CN103113455A - Preparation and application of hemagglutinin peptide mark recombinant protein immunoaffinity purification enriching column - Google Patents
Preparation and application of hemagglutinin peptide mark recombinant protein immunoaffinity purification enriching column Download PDFInfo
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- CN103113455A CN103113455A CN2012104815430A CN201210481543A CN103113455A CN 103113455 A CN103113455 A CN 103113455A CN 2012104815430 A CN2012104815430 A CN 2012104815430A CN 201210481543 A CN201210481543 A CN 201210481543A CN 103113455 A CN103113455 A CN 103113455A
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Abstract
The invention provides a high-efficiency specificity enriched hemagglutinin peptide HA mark recombinant protein immunoaffinity purification enriching column as well as a preparation method and an application thereof. The immunoaffinity purification enriching column provided by the invention comprises an activated agarose filler coupled with HA specificity polyclonal antibody which is subjected to immunoaffinity purification and a plastic column carrying the immunoaffinity filler. The immunoaffinity purification filler coupled HA specificity polyclonal antibody is extracted by using an immunoaffinity method; and the immunoaffinity purification enrichment column is obtained by carrying the immunoaffinity filler into a specific plastic column. The immunoaffinity purification enrichment column enriched HA mark recombinant protein is high in efficiency, strong in specificity and temperate in elution condition, can be repeatedly utilized and is an ideal material for separating, purifying and enriching the HA mark recombinant protein at present.
Description
Technical field
The present invention relates to a kind of bioseparation, purification enrichment device, preparation method and the application thereof of the immunoaffinity purification enriching column of especially a kind of high efficiency separation purifying, enrichment hemagglutinin peptide HA label recombinant protein.
Background technology
Molecular biology and proteomics are the Hot subjects in life science field, and the use of recombinant protein is increasing in recent years greatly.For convenience of mark, tracking, evaluation and the purifying of gene recombinant protein, can artificially introduce a segment mark label polypeptide and form recombinant protein in target protein is expressed.The technology of therefore separation and purification, enrichment recombinant protein more and more shows its importance.Separation and purification from the expression system of complicated, enrichment target protein are an arduousness and heavy task, and affinity chromatography is the method for separation and purification the most widely at present, enrichment recombinant protein.
Owing at present mainly containing the affine post of metal-chelating for separating of the affinity column of purifying, enrichment HA recombinant protein and utilizing the substrate, reactive fuel of enzyme to be the affinity column of aglucon, there are the remarkable shortcomings such as non-specific adsorption, elution requirement is violent, refining effect is not good in these affinity columns, and the affine post of metal-chelating also exists metal ion to be shed to defective in elutriant, and these all will limit separating-purifying target protein out follow-up application, detection.
Summary of the invention
The purpose of this invention is to provide immunoaffinity purification enriching column preparation method and the application thereof of a kind of efficient, specific isolation purifying, enrichment HA label recombinant protein.
For reaching above-mentioned purpose, the present invention adopts specific reaction and the dissociable characteristic Ag+Ab (solid phase) of antigen-antibody for 2 times
Ag-Ab (solid phase) principle, specific as follows:
At first utilize this principle that HA small peptide hapten conjugation is made HA-Agarose affinity column on Agarose, be used for extracting the HA specific polyclonal antibody.
Recycle this principle and will extract the anti-HA specific antibody Anti-HA of gained and be coupled to and make Anti-HA-Agarose immune affinity column on Agarose, for separating of the HA label recombinant protein in purifying, enrichment of cell lysate.
According to above-mentioned mechanism, the present invention adopts following technical scheme:
A kind of preparation method of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column is characterized in that:
1) the chemical coupling reaction of the hemagglutinin peptide HA specific polyclonal antibody of immunoaffinity purification being adopted streaming cross post is coupled on the agarose Agarose of sodium periodate oxidation, use sodium borohydride reduction, clean unconjugated antibody with distilled water, clean filler with phosphoric acid buffer PBS, 10 times of packing volumes/time, wash 4 times, add equal-volume glycerine after drip-dry PBS, mixing namely gets the affine filler of HA label recombinant protein immunity;
2) the affine filler of HA label recombinant protein immunity is packed in plastic column, volume dress post on demand namely gets described HA label recombinant protein immunoaffinity purification enriching column.
Made HA label recombinant protein immunoaffinity purification enriching column comprise coupling have immunoaffinity purification the HA specific polyclonal antibody the agarose filler and load the plastic column of the affine filler of this immunity.
The HA specific polyclonal antibody of described immunoaffinity purification is to make the immunogen immune animal with the carrier proteins that the coupling of HA small peptide activates, obtain containing antibody serum, to contain on antibody serum to coupling the immune affinity chromatographic column of HA small peptide will be arranged, clean unconjugated impurity, with HA specific polyclonal antibody under the weak acid wash-out, desalination, freeze-drying are standby.
Described HA small peptide is the small peptide that tyrosyl-prolyl-tyrosyl-aspartoyl-valyl-prolyl-aspartoyl-tyrosyl-L-Ala forms by 9 amino acid, adopt these 9 amino-acid residues to be divided into 2 groups and introduce in the opposite direction the HA small peptide that cysteine sulfydryl SH group makes sulfhydrylation, its sequence is: 1) cysteinyl-valyl-prolyl-aspartoyl-tyrosyl-L-Ala is C-VPDYA, 2) and be tyrosyl-prolyl-tyrosyl-aspartoyl-halfcystine YPYD-C.See sequence table.
The immunogen that the carrier proteins of described HA small peptide coupling activation makes; with common carrier albumen such as sodium laurylsulfonate SDS sex change hemocyanin KLH or ovalbumin OVA; be that iodacetyl-NHS reaction generates iodacetyl KLH or OVA with iodacetyl-hydroxysuccinimide eater again; after removing excessive iodoacetic acid by sephadex G 25 Sephadex desalinations; sulfhydrylation HA small peptide is mixed with KLH or the OVA of iodacetyl; transfer pH=8.5; shake reaction 60 minutes in room temperature, what obtain after G25 Sephadex desalination is immunogen.
Described coupling has the immune affinity chromatographic column of HA small peptide; the HA small peptide haptens of 2 kinds of opposite direction sulfhydrylations and the Agarose of iodacetyl are reacted; form chemical covalent linkage complex body filler HA-Agarose, HA-Agarose is filled post namely get the immune affinity chromatographic column that coupling has the HA small peptide.
A kind of application of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column is characterized in that: the HA specific polyclonal antibody coupling of the immunoaffinity purification of immunoaffinity purification enriching column and use horseradish peroxidase HRP mark.
Advantage of the present invention:
The prepared immunoaffinity purification enriching column of the present invention has following significant advantage:
(1) 4-5 the amino acid whose HA small peptide that only have that adopts as the HA specific polyclonal antibody of aglucon is that the immunogen stimulating animal produces, and antibody is stronger to the HA label protein recognition capability in expression system; And antibody adopts immune-affinity chromatography to purify, purification gained antibodies specific is high, make antibody and sepharose 4B coupling density large, the coupling rate is high, obviously improves with the HA label recombinant protein efficient of the affine filler recognition expression of the prepared immunity of the antibody of this high specific system;
(2) adopt the Agarose of sodium periodate activation, make antibody and Agarose form stable chemical bond, the antibody that is coupled on filler is not easy to come off, and can more accurately separate, enrichment HA label recombinant protein;
(3) horseradish peroxidase HRP on the HA specific polyclonal antibody mark of the immunoaffinity purification of gained is obtained Anti-HA HRP, directly to be used for detecting the concentration effect of immunoaffinity purification enriching column more accurate for the mark primary antibodie; This immunoaffinity purification enriching column elution requirement is gentle simultaneously, and the energy Reusability reduces costs.Present separating-purifying, the desirable material of enrichment HA label recombinant protein.
Description of drawings
Fig. 1 detects HA label recombinant protein immunoaffinity purification enriching column absorption target protein figure with immuno-precipitation.In figure: 1 is molecular weight marker;
25 times of diluents of bacterial lysate stoste supernatant, the 10 μ L for restructuring HA label protein;
The 3 filtrate 10 μ Ls of 5 times of diluents of bacteria lysis stoste supernatant after the enrichment of HA label recombinant protein immunoaffinity purification enriching column for restructuring HA label protein;
The 4 elutriant 5 μ Ls of 5 times of diluents of bacteria lysis stoste supernatant after the enrichment of HA label recombinant protein immunoaffinity purification enriching column for restructuring HA label protein;
The 5 elutriant 10 μ Ls of 5 times of diluents of bacteria lysis stoste supernatant after the enrichment of HA label recombinant protein immunoaffinity purification enriching column post for restructuring HA label protein.
Accompanying drawing 1 illustrates HA label recombinant protein immunoaffinity purification enriching column can identify HA label protein in the bacterial lysate of restructuring HA label protein, and can be with the absorption of HA label protein, and the HA label protein that is adsorbed on filler can be used the weak acid wash-out.
Accompanying drawing 2 is to detect HA label recombinant protein immunoaffinity purification enriching column concentration effect figure with immuno-precipitation.In figure:
1 is molecular weight marker;
2 50 times of diluents of bacterial lysate stoste supernatant, the 10 μ L for restructuring HA label protein;
The 3 filtrate 10 μ Ls of 50 times of diluents of bacterial lysate stoste supernatant after HA label recombinant protein immunoaffinity purification enriching column enrichment HA for restructuring HA label protein;
The 4 elutriant 10 μ Ls of 50 times of diluents of bacterial lysate stoste supernatant after the enrichment of HA label recombinant protein immunoaffinity purification enriching column for restructuring HA label protein.
Wherein 4 roads, the left side do not add the inhibition of HA polypeptide, and 4 roads, the right add the HA polypeptide to suppress.
Accompanying drawing 2 illustrate HA label recombinant protein immunoaffinity purification enriching column can be from contain the very low bacterial lysate of restructuring HA label protein enrichment HA label protein.Can suppress fully with the HA polypeptide, illustrate that the immune marking signal that obtains is the HA specific signals.
Accompanying drawing 3 is for using HA label recombinant protein immunoaffinity purification enriching column and Anti-HA-HRP coupling design sketch.In figure:
:
The NZHUS art.1 is molecular weight marker;
The 2 elutriant 5 μ Ls of 10 times of diluents of bacteria lysis stoste supernatant after the enrichment of HA label recombinant protein immunoaffinity purification enriching column for restructuring HA label protein
The 3 elutriant 15 μ Ls of 10 times of diluents of bacteria lysis stoste supernatant after the enrichment of HA label recombinant protein immunoaffinity purification enriching column for restructuring HA label protein
Annotate: the bacterial lysate of accompanying drawing 1, accompanying drawing 2, accompanying drawing 3 experiment restructuring used HA label proteins is from same pipe, available from Wuhan Sanying Bio-Technology Co., Ltd..
Embodiment
It is in order further to understand better the present invention that following embodiment is provided, and never content of the present invention and protection domain is consisted of any restriction.
The preparation of the HA specific polyclonal antibody of embodiment 1 immunoaffinity purification
1.1 HA small peptide hapten design
Employing is the small peptide that tyrosyl-prolyl-tyrosyl-aspartoyl-valyl-prolyl-aspartoyl-tyrosyl-L-Ala forms by 9 amino-acid residues, is divided into 2 groups with these 9 amino-acid residues and introduces in the opposite direction the HA small peptide that cysteine sulfydryl SH group makes sulfhydrylation.
1.2 HA small peptide immunogen preparation
Get 5 mg/mL KLH 10 mL, add the 100 saturated Na of μ L
2CO
3Boil sex change in 5 minutes after adding again the SDS mixing of 1 mL 10%; generate iodacetyl KLH with iodacetyl-NHS reaction again; after removing excessive iodoacetic acid by G25 Sephadex; the HA small peptide haptens that adds 55 mg is transferred pH=8.5, and room temperature reaction is after 60 minutes; obtain HA-KLH covalent linkage complex body, what obtain after G25 Sephadex desalination is immunizing antigen.Using OVA instead is that carrier proteins prepares detection envelope antigen HA-OVA. with same method
1.3 polyclonal antiserum preparation: be immune animal with new zealand white rabbit, vaccine concentration is 1mg/mL, first immunisation adds 1mL Freund's complete adjuvant mixing multi-point injection with the 0.5mL vaccine, the each every rabbit of booster immunization adds 0.75mLPBS with the 0.25mL vaccine and adds 1mL Freunds incomplete adjuvant mixing and divide 2 injections, after first immunisation, at interval of 2 all booster immunizations once, gathered serum detection titre in 35-40 days, serum titer carries out the preparation of productivity serum after reaching 1:6400.
1.4 HA small peptide-Agarose affinity column preparation.
Be dissolved in 10 mL 0.1 mol/L Na 1.4.1 get 2 g Methionins
2CO
3,Fully join 20 mL after dissolving with mixing in the Agarose of sodium periodate activation, 60 ℃ were reacted 5 minutes, and shook up and again react 10 minutes, turn 4 ℃ standing 20 minutes, then add sodium borohydride 100 mg mixings, shaken over night on the room temperature shaking table obtains amino-agarose 20 ml.
1.4.2 amino-agarose of 20 ml is transferred to void column of 50ml cleaning, cleans for 5 times with 500ml distillation moisture.Drain water standby.
1.4.3 clean amino-agarose with cold acetone, then clean with the 25ml acetone soln that contains the 0.5ml triethylamine, also surplus 1/3 during on the filler upper strata, repeatedly shake resuspended amino-agarose when solution.
1.4.4 with the iodoacetic acid of 1 ml dimethyl fumarate DMF dissolving 75 mg NHS activation, thoroughly after dissolving, 1000 rev/mins centrifugal 1 minute, get supernatant and dropwise add the resuspended good amino-agarose of 1.4.3 while vibrate, cover tightly, the room temperature lucifuge was reacted 60 minutes.
1.4.5 amino-agarose is filled post, and drip-dry washes excessive iodoacetic acid, moves to after drip-dry in the clean tube of 50 ml;
1.4.6 contain the 0.1 mol/L Na of 10 mmol/L disodium ethylene diamine tetraacetate EDTA with 1 ml
2CO
3Solution dissolving 10 mg HA small peptides, on amino after joining 1.4.5 and cleaning-Agarose room temperature middling speed shaking table, reaction is 60 minutes, obtains HA-Agarose, with this HA-Agarose dress post, after fully cleaning with the phosphate buffered saline buffer PBSt that contains 0.05% tween, then be neutralized to PH=7 with PBS.
1.5 the HA specific polyclonal antibody is purified: the immune serum of 500 mL is flow through the made immune affinity chromatographic column of step 1.4.6.Clean unconjugated impurity, the HA specific polyclonal antibody on post is with 1.6% acetic acid wash-out.The antibody of wash-out removes acetic acid with the dialysis tubing dialysis, and freeze-drying is standby.
Detect through indirect ELISA, result shows that the HA specific polyclonal antibody of the immunoaffinity purification that the present invention is made tires very highly, and specificity is very high.
Embodiment 2 high-density HA label recombinant protein immunoaffinity purification enriching column preparations.
Agarose 3 mL of sodium periodate activation change in pillar 2.1 learn from else's experience, and wash with 100 mL distillations, and drip-dry is after 0.1 mol/L Na of 2 times of volumes of Agarose
2CO
3, drip-dry is standby;
2.2 with 300 mg through the HA of immunoaffinity purification specific polyclonal antibody with 0.1 mol/L Na
2CO
3Dissolving makes concentration reach 20 mg/mL, fully centrifuging and taking supernatant after the dissolving;
2.3 supernatant adds in the affinity column of step 2.1, collects effluent liquid and again crosses post, 3-5 time repeatedly;
2.4 with 20 mL distilled water, unconjugated antibody is come out, is collected together;
2.5 be sodium borohydride solution 1.2 mL of 10 mg/mL with the distilled water compound concentration, add in pillar, the vibration mixing, 4 ℃ were reacted 30 minutes;
2.6 the PBS with 10 times of packing volumes washes filler, drip-dry PBS moves into filler in clean test tube, adds the glycerine that equates with packing volume, namely gets the affine filler of D-tag label recombinant protein immunity.
2.7 filtering membrane is loaded onto in the lower end in the tailormadepiston post, and the affine filler of HA label recombinant protein immunity is measured in the post of packing on demand, the filler upper strata covers filtering membrane, namely gets described HA label recombinant protein immunoaffinity purification enriching column.
This immunoaffinity purification enrichment affinity column preservation condition is :-20 ℃.
Embodiment 3 HA specific antibody mark horseradish peroxidases
3.1 claim the HA specific polyclonal antibody of 5 mg immunoaffinity purifications, be dissolved in 100 μ L 0.1 mol/L Na
2CO
3, reacted 10 minutes, add the activated horseradish peroxidase of 5 mg, mixing by antibody amount: horseradish peroxidase=1:1;
3.2 add 1 mg sodium borohydride, the mixing final vacuum, move to after placing 1 minute in-20 ℃ 4 ℃ standing 2 hours;
3.3 through the desalination of G25 Sephadex desalting column;
3.4 adding BSA to make the BSA final concentration is 1mg/mL, then adds 50% glycerine, detects the use titre of institute's traget antibody with the ELISA method.
Embodiment 4 use immuno-precipitations detect HA label recombinant protein immunoaffinity purification enriching column absorption target protein
4.1 get HA label recombinant protein immunoaffinity purification enrichment filler 100 μ L dress posts, wash pillar with 500 μ L 0.05 mol/L HCl, PBS is washed till neutrality;
4.2 the bacterial lysate centrifuging and taking supernatant 2ml of restructuring HA label protein dilutes the 5 times of rear HA of mistake label recombinant protein immunoaffinity purification enriching columns with distilled water with supernatant liquor;
4.3 wash pillar with 3 mL PBSt, then with 3 mL distillation washing pillars, with the PBSt eluant solution of 300 μ L 0.05 mol/L HCl, collect 300 μ L elutriants, add 10 μ L saturated sodium carbonate neutralizations and make PH=7.
4.4 each 300 μ L of bacterial lysate stoste supernatant 5 times of diluents, filtered solution and elutriants that get restructuring HA label protein equivalent respectively add the 5 sample-loading buffer 5*loading buffer that take advantage of, boiled 5 minutes, centrifugal, get supernatant point sample leakage of electricity swimming, do immunoblot experiment.Experimental result such as Fig. 1.
Embodiment 5 use immuno-precipitations detect the concentration effect of HA label recombinant protein immunoaffinity purification enriching column
5.1 get HA label recombinant protein immunoaffinity purification enrichment filler 100 μ L dress posts, wash pillar with 500 μ L 0.05 mol/L HCl, PBS is washed till neutrality;
5.2 the bacterial lysate stoste centrifuging and taking supernatant 0.2ml of restructuring HA label protein dilutes the 50 times of rear HA of mistake label recombinant protein immunoaffinity purification enriching columns with distilled water with supernatant liquor;
5.3 wash pillar with 3 mL PBSt, then with 3 mL distillation washing pillars, with the PBSt eluant solution of 300 μ L 0.05 mol/L HCl, collect 300 μ L elutriants, add 10 μ L saturated sodium carbonate neutralizations and make PH=7.
5.4 each 300 μ L of bacterial lysate stoste supernatant 50 times of diluents, filtered solution and elutriants that get restructuring HA label protein equivalent respectively add 5*loading buffer, boil 5 minutes, and are centrifugal, get supernatant point sample leakage of electricity swimming, do immunoblot experiment., do 2 repetitions, after doing the immunoblotting transferring film, one is repeated not add the inhibition of HA polypeptide, and another repeats to add the inhibition of HA polypeptide, experimental result such as Fig. 2.
The HA specific polyclonal antibody coupling of the immunoaffinity purification of embodiment 6 HA label recombinant protein immunoaffinity purification enriching columns and use horseradish peroxidase-labeled
6.1 get HA label recombinant protein immunoaffinity purification enrichment filler 50 μ L dress posts, wash pillar with 250 μ L 0.05 mol/L HCl, PBS is washed till neutrality;
6.2 get the bacterial lysate stoste centrifuging and taking supernatant 1ml of restructuring HA label protein, with distilled water, supernatant liquor diluted the 10 times of rear HA of mistake label recombinant protein immunoaffinity purification enriching columns;
6.3 wash pillar with 1.5 mL PBSt, then with 1.5 mL distillation washing pillars, with the PBSt eluant solution of 150 μ L 0.05 mol/L HCl, collect 150 μ L elutriants, add 10 μ L saturated sodium carbonate neutralizations and make PH=7;
Add equivalent 5*loading buffer 6.4 get elutriant 150 μ L, boil 5 minutes, centrifugal, get supernatant point sample leakage of electricity swimming, do immunoblot experiment.
6.5 when doing on immunoblotting antibody, directly go up Anti-HA-HRP, experimental result such as Fig. 3.
Annotate: the bacterial lysate of embodiment 4, embodiment 5, embodiment 6 experiment restructuring used HA label proteins is from same pipe, available from Wuhan Sanying Bio-Technology Co., Ltd..
＜110〉Nanning City's blue light Bioisystech Co., Ltd
＜120〉preparation of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column and application thereof
<130> 2012
<140> 2012
<141> 2012-11-08
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 6
<212> PRT
＜213〉artificial sequence
<400> 1
Cys Val Pro Asp Tyr Ala
1 5
<210> 2
<211> 5
<212> PRT
＜213〉artificial sequence
<400> 2
Tyr Pro Tyr Asp Cys
1 5
SEQUENCE LISTING
＜110〉Nanning City's blue light Bioisystech Co., Ltd
＜120〉preparation of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column and application thereof
<130> 2012
<140> 2012
<141> 2012-11-08
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 6
<212> PRT
＜213〉artificial sequence
<400> 1
Cys Val Pro Asp Tyr Ala
1 5
<210> 2
<211> 5
<212> PRT
＜213〉artificial sequence
<400> 2
Tyr Pro Tyr Asp Cys
1 5
Claims (7)
1. the preparation method of a hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column is characterized in that:
1) the chemical coupling reaction of the hemagglutinin peptide HA specific polyclonal antibody of immunoaffinity purification being adopted streaming cross post is coupled on the agarose Agarose of sodium periodate oxidation, use sodium borohydride reduction, clean unconjugated antibody with distilled water, clean filler with phosphoric acid buffer PBS, 10 times of packing volumes/time, wash 4 times, add equal-volume glycerine after drip-dry PBS, mixing namely gets the affine filler of HA label recombinant protein immunity;
2) the affine filler of HA label recombinant protein immunity is packed in plastic column, volume dress post on demand namely gets described HA label recombinant protein immunoaffinity purification enriching column.
2. the preparation method of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column according to claim 1 is characterized in that: made HA label recombinant protein immunoaffinity purification enriching column comprise coupling have immunoaffinity purification the HA specific polyclonal antibody the agarose filler and load the plastic column of the affine filler of this immunity.
3. the preparation method of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column according to claim 1, it is characterized in that: the HA specific polyclonal antibody of described immunoaffinity purification is to make the immunogen immune animal with the carrier proteins that the coupling of HA small peptide activates, obtain containing antibody serum, to contain on antibody serum to coupling the immune affinity chromatographic column of HA small peptide will be arranged, clean unconjugated impurity, with HA specific polyclonal antibody under the weak acid wash-out, desalination, freeze-drying are standby.
4. the preparation method of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column according to claim 3, it is characterized in that: described HA small peptide is the small peptide that tyrosyl-prolyl-tyrosyl-aspartoyl-valyl-prolyl-aspartoyl-tyrosyl-L-Ala forms by 9 amino acid, adopt these 9 amino-acid residues to be divided into 2 groups and introduce in the opposite direction the HA small peptide that cysteine sulfydryl SH group makes sulfhydrylation, its sequence is: 1) cysteinyl-valyl-prolyl-aspartoyl-tyrosyl-L-Ala is C-VPDYA, 2) be tyrosyl-prolyl-tyrosyl-aspartoyl-halfcystine YPYD-C.
5. the preparation method of hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column according to claim 3, it is characterized in that: the immunogen that the carrier proteins of described HA small peptide coupling activation makes, with sodium laurylsulfonate SDS sex change hemocyanin KLH or ovalbumin OVA common carrier albumen, be that iodacetyl-NHS reaction generates iodacetyl KLH or OVA with iodacetyl-hydroxysuccinimide eater again, after removing excessive iodoacetic acid by sephadex G 25 Sephadex desalinations, sulfhydrylation HA small peptide is mixed with KLH or the OVA of iodacetyl, transfer pH=8.5, shake reaction 60 minutes in room temperature, what obtain after G25 Sephadex desalination is immunogen.
6. the preparation method of hemagglutinin peptide HA label recombinant protein immunoaffinity purification enriching column according to claim 3; it is characterized in that: described coupling has the immune affinity chromatographic column of HA small peptide; the HA small peptide haptens of 2 kinds of opposite direction sulfhydrylations and the Agarose of iodacetyl are reacted; form chemical covalent linkage complex body filler HA-Agarose, HA-Agarose is filled post namely get the immune affinity chromatographic column that coupling has the HA small peptide.
7. the application of a hemagglutinin peptide tag recombinant protein immunoaffinity purification enriching column is characterized in that: the HA specific polyclonal antibody coupling of the immunoaffinity purification of immunoaffinity purification enriching column and use horseradish peroxidase HRP mark.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103480340A (en) * | 2013-09-11 | 2014-01-01 | 广西壮族自治区粮油科学研究所 | Immunoaffinity absorbent for enriching ochratoxin A, preparation method and application thereof |
| CN107247132A (en) * | 2017-05-22 | 2017-10-13 | 东莞市儿童医院 | A kind of screening appraisal procedure of polypeptide vaccine |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101072794A (en) * | 2004-12-02 | 2007-11-14 | 联合利华公司 | Method for affinity purification |
| CN102190701A (en) * | 2010-03-05 | 2011-09-21 | 西北大学 | Method for separating and purifying influenza virus hemagglutinin on large scale |
-
2012
- 2012-11-23 CN CN201210481543.0A patent/CN103113455B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101072794A (en) * | 2004-12-02 | 2007-11-14 | 联合利华公司 | Method for affinity purification |
| CN102190701A (en) * | 2010-03-05 | 2011-09-21 | 西北大学 | Method for separating and purifying influenza virus hemagglutinin on large scale |
Non-Patent Citations (3)
| Title |
|---|
| JEFFREY FIELD ET AL.: "Purification of a RAS-respons...n epitope addition method", 《MOLECULAR AND CELLULAR BIOLOGY》, vol. 8, no. 5, 31 May 1988 (1988-05-31), pages 2159 - 2165 * |
| 应国清等: "免疫亲和层析技术及其医药应用进展", 《药物生物技术》, vol. 12, no. 05, 30 October 2005 (2005-10-30), pages 342 - 346 * |
| 龚雄麒: "蛋白质交联方法及其应用", 《国外医学.药学分册》, no. 02, 1 March 1988 (1988-03-01), pages 65 - 73 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103480340A (en) * | 2013-09-11 | 2014-01-01 | 广西壮族自治区粮油科学研究所 | Immunoaffinity absorbent for enriching ochratoxin A, preparation method and application thereof |
| CN107247132A (en) * | 2017-05-22 | 2017-10-13 | 东莞市儿童医院 | A kind of screening appraisal procedure of polypeptide vaccine |
| CN107247132B (en) * | 2017-05-22 | 2019-06-11 | 东莞市儿童医院 | A kind of screening appraisal procedure of polypeptide vaccine |
| CN111138538A (en) * | 2020-02-25 | 2020-05-12 | 四川农业大学 | Goose pituitary prolactin protein immunoaffinity chromatography column, preparation method and purification method |
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