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Preparation method of recombinant human alpha interferon

A technology of interferon-alpha and interferon-alpha, applied in the field of preparation of recombinant human interferon-alpha, which can solve the problems of large refolding container, long time-consuming dialysis and high production cost

Inactive Publication Date: 2006-12-13
SHENYANG SUNSHINE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the common problems in the production of recombinant protein are: (1) low renaturation efficiency
Dilution renaturation method will dilute the sample dozens of times or even hundreds of times, which will greatly increase the volume of the sample, which will bring great difficulties to the subsequent separation and purification, and a larger renaturation container is required in the renaturation process
Dialysis is time-consuming and requires multiple changes of the dialysis solution
The common disadvantage of these two methods is that the protein will aggregate during the renaturation process and produce a large amount of precipitation, and the renaturation efficiency is low. Usually, the activity recovery rate of the protein is only 5-20%, and the protein solution after renaturation contains a large amount of Miscellaneous protein, need further separation and purification
(2) High production cost and large equipment investment
However, the method of simultaneously refolding and purifying recombinant human alpha interferon by metal chelation chromatography has not been reported yet.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: fermentation

[0032] The following steps relate to the method of fermentation and induction of recombinant human α2a interferon.

[0033] Material:

[0034] The medium consists of the following (per 1L):

[0035] Peptone 12g

[0036] Yeast Extract 24g

[0037] Glycerin 4ml

[0038] K H 2 PO 4 2.31g

[0039] K 2 HPO 4 12.54g

[0040] 50% MgSO 4 .7H 2 O 4ml

[0041] Add an appropriate amount of purified water to 1L.

[0042] E. coli strain is DH5αpETIFN.

[0043] Vaccination:

[0044] Take one tube of freeze-dried DH5αpETIFN and suspend it in 1ml of the above medium. The suspension is coated on LB plates, cultured at 37°C for 35-50 hours, then inoculated in the above-mentioned medium containing 50 μg / ml ampicillin, placed on a shaker at 100-300 rpm, cultured at 37°C for 5-10 hours, and then expanded to Inoculate the required amount of the fermenter.

[0045] Fermentation is carried out under the following con...

Embodiment 2

[0057] Example 2: Extraction and purification of inclusion bodies

[0058] Material:

[0059] Lysis solution: 0.05mol / L Tris.HCl-0.001mol / L EDTA-0.001mol / L DTT (pH7.5)

[0060] Denaturing solution: 0.05mol / L Tris.HCl-8mol / L urea-0.001mol / L DTT (pH7.5)

[0061] Cells were lysed by sonication. Add lysate to the lysed cell suspension. Centrifuge at low temperature (2-10° C.) at high speed (14000 rpm) for 30 minutes, discard the supernatant, and suspend the precipitate with lysate. Repeat the centrifugation and suspension steps 3 times. The obtained inclusion bodies were dissolved with a denaturing solution.

Embodiment 3

[0062] Example 3: Refolding and purification of inclusion bodies

[0063] Materials and parameters:

[0064] Wavelength: 280nm

[0065] Chromatography medium: Copper Chelate Sepharose

[0066] Column sample: dissolved inclusion bodies, the volume does not exceed 10 times the column volume

[0067] Flow rate: 1cm / min

[0068] Buffer A: 0.05mol / L Tris.HCl-8mol / L Urea-0.001mol / L DTT (pH7.5)

[0069] Buffer B: 0.05mol / L Tris.HCl-0.05mol / L Urea-0.005mol / L Glutathione (pH7.5)

[0070] Buffer C: 0.15mol / L NaCl (pH2.5)

[0071] The dissolved inclusion bodies are loaded into a metal chelate chromatography column. Wash and equilibrate the column with Buffer A. Then use a linear gradient of 15 times the column volume to transition from buffer A to buffer B, and finally use buffer C to elute the protein. Elution peaks were collected by measuring absorbance at 280 nm.

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Abstract

The invention discloses a method for preparation of interferon. The method includes extracting inclusion body after the fermentation of bacillus coli having coding interferon alpha ribonucleotide sequence, applying affinity tomographic column to perform reclamation and purification for the inclusion body, then performing ion-exchange chromatography, anti-phase high performance liquid chromatography and gel exclusion chromatography to obtain high-purity recombination alpha interferon.

Description

technical field [0001] The invention relates to a preparation method of interferon, in particular to a method for preparing recombinant human alpha interferon by applying the method of renaturation on a column. Background technique [0002] Interferon is a small molecular polypeptide secreted by cells, which has broad-spectrum anti-virus, anti-tumor and immune regulation and other biological activities. It is one of the biotherapeutic drugs with relatively broad application prospects today. It is the first cytokine that has been produced in large-scale commercial production using genetic engineering technology and has been verified by long-term clinical application so far. According to the biochemical characteristics of interferon and the different roles it plays in the body's immunity, it is divided into three categories: α, β, and γ. The most widely used interferon on the market is recombinant human alpha interferon. [0003] Methods for producing interferon alpha by bac...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/70C12N15/21C12P21/02C07K1/14
Inventor 陆军赵会林娄竞
Owner SHENYANG SUNSHINE PHARMA
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