CN1229389C - Immunomagnetic Separation Technology of Purifying Gene Engineering Recombinant Interferon - Google Patents

Abstract

The invention relates to a separation technology of genetic engineering recombinant protein, in particular to the separation and purification of genetic engineering recombinant interferon (interferon, IFN) by using immunomagnetic separation technology, and belongs to the field of biotechnology. The present invention uses different materials to prepare magnetic microspheres containing hydroxyl groups. After being activated by epichlorohydrin, active amino groups are introduced, and glutaraldehyde is used as a linking arm to connect with anti-interferon antibodies to obtain an immunomagnetic microsphere that can be used for the separation and purification of interferon. Microspheres. The immunomagnetic microspheres and magnetic separation device were used to separate and purify the fermented cell lysate of genetically engineered recombinant interferon, the activity recovery rate was over 50%, and the specific activity of the purified interferon was higher than 1.0×10 ⁸ IU/mg, The purity of SDS-PAGE gel electrophoresis analysis was close to 100%.

Description

Immunomagnetic Separation Technology of Purifying Gene Engineering Recombinant Interferon

technical field

The invention relates to a separation technology of genetically engineered recombinant protein, in particular to the purification of interferon by using the immunomagnetic separation technology, and belongs to the field of biotechnology.

Background technique

Interferon (Interferon, IFN) is a kind of regulatory protein produced by the body in response to exogenous stimuli, and has broad-spectrum antiviral, antitumor and immunomodulatory activities. Clinically, it has a certain curative effect on viral hepatitis, herpes zoster, lymphoma, leukemia, etc. Gene recombinant human interferon α-2b (α-2bIFN), as one of the earliest gene recombinant protein drugs, was launched in Europe and America in 1986 (Schering-Plough Company, USA). In 1990, through independent research and introduction of foreign technology in China, many enterprises successively put into commercial production. At present, the annual sales in the domestic market have reached 1 billion yuan, and the annual sales in the United States have exceeded 2 billion US dollars. Recombinant human interferon is mainly produced by the fermentation of genetically engineered bacteria. It is an active expression product in cells. The separation and purification process is complicated. gel filtration chromatography etc. Due to the many steps in the separation process, the product yield is low and the cost is high. At present, some companies usually use affinity chromatography to improve purification efficiency and specificity, but prior to affinity chromatography, salting-out, hydrophobic or ion-exchange chromatography must be performed to obtain higher purification efficiency. In addition, highly toxic cyanogen bromide is often used as a coupling agent for the connection between the affinity chromatography medium and the specific antibody, which increases the risk of the process. At present, all enterprises at home and abroad are committed to the development of new technologies for the separation and purification of interferon and the update of production processes.

Contents of the invention

The purpose of the present invention is to open up a new approach for the separation and purification of protein genetic engineering products, which involves the preparation, activation and sensitization of magnetic microspheres for purifying genetic engineering recombinant interferon, and the preparation of magnetic separation devices.

The present invention uses agarose, polyvinyl alcohol, cellulose and other materials to prepare magnetic microspheres with hydroxyl groups on the surface, activates the microspheres with epichlorohydrin, introduces active amino groups, and uses glutaraldehyde as the connecting arm Link with anti-interferon IgG antibody to obtain immunomagnetic microspheres that can be used for separation and purification of interferon. The anti-interferon IgG antibody used may be a purified anti-interferon monoclonal or polyclonal IgG antibody.

Use the above-mentioned immunomagnetic microspheres and magnetic separation device to carry out immunomagnetic separation on the fermented cell lysate or pretreated cell lysate of interferon, the activity recovery rate exceeds 50%, and the specific activity of purified human interferon (IFN) Higher than 1.0×10 ⁸ IU/mg, the purity of SDS-PAGE gel electrophoresis analysis is close to 100%, which meets the national standard. The animal-derived antibody content in the purified sample is lower than the national requirement of 100ng per dose.

The beneficial effects of the present invention are: using the immunomagnetic separation technology to purify the genetically engineered recombinant interferon (IFN), the operation is simple and fast, and there is no need to perform complicated pretreatment on the sample, and it can be directly purified in one step from the lysate of fermented cells The high-purity target protein is obtained, the steps of separation and purification are greatly simplified, and the product yield is improved. The separation and purification conditions are mild, and the immunomagnetic microspheres can be reused more than 10 times. The invention is expected to replace the immunoaffinity chromatography column and become a new technology for fast and efficient separation and purification of interferon.

Description of drawings

Fig.1 Scanning electron micrograph of magnetic microspheres

Among them, picture A shows agarose magnetic microspheres (×1500); picture B shows cellulose magnetic microspheres (×1200); picture C shows polyvinyl alcohol magnetic microspheres (×300).

Figure 2 Schematic diagram of magnetic separation device

Wherein (1) a columnar reaction sealed container; (2) immunomagnetic microspheres; (3) a magnet; (4) a liquid outlet valve at the end; (5) a reaction liquid; (6) an upper vent valve; (7) a collection container.

Figure 3 SDS-PAGE gel electrophoresis analysis of interferon

The conditions of each lane in the figure are: lane a: standard molecular weight protein; lane b: lysate of α-2b interferon fermented bacteria; lane c: lysate of bacteria treated with thiamine precipitation; lane d: agarose immunomagnetic microspheres Separation results; lane e: separation results of cellulose immunomagnetic microspheres; lane f: separation results of polyvinyl alcohol immunomagnetic microspheres.

Detailed ways

1. Preparation of anti-interferon IgG antibody:

1) Preparation of Anti-IFN serum:

Take human interferon (IFN) standard substance, fully mix and emulsify with an equal volume of Freund's complete adjuvant to obtain human interferon (IFN) antigen, take human interferon (IFN) antigen, and treat 2 Japanese white-eared males respectively Rabbits (body weight 2Kg) were intramuscularly and subcutaneously injected at multiple points. The additional injection was prepared by emulsifying the antigen with Freund's incomplete adjuvant, and the booster injection was performed once every two weeks, for a total of three times, with the dose decreasing. Three days after the last immunization, blood was taken from the vein, and the antibody titer was determined by indirect ELISA method. The neutralizing titer of the antibody in the serum was measured to be greater than 1:10000. After that, the abdominal artery was bled, and the whole blood was left to stand for 2 hours and centrifuged at 3000rpm for 20 minutes. , collect the serum and inactivate it in a water bath at 55°C for 30 minutes, then freeze it at -20°C after aliquoting;

2) Purification of Anti-IFN IgG antibody:

Take Anti-IFN serum, add an equal volume of PBS to mix well, add an equal volume of saturated ammonium sulfate to precipitate, centrifuge to collect the precipitate, dissolve the precipitate in PBS, and dialyze overnight at 4°C to obtain crude anti-IFN immune spheres For the protein, the crude immunoglobulin was subjected to gradient elution using DEAE-cellulose 52 ion exchange chromatography, and the combined IgG elution peaks were collected, dialyzed with distilled water, and freeze-dried to obtain a refined rabbit anti-interferon IgG polyclonal antibody.

2. Preparation of immunomagnetic microspheres:

Magnetic microspheres, including agarose magnetic microspheres and cellulose magnetic microspheres prepared by reverse-phase physical embedding method, polyvinyl alcohol magnetic microspheres prepared by reverse-phase suspension cross-linking method and other magnetic microspheres containing active hydroxyl groups; The preparation process includes the following processes:

1) Preparation of magnetic fluid:

Dissolve FeCl ₂ 4H ₂ O in deionized water, filter through a microporous membrane to remove impurities, then add a certain amount of polyethylene glycol and hydrogen peroxide, mix well, adjust the pH to 9~11 with NaOH solution, and set the temperature at 50~80℃ React in a water bath for 3 to 6 hours to obtain a stable suspension of brown-black magnetic colloidal particles, that is, magnetic fluid;

2) Preparation of agarose magnetic microspheres:

Take toluene, carbon tetrachloride and Span-80, preheat to 60 ~ 80 ℃, as the organic phase; add a certain amount of magnetic fluid to the agarose solution, heat to dissolve in a boiling water bath, and quickly transfer to the organic phase After reacting for 5-30 minutes under stirring at 2000-5000rpm, cool down to room temperature rapidly; finally wash with ether and distilled water, sieve the agarose magnetic microspheres with a particle size between 100-300μm, and store them at 2-8°C spare.

3) Preparation of polyvinyl alcohol magnetic microspheres:

Mix 200 ^# gasoline, carbon tetrachloride and Span-80 as the organic phase; prepare a certain concentration of polyvinyl alcohol solution, add a certain amount of magnetic fluid, ice bath for 5 to 20 minutes, add glutaraldehyde solution and hydrochloric acid, After mixing, quickly pour into the organic phase, disperse at 2000-5000rpm for 0.5-2 hours, slowly raise the temperature to 60-80°C and continue the reaction for 1-3 hours; finally rinse with ether and distilled water, and sieve the particle size at 100 Polyvinyl alcohol magnetic microspheres between ～300 μm, stored at 2～8 ℃ for later use;

4) Preparation of cellulose magnetic microspheres:

Add NaOH solution to a certain amount of absorbent cotton, soak for 1-4 hours, squeeze out the lye, after aging at 10-30°C, add a certain amount of carbon disulfide and mix well, continue to react for 2-8 hours after sealing, and then add some Concentrated NaOH solution was reacted for 5 to 10 hours to obtain orange-red xanthate viscose; take chlorobenzene, carbon tetrachloride and potassium oleate, stir and mix in a water bath at 20 to 50°C, and then add a certain amount of dissolved Xanthate viscose solution of magnetic fluid, disperse at 2000-5000rpm for 20-60 minutes, then heat up to 75-100°C, and continue to react for 1-4 hours; finally rinse with ether and distilled water, and sieve the particle size at Cellulose magnetic microspheres between 100-300 μm are stored at 2-8°C for later use.

5) Activation of magnetic microspheres:

Take a certain amount of magnetic microspheres, add a certain volume of epichlorohydrin under alkaline conditions, activate at 25-40°C for 2-4 hours, add 2-20% ammonia water after washing the epichlorohydrin, and react at room temperature After 3-9 hours, fully rinse and then add 2-20% glutaraldehyde solution, react at room temperature for 1-5 hours, and finally wash off excess glutaraldehyde with distilled water.

6) Sensitization of magnetic microspheres:

At 2-8°C, take a certain amount of activated magnetic microspheres, add NaHCO ₃ -NaCO ₃ buffer solution containing anti-interferon monoclonal or polyclonal IgG antibodies, fully react, rinse with PBS, add NaBH _4. Perform reduction, and then wash with PBS to obtain immunomagnetic microspheres that can be used to purify interferon (IFN), and the antibody coupling amount is 3-15 mg/mL spheres.

3. Immunomagnetic separation of interferon (IFN):

Take the immunomagnetic microspheres and wash them thoroughly with PBS, then place them in the container of the magnetic separation device; add the fermented cell lysate or pretreated lysate of the genetically engineered recombinant interferon (IFN) diluted with buffer solution, and in 2～ Slowly shake and react at 8°C for 3 to 24 hours. After the reaction, place the container on a magnetic separation device, absorb and fix the magnetic microspheres, discard the reaction solution, wash with PBS, and then add glycine-hydrochloric acid buffer (pH1.5 to 3.5) , slowly shaking and reacting at 2-8°C for 1-5 hours to dissociate the adsorbed interferon (IFN), then place the glass container on the separation device, absorb the magnetic microspheres, collect the dissociation solution, and immediately use high-concentration PB buffer (pH 6.5-8.0) is neutralized to obtain purified genetically engineered recombinant interferon (IFN). After washing with PBS, the immunomagnetic microspheres can be stored at 2-8°C and reused.

The immunological activity of purified interferon (IFN) was measured by ELISA method, and its biological activity was measured by cytostatic lesion method. The purity indexes of the purified interferon (IFN) and the content of animal-derived IgG all reached the national standard. The interferon (IFN) adsorption capacity of directly purified fermentation cell lysates and lysates pretreated by ammonium sulfate precipitation can reach 9.6×10 ⁵ IU/ml and 8.0×10 ⁵ IU/ml. Multiple purification experiments were carried out with immunomagnetic microspheres, the average activity recovery rate was greater than 50%, and the average specific activity was greater than 1.0×10 ⁸ IU/mg. SDS-PAGE gel electrophoresis analysis showed that the purity of interferon (IFN) purified by immunomagnetic separation was close to 100%.

Embodiment one.

1. Preparation of anti-IFNα-2b antibody IgG:

1. Preparation of antiserum:

Take 2 mL of 1 mg/mL IFNα-2b standard substance, mix well with an equal volume of Freund's complete adjuvant and emulsify to obtain IFNα-2b emulsified antigen. Take 2mL of the emulsified antigen of IFNα-2b, and inject multi-point intramuscularly and subcutaneously to 2 rabbits (Japanese big-eared white, male, body weight 2Kg). The additional injection was prepared by emulsifying the antigen with Freund's incomplete adjuvant, and the booster injection was performed once every two weeks, for a total of three times, with the dose decreasing. Six days after the last immunization, blood was collected from the abdominal artery. The whole blood was left to stand for 2 hours, centrifuged at 3000rpm for 20 minutes, and the serum was collected and inactivated in a water bath at 55°C for 30 minutes.

2. Purification of anti-IFNα-2b IgG antibody:

Take 10 mL of anti-IFNα-2b serum, add an equal volume of PBS and mix well, carry out fractional salting out with 50% and 33% saturated ammonium sulfate respectively, collect the precipitate by centrifugation, dissolve the precipitate in PBS, and dialyze overnight at 4°C to obtain Crude anti-IFNα-2b immunoglobulin. The crude immunoglobulin obtained from the above graded salting-out was subjected to DEAE-cellulose 52 ion-exchange chromatography, eluted with 0.02M PB buffer (pH 8.0), and the combined IgG fraction was collected, dialyzed with deionized water, and freeze-dried The purified rabbit anti-IFNα-2b (Anti-IFNα-2b) IgG antibody was detected as a single band by SDS-PAGE.

2. Preparation of agarose immunomagnetic microspheres:

1. Preparation of ferrofluid:

6 g of FeCl ₂ ·4H ₂ O was dissolved in 400 mL of deionized water, and filtered through a microporous membrane to remove impurities. Then add 150 grams of polyethylene glycol and 100 μL of 30% H ₂ O _{2 and} mix well, adjust the pH of the solution to 11 with 3M NaOH, and react in a water bath at 50-60°C for 4 hours to obtain a stable suspension of brown-black magnetic colloidal particles, that is, a magnetic fluid.

2. Preparation of agarose magnetic microspheres:

Add 75mL toluene, 30mL carbon tetrachloride and 1.2mL Span-80 to a 250ml Erlenmeyer flask, preheat to 70°C, as the organic phase; take 1 g of magnetic fluid, add it to 30mL 6% agarose solution, at 100°C Heat and dissolve in a water bath as the water phase. The aqueous phase was quickly transferred to the organic phase, stirred at 3000 rpm for 15 minutes, and then rapidly cooled to room temperature to obtain agarose magnetic microspheres. Rinse with diethyl ether and distilled water respectively, sieve the magnetic microspheres with a particle size ranging from 100 to 300 μm, and store them at 4°C for later use.

3. Activation and sensitization of agarose magnetic microspheres:

Take 1 mL of agarose magnetic microspheres, add 2 mL of 2M NaOH and 1 mL of epichlorohydrin, shake and react for 2 hours at 30 ° C, wash with acetone and distilled water after suction filtration. 2 mL of 10% ammonia water was added, and the reaction was allowed to stand at room temperature for 6 hours. After fully washing, add 2 mL of 5% glutaraldehyde, shake at room temperature for 2 hours, then wash off excess glutaraldehyde with distilled water to obtain activated agarose magnetic microspheres. Add 5 mL of 0.1 M NaHCO ₃ -NaCO ₃ buffer solution (pH 9.6) containing 15 mg of Anti-IFNα-2b polyclonal IgG antibody to 1 mL of the above-mentioned microspheres, and react overnight at 4°C. After fully washing with PBS, add 2mL of 0.5% NaBH ₄ for reduction, and then fully wash with PBS to obtain agarose immunomagnetic microspheres that can be used to purify IFNα-2b. The coupling amount of antibody was determined by Lowry method to be 12.8 mg/mL ball.

3. Immunomagnetic separation of α-2bIFN:

Take 1 mL of sensitized agarose magnetic microspheres and place them in the glass container of the magnetic separation device; take 1 mL of IFNα-2b fermentation cell lysate, centrifuge at 10,000 rpm for 10 minutes, and take 200 μL of supernatant in 10 mL of PBS (diluted 50 times), then added to a glass container, and reacted overnight at 4°C with slow shaking. After the reaction, the glass container was placed on a magnetic separation device, the magnetic microspheres were adsorbed and fixed, the reaction solution was discarded, and then washed 6 times with PBS. Add 4 mL of 0.1M glycine-hydrochloric acid buffer (pH 2.5) and shake at 4°C for 2 hours to dissociate the IFNα-2b adsorbed by the immunomagnetic microspheres. Place the glass container on the separation device, absorb the magnetic microspheres, collect the dissociated solution, and immediately neutralize it with a small amount of 1M PB buffer (pH 7.0) to obtain purified IFNα-2b. Immunomagnetic microspheres were washed with PBS and stored at 4°C for reuse.

The immunological activity of purified IFNα-2b was detected by ELISA method, and its biological activity was measured by cytostatic lesion method. Using sensitized agarose magnetic microspheres to directly purify the lysate of α-2bIFN fermented cells, repeated the experiment 3 times, the average adsorption capacity was 9.6×10 ⁵ IU/ml, the average activity recovery rate was 88.2%, and the average specific activity was 1.5 ×10 ⁸ IU/mg, analyzed by SDS-PAGE gel electrophoresis, the purity is close to 100%, all indicators and rabbit IgG content have reached the national standard, and showed good reproducibility.

Embodiment two.

1. Preparation of cellulose immunomagnetic microspheres:

1. Preparation of cellulose magnetic microspheres:

Take 100g of absorbent cotton soaked in 800mL of 19% NaOH solution for 2 hours, squeeze out the lye, and age at 25°C for 3 days; add 50mL of carbon disulfide and mix well, seal and shake at 28°C for 5 hours, then add 750-850mL of 6% NaOH solution to mix Evenly, shake at 28°C for 5 hours to obtain orange-red xanthate viscose. Add 180mL of chlorobenzene, 30mL of carbon tetrachloride and 0.5g of potassium oleate into a 500ml three-neck flask, stir and mix in a water bath at 40°C as the organic phase. Take 70 mL of xanthate viscose solution containing 1 gram of magnetic fluid, add it to the organic phase, disperse at 3000 rpm for 30 minutes, then rapidly raise the temperature to 90° C., and continue to react for 2 hours to obtain cellulose magnetic microspheres. Rinse with ether and distilled water respectively, sieve and screen the cellulose magnetic microspheres with a particle size between 100 and 300 μm, and store them at 4°C for later use.

2. Activation and sensitization of cellulose magnetic microspheres:

Take 1 mL of cellulose magnetic microspheres, add 2 mL of 3M NaOH and 1 mL of epichlorohydrin, shake and react at 40°C for 2.5 hours, wash with acetone and distilled water after suction filtration. 4 mL of 5% ammonia water was added, and the reaction was allowed to stand at room temperature for 9 hours. After fully washing, add 5 mL of 2% glutaraldehyde, shake at room temperature for 5 hours, and then wash off excess glutaraldehyde with distilled water to obtain activated cellulose magnetic microspheres. Add 5 mL of 0.1M NaHCO ₃ -NaCO ₃ buffer solution (pH 9.6) containing 12 mg of Anti-IFNα-2bIgG polyclonal antibody to 1 mL of the above-mentioned microspheres, and react overnight at 4°C. After being fully washed with PBS, 2 mL of 0.5% NaBH ₄ was added for reduction, and then fully washed with PBS to obtain cellulose immunomagnetic microspheres that can be used for purifying IFNα-2b. The coupling amount of the antibody was determined by the Lowry method to be 11.8 mg/mL ball.

2. Immunomagnetic separation of α-2bIFN:

Take 1mL of cellulose magnetic microspheres and place them in the glass container of the magnetic separation device; take 5mL of α-2bIFN fermentation cell lysate, centrifuge at 10,000rpm for 10 minutes, then take the supernatant and add an equal volume of PBS to mix, slowly add etc. Volume of pre-cooled saturated ammonium sulfate solution, mix well, place on ice bath, stand for 4 hours. Centrifuge at 6000rpm at 4°C for 15 minutes, discard the supernatant, dissolve the pellet in 5ml PBS, dialyze with 0.1M PBS, take 500μL dialysate in 10mL PBS (diluted 20 times), then add it to a glass container, and slowly Shake the reaction overnight. After the reaction, the glass container was placed on a magnetic separation device, the magnetic microspheres were adsorbed and fixed, the reaction solution was discarded, and then washed 5 times with PBS. Add 4 mL of 0.1M glycine-hydrochloric acid buffer (pH 2.5) and shake at 4°C for 2 hours to dissociate the IFNα-2b adsorbed by the immunomagnetic microspheres. Place the glass container on the separation device, absorb the magnetic microspheres, collect the dissociated solution, and immediately neutralize it with a small amount of 1M PB buffer (pH 7.0) to obtain purified IFNα-2b. Immunomagnetic microspheres were washed with PBS and stored at 4°C for reuse.

The immunological activity of purified IFNα-2b was detected by ELISA method, and its biological activity was measured by cytostatic lesion method. Using sensitized cellulose magnetic microspheres to purify the lysate of IFNα-2b fermented cells pretreated by thiamine precipitation, the experiment was repeated three times, the average adsorption capacity was 8.0×10 ⁵ IU/ml, and the average activity recovery rate was 50.3 %, the average specific activity is 1.4×10 ⁸ IU/mg, the purity is analyzed by SDS-PAGE gel electrophoresis, the purity is close to 100%, all the indicators and rabbit IgG content have reached the national standard, and have shown good reproducibility sex.

Embodiment three.

1. Preparation of polyvinyl alcohol immunomagnetic microspheres:

1. Preparation of polyvinyl alcohol magnetic microspheres:

Add 140mL 200 ^# gasoline, 110mL carbon tetrachloride and 5mL Span-80 into a 500ml three-necked bottle and mix well as the organic phase; prepare 50ml of 10% polyvinyl alcohol, add 0.6g of magnetic fluid and ice bath for 15 minutes, then add 25 % glutaraldehyde 5mL and 1N HCL 6mL; after mixing, quickly add to the organic phase, stir at 3000rpm for 30 minutes, then raise the temperature to 70°C, continue to stir and react for 2 hours to obtain polyvinyl alcohol magnetic microspheres. Rinse with ether and distilled water respectively, screen the polyvinyl alcohol magnetic microspheres with a particle size between 100 and 300 μm, and store them at 4°C for later use.

2. Activation and sensitization of polyvinyl alcohol magnetic microspheres:

Take 1ml of polyvinyl alcohol magnetic microspheres, add 10mL of dimethyl sulfoxide and 3mL of epichlorohydrin, slowly add 10mL of 2M NaOH dropwise at 40°C, continue to react for 4 hours, filter with suction, and wash with acetone and distilled water respectively. Add 1.5 mL of 15% ammonia water and let it stand at room temperature for 3 hours. After thorough washing, add 1 mL of 10% glutaraldehyde, shake at room temperature for 1 hour, and then wash off excess glutaraldehyde with distilled water to obtain activated polyvinyl alcohol magnetic microspheres. Add 5 mL of 0.1 M NaHCO ₃ -NaCO ₃ buffer solution (pH 9.6) containing 8 mg of Anti-IFNα-2b Mab IgG antibody to 1 mL of the above-mentioned microspheres, and react overnight at 4°C. After being fully washed with PBS, 2 mL of 0.5% NaBH ₄ was added for reduction, and then fully washed with PBS to obtain polyvinyl alcohol immunomagnetic microspheres that can be used for purifying IFNα-2b. The coupling amount of antibody determined by Lowry method was 3.7mg/mL ball.

2. Immunomagnetic separation of α-2bIFN:

Take 1mL of polyvinyl alcohol magnetic microspheres and place them in the glass container of the magnetic separation device; take 1mL of α-2bIFN fermentation cell lysate, centrifuge at 10000rpm for 10 minutes, take 100μL of supernatant in 10mL of PBS (diluted 100 times), Then it was added into a glass container and reacted overnight at 4°C with slow shaking. After the reaction, the glass container was placed on a magnetic separation device, the magnetic microspheres were adsorbed and fixed, the reaction solution was discarded, and then washed 6 times with PBS. Add 4 mL of 0.1M glycine-hydrochloric acid buffer (pH 2.5) and shake at 4°C for 2 hours to dissociate the IFNα-2b adsorbed by the immunomagnetic microspheres. Place the glass container on the separation device, absorb the magnetic microspheres, collect the dissociated solution, and immediately neutralize it with a small amount of 1M PB buffer (pH 7.0) to obtain purified IFNα-2b. Immunomagnetic microspheres were washed with PBS and stored at 4°C for reuse.

The immunological activity of purified IFNα-2b was detected by ELISA method, and its biological activity was measured by cytostatic lesion method. Using sensitized polyvinyl alcohol magnetic microspheres to directly purify the lysate of α-2bIFN fermented cells, repeated the experiment three times, the average adsorption capacity was 4.5×10 ⁵ IU/ml, the average activity recovery rate was 93.9%, and the average specific activity was 1.2×10 ⁸ IU/mg, analyzed by SDS-PAGE gel electrophoresis, the purity is close to 100%, all indicators and mouse IgG content reach the national standard, and show good reproducibility.

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.

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