CN1229389C - Immunization magnetic separation technology for purifying genetic engineering recombinant interferon - Google Patents

Immunization magnetic separation technology for purifying genetic engineering recombinant interferon Download PDF

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CN1229389C
CN1229389C CN 03144274 CN03144274A CN1229389C CN 1229389 C CN1229389 C CN 1229389C CN 03144274 CN03144274 CN 03144274 CN 03144274 A CN03144274 A CN 03144274A CN 1229389 C CN1229389 C CN 1229389C
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magnetic microsphere
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interferon
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CN1524878A (en
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白钢
杨文博
陈家琪
曹宇
曹学琳
张磊
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Nankai University
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Nankai University
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Abstract

The present invention relates to a separation technique of gene engineering recombination proteins, particularly to the separation and the purification of gene engineering recombination interferon (interferon, IFN) by an immunization magnetic separation technique, which belongs to the technical field of biology. In the present invention, a magnetic microsphere containing a hydroxyl group is prepared by using different materials; an immunization magnetic microsphere is formed by that after the magnetic microsphere is activated by epoxy chloropropane, active amino groups are introduced and connected with anti-interferon antibodies by using glutaraldehyde as a link arm. The prepared immunization magnetic microsphere can be used for the separation and the purification of interferon. If the fermentation biomass lysates of the gene engineering recombination interferon are separated and purified with the immunization magnetic microsphere and a magnetic separation device, the activity recovery rate exceeds 50%, the specific activity of the purified interferon is higher than 1.0*10<8> IU/mg, and the analysis purity of SDS-PAGE gel electrophoresis is close to 100%.

Description

The immunomagnetic isolation technology of purifying gene engineering recombinant interferon
Technical field
The present invention relates to a kind of isolation technique of genetically engineered recombinant protein, particularly utilize immunomagnetic isolation technology purifying alpha-interferon, belong to biological technical field.
Background technology
(Interferon is that the class that body is produced by exogenous stimulation is regulated albumen IFN) to Interferon, rabbit, has broad-spectrum antiviral, antitumor and immunoregulatory activity.Clinically viral hepatitis, zoster, lymphoma, leukemia etc. all there is certain curative effect.(α-2bIFN) is as one of the earliest gene recombinant protein class medicine, and 1986 promptly in America and Europe's listing (U.S. Schering-Plough company) for human recombinant interferon α-2b.Nineteen ninety, domestic by independent development and introduction of foreign technology, there are many enterprises to drop in succession and commercially produce, the domestic market annual sales amount has reached 1,000,000,000 yuan, more than 20 hundred million dollars of U.S.'s annual sales amounts at present.Human recombinant interferon mainly utilizes the genetic engineering bacterium fermentative production, it is activity expression product in the born of the same parents, separation and purification process complexity will be passed through processes such as cytoclasis, centrifugation, salt precipitation, dissolution precipitation, hydrophobic chromatography, zwitterion displacement chromatography and gel permeation chromatography.Because the sepn process step is many, cause that product yield is lower, cost is high.At present, some companies adopt affinity chromatography improving purification efficiency and specificity usually, could obtain higher purification efficiency but also will handle through oversalting, hydrophobic or ion exchange chromatography etc. before affinity chromatography.In addition, the connection of the affinity chromatography medium homospecificity antibody cyanogen bromides that adopt huge poison have increased the danger of technology as coupling agent more.At present both at home and abroad each enterprise all is devoted to the exploitation of new technology of Interferon, rabbit separation and purification and the renewal of production technique.
Summary of the invention
The objective of the invention is to open up a new approach, relate to preparation, activation and the sensitization of the magnetic microsphere of purifying gene engineering recombinant interferon for the separation and purification of protein genetic engineered product, and the preparation of magnetic resolution device.
The present invention adopts materials such as agarose, polyvinyl alcohol, Mierocrystalline cellulose to prepare the surperficial magnetic microsphere that has hydroxyl respectively, with epoxy chloropropane microballoon is activated, after importing active amino, and adopt glutaraldehyde to be connected with anti-interferon IgG antibody as connecting arm, obtain can be used for the immune magnetic microsphere of Interferon, rabbit separation and purification.Employed anti-interferon IgG antibody can be purified purified anti-interferon mono-clonal or polyclone IgG antibody.
With above-mentioned immune magnetic microsphere and magnetic resolution device the fermentation thalline lysate or the pretreated cellular lysate liquid of Interferon, rabbit is carried out immunomagnetic isolation, activity recovery surpasses 50%, and the human interferon behind the purifying (IFN) specific activity is higher than 1.0 * 10 8IU/mg, SDS-PAGE gel electrophoresis analysis purity meets national standard near 100%.Behind the purifying in the sample animal derived anti-body contg be lower than the following requirement of each dosage 100ng of national regulation.
The invention has the beneficial effects as follows: utilize this immunomagnetic isolation technology purifying gene engineering recombinant interferon (IFN), easy and simple to handle, quick, need not carry out complicated pre-treatment to sample, can be directly from the lysate of fermentation thalline, single step purification obtains highly purified target protein, simplify the step of separation and purification greatly, improve product yield.The separation and purification mild condition, this immune magnetic microsphere is reusable more than 10 times.The present invention is expected to substitute immune affinity chromatographic column, become a kind of fast, the new technology of high efficiency separation purifying alpha-interferon.
Description of drawings
The stereoscan photograph of Fig. 1 magnetic microsphere
Wherein A figure is agarose magnetic microsphere (* 1500); B figure is cellulose magnetic microsphere (* 1200); C figure is polyvinyl alcohol magnetic microsphere (* 300).
Fig. 2 magnetic resolution device synoptic diagram
The reaction sealed vessel of (1) column wherein; (2) immune magnetic microsphere; (3) magnet; (4) terminal liquid-outlet valve; (5) reaction solution; (6) upper end breather valve; (7) collection container.
The SDS-PAGE gel electrophoresis analysis of Fig. 3 Interferon, rabbit
Each swimming lane condition is among the figure: swimming lane a: standard molecular weight albumen; Swimming lane b: α-2b Interferon, rabbit fermentation thalline lysate; Swimming lane c: the cellular lysate liquid of thiamines precipitation process; Swimming lane d: agarose immune magnetic microsphere separating resulting; Swimming lane e: Mierocrystalline cellulose immune magnetic microsphere separating resulting; Swimming lane f: polyvinyl alcohol immune magnetic microsphere separating resulting.
Embodiment
One. the preparation of anti-interferon IgG antibody:
1) preparation of Anti-IFN serum:
Get human interferon (IFN) standard substance, promptly get human interferon (IFN) antigen with the emulsification of isopyknic Freund's complete adjuvant thorough mixing, get human interferon (IFN) antigen, respectively to 2 the big ear of Japan white male rabbit (body weight 2Kg) muscle and the subcutaneous multi-point injection that carries out.Append injection and adopt Freund's incomplete adjuvant emulsification to prepare antigen, every two all booster shots once, totally three times, dosage successively decreases.3 days posterior veins of last immunity are got blood, measure antibody titer with indirect elisa method, the antibody neutralization that records serum is tired to greater than after 1: 10000, the abdomen arterial blood letting, whole blood was left standstill 2 hours, centrifugal 20 minutes of 3000rpm collects serum deactivation 30 minutes in 55 ℃ of water-baths, after the packing in-20 ℃ of freezing preservations;
2) Anti-IFN IgG purifying antibody:
Get Anti-IFN serum, after adding isopyknic PBS mixing, add isopyknic saturated sulphur ammonium precipitation respectively, centrifugal collecting precipitation, throw out is dissolved among the PBS, 4 ℃ of dialysed overnight, the Anti-IFN immunoglobulin (Ig) of slightly being carried is with above-mentioned immunoglobulin (Ig) crude product, adopt DEAE-cellulose 52 ion exchange chromatographies to carry out gradient elution, collect merging IgG elution peak,, get refined rabbit anti-interferon IgG polyclonal antibody after the lyophilize through distill water dialysis.
Two. the preparation of immune magnetic microsphere:
Magnetic microsphere comprises agarose magnetic microsphere and cellulose magnetic microsphere that anti-phase physically trapping method prepares, and the polyvinyl alcohol magnetic microsphere of inverse suspension crosslinking method preparation etc. contains the magnetic microsphere of activity hydroxy group; Preparation technology comprises subordinate's process:
1) preparation of magnetic fluid:
Get FeCl 24H 2O is dissolved in the deionized water, through the filtering with microporous membrane removal of impurity, add a certain amount of polyoxyethylene glycol and hydrogen peroxide then, transfer to pH9～11 with NaOH solution behind the mixing, reacted 3～6 hours in 50～80 ℃ of water-baths, the stable suspension that obtains brownish black magnetic colloid particle is a magnetic fluid;
2) preparation of agarose magnetic microsphere:
Get toluene, tetracol phenixin and Span-80 are preheated to 60～80 ℃, as organic phase; A certain amount of magnetic fluid is joined in the agarose solution, heating for dissolving in boiling water bath, and be transferred to rapidly in the organic phase, after reacting 5～30 minutes under 2000～5000rpm stirring, be cooled to room temperature rapidly; Clean with ether, distilled water at last, the agarose magnetic microsphere of screening particle diameter between 100～300 μ m, standby in 2～8 ℃ of preservations.
3) preparation of polyvinyl alcohol magnetic microsphere:
Get 200 #Gasoline, tetracol phenixin and Span-80 mixing are as organic phase; Preparation finite concentration polyvinyl alcohol solution adds a certain amount of magnetic fluid, ice bath 5～20 minutes, add glutaraldehyde solution and hydrochloric acid, pour into rapidly behind the mixing in the organic phase, under 2000～5000rpm, disperseed 0.5～2 hour, slowly be warming up to 60～80 ℃ and continue reaction 1～3 hour again; Use ether, distilled water drip washing at last, the polyvinyl alcohol magnetic microsphere of screening particle diameter between 100～300 μ m, standby in 2～8 ℃ of preservations;
4) preparation of cellulose magnetic microsphere:
NaOH solution is joined in a certain amount of absorbent cotton, soaked 1～4 hour, squeeze lixiviating liquid, after wearing out under 10～30 ℃, add a certain amount of dithiocarbonic anhydride mixing, reaction 2～8 hours is continued in the sealing back, adds certain density NaOH solution reaction again 5～10 hours, obtains orange-red xanthate viscose; Get chlorobenzene, tetracol phenixin and potassium oleate, behind 20～50 ℃ of stirring in water bath mixings, add a certain amount of xanthate viscose that is dissolved with magnetic fluid, under 2000～5000rpm, disperse to be warming up to 75～100 ℃ after 20～60 minutes, continue reaction 1～4 hour again; Use ether, distilled water drip washing at last, the cellulose magnetic microsphere of screening particle diameter between 100～300 μ m, standby in 2～8 ℃ of preservations.
5) activation of magnetic microsphere:
Get a certain amount of magnetic microsphere adds certain volume under alkaline condition epoxy chloropropane, under 25～40 ℃, activate 2～4 hours, add 2～20% ammoniacal liquor after cleaning epoxy chloropropane, room temperature reaction 3～9 hours, fully add 2～20% glutaraldehyde solutions again after the drip washing, room temperature reaction 1～5 hour is washed excessive glutaraldehyde off with distilled water at last.
6) sensitization of magnetic microsphere:
Under 2～8 ℃, get the good magnetic microsphere of a certain amount of activation, add and contain the mono-clonal of anti-interferon or the NaHCO of polyclone IgG antibody 3-NaCO 3Damping fluid, fully reaction is after PBS drip washing adds NaBH 4Reduce, clean the immune magnetic microsphere that promptly obtains can be used for purifying alpha-interferon (IFN) with PBS then, the antibody coupling amount is 3～15mg/mL ball.
Three. the immunomagnetic isolation of Interferon, rabbit (IFN):
Get immune magnetic microsphere after PBS fully cleans, place the container of magnetic resolution device; Adding is with the fermentation thalline lysate or the pretreated lysate of the genetically engineered recombinant interferon (IFN) of damping fluid dilution, in 2～8 ℃ of slow oscillatory reactions 3～24 hours, after the reaction container is placed on the magnetic resolution device, absorption fixed magnetic microballoon, discard reaction solution, after cleaning, PBS adds glycine-hydrochloride buffer (pH1.5～3.5) again, be used to the Interferon, rabbit (IFN) of absorption that dissociates in 1～5 hour in 2～8 ℃ of slow oscillatory reactions, again Glass Containers is placed on the tripping device, the absorption magnetic microsphere, collect dissociation solution, use PB damping fluid (pH6.5～8.0) neutralization of high density immediately, obtain the genetically engineered recombinant interferon (IFN) of purifying.After immune magnetic microsphere cleans with PBS, reusable in 2～8 ℃ of preservations.
Adopt Interferon, rabbit (IFN) immunologic competence after the ELISA method is measured purifying, suppress the pathology method with cell and measure its biologic activity.The every purity index and the animal source IgG content of Interferon, rabbit behind the purifying (IFN) all are up to state standards.Direct purification fermentation thalline lysate and can reach 9.6 * 10 through Interferon, rabbit (IFN) adsorptive capacity that the sulphur ammonium precipitates pretreated lysate 5IU/ml and 8.0 * 10 5IU/ml.Carry out repeatedly the purifying experiment with immune magnetic microsphere, the average activity rate of recovery is greater than 50%, and the average specific activity is greater than 1.0 * 10 8IU/mg.Interferon, rabbit (IFN) purity of SDS-PAGE gel electrophoresis analysis immunomagnetic isolation purifying is near 100%.
Embodiment one.
One. the preparation of anti-IFN α-2b IgG antibody:
1. sero-fast preparation:
Get IFN α-2b standard substance 2mL of 1mg/mL, with after the emulsification of isopyknic Freund's complete adjuvant thorough mixing IFN α-2b emulsification antigen.Get the emulsification antigen of 2mL IFN α-2b, respectively 2 rabbit (the big ear of Japan is white, male, body weight 2Kg) are carried out muscle and subcutaneous multi-point injection.Append injection and adopt Freund's incomplete adjuvant emulsification to prepare antigen, every two all booster shots once, totally three times, dosage successively decreases.Last immunity is after 6 days, and the abdomen arterial blood drawing leaves standstill whole blood 2 hours, and centrifugal 20 minutes of 3000rpm collects serum deactivation 30 minutes in 55 ℃ of water-baths, after the packing in-20 ℃ of freezing preservations.
2. anti-IFN α-2b IgG purifying antibody:
Get anti-IFN α-2b serum 10mL, add isopyknic PBS mixing after, carry out salt fractionation with 50% and 33% saturated sulphur ammonium respectively, centrifugal collecting precipitation, throw out are dissolved among the PBS, 4 ℃ of dialysed overnight, anti-IFN α-2b immunoglobulin (Ig) of slightly being carried.Immunoglobulin (Ig) crude product with above-mentioned salt fractionation, carry out DEAE-cellulose 52 ion exchange chromatographies, with 0.02M PB damping fluid (pH8.0) wash-out, collect and merge the IgG component, dialyse with deionized water, after the lyophilize the anti-IFN α-2b of refined rabbit (the IgG antibody of Anti-IFN α-2b) detects through SDS-PAGE and to be single band.
Two. the preparation of agarose immune magnetic microsphere:
1. the preparation of magnetic fluid:
Get 6 gram FeCl 24H 2O is dissolved in the 400mL deionized water, removes impurity through filtering with microporous membrane.Add 150 gram polyoxyethylene glycol and 100 μ L 30%H then 2O 2Mixing is transferred pH value of solution 11 with 3M NaOH, and reaction is 4 hours in 50～60 ℃ of water-baths, and the stable suspension that obtains brownish black magnetic colloid particle is a magnetic fluid.
2. the preparation of agarose magnetic microsphere:
Add 75mL toluene in the 250ml triangular flask, 30mL tetracol phenixin and 1.2mL Span-80 are preheated to 70 ℃, as organic phase; Get 1 gram magnetic fluid, join in 30mL 6% agarose solution, heating for dissolving is as water in 100 ℃ of water-baths.Water is transferred to rapidly in the organic phase, and 3000rpm stirred 15 minutes, was cooled to room temperature then rapidly, got the agarose magnetic microsphere.Use ether, distilled water drip washing respectively, the magnetic microsphere of screening particle size range between 100～300 μ m, standby in 4 ℃ of preservations.
3. the activation of agarose magnetic microsphere and sensitization:
Get agarose magnetic microsphere 1mL, add 2mL 2M NaOH and 1mL epoxy chloropropane, under 30 ℃, acetone is used in oscillatory reaction 2 hours respectively behind the suction filtration, and distilled water cleans.Add 10% ammoniacal liquor 2mL, room temperature standing and reacting 6 hours.Behind the thorough washing, add 5% glutaraldehyde 2mL, the excessive glutaraldehyde of distilled water flush away is used in room temperature oscillatory reaction 2 hours again, gets activatory agarose magnetic microsphere.In above-mentioned microballoon 1mL, add the 0.1M NaHCO that 5mL contains 15mg Anti-IFN α-2b polyclone IgG antibody 3-NaCO 3Damping fluid (pH9.6), 4 ℃ of reactions are spent the night.Behind the PBS thorough washing, add 2mL0.5%NaBH 4Reduce, behind the PBS thorough washing, promptly obtain can be used for the agarose immune magnetic microsphere of purifying IFN α-2b again.The coupling amount of measuring antibody with the Lowry method is the 12.8mg/mL ball.
Three. the immunomagnetic isolation of α-2bIFN:
Get the agarose magnetic microsphere 1mL after the sensitization, place the Glass Containers of magnetic resolution device; Get IFN α-2b fermentation thalline lysate 1mL, centrifugal 10 minutes of 10000rpm gets 200 μ L supernatant liquors and (dilute 50 times) in 10mL PBS, joins then in the Glass Containers, spends the night in 4 ℃ of slow oscillatory reactions.After the reaction Glass Containers is placed on the magnetic resolution device, absorption fixed magnetic microballoon discards reaction solution, cleans 6 times with PBS then.Add 4mL 0.1M glycine-hydrochloride buffer (pH2.5) again, 4 ℃ of vibrations 2 hours, the IFN α-2b that dissociates and adsorbed by immune magnetic microsphere.Glass Containers is placed on the tripping device, the absorption magnetic microsphere, and collect dissociation solution, and use a small amount of 1M PB damping fluid (pH7.0) neutralization immediately, get the IFN α-2b of purifying.Immune magnetic microsphere in 4 ℃ of preservations, reuses after PBS cleans fully.
Adopt the IFN α-2b immunologic competence after the ELISA method detects purifying, suppress the pathology method with cell and measure its biologic activity.Adopt the agarose magnetic microsphere direct purification α-2bIFN fermentation thalline lysate of sensitization, repeated experiments 3 times, average adsorptive capacity is 9.6 * 10 5IU/ml, the average activity rate of recovery is 88.2%, the average specific activity is 1.5 * 10 8IU/mg carries out purity check through the SDS-PAGE gel electrophoresis, and purity is near 100%, and every index and rabbit IgG content all are up to state standards, and have shown good circulation ratio.
Embodiment two.
One. the preparation of Mierocrystalline cellulose immune magnetic microsphere:
1. the preparation of cellulose magnetic microsphere:
Get 100g absorbent cotton and soaked 2 hours with 19%NaOH solution 800mL, squeeze lixiviating liquid, 25 ℃ were worn out 3 days; Add 50mL dithiocarbonic anhydride mixing, seal back 28 ℃ of oscillatory reactions 5 hours, add the 6%NaOH solution mixing of 750～850mL again, 28 ℃ of vibrations then obtained orange red xanthate viscose in 5 hours.Add 180mL chlorobenzene, 30mL tetracol phenixin and 0.5 gram potassium oleate in the 500ml three-necked bottle, 40 ℃ of stirring in water bath mixings are as organic phase.Get the xanthate viscose 70mL that contains 1 gram magnetic fluid, join in the organic phase, 3000rpm disperses to be warming up to 90 ℃ rapidly after 30 minutes, continues reaction 2 hours, gets cellulose magnetic microsphere.Use ether, distilled water drip washing respectively, the cellulose magnetic microsphere of screening screening particle diameter between 100～300 μ m, standby in 4 ℃ of preservations.
2. the activation of cellulose magnetic microsphere and sensitization:
Extracting cellulose magnetic microsphere 1mL adds 2mL 3M NaOH and 1mL epoxy chloropropane, and acetone is used in 40 ℃ of following oscillatory reactions 2.5 hours respectively behind the suction filtration, and distilled water cleans.Add 4mL 5% ammoniacal liquor, room temperature standing and reacting 9 hours.Add 5mL 2% glutaraldehyde behind the thorough washing, the excessive glutaraldehyde of distilled water flush away is used in room temperature vibration 5 hours again, gets the activatory cellulose magnetic microsphere.In above-mentioned microballoon 1mL, add the 0.1MNaHCO that 5mL contains 12mg Anti-IFN α-2bIgG polyclonal antibody 3-NaCO 3Damping fluid (pH9.6), 4 ℃ of reactions are spent the night.Behind the PBS thorough washing, add 2mL 0.5%NaBH 4Reduce, behind the PBS thorough washing, promptly obtain can be used for the Mierocrystalline cellulose immune magnetic microsphere of purifying IFN α-2b again.The coupling amount of measuring antibody with the Lowry method is the 11.8mg/mL ball.
Two. the immunomagnetic isolation of α-2bIFN:
Extracting cellulose magnetic microsphere 1mL places the Glass Containers of magnetic resolution device; Get α-2bIFN fermentation thalline lysate 5mL, centrifugal 10 minutes of 10000rpm gets supernatant liquor again and adds isopyknic PBS mixing, slowly adds the saturated ammonium sulfate solution of equal-volume precooling, and mixing places on the ice bath, places 4 hours.4 ℃, centrifugal 15 minutes of 6000rpm abandons supernatant liquor, and precipitation is dissolved in 5ml PBS, with 0.1M PBS dialysis, gets 500 μ L dialyzates and (dilute 20 times) in 10mL PBS, joins then in the Glass Containers, spends the night in 4 ℃ of slow oscillatory reactions.After the reaction Glass Containers is placed on the magnetic resolution device, absorption fixed magnetic microballoon discards reaction solution, cleans 5 times with PBS then.Add 4mL 0.1M glycine-hydrochloride buffer (pH2.5) again, 4 ℃ of vibrations 2 hours, the IFN α-2b that dissociates and adsorbed by immune magnetic microsphere.Glass Containers is placed on the tripping device, the absorption magnetic microsphere, and collect dissociation solution, and use a small amount of 1M PB damping fluid (pH7.0) neutralization immediately, get the IFN α-2b of purifying.Immune magnetic microsphere in 4 ℃ of preservations, reuses after PBS cleans fully.
Adopt the IFN α-2b immunologic competence after the ELISA method detects purifying, suppress the pathology method with cell and measure its biologic activity.Adopt the cellulose magnetic microsphere purifying of sensitization to precipitate pretreated IFN α-2b fermentation thalline lysate through thiamines, repeated experiments 3 times, average adsorptive capacity is 8.0 * 10 5IU/ml, the average activity rate of recovery is 50.3%, the average specific activity is 1.4 * 10 8IU/mg carries out purity check through the SDS-PAGE gel electrophoresis, and purity is near 100%, and every index and rabbit IgG content all are up to state standards, and have shown good circulation ratio.
Embodiment three.
One. the preparation of polyvinyl alcohol immune magnetic microsphere:
1. the preparation of polyvinyl alcohol magnetic microsphere:
In the 500ml three-necked bottle, add 140mL 200 #Gasoline, 110mL tetracol phenixin and 5mL Span-80 mixing are as organic phase; Prepare 10% polyvinyl alcohol 50ml, ice bath is 15 minutes behind the adding 0.6 gram magnetic fluid, adds 25% glutaraldehyde 5mL and 1N HCL 6mL again; Add rapidly in the organic phase behind the mixing, 3000rpm stirred 30 minutes, was warming up to 70 ℃ then, continued stirring reaction 2 hours, got the polyvinyl alcohol magnetic microsphere.Use ether, distilled water drip washing respectively, the polyvinyl alcohol magnetic microsphere of screening particle diameter between 100～300 μ m, standby in 4 ℃ of preservations.
2. the activation of polyvinyl alcohol magnetic microsphere and sensitization:
Get polyvinyl alcohol magnetic microsphere 1ml, add 10mL dimethyl sulfoxide (DMSO) and 3mL epoxy chloropropane, 40 ℃, slowly drip 2M NaOH 10mL, continue reaction after 4 hours, suction filtration is used acetone respectively, and distilled water cleans.Add 1.5mL15% ammoniacal liquor, room temperature standing and reacting 3 hours.Add 1mL 10% glutaraldehyde behind the thorough washing, the excessive glutaraldehyde of distilled water flush away is used in room temperature vibration 1 hour again, gets activatory polyvinyl alcohol magnetic microsphere.In above-mentioned microballoon 1mL, add the 0.1M NaHCO that 5mL contains 8mgAnti-IFN α-2b Mab IgG antibody 3-NaCO 3Damping fluid (pH9.6), 4 ℃ of reactions are spent the night.Behind the PBS thorough washing, add 2mL 0.5%NaBH 4Reduce, behind the PBS thorough washing, promptly obtain can be used for the polyvinyl alcohol immune magnetic microsphere of purifying IFN α-2b again.The coupling amount of measuring antibody with the Lowry method is the 3.7mg/mL ball.
Two. the immunomagnetic isolation of α-2bIFN:
Get polyvinyl alcohol magnetic microsphere 1mL, place the Glass Containers of magnetic resolution device; Get α-2bIFN fermentation thalline lysate 1mL, centrifugal 10 minutes of 10000rpm gets 100 μ L supernatant liquors and (dilute 100 times) in 10mL PBS, joins then in the Glass Containers, spends the night in 4 ℃ of slow oscillatory reactions.After the reaction Glass Containers is placed on the magnetic resolution device, absorption fixed magnetic microballoon discards reaction solution, cleans 6 times with PBS then.Add 4mL 0.1M glycine-hydrochloride buffer (pH2.5) again, 4 ℃ of vibrations 2 hours, the IFN α-2b that dissociates and adsorbed by immune magnetic microsphere.Glass Containers is placed on the tripping device, the absorption magnetic microsphere, and collect dissociation solution, and use a small amount of 1M PB damping fluid (pH7.0) neutralization immediately, get the IFN α-2b of purifying.Immune magnetic microsphere in 4 ℃ of preservations, reuses after PBS cleans fully.
Adopt the IFN α-2b immunologic competence after the ELISA method detects purifying, suppress the pathology method with cell and measure its biologic activity.Adopt the polyvinyl alcohol magnetic microsphere direct purification α-2bIFN fermentation thalline lysate of sensitization, repeated experiments 3 times, average adsorptive capacity is 4.5 * 10 5IU/ml, the average activity rate of recovery is 93.9%, the average specific activity is 1.2 * 10 8IU/mg carries out purity check through the SDS-PAGE gel electrophoresis, and purity is near 100%, and every index and mouse IgG content all are up to state standards, and have shown good circulation ratio.

Claims (1)

1. the immunomagnetic isolation technology of a purifying gene engineering recombinant interferon, it comprises the separation and purification process of genetically engineered recombinant interferon in the sensitization of activation, magnetic microsphere of preparation, the magnetic microsphere of magnetic microsphere and the fermentation thalline lysate; It is characterized in that: the preparation of magnetic microsphere is the magnetic microsphere that the hydroxyl activity group is contained on the surface, the agarose magnetic microsphere, the cellulose magnetic microsphere that comprise the preparation of anti-phase suspension entrapping method, with the polyvinyl alcohol magnetic microsphere of inverse suspension crosslinking method preparation, preparation technology comprises following process:
1) preparation of magnetic fluid:
Get FeCl 24H 2O is dissolved in the deionized water, through the filtering with microporous membrane removal of impurity, add a certain amount of polyoxyethylene glycol and hydrogen peroxide then, transfer to pH 9～11 with NaOH solution behind the mixing, reacted 3～6 hours in 50～80 ℃ of water-baths, the stable suspension that obtains brownish black magnetic colloid particle is a magnetic fluid;
2) preparation of agarose magnetic microsphere:
Add toluene in container, tetracol phenixin and Span-80 are preheated to 50～80 ℃, as organic phase; A certain amount of magnetic fluid is joined in the agarose solution, heating for dissolving in 60～100 ℃ of water-baths, and be transferred to rapidly in the organic phase, after stirring fast, be cooled to room temperature rapidly; Clean with ether, distilled water at last, after screening is handled, standby in 2～8 ℃ of preservations;
3) preparation of polyvinyl alcohol magnetic microsphere:
In container, add gasoline, tetracol phenixin and Span-80 mixing, as organic phase; Polyvinyl alcohol solution with distilled water preparation 5～20% adds a certain amount of magnetic fluid, ice bath 5～20 minutes, add glutaraldehyde solution and 1N HCl, pour into rapidly behind the mixing in the organic phase, after fully stirring, slowly be warming up to 60～80 ℃ and continue reaction 1～5 hour again; Use ether, distilled water drip washing at last, after screening is handled, standby in 2～8 ℃ of preservations;
4) preparation of cellulose magnetic microsphere:
In a certain amount of cellulose materials, add 10～30%NaOH solution, soaked 1～4 hour, squeeze lixiviating liquid, 10～50 ℃ aging down, adds a certain amount of dithiocarbonic anhydride mixing, sealing, continue reaction 2～10 hours after 10～50 ℃, add 2～20%NaOH solution again, 10～50 ℃ of oscillatory reactions 2～10 hours obtain orange-red xanthate viscose; Add chlorobenzene, tetracol phenixin and potassium oleate in container, 10～50 ℃ of water-baths behind the stirring and evenly mixing, add a certain amount of xanthate viscose that is dissolved with magnetic fluid, after fully stirring, are warming up to 75～100 ℃, continue reaction 1～4 hour again; Use ether, distilled water drip washing at last, after screening is handled, standby in 2～8 ℃ of preservations.
CN 03144274 2003-09-15 2003-09-15 Immunization magnetic separation technology for purifying genetic engineering recombinant interferon Expired - Fee Related CN1229389C (en)

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CN101274985B (en) * 2008-05-12 2011-04-20 武汉大学 Magnetic cellulose microsphere, preparation thereof and use thereof
CN101782578B (en) * 2009-01-21 2013-02-06 王树森 Preparation method and application of immunomagnetic microspheres coated with staphylococal protein
CN104475041A (en) * 2014-10-22 2015-04-01 哈尔滨工业大学 A novel method of preparing agarose magnetic microspheres and uses of the agarose magnetic microspheres in separation and purification of an IgG antibody
CN105170042B (en) * 2015-09-17 2017-10-20 湖州师范学院 A kind of method of the antibody separation based on magnetic microsphere
CN108414305B (en) * 2018-01-24 2020-08-04 广州市丰华生物工程有限公司 Sample processing method and treating agent for determination of tuberculosis infection T cells
CN108287235B (en) * 2018-02-07 2021-03-09 常州天地人和生物科技有限公司 Preparation and application of efficient and stable magnetic immune microspheres

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