Immunization magnetic separation technology for purifying genetic engineering recombinant interferon

An immunomagnetic separation and recombinant interferon technology, applied in the direction of interferon, cytokines/lymphokines/interferons, animal/human proteins, etc., can solve the problems of increasing the risk of the process, and achieve simplified separation and purification steps, high efficiency The effect of separation and purification, easy operation

Inactive Publication Date: 2005-11-30
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Immunization magnetic separation technology for purifying genetic engineering recombinant interferon
  • Immunization magnetic separation technology for purifying genetic engineering recombinant interferon
  • Immunization magnetic separation technology for purifying genetic engineering recombinant interferon

Examples

Experimental program
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Effect test

preparation example Construction

[0013] 1. Preparation of anti-interferon IgG antibody:

[0014] 1) Preparation of Anti-IFN serum:

[0015] 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 hour...

Embodiment 1

[0036] 1. Preparation of anti-IFNα-2b antibody IgG:

[0037] 1. Preparation of antiserum:

[0038] 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 it into two rabbits (Japanese big-eared white, male, body weight 2Kg) at multiple points intramuscularly and subcutaneously. 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.

[0039] 2. Purification of anti-IFNα-2b IgG antibody:

[0040] Take 10 mL ...

Embodiment 2

[0052] 1. Preparation of cellulose immunomagnetic microspheres:

[0053] 1. Preparation of cellulose magnetic microspheres:

[0054] 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 magne...

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Abstract

The invention relates to an isolation process for recombination protein in gene engineering, in particular a process for isolating purification gene engineering recombination interferon (IFN) by using immunity isolation technique, which belongs to the field of biological technology, wherein dissimilar materials are employed for preparation of magnetic micro-balloons containing hydroxyl, after epichlorohydrin activating, active amino is introduced and glutaric dialdehyde is used as link arm for joining the antiinterferon antibody.

Description

technical field [0001] 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 [0002] 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 bil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14C07K14/555
Inventor 白钢杨文博陈家琪曹宇曹学琳张磊
Owner NANKAI UNIV
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