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%.