol sulfuric acid standard measure, the sugared total amount CV ＜ 10% obtained after purification；.
All there is single absworption peak in HPLC purity analysis；
SDS-PAGE foreign protein content analysis is all without foreign protein band.
The repeated the result of table 1 Candida mannan immune affinity chromatographic column
B. response rate checking, specifically comprises the following steps that
(1) 5 Candida mannan Mn immune affinity chromatographic columns are prepared by embodiment 2 step.
(2) in Example 3, the Candida mannan Mn of preparation is as purification standard specimen, and concentration is 3.65mg/mL.
(3) 2mL purification standard specimen solution is added in the chromatographic column of 5 parallel tests, carry out pure by the operation of embodiment 3
Change.
(4) detect the sugared total amount of standard specimen after purification, calculate the response rate between 96.5%-100.5%.
The response rate the result of table 2 Candida mannan Mn immune affinity chromatographic column
C. stability checking, specifically comprises the following steps that
(1) 1 Candida mannan Mn immune affinity chromatographic column is prepared by the step of embodiment 2.
(2) in Example 3, the Candida mannan Mn of preparation is as purification standard specimen, and concentration is 3.65mg/mL.
(3) being added in chromatographic column by 1mL purification standard specimen solution, be purified by the operation of embodiment 3, same pillar repeats
Sample introduction 10 times, (regenerating after each eluting).
(4) the sugared total amount of 10 standard specimens after purification of detection, calculates the response rate between 80.16%-108.56%.
The stability the result of table 3 Candida mannan Mn immune affinity chromatographic column
From the above results, the Candida mannan Mn immune affinity chromatographic column prepared by embodiment 2 step is to mesh
The mark specificity of compound and the response rate are the highest, and repeatability and stability preferable, pure for Candida mannan Mn
Change preparation can effectively reduce purification preparation cost, simplify preparation process and improve preparation efficiency.
Claims (13)
1. the preparation method of a Candida mannan Mn immune affinity chromatographic column, it is characterised in that comprise the steps:
(1) Candida mannan Mn monoclonal antibody is dissolved in solution, and is mixed into homogenate with affinity chromatography matrices；
(2) homogenate obtained with cross-linking buffer washing step (1), and be centrifuged, obtain the mixing of antibody and affinity chromatography matrices
Thing；
(3) antibody obtained with the resuspended step of cross-linking buffer (2) and the mixture of affinity chromatography matrices, to the suspension obtained
The difunctional bonding agent of middle addition, incubated at room 20-40 minute, and mix, solid-liquor separation, obtain affinity chromatography matrices-antibody and hand over
Connection complex；
(4) affinity chromatography matrices-antibody linked complex obtained with lock solution washing step (3)；
(5) affinity chromatography matrices-antibody linked complex that step (4) obtains is resuspended in lock solution, incubated at room
1.5-2.5 hour, mixing；
(6) dress post: affinity chromatography matrices-antibody linked complex step (5) obtained is packed in chromatographic column, prepares beads
Bacterium mannan Mn immune affinity chromatographic column.
2. the preparation method of Candida mannan Mn immune affinity chromatographic column as claimed in claim 1, it is characterised in that step
Suddenly the affinity chromatography matrices described in (1) is selected from protein A microballon, Protein G microballon, activity microballon, optimization protein A microballon；
And/or, the solution described in step (1) is selected from carbonate buffer solution, Acetic acid-sodium acetate buffer；
In step (1), the ratio of affinity chromatography matrices and solution is: add 0.5-2.0mL affinity chromatograph base in every 10mL solution
Matter, affinity chromatography matrices with the ratio of antibody is: every 1mL affinity chromatography matrices is combined 1-4mg monoclonal antibody.
3. the preparation method of Candida mannan Mn immune affinity chromatographic column as claimed in claim 1, it is characterised in that step
Suddenly the cross-linking buffer described in (2) is the dobell's solution of pH8.0-9.5 of 0.1-0.3mol/L, consumption be 5-15 times affine
Chromatography substrate volume；
And/or, cross-linking buffer described in step (3) is the dobell's solution of the pH8.3-9.5 of 0.1-0.3mol/L, and consumption is
5-15 times of affinity chromatography matrices volume.
4. the preparation method of Candida mannan Mn immune affinity chromatographic column as claimed in claim 1, it is characterised in that step
Suddenly the difunctional bonding agent described in (3) is selected from dimethyl pimelate, carbonyl dimidazoles, Bromine cyanide., hydroxysuccinimide, second
Acid iodide, preferably dimethyl pimelate, consumption is the final concentration of 15-25mmol/L making it in suspension.
5. the preparation method of Candida mannan Mn immune affinity chromatographic column as claimed in claim 1, it is characterised in that step
Suddenly the lock solution described in (4) and (5) is selected from ethanolamine, aminoethane solution, preferably ethanolamine solutions, further preferably
Being 0.1-0.25mol/L for concentration, pH value is the ethanolamine solutions of 7.5-8.5.
6. a Candida mannan Mn immune affinity chromatographic column, it is characterised in that described Candida mannan Mn exempts from
Epidemic disease affinity column is by the preparation method of the Candida mannan Mn immune affinity chromatographic column described in any one of claim 1-5
Prepare.
7. the preparation method of a Candida mannan Mn, it is characterised in that comprise the steps:
(1) extraction of Candida mannan Mn；
(2) the Candida mannan Mn immune affinity chromatographic column described in claim 6 is utilized to carry out Candida mannan Mn
Purification.
8. the preparation method of Candida mannan Mn as claimed in claim 7, it is characterised in that described candidiasis manna
The extraction of polysaccharide Mn, comprises the steps:
1) Candida albicans is cultivated and is used general Candida albicans condition of culture, specifically culture medium to use YM meat soup to cultivate
Base, composition is every liter of culture medium yeast extract Han 3g, 5g peptone, 3g maltose extract, 10g D-Glucose；Cultivate temperature
Degree is 30 DEG C, and concussion speed is 200rpm, incubation time about 28h；
2) above-mentioned culture fluid is centrifuged, precipitation i.e. albicans cell 0.9wt% NaCl twice, 56 DEG C of heating
2h kills cell, obtains Candida albicans mycopowder with acetone drying；
3) gained Candida albicans mycopowder is at pH=6.8-7.2, and concentration is the citrate buffer mesohigh sterilizing of 0.02M
90min, centrifugal, retain supernatant, sterilizing under precipitation jelly the same terms, be centrifuged, merge supernatant；
4) above-mentioned supernatant joins in methanol, precipitation occurs；Being centrifugally separating to obtain precipitation, precipitation is dissolved in deionized water, spends
Ionized water is dialysed, and lyophilizing obtains rough mannan；
5) rough mannan is dissolved in deionized water, adds the aqueous solution of cetyl trimethylammonium bromide, and mixed liquor is static
There is precipitation in a period of time；
6) centrifugation precipitation, precipitate with deionized water is washed, and merges cleaning mixture and 5) step supernatant, amalgamation liquid adds 1wt%
Boric acid solution；
7) above-mentioned mixed liquor adds 2M sodium hydroxide while stirring, regulates pH to 8.6-9.0, generates cetyl trimethyl bromination
Ammonium-mannan complex compound sediment, collects precipitation, with the 0.5% sodium acetate washing of pH=8.8, then uses 2% acetic acid,
Gained solution precipitates in ethanol；
8) centrifugal, abandon supernatant, precipitate and wash with the ethanol acetate solution of 2%, neutralize with 2M sodium hydroxide after being dissolved in water, distillation
Water is dialysed, lyophilizing, obtain Candida albicans mannan product.
9. the preparation method of Candida mannan Mn as claimed in claim 7, it is characterised in that the described right that utilizes is wanted
The Candida mannan Mn immune affinity chromatographic column described in 6 is asked to carry out the purification of Candida mannan Mn, including walking as follows
Rapid:
(1) with the Candida mannan Mn immunity described in the wash buffer claim 6 identical with sample solution to be purified
Affinity column；
(2) sample solution to be purified is made to flow through the chromatographic column after step (1) processes；
(3) post is washed with combining buffer；
(4) post is washed with pre-elution buffer；
(5) use stepwise elution, flow through chromatographic column with elution buffer continuously, be in charge of each component of collection；
(6) detect the Mn content of often pipe, each pipe high for concentration is merged.
10. the preparation method of Candida mannan Mn as claimed in claim 9, it is characterised in that described in step (2)
The flow velocity of sample to be purified is 0.5-1.5mL/h, uses pump coutroi velocity, peristaltic pump is preferably used.
The preparation method of 11. Candida mannan Mn as claimed in claim 9, it is characterised in that described in step (3)
In conjunction with buffer one in PBS, Tris-HCl buffer, Acetic acid-sodium acetate buffer, preferably PBS buffers
Liquid, consumption is 10-25 times of bed volume；
And/or, the pre-elution buffer described in step (4) is carbonate buffer solution, and consumption is 10-25 times of bed volume；
And/or, the buffer that elution buffer is pH3.0 described in step (5), the 0.1M glycine buffer of preferably pH3.0
Liquid, the citrate-phosphate salt buffer of pH3.0, the citric acid-sodium citrate buffer of pH3.0, the Acetic acid-sodium acetate of pH3.0
Buffer, consumption is 0.4-0.8 bed volume.
12. 1 kinds of Candida mannan Mn, it is characterised in that described Candida mannan Mn is appointed by claim 7-11
The preparation method of one described Candida mannan Mn prepares.
13. 1 kinds of Candida mannan Mn as claimed in claim 12 answering in the early diagnosis reagent of monilial infection
With.
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Cited By (2)
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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 |
| CN113461832A (en) * | 2020-03-30 | 2021-10-01 | 湖南斯奇生物制药有限公司 | Bacillus calmette-guerin mannan, preparation method and application thereof |
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