CN108129558A - The extraction of main allergic protein Bet v 8 of birch pollen a kind of and isolation and purification method and application - Google Patents
The extraction of main allergic protein Bet v 8 of birch pollen a kind of and isolation and purification method and application Download PDFInfo
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- CN108129558A CN108129558A CN201711395712.8A CN201711395712A CN108129558A CN 108129558 A CN108129558 A CN 108129558A CN 201711395712 A CN201711395712 A CN 201711395712A CN 108129558 A CN108129558 A CN 108129558A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
Extraction and isolation and purification method and application the present invention provides a kind of main allergic protein Bet v 8 of birch pollen.The acetate buffer that the extracting method of the Bet v 8 includes the use of pH 4.5 extracts birch pollen allergic protein as extracting solution, the isolation and purification method includes first preparing birch pollen aqueous soluble protein extracting solution, it is then further isolated and purified by cation-exchange chromatography and Con A Ago-Gels 4B chromatographies, the final main allergic protein Bet v 8 for obtaining purifying.Extracting solution or purifying protein prepared by the present invention can be used for the detection of main allergic protein in patient serum sample, it can also be further prepared into Allergic skin test reagent or desensitization preparation simultaneously, there is extensive use in terms of the skin test positive rate of birch pollen and desensitization curative effect is improved.
Description
Technical field
Isolation and purification method and application the present invention relates to albumen, and in particular to the main allergic protein Bet v of birch pollen
8 isolation and purification method and application.
Background technology
Allergy is to caused by the immune response of usually harmless macromolecular (such as protein) substance as body.Industrialization
Population in country more than 25% increases with I types allergy and quantity in stable increase, and birch pollen disease is type i allergic reaction
Very common form.In China, birch pollen is one of northern area spring main pollen (Ye Shitai, climate in china
Pollen is investigated, and 1991).
The birch pollen allergic protein named at present has 7 kinds, respectively Bet v 1, Bet v 2, Bet v3, Bet v
4th, Bet v 6, Bet v 7, Bet v 8, wherein Bet v 1 are the main allergic proteins of birch pollen, the people in different regions
In group, Bet v 1 can be identified (Moverare R et al, Int Arch by 62-98% birch allergy patients serums
Allergy Immunol,2002,128(4):325-35；Sekerkova A et al,Int Arch Allergy
Immunol,2011,154(4):278-85), the more information about 1 anaphylactogens of Bet v, its isomery anaphylactogen and variant, in detail
See www.allergen.org.
However, from Ipsen etc. isolated from birch pollen molecular weight be 17kDa main allergic protein Bet v 1 with
Come, the research for birch pollen allergen protein, also mainly concentrate on Bet v 1, related its gene coded sequence, amino acid sequence
Row, protein three-dimensional structure and the cross reactivity between various homologous allergens there is now lot of documents report.It is but right
Other main allergic proteins are identified and isolated from purifying in birch pollen, do not provide specific report in the prior art.
Invention content
The main object of the present invention is to identify another main allergic protein Bet v 8 in birch pollen, and provide needle
Extraction and isolation and purification method to above-mentioned 8 albumen of Bet v.
The extracting method of 8 albumen of Bet v in the present invention, which is characterized in that including using pH as 4.5 acetate buffer
8 albumen of birch pollen Bet v is extracted as extracting solution；Preferably, the acetate buffer is sodium acetate.
The isolation and purification method of Bet v8 albumen in the present invention, which is characterized in that the isolation and purification method contain with
Lower step：
1) preparation of birch pollen aqueous soluble protein extracting solution；
2) cation-exchange chromatography, including by above-mentioned steps 1) prepared by birch pollen aqueous soluble protein extracting solution loading it
Afterwards, be rinsed first using combination buffer, subsequent secure bond buffer solution and gradually increase the concentration of elution buffer into
Row elution, collects the 1st eluting peak of eluent；Wherein combination buffer is the sodium-acetate buffer that pH is 4.5, elution buffer
For the pH sodium-acetate buffers for being 4.5 and NaCl solution.
3) Con A Ago-Gels 4B is chromatographed, including by above-mentioned steps 2) on the eluent containing destination protein for preparing
After sample, pillar first is rinsed using combination buffer, is then eluted using elution buffer, collect the 1st eluting peak of eluent；
Wherein the ingredient of combination buffer is Tris HCl, NaCl, MnCl2, CaCl2, pH 7.4；The ingredient of elution buffer is：First
Base-α-D- glucopyranosides, Tris-HCl, NaCl, pH 7.4.
Further, this field routine may be used in the preparation of the birch pollen aqueous soluble protein extracting solution involved in the present invention
Method extract, it is preferable that the present invention use pH be 4.5 acetate buffers as extracting solution to birch pollen water-soluble egg
It extracts in vain.
Further, the present invention involved in cation-exchange chromatography the specific steps are：It is slow including 10mM is first used to combine
Fliud flushing balances pillar, then will carry out loading after prepared pollen aqueous soluble protein extracting solution concentration, is continuing with above-mentioned knot
Wash buffer about 20mL is closed, subsequent secure bond buffer solution gradually increases the concentration of elution buffer to 50%, rinses three
Column volume is collected simultaneously the 1st eluting peak of eluent；Wherein combination buffer is the 20mM sodium-acetate buffers that pH is 4.5, is washed
De- buffer solution is the 20mM sodium-acetate buffers and 1M NaCl solutions that pH is 4.5.
Further, the present invention involved in Con A Ago-Gels 4B chromatography the specific steps are：It is first combined with 5mL slow
Fliud flushing balances pillar, then loading after the eluent concentration that cation-exchange chromatography is collected is rushed using 5mL combination buffers
Pillar is washed, finally elutes pillar with elution buffer, collects the 1st eluting peak of eluent；Wherein, combination buffer is：20mM
Tris HCl,0.5M NaCl,1mM MnCl2,1mM CaCl2,pH 7.4；Elution buffer is：0.1M methyl-α-D- pyrans Portugal
Glucosides, 20mM Tris-HCl, 0.5M NaCl, pH 7.4.
Further, it in main 8 isolation and purification methods of allergic protein Bet v of the invention, further comprises to described
8 Identification of Fusion Protein steps of Bet v, it is preferable that pass through Bet v8 described in SDS-PAGE, isoelectric focusing electrophoresis or western blotting qualification
Albumen.
The present invention also provides a kind of allergen detection in vitro method, include the use of above-mentioned 8 protein extracts of Bet v or
8 albumen of Bet v isolated and purified is detected blood serum sample.
The present invention also provides the applications by above-mentioned Bet v8 albumen in Allergic skin test reagent is prepared, it is preferable that mistake
Quick original detection reagent is anaphylactogen solution for skin test.
The present invention also provides the applications by above-mentioned 8 albumen of Bet v in desensitizing agent is prepared.
Beneficial effects of the present invention：
(1) using birch pollen aqueous soluble protein as substrate, birch pollen disease patients serum is carried out by western blotting method
Analysis finds that the albumen that a molecular weight is about 70kDa is up to 51% with SERUM IgE positive reaction rate, meets main cause for the first time
Quick albumen defines standard.
(2) isolation and purification method of main allergic protein provided by the invention, by improveing pollen protein extracting solution ingredient,
The content of main allergic protein Bet v 8 in natural birch pollen crude extract can be significantly improved, then passes through cation exchange
Chromatography and Con A Ago-Gels 4B chromatographies for the first time isolate and purify destination protein in birch pollen crude extract.
(3) in the allergen detection in vitro method-ImmonoCAP used in clinic at present and not comprising Bet v 8, sheet
Invention finds its higher IgE positive reaction rate for the first time, helps to be applied to prepare by the crude extract of the albumen or purifying protein
Anaphylactogen solution for skin test or desensitization preparation can improve the skin test positive rate of birch pollen and desensitization curative effect.In addition, the Bet v of purifying
8 albumen are expected to the outer detection reagent of exploitation adult.
Description of the drawings
Main allergic protein (about 70kDa) content balance in birch pollen crude extract in two kinds of extracting methods of Fig. 1.Wherein,
L1：Using phosphate buffer (pH 7.5) as extracting solution；L2：Using acetate buffer (pH 4.5) as extracting solution.
Fig. 2 birch pollen crude extract immunoblot results.Wherein, Fig. 2 a, Fig. 2 b, Fig. 2 c are respectively to use different birches
Pollinosis disease patients serum (L1 to L41 be different blood serum samples) carry out immunoblotting as a result, substrate is to be delayed with acetate
The birch pollen crude extract of fliud flushing extraction.
Fig. 3 cation-exchange chromatography results.Arrow meaning is the collection peak containing destination protein；M：Albumen marker；L1-
12：The visible apparent 70kDa protein bands (i.e. destination protein) of 2-13 collection liquids, wherein 4-10 collection liquids SDS-PAGE.
Fig. 4 .HiTrapTMThe separating resulting of Con A 4B chromatographies.Arrow meaning is the collection peak containing destination protein；M：Egg
White marker；flow:Uncollected eluent；1-14 is 1-14 collection liquids, and wherein 5-14 collection liquids SDS-PAGE is visible
The 70kDa protein bands (i.e. destination protein) of purification.
Fig. 5 purifying proteins immunoblottings and Inhibition test.L1-2：Washing rich in 70kDa albumen in cation-exchange chromatography
De- liquid SDS-PAGE；L3-4：HiTrapTMCon A Ago-Gel 4B eluents SDS-PAGE；L5：Using purifying protein the bottom of as
Object, birch pollen allergic patients sera carry out immunoblotting for primary antibody (serum used in L29 in Fig. 2)；L6：It is slightly carried with birch pollen
Liquid is substrate, and birch pollen allergic patients sera carries out immunoblotting for primary antibody；L7：Using birch pollen crude extract as substrate, birch
It is primary antibody to set pollen allergic patients' serum, isolates and purifies albumen as mortifier, carries out immunoblotting Inhibition test, egg after purification
70kDa bands and seroreaction can partly be inhibited in vain (70kDa bands die down compared with the band in L6 in L7).Wherein horizontal line mark portion
It is divided into 70kDa albumen (i.e. destination protein).
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
Unless otherwise instructed, the operating method employed in the present invention refers to《Gene molecule biology, James D Watson, the 6th
Version》.
The extraction of main allergic protein (about 70kDa) is rich in 1 birch pollen of embodiment
1.1 pollen raw material
Chinese birch pollen is acquired to be acquired in birch pollen season in April, 2014 in Inner Mongol Chifeng forest of white birch after -80
DEG C freezen protective.
1.2 experimental procedure
1.2.1 Birch pollen 2g is dissolved in 20mL 20mM sodium acetates (pH 4.5), is added in 250 μ L compound proteases and is inhibited
Agent, vortex oscillation 1 minute, 4 DEG C of shaken overnights；At 4 DEG C, 6000g centrifugation pollen suspension 30 minutes stays supernatant, with 0.2 μm
Filter filters supernatant, and 3kDa Millipore super filter tubes are concentrated into 5mL.- 20 DEG C of preservations.
1.2.2 using protein concentration in BCA kits detection birch pollen protein extract
Reagent A in BCA kits and reagent B are pressed 50:1 ratio mixing, is made into BCA working solutions.Take each dilution
Each 25 μ L of Protein standards and sample to be tested of concentration, are added in 96 orifice plates, add in 200 μ L BCA work per hole later
Liquid, gently horizontal concussion, is allowed to mixing.By 96 pore plate by sealing, it is incubated 30 minutes in 37 DEG C of incubators.Sample is measured using microplate reader
Light absorption value at 562nm.By standard items and the OD of sample to be tested562Value subtracts the OD of zero standard product562Value, uses standard items
OD562Standard curve is drawn to its concentration (μ g/mL), equation and linearly dependent coefficient is obtained, brings sample to be tested OD into562It can count
Calculate its protein concentration.
1.2.3 compared with the main allergic protein extracting method of conventional birch pollen
Our common pollen aqueous soluble protein Extraction buffers are the phosphate buffer that pH is 7.5 at present, by above-mentioned step
Sodium-acetate buffer in rapid 1.2.1 replaces with phosphate buffer (pH 7.5), other steps are identical, pass through SDS-PAGE pairs
Than the content of allergic protein main in two kinds of Different Extraction Methods.
1.3 result
Main allergic protein content balance is shown in Fig. 1 in birch crude extract in two kinds of extracting methods, uses traditional phosphate
When buffer solution (pH 7.5) extracts, 1 content highests (17kDa) of Bet v in crude extract, and another main allergic protein
(Bet v 8, about 70kDa) less (band 1), and the present invention utilizes vinegar by adjusting the ingredient and acid-base value of Extraction buffer
Phthalate buffer (pH 4.5) is extracted as extracting solution, and higher Bet v 1 (17kDa) are not only contained in crude extract,
The content (band 2) of main allergic protein Bet v 8 can also be significantly improved.
2 birch pollen aqueous soluble protein immunoblotting (Western blot) of embodiment
(what in embodiment 1 prepared by 1.2.1 steps is thick for the 2.1 birch pollen protein crude extracts extracted with acetate buffer
Extract) carry out SDS-PAGE electrophoresis：Operation is the same.After electrophoresis, transferring film " sandwich " is assembled, is sequentially followed successively by cathode -2
Layer filter paper-separation gel-nylon membrane -2 layer filter paper-anode, nylon membrane is transferred to half-dried transferring film method by albumen from gel
On.Transferring film buffer solution is 250mM Tris pure, 1.92M glycine.Nylon membrane is dyed using Ponceau S, according to dye
Color is as a result, cutting nylon membrane and numbering, for being incubated from different serum.Nylon membrane slitting is put into and is incubated in box, is added in
Confining liquid is placed on side-sway shaking table, and room temperature is closed 1 hour.Confining liquid is 5% milk.After closing, washed and cut with TBST
Item 3 times.Birch pollen disease patient and normal healthy controls serum are pressed 1:10 dilutions, slitting are respectively placed in incubation slot, are added in per slot
1ml dilute serums.Incubation slot is closed, is placed on side-sway shaking table, 4 DEG C overnight.It is cut 3 times using TBST washings afterwards.1:1000 is dilute
The sheep antihuman IgE antibody of HRP labels is released, 1ml dilution secondary antibodies are entered per slot glue, is placed on side-sway shaking table, is incubated at room temperature 1 hour.Afterwards
It is cut 3 times using TBST washings.ECL develops the color.
2.2 result
Birch pollen crude extract immunoblot results are shown in that Fig. 2, wherein L1-L41 are different blood serum sample immunoblot results.
The birch pollen crude extract extracted using acetate buffer carries out immunoblotting point in 41 parts of birch pollen disease patients serums
As a result analysis shows that the serum of 58.5% (n=24) can be combined with Bet v 1 (17kDa bands), the blood of 51.2% (n=21)
The standard for defining main allergic protein can be reached with 70kDa protein bindings, the serum discrimination of 70kDa albumen clearly.
3 cation-exchange chromatography of embodiment
3.1 solution are prepared
3.1.1Cation A：20mM sodium acetates, 4.5,0.2 μm of filter paper filtering solution of pH, room temperature preservation.
3.1.2Cation B：4.5,0.2 μm of 20mM sodium acetates, 1M NaCl, pH filter paper filtering solution, room temperature preservation.
It is prepared by 3.2 protein samples：The birch for being prepared step 1.2.1 in embodiment 1 using 3kDa Millipore super filter tubes
It sets pollen crude extract and carries out 3 times of concentrations, it is spare.
3.3 dress columns：Pillar is cleaned with Milli-Q water using preceding.Pillar is assembled, is fixed on tripod, and even
It is connected on peristaltic pump.All pipelines are cleaned with Milli-Q water first, and check whether there is leakage simultaneously, later, are added into pillar
Enter the Milli-Q water of 1/3 volume, then pour into about 3mL cation-exchangers (SP-sepherose), fill it up with pillar.
3.4 loadings, elution and collection：It is pumped with Milli-Q water cleaning system, is then connected to the pillar assembled first
On AKTA FPLC instruments, then cleaning sample pump.Then pillar is balanced with 10ml Cation-A.Loading is pumped by sample, is continued
About 20ml is rinsed using Cation-A.Fixed Cation-A, gradually increases Cation-B concentration to 50%.Rinse 3 column volumes.
It is collected simultaneously eluent.Different eluents progress SDS-PAGE electrophoresis is selected to analyze Protein Separation effect according to peak is collected.
3.5 result
Cation-exchange chromatography and SDS-PAGE electrophoresis results are shown in Fig. 3, and wherein arrow meaning is the collection containing destination protein
Peak；2-13 collection liquids is selected to carry out SDS-PAGE analyses, the visible apparent 70kDa albumen of wherein 4-10 collection liquids SDS-PAGE
Band (i.e. destination protein).
Embodiment 4HiTrapTMCon A Ago-Gels 4B is chromatographed
4.1 solution are prepared：Corresponding solution is prepared according to prepacked column operating instruction, it is specific as follows：
4.1.1Binding buffer:20mM Tris HCl,0.5M NaCl,1mM MnCl2,1mM CaCl2,pH
7.4。
4.1.2Elution buffer:0.1M methyl-α-D-glucopyranosides, 20mM Tris-HCl, 0.5M NaCl,
pH 7.4。
4.2 preparation of samples：One will be mixed into through the isolated eluent containing destination protein of cation-exchange chromatography
It rises, is dialysed 12 hours using 1000Da bag filters, Binding buffer are during which replaced for several times, with balance sample.Use 10kDa
Super filter tube concentrates its 5 times.
4.3 loadings, elution and collection：It is pumped respectively with Milli-Q water cleaning system first, the solvent resistant column of prepackage is pacified
It is attached on AKTA FPLC instruments.Then pillar is rinsed with Milli-Q water respectively, is balanced afterwards using 5ml Binding buffer
Pillar.Loading is pumped by sample.Pillar is rinsed using 5ml Binding buffer, rear Elution buffer elute pillar,
Collect eluent.According to collection peak, it would be possible to which the eluent for also having purpose band carries out SDS-PAGE electrophoresis and Isoelectric Focusing
Swimming.
4.4 result
HiTrapTMCon A Ago-Gels 4B is chromatographed and SDS-PAGE results are shown in Fig. 4, and wherein arrow meaning is containing purpose
The collection peak of albumen；M：Albumen marker；flow:Uncollected eluent；1-14 is 1-14 collection liquids, and wherein No. 5-14 is received
The 70kDa protein bands (i.e. destination protein) of the visible purifications of liquid collecting SDS-PAGE
5 immunoblotting Inhibition test of embodiment
The present invention also further verifies separated purify in obtained albumen and embodiment 2 by immunoblotting Inhibition test
The albumen identified by birch patients serum is same protein.
Major experimental step with embodiment 2 immunoblotting, unique difference for patients serum in advance respectively with implementation
The obtained albumen isolated and purified is incubated 1 hour under room temperature in example 4, a concentration of 100 μ g/mL of mortifier.
Immunoblotting inhibition assay result is as shown in figure 5, from fig. 5, it can be seen that birch pollen extracting solution is through ion exchange
A protein band (L1, L2) for being rich in 70kDa can be obtained after chromatographic purifying, in eluent；The eluent is again through HiTrapTM
Con A Ago-Gel 4B chromatographies purify, and single goal protein band (L3, L4), and the purifying can be obtained in eluent
Albumen can specifically bind (L5) with birch pollen allergic patients sera and emulative can inhibit in birch pollen
The albumen of 70kDa and above-mentioned seroreaction (L6, L7), it is identical albumen to prompt the two.
6 isoelectric focusing electrophoresis of embodiment and immunoblotting
6.1 solution are prepared
6.1.1IEF sample buffer：7M urea, 2M thiocarbamides, 2%CHAPS, 0.5%IPG buffer pH 3-7, bromine phenol
0.002%, 65mM of indigo plant DTT.
6.1.2SDS-PAGE equilibration buffer：50mM Tris-HCl (pH 8.8), 6M urea, 2%SDS, 30% glycerine,
0.0002% bromophenol blue adds Milli-Q water to be settled to 400ml.By two parts of storing liquid average out to, respectively I He of equilibration buffer
Ⅱ.Equilibration buffer I uses preceding addition 65mM DTT, matching while using.5g iodohydrin amine (25mg/ is added in equilibration buffer II
mL).Then -20 DEG C of preservations are dispensed.
6.1.3 agarose confining liquid：0.25g agaroses are weighed, 100 μ L bromophenol blue concentrates is added in, is dissolved in 50mL SDS-
In PAGE electrophoretic buffers, dissolve by heating, when being cooled to 60 DEG C or so, packing, room temperature preservation.
6.2 second prepare to gel：PAGE gel separation gel is prepared, selects the glass plate of 1.5 cm thicks, separation
Remaining plate height is made to be about 0.8 centimetre or so after glue perfusion, for placing IPG adhesive tape.It does not need to perfusion concentration glue and is inserted into comb.
6.3 isoelectric focusing sample preparations：By HiTrapTMThe isolated eluents containing destination protein of Con A 4B make
It is dialysed with 1000Da bag filters, during which repeatedly replaces Milli-Q water, with abundant desalination.It is dense that BCA measures albumen in eluent
Degree.150mg albumen is calculated according to protein concentration and corresponds to extracting liquid volume, adds in the acetone of -20 DEG C of precoolings of 3 times of volumes to precipitate
Albumen, and demineralization can be played, -20 DEG C are placed 3 hours.After taking-up, 15000g, 4 DEG C centrifuge 30 minutes, visible albumen at this time
Precipitation and tube bottom.Acetone is abandoned, precipitation is washed 3 times with the acetone of 50 μ L 95%, is placed in draught cupboard, treats that acetone volatilization is complete.It will
The IEF sample-loading buffers frozen thaw at room temperature, add in 1M DTT (65 μ l/mL) at this time.It will with 125 μ L IEF sample-loading buffers
Albumen precipitation is resuspended, and is subsequently placed into heating oscillator, 22 DEG C, middling speed concussion, 15 minutes.10000g Centrifuge A samples 5 minutes.
6.4 loadings and first are to electrophoresis
6.4.1 the prefabricated adhesive tape of IPG (pH3-10) by -20 DEG C of preservations is placed 10 minutes under room temperature.Pipettor is by sample
Product add to isoelectric focusing slot center.The protective film of the prefabricated adhesive tape of IPG is removed with tweezers, glue surface is downward, and positive terminal first is put into glue
It in slot, gently pushes, is finally that negative pole end is also placed in isoelectric focusing slot.400 μ L are slowly added dropwise above adhesive tape
DryStrip.Glue groove lid is covered, is put it into isoelectric focusing electrophoresis instrument.
6.4.2, electrophoretic parameters are set：Temperature -19 DEG C, maximum current -0.05mA/ items, adhesive tape length -7 centimetre, 30V
13 hours, 200V 1 hour, 500V 1 hour, 500 to 8000V 30 minutes, 8000V 2 hours.
6.5 balance adhesive tape：The equilibration buffer I and II of -20 DEG C of preservations is taken out, is dissolved at room temperature.Into equilibration buffer I
Add in 65mM DTT.When the 1st to electrophoresis after, take out adhesive tape, be put into 15mL centrifuge tubes, add in 5mL equilibration buffers I
In, room temperature rolls to be balanced 15 minutes in concussion instrument.It is then placed in containing in II 15mL centrifuge tubes of 5mL equilibration buffers, room temperature is rolled
It is balanced 15 minutes on wavy instrument.
6.6 dissolve by heating agarose sealing fluid.After adhesive tape balances, quick wash 1-2 seconds in Milli-Q water,
It is placed on filter paper, to remove surplus liquid.Adhesive tape is put into PAGE gel upper surface with tweezers, by 5 μ L albumen
Marker is added on filter paper, and filter paper is inserted into along one end between glass plate.Then the agarose melted is sealed with pipettor
Liquid is added to adhesive tape surface.After agarose solidification, appropriate electrophoretic buffer is added in into electrophoresis tank, starts electrophoresis.Initial voltage
80V is set as, when Bromophenol Blue dye moves down 1 centimetre or so, voltage is adjusted to 150V.About 50 minutes or so, electrophoresis
Terminate.
6.7 immunoblotting：Gel is removed after isoelectric focusing, carries out immunoblotting, method is the same.Suffered from using birch pollen disease
Whether person's Virus monitory protein isolate is purpose albumen.
6.8 further verify through immunoblot experiment, and the present invention is obtained through ion exchange series of strata and gel permeation chromatography separation
The single protein band of molecular weight, for the destination protein with the IgE positives.
Claims (10)
1. a kind of extracting method of the main allergic protein Bet v 8 of birch pollen, which is characterized in that the method includes using pH
Acetate buffer for 4.5 extracts birch pollen allergic protein as extracting solution.
2. extracting method as described in claim 1, which is characterized in that the acetate buffer is sodium acetate.
3. a kind of isolation and purification method of the main allergic protein Bet v 8 of birch pollen, which is characterized in that the method includes such as
Lower step：
1) preparation of birch pollen aqueous soluble protein extracting solution；
2) cation-exchange chromatography, including by above-mentioned steps 1) prepared by birch pollen aqueous soluble protein extracting solution loading after,
It is rinsed first using combination buffer, subsequent secure bond buffer solution simultaneously gradually increases the concentration of elution buffer and washed
It is de-, collect eluent；Wherein combination buffer is the sodium-acetate buffer that pH is 4.5, and elution buffer is the acetic acid that pH is 4.5
Sodium buffer solution and NaCl solution.
3) Con A Ago-Gels 4B is chromatographed, including by above-mentioned steps 2) the eluent loading containing destination protein for preparing it
Afterwards, pillar first is rinsed using combination buffer, is then eluted using elution buffer, collect eluent；Wherein combination buffer
Ingredient be Tris HCl, NaCl, MnCl2, CaCl2, pH 7.4；The ingredient of elution buffer is：Methyl-α-D- glucopyranoses
Glycosides, Tris-HCl, NaCl, pH 7.4.
4. isolation and purification method as claimed in claim 3, which is characterized in that the 20mM that pH is 4.5 is used in the step 1)
Acetate buffer extracts birch pollen aqueous soluble protein as extracting solution；
Cation-exchange chromatography concrete operations is first sub using 10mM binding buffer balance columns in the step 2), then by institute
Loading is carried out after the pollen aqueous soluble protein extracting solution concentration of preparation, above-mentioned combination buffer is continuing with and rinses about 20mL, with
Secure bond buffer solution afterwards gradually increases the concentration of elution buffer to 50%, rinses three column volumes, be collected simultaneously elution
Liquid；Wherein combination buffer is the 20mM sodium-acetate buffers that pH is 4.5, and elution buffer is the 20mM sodium acetates that pH is 4.5
Buffer solution and 1M NaCl solutions；
Con A Ago-Gels 4B chromatographs concrete operations to be first sub with 5mL binding buffer balance columns in the step 3), will be positive
Loading after the eluent concentration that ion-exchange chromatography is collected, then rinses pillar using 5mL combination buffers, finally with elution
Buffer solution elutes pillar, collects eluent；Wherein, combination buffer is：20mM Tris HCl,0.5M NaCl,1mM
MnCl2,1mM CaCl2,pH 7.4；Elution buffer is：0.1M methyl-α-D-glucopyranosides, 20mM Tris-HCl, 0.5M
NaCl,pH 7.4。
5. main 8 extracting solutions of allergic protein Bet v of birch pollen prepared using the extracting method described in claims 1 or 22.
6. the main allergic protein Bet v 8 of birch pollen prepared using the isolation and purification method described in claim 3 or 4.
7. a kind of allergen detection in vitro method includes the use of main 8 extracting solutions of allergic protein Bet v described in claim 5
Or the main allergic protein Bet v 8 described in claim 6 are detected blood serum sample.
8. the main allergic protein described in main 8 extracting solutions of allergic protein Bet v or claim 6 described in claim 5
Applications of the Bet v 8 in Allergic skin test reagent is prepared.
9. application according to claim 8, which is characterized in that the Allergic skin test reagent is anaphylactogen solution for skin test.
10. applications of the main allergic protein Bet v 8 in desensitizing agent is prepared described in claim 6.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711395712.8A CN108129558B (en) | 2017-12-21 | 2017-12-21 | Extraction, separation and purification method and application of main allergenic protein Bet v8 of birch pollen |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711395712.8A CN108129558B (en) | 2017-12-21 | 2017-12-21 | Extraction, separation and purification method and application of main allergenic protein Bet v8 of birch pollen |
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| CN108129558A true CN108129558A (en) | 2018-06-08 |
| CN108129558B CN108129558B (en) | 2021-10-12 |
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