CN1325516C - Biomimetic affinity purification method of vitellus immune globulin - Google Patents
Biomimetic affinity purification method of vitellus immune globulin Download PDFInfo
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- CN1325516C CN1325516C CNB2005100306573A CN200510030657A CN1325516C CN 1325516 C CN1325516 C CN 1325516C CN B2005100306573 A CNB2005100306573 A CN B2005100306573A CN 200510030657 A CN200510030657 A CN 200510030657A CN 1325516 C CN1325516 C CN 1325516C
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Abstract
The present invention relates to a bionics affinity purification method of yolk immune globulins, which belongs to the technical field of biology. The present invention comprises the following steps: (1), basic chromatography media respectively reacts with p-aminobenzoic acid, tyrosine and arginine for synthesizing bionics affinity separating materials after reacts or being activated with tri-chloro tri-nitrogen zin; (2), the synthesized affinity chromatography media are prepared into affinity chromatography columns; the samples comprising yolk immune globulins flow through the affinity chromatography columns; yolk immune globulins are adsorbed on the affinity chromatography columns; the affinity chromatography columns are washed; hybrid-proteins not adsorbed on the affinity chromatography columns are removed; then, the buffer solution conditions are changed, and the affinity chromatography columns are washed for eluting combined yolk immune globulins for obtaining purified yolk immune globulins. The present invention can be used for the large-scale production of high purity yolk immune globulins with high efficiency and low cost; hen yolk immunity IgY has the recovery rate of one step purification larger than 50% and the purity larger than 99.5%; the duck IgY and IgY(deltaFc) have the recovery rate of one step purification larger than 55% and the purity larger than 75%.
Description
Technical field
What the present invention relates to is a kind of method of biological technical field, specifically, is a kind of bio-affinity purifying method of Yolk immunoglobulin.
Background technology
Also contain IgY (yolk antibody or Yolk immunoglobulin) in the yolk of birds, reptiles, batrachians and fish, in the yolk of duck, goose, green turtle and lung fish, also found a kind of IgY that is sheared (Δ Fc) (Immunol Today of weak point, 1995,16 (8), 392-8.).Aspect immunodiagnosis; because IgY is activating complement not; the debond Rheumatoid factors, polyclonal; not with albumin A; Protein G; Mammals Fc acceptor and complement combination; with several no cross reactions of IgG; so can be used as the immunological experiment testing tool; be used to measure circulating complexe; Rheumatoid factors, polyclonal and complement etc.; reduce false positive (J Immunol Met; 1992; 156; 79-83.) Yolk immunoglobulin analyzing; diagnosis; critical role is occupied in preventing disease and treatment disease field; Yolk immunoglobulin is compared with mammiferous antibody has many tangible advantages, and its mass-producing purification technique technology has significant application prospect.
Lipid and the lipoprotein in the Ovum Gallus domesticus Flavus is removed in the general traditionally first dilute with water method of the purifying of Yolk immunoglobulin, chloroform extracting or sad extracting, the Yolk immunoglobulin crude product that obtains through ammonium sulfate precipitation, T 500 precipitation or polyethylene glycol precipitation again, use chromatography method at last, be further purified as thiophilic absorption chromatography (containing the 2-mercaptopyridine).Because complex process, make the purifying of IgY consuming time, consumption power, wastage of material, the rate of recovery is low, cost is high, is unfavorable for the scale operation of IgY and the application of medical aspect.
Find by prior art documents, " utilizing a kind of new synthetic ligands affinity purification Immunoglobulin of Yolk " paper that Giorgio Fassina delivers on " chromatography impurity B series " (Affinitypurification of immunoglobulins from chicken egg yolk using a new syntheticligand, J Chromatogr B, 2000,749:233-242.).This paper utilizes the affine parting material of a peptide species aglucon (TG19318) preparation to extract IgY from the Ovum Gallus domesticus Flavus solution of removing lipid and lipoprotein.At Bis-Tris[Bis (2-Hydroxyethyl) Imimotris (Hydroxymethyl)-Methane] buffer system purifying IgY, purity can reach 95%.The TG19318 preparation needs the synthetic and high-efficient liquid phase chromatogram purification of solid-phase polypeptide, is fixed on the medium complex steps, cost height again; TG19318 itself is a polypeptide character instability, can not tolerate the on-line cleaning condition.Though use TG19318 that complicated IgY purifying process is simplified, above drawbacks limit TG19318 be used for the large scale purification application.Therefore, in this area, the method and the material of exploitation high-level efficiency, low cost, scale operation Yolk immunoglobulin that step is easy can promote the industrialization paces.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of bio-affinity purifying method of Yolk immunoglobulin is provided, make it overcome shortcoming in the Yolk immunoglobulin large scale purification, when extensive separation and purification, step is few, cost is low, efficient is high, utilize the single-minded affinity ligand of Yolk immunoglobulin, can be quick, easy, purifying Yolk immunoglobulin low-costly and in high volume.
The present invention is achieved by the following technical solutions, after the present invention is raw material and trichloride and triazine reaction activation with basic chromatography media, react with para-amino benzoic acid, again with tyrosine or arginine reaction, prepare bionical affine parting material, with the bionical affine parting material purifying Yolk immunoglobulin of preparation.
The present invention includes following steps:
(1) after basic chromatography media and the trichloride and triazine reaction activation,, with tyrosine or arginine reaction, prepares bionical affine parting material again with the para-amino benzoic acid reaction;
Described step (1) specifically is meant: with amino basic chromatography media, activate with the trichloride and triazine reaction; Perhaps with the conventional chemical method to not activating with amino basic chromatography media, generate amino with ammoniacal liquor or aminocompound reaction back after, again with trichloride and triazine reaction activation;
Describedly do not comprise: dextrane gel, crosslinked dextran bead, allyl group dextran and N, the cross-linking copolymer of the cross-linking copolymer of N '-methylene-bisacrylamide, sepharose, agarose and dextran, polyacrylamide/agarose mixture pearl, cellulose bead and scribble silica gel, sintered glass and the pottery of chemical active radical with amino basic chromatography media.
Described conventional chemical method is meant: C.R.Lowe is at " affinity chromatography introduction " (Lip river (C.R.Lowe) work; Liu Yuxiu translates, Science Press, May nineteen eighty-three the 1st edition, the 61-84 page or leaf) matrix activation and the functional method mentioned in, as: for other matrix of polysaccharide base and hydroxyl, available halogen cyan, triazine (all dichlorotriazine or three chlorotriazines), periodate oxidation, oxyethane (1,4-dihydroxyl normal butane bisglycidyl ether), epoxy chloropropionate alcohol, epoxy bromopropyl alcohol, two aziridine, divinyl sulfone, benzoquinone compound such as quinone, bromoacetyl bromide activate and functionalization; For polyacrylamide matrix, available anhydrous ethylenediamine, the hydrazides posthydrolysis, directly basic hydrolysis activates and functionalization; Activate and functionalization for silica gel, sintered glass and ceramic available silylating reagent; Thereby on basic chromatography media, introduce the group that needs.
Described bionical affine parting material is meant the structure that contains the 4-benzaminic acid, perhaps contains arginic structure, perhaps contains the structure of tyrosine.
(2) prepare affinity column with the bionical affine parting material of above-mentioned synthetic, the sample flow that will contain Yolk immunoglobulin is crossed this affinity column, Yolk immunoglobulin is adsorbed on the affinity column, the flushing affinity column, the foreign protein that is not adsorbed on the affinity column is removed, and conversion buffer conditions is then washed affinity column again, the bonded Yolk immunoglobulin is eluted, obtain the Yolk immunoglobulin of purifying.
In the described step (2), the condition of affinity media absorption immunoglobulin (Ig) is pH 4.5-9.0, and ionic strength is the damping fluid of 0.0-0.2 M; Elution requirement is pH 2.0-12, and ionic strength is the damping fluid of 0.1-0.5M.
It is loaded down with trivial details that the present invention had both overcome affine parting material synthesis step, the cost height, the character instability, can not tolerate the shortcoming of on-line cleaning condition, reduced the step of Yolk immunoglobulin large scale purification again, reduce production costs, improved purification efficiency, make that Yolk immunoglobulin is quick, easy, purifying becomes possibility low-costly and in high volume.The rate of recovery of the single step purification of chicken yolk immune IgY＞50%, purity is greater than 99.5%.The rate of recovery＞55% of the single step purification of duck IgY and IgY (Δ Fc), purity is greater than 75%.
Embodiment
Embodiment 1
(1) preparation parting material.
Get NH
2-Sepharose (200ml), with the NaCl washing of the 1M of 5 times of volumes, use the distilled water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the water of 150ml, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (40g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (180ml), take by weighing benzaminic acid (20g) with deionized water (50ml) dissolving, with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine B mixings placed 50 ℃ of stirring reactions of temperature 24 hours.After reaction finishes, take out reactant,, use the distilled water wash of 10 times of volumes then with the NaCl of 3 times of volumes.Obtain the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (160ml).
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing tyrosine (10g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 95 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-tyrosine-2,4, the 6-affine parting materials of three nitrogen piperazines (43ml) store stand-by with 20% ethanol.
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing arginine (12g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 90 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (45ml) store stand-by with 20% ethanol.
(2) with affine parting material purifying chicken yolk immune IgY.
Egg yellow liquor: distilled water: chloroform=mix fully vibration at 1: 4: 1, centrifugal, sucking-off supernatant water discards precipitation and chloroform.With the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml) are packed chromatography column into (in 1.5 * 5.0cm).After 20ml phosphate buffered saline buffer (25mM, pH7.2,0.05M NaCl) balance, the above-mentioned supernatant water of 15ml (pH is identical with phosphate buffered saline buffer with specific conductivity for accent) is added on the post.Behind the material that does not adsorb with 10ml phosphate buffered saline buffer (25mM, pH7.2,0.05M NaCl) flush away, use 10ml 0.1M acetum (pH2.0) wash-out again, collect the wash-out component.Detect the purity of antibody with the non-reduced SDS-PAGE of the sex change of 10-15%.The rate of recovery of the single step purification of chicken yolk immune IgY＞50%, purity is greater than 99.5%.
Embodiment 2
(1) preparation parting material.
Get NH
2-Sepharose (200ml), with the NaCl washing of the 1M of 5 times of volumes, use the distilled water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the water of 150ml, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (40g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (180ml), take by weighing benzaminic acid (20g) with deionized water (50ml) dissolving, with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine B mixings placed 50 ℃ of stirring reactions of temperature 24 hours.After reaction finishes, take out reactant,, use the distilled water wash of 10 times of volumes then with the NaCl of 3 times of volumes.Obtain the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (160ml).
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing tyrosine (10g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 95 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-tyrosine-2,4, the 6-affine parting materials of three nitrogen piperazines (43ml) store stand-by with 20% ethanol.
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing arginine (12g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 90 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (45ml) store stand-by with 20% ethanol.
(2) with the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4,6-three nitrogen piperazines affine parting material purifying duck IgY and IgY (Δ Fc).
The duck's egg yellow liquor: distilled water=mix fully vibration at 1: 6,4 ℃ left standstill 6 hours, centrifugal, sucking-off supernatant water.With the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml) are packed chromatography column into (in 1 * 5.0cm).After 50ml phosphate buffered saline buffer (25mM, pH7.2,0.03M NaCl) balance, the above-mentioned supernatant water of 20ml (pH is identical with phosphate buffered saline buffer with specific conductivity for accent) is added on the post.Behind the material that does not adsorb with 5ml phosphate buffered saline buffer (25mM, pH7.2,0.03M NaCl) flush away, use 5ml 0.1M acetum wash-out again, collect the wash-out component.Detect the purity of antibody with the non-reduced SDS-PAGE of the sex change of 10-15%.The rate of recovery＞55% of the single step purification of duck IgY and IgY (Δ Fc), purity is greater than 75%.
Embodiment 3
(1) preparation parting material.
Get NH
2-Sepharose (200ml), with the NaCl washing of the 1M of 5 times of volumes, use the distilled water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the water of 150ml, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (40g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (180ml), take by weighing benzaminic acid (20g) with deionized water (50ml) dissolving, with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine B mixings placed 50 ℃ of stirring reactions of temperature 24 hours.After reaction finishes, take out reactant,, use the distilled water wash of 10 times of volumes then with the NaCl of 3 times of volumes.Obtain the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (160ml).
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing tyrosine (10g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 95 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-tyrosine-2,4, the 6-affine parting materials of three nitrogen piperazines (43ml) store stand-by with 20% ethanol.
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing arginine (12g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 90 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (45ml) store stand-by with 20% ethanol.
(2) with the amino Sepharose-3-benzaminic acid of 1--5-tyrosine-2,4, the affine parting material purifying of 6-three nitrogen piperazines chicken yolk immune IgY.
Egg yellow liquor: distilled water: chloroform=mix fully vibration at 1: 4: 1, centrifugal, sucking-off supernatant water discards precipitation and chloroform.With the amino Sepharose-3-benzaminic acid of 1--5-tyrosine-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml) are packed chromatography column into (in 1.5 * 5.0cm).After 20ml phosphate buffered saline buffer (25mM, pH7.5,0.05M NaCl) balance, the above-mentioned supernatant water of 15ml (pH is identical with phosphate buffered saline buffer with specific conductivity for accent) is added on the post.Behind the material that does not adsorb with 10ml phosphate buffered saline buffer (25mM, pH7.5,0.05M NaCl) flush away, use 10ml 0.1M acetum wash-out again, collect the wash-out component.Detect the purity of antibody with the non-reduced SDS-PAGE of the sex change of 10-15%.The rate of recovery of the single step purification of chicken yolk immune IgY＞50%, purity is greater than 95%.
Embodiment 4
(1) preparation parting material
Get NH
2-Sepharose (200ml), with the NaCl washing of the 1M of 5 times of volumes, use the distilled water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the water of 150ml, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (40g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (180ml), take by weighing benzaminic acid (20g) with deionized water (50ml) dissolving, with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine B mixings placed 50 ℃ of stirring reactions of temperature 24 hours.After reaction finishes, take out reactant,, use the distilled water wash of 10 times of volumes then with the NaCl of 3 times of volumes.Obtain the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (160ml).
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing tyrosine (10g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 95 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-tyrosine-2,4, the 6-affine parting materials of three nitrogen piperazines (43ml) store stand-by with 20% ethanol.
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing arginine (12g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 90 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (45ml) store stand-by with 20% ethanol.
(2) with affine parting material purifying yolk immunity IgY.
Egg yellow liquor: distilled water: chloroform=mix fully vibration at 1: 4: 1, centrifugal, sucking-off supernatant water discards precipitation and chloroform.With the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml) are packed chromatography column into (in 1.5 * 5.0cm).(25mM pH4.5) after the balance, is added to the above-mentioned supernatant water of 15ml (pH is identical with phosphate buffered saline buffer with specific conductivity for accent) on the post with the 20ml phosphate buffered saline buffer.(25mM pH4.5) behind the material that flush away does not adsorb, uses 10ml 0.1M glycine-hydrochloric acid soln (pH1.0) wash-out again, collects the wash-out component with the 10ml phosphate buffered saline buffer.Detect the purity of antibody with the non-reduced SDS-PAGE of the sex change of 10-15%.The rate of recovery of the single step purification of chicken yolk immune IgY is about 35%, and purity is greater than 90%.
Embodiment 5
(1) preparation parting material
Get NH
2-Sepharose (200ml), with the NaCl washing of the 1M of 5 times of volumes, use the distilled water thorough washing again after, take out branchs that anhydrate, pour in the reaction vessels, add the water of 150ml, be put in precooling in the cryosel bath.Treat that temperature reduces to 5 ℃, begin to stir.Add in the reaction vessel with precooling acetone solution three nitrogen piperazines (40g) back.Use saturated NaHCO
3The pH of solution is remained between 6～7, and 5 ℃ are reacted 3h down, take out, the water/acetone of 3 * 10 times of volumes (1: 1,1: 3,0: 1,1: 1,3: 1,1: 0) washing successively, obtain 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (190ml).
Get 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazines (180ml), take by weighing benzaminic acid (20g) with deionized water (50ml) dissolving, with 1-amino-Sepharose-3,5-two chloro-2,4,6-three nitrogen piperazine B mixings placed 50 ℃ of stirring reactions of temperature 24 hours.After reaction finishes, take out reactant,, use the distilled water wash of 10 times of volumes then with the NaCl of 3 times of volumes.Obtain the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (160ml).
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing tyrosine (10g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 95 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-tyrosine-2,4, the 6-affine parting materials of three nitrogen piperazines (43ml) store stand-by with 20% ethanol.
Get the amino Sepharose-3-benzaminic acid of 1--5-chloro-2,4,6-three nitrogen piperazines (50ml) take by weighing arginine (12g), with methyl-sulphoxide and deionized water mixed dissolution, the pH of solution is transferred to about 5-8, preferable pH7 pours the amino Sepharose-3-benzaminic acid of reactor and 1--5-chloro-2,4 into, 6-three nitrogen piperazines mix, and are put in temperature and are under 90 ℃ the isoperibol reaction 24 hours.After reaction finishes, take out reactant, with the NaCl of 4 times of volumes, 3 volume methyl-sulphoxides wash, and use the deionized water wash of 10 times of volumes then.Obtain the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (45ml) store stand-by with 20% ethanol.
(2) with affine parting material purifying yolk immunity IgY.
Egg yellow liquor: distilled water: chloroform=mix fully vibration at 1: 4: 1, centrifugal, sucking-off supernatant water discards precipitation and chloroform.With the amino Sepharose-3-benzaminic acid of 1--5-arginine-2,4, the 6-affine parting materials of three nitrogen piperazines (5ml) are packed chromatography column into (in 1.5 * 5.0cm).After 20ml phosphate buffered saline buffer (25mM, pH9.0,0.2M NaCl) balance, the above-mentioned supernatant water of 15ml (pH is identical with phosphate buffered saline buffer with specific conductivity for accent) is added on the post.(25mM, pH9.0 0.2MNaCl) behind the material that flush away does not adsorb, use 10ml 0.1M glycine-sodium hydroxide solution (pH12.0,0.5M NaCl) wash-out again, collect the wash-out component with the 10ml phosphate buffered saline buffer.Detect the purity of antibody with the non-reduced SDS-PAGE of the sex change of 10-15%.The rate of recovery of the single step purification of chicken yolk immune IgY is about 45%, and purity is greater than 85%.
Claims (9)
1. the bio-affinity purifying method of a Yolk immunoglobulin, it is characterized in that, with basic chromatography media is after raw material and trichloride and triazine reaction activate, react with para-amino benzoic acid, again with tyrosine or arginine reaction, prepare bionical affine parting material, with the bionical affine parting material purifying Yolk immunoglobulin of preparation.
2. the bio-affinity purifying method of Yolk immunoglobulin according to claim 1 is characterized in that, may further comprise the steps:
(1) after basic chromatography media and the trichloride and triazine reaction activation,, with tyrosine or arginine reaction, prepares bionical affine parting material again with the para-amino benzoic acid reaction;
(2) prepare affinity column with the bionical affine parting material of above-mentioned synthetic, the sample flow that will contain Yolk immunoglobulin is crossed this affinity column, Yolk immunoglobulin is adsorbed on the affinity column, the flushing affinity column, the foreign protein that is not adsorbed on the affinity column is removed, and conversion buffer conditions is then washed affinity column again, the bonded Yolk immunoglobulin is eluted, obtain the Yolk immunoglobulin of purifying.
3. the bio-affinity purifying method of Yolk immunoglobulin according to claim 2 is characterized in that, in the described step (1), with amino basic chromatography media, activates with the trichloride and triazine reaction.
4. the bio-affinity purifying method of Yolk immunoglobulin according to claim 2, it is characterized in that, in the described step (1), with the conventional chemical method to not activating with amino basic chromatography media, behind ammoniacal liquor or aminocompound reaction back generation amino, again with trichloride and triazine reaction activation; Describedly do not comprise: dextrane gel, crosslinked dextran bead, allyl group dextran and N, the cross-linking copolymer of the cross-linking copolymer of N '-methylene-bisacrylamide, sepharose, agarose and dextran, polyacrylamide/agarose mixture pearl, cellulose bead and scribble silica gel, sintered glass and the pottery of chemical active radical with amino basic chromatography media.
5. the bio-affinity purifying method of Yolk immunoglobulin according to claim 1 and 2 is characterized in that, described bionical affine parting material is meant the structure that contains the 4-benzaminic acid.
6. the bio-affinity purifying method of Yolk immunoglobulin according to claim 1 and 2 is characterized in that, described bionical affine parting material is meant and contains arginic structure.
7. the bio-affinity purifying method of Yolk immunoglobulin according to claim 1 and 2 is characterized in that, described bionical affine parting material is meant the structure that contains tyrosine.
8. the bio-affinity purifying method of Yolk immunoglobulin according to claim 2 is characterized in that, in the described step (2), the condition of affinity column absorption immunoglobulin (Ig) is pH 4.5-9.0, and ionic strength is the damping fluid of 0.0-0.2M.
9. the bio-affinity purifying method of Yolk immunoglobulin according to claim 2 is characterized in that, in the described step (2), elution requirement is pH 2.0-12, and ionic strength is the damping fluid of 0.1-0.5M.
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| CN1970745B (en) * | 2006-12-07 | 2010-05-12 | 上海交通大学 | Bio-affinity purifying method for lipase |
| CN102276718A (en) * | 2011-07-26 | 2011-12-14 | 广州格雷特生物科技有限公司 | Process for extracting yolk immunoglobulin by water-caprylic acid two-step method |
| CN104419695B (en) * | 2013-08-22 | 2019-11-15 | 上海亨臻实业有限公司 | The preparation of the bionical affinitive material of chymotrypsinogen and chymotrypsin purification process |
| CN104419689B (en) * | 2013-08-22 | 2019-11-15 | 上海亨臻实业有限公司 | The bio-affinity purifying method of hyaluronidase |
| CN104761638B (en) * | 2015-04-16 | 2018-08-03 | 西北农林科技大学 | A kind of method of albumen affinity purification IgY |
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