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Method for purifying composite interferon

A technology of compound interferon and purification method, which is applied in the field of one-step purification of recombinant compound interferon with cation exchange medium, can solve the problems of low purity, uneven product structure and the like, achieves simple steps, saves process and operator, and is easy to use. The effect of promotion

Inactive Publication Date: 2006-06-14
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to overcome the defects of the existing compound interferon purification method that the purity of the purified product is not very high and the structure of the purified product is not uniform, so as to provide a kind of protein that can not only completely remove the impurity protein with a molecular weight of 30,000 to 40,000, but also A purification method for compound interferon that can basically separate the renatured intermediate from the fully renatured protein, so that the resulting product is not only of high purity, but also has a more uniform product structure

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  • Method for purifying composite interferon

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Embodiment l

[0022] Embodiment 1, the production of composite interferon

[0023] The unpurified compound interferon used in the present invention is obtained by artificially constructing the compound interfering gene by conventional methods and fermenting the host cell containing the DNA sequence expression vector. The specific fermentation process is as follows:

[0024] Escherichia coli BL21 was selected as the host bacteria, and the strains were stored at -80°C. Pick compound interferon engineering bacteria (provided by researcher Tang Hong, Institute of Microbiology, Chinese Academy of Sciences) to streak on ammonia-resistant LB plates, and culture them in a greenhouse at 37°C for 12-24 hours. Pick a single colony, inoculate in 3 ml of liquid LB medium (ammonia, 1-50 mg / ml), culture on a shaker at 37°C at a speed of 240 rpm for 12-24 hours, measure OD 600 When it is 1.0-2.0, it is transferred to the seed solution twice, cultivated under the same conditions, and the OD 600 When it re...

Embodiment 2

[0029] Embodiment 2, the purification of recombinant composite interferon

[0030] The compound interferon with a purity of 65% obtained by fermenting and collecting inclusion body denaturation by conventional methods in Example 1 was added buffer D (100mM Tris+0.5M arginine+0.2mM EDTA (ethylenediamine disodium tetraacetate), adjust the pH to 8.3 with HCl), the volume ratio of the added buffer solution D to the composite interferon is 40:1, and then the mixed solution is allowed to stand overnight.

[0031] Then put this mixed solution into a dialysis bag with a molecular weight of 8000, and dialyze in buffer E (25mM Tris-HCl, pH8.3) for 12 hours to perform renaturation of the composite interferon protein. The volume ratio is 5:1; the white aggregates are removed by centrifugation or filtration, and the supernatant is separated, which is the solution of refolded compound interferon. The concentration of refolded protein is 0.3mg / ml. The protein purity is 90%, and the protein ...

Embodiment 3

[0034] Embodiment 3, the purification of recombinant composite interferon

[0035] Composite interferon with a purity of 65% obtained by fermenting and collecting the inclusion bodies denatured by conventional methods in Example 1, adding buffer D (100mM Tris+0.5M arginine+0.2mM EDTA, adjusted with HCl) while stirring pH to 8.3), the volume ratio of the added buffer D and composite interferon is 100:1, and then the mixed solution is allowed to stand overnight;

[0036] Then put this mixed solution into a dialysis bag with a molecular weight of 50,000, and dialyze in buffer E (25mM Tris-HCl, pH8.3) for 24 hours to perform renaturation of the complex interferon protein. The volume ratio is 20:1; centrifuge or filter to remove white aggregates, separate the supernatant, which is the solution of refolded compound interferon, the concentration of refolded protein is 0.8mg / ml, and the compound interfered by SDS-PAGE The protein purity is 90%, and the protein refolding rate is 70%. ...

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Abstract

The invention provides a method for purifying a compound interferon, which comprises the following steps: adding buffer D to the compound interferon obtained by denaturing the inclusion bodies fermented and collected by conventional methods, adding buffer D while stirring, and then adding the compound interferon that can permeate the molecular weight below 50000 In the dialysis bag, dialyze in the buffer E to carry out the renaturation of the complex interferon protein; separate the supernatant, and use buffer F and buffer G gradient elution on the chromatographic column equipped with strong cation exchange medium, Obtain purified compound interferon. The method can not only completely remove impurity proteins with a molecular weight of 30,000 to 40,000, but also basically separate the refolded intermediates from the fully refolded proteins, so that the obtained compound interferon has high purity and a more uniform product structure.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a preparation process of recombinant composite interferon (Consensus interferon, IFN-α-conl), in particular to a method for one-step purification of recombinant composite interferon by using a cation exchange medium. Background technique [0002] Interferon (IFN, Interferon) is a general term for a class of cytokines with broad-spectrum anti-virus, anti-proliferation and immune regulation, which can activate natural killer cells (NK cells) and other functions. It is a low-molecular-weight glycoprotein with similar structure, similar function and biological activity produced by cells induced by various inducers, and is an important class of cytokines. Interferons are generally divided into type I and type II. Type I includes IFN-α, IFN-β and IFN-ω, and IFN-α can be divided into more than 25 subtypes with different primary structures; type II includes IFN- γ . These interferons are enc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/555C07K1/14
Inventor 刘永东李京京王芳薇苏志国
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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