Method for producing recombinant human interferon beta 1b protein through large-scale fermentation

A recombinant human interferon and a fermentor technology, which is applied in the field of large-scale fermentation production of recombinant human interferon beta 1b protein, can solve the problems of inability to use beta interferon for industrialized production, low yield of fermentation products, etc., and achieves improved bacterial harvest. The effect of improving fermentation yield and high-efficiency expression

Active Publication Date: 2017-08-29
BEIJING BIOLOGICAL PROD INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Interferon-beta drugs are genetically engineered protein drugs, and their industrialization needs to first solve the problem of large-scale fermentation production. However, the fermentation research on interferon-beta protein in the existing technology has only reached the pilot test level at most, and the scale is basically using a 50L fermenter. Complete the fermentation of 30L culture, the yield of fermentation products is low, and it cannot be applied to the industrial production of beta interferon

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. Prepare 300L 2YT medium in a 500L fermenter, sterilize at 121°C and cool down to 35°C.

[0019] 2. Inoculate 15L of mature recombinant Escherichia coli seed solution for cultivation. During the entire fermentation and cultivation stage, the temperature is maintained at 35±1°C, the rotation speed is 200r / min, the ventilation rate is 200L / min, and the pH is 5.0.

[0020] 3. During the fermentation and cultivation process, take samples regularly to measure the OD600 value of the cell density of the bacterial liquid; cultivate until the OD600 of the cell density reaches 2, and then add isopropylthiogalactopyranoside (IPTG) to a final concentration of 0.2mM for induction.

[0021] 4. Start feeding after induction, and the feeding medium is also 2YT medium. Feeding rate Feed-feeding was carried out at a rate of 5% of the original medium volume per hour. Continue to cultivate for 4 hours and the fermentation ends.

[0022] 5. Sampling was used to determine the expression ...

Embodiment 2

[0024] 1. Prepare 300L 2YT medium in a 500L fermenter, sterilize at 121°C and cool down to 37°C.

[0025] 2. Inoculate 30L of mature recombinant Escherichia coli seed solution for cultivation. During the entire fermentation and cultivation stage, the temperature is maintained at 37±1°C, the rotation speed is 300r / min, the ventilation rate is 300L / min, and the pH is 8.0.

[0026] 3. During the fermentation and cultivation process, take samples regularly to measure the OD600 value of the cell density of the bacterial liquid; cultivate until the OD600 of the cell density reaches 8, and then add isopropylthiogalactopyranoside (IPTG) to a final concentration of 0.8mM for induction.

[0027] 4. Start feeding after induction, and the feeding medium is also 2YT medium. Feeding rate Feed-feeding was carried out at a rate of 10% of the original medium volume per hour. Continue to cultivate for 2 hours and the fermentation ends.

[0028] 5. Sampling was used to determine the expression...

Embodiment 3

[0030] 1. Prepare 300L 2YT medium in a 500L fermenter, sterilize at 121°C and cool down to 40°C;

[0031] 2. Inoculate 45L of mature recombinant Escherichia coli seed solution for cultivation, and maintain the temperature at 40±1°C, rotating speed at 500r / min, ventilation rate at 500L / min, and pH at 7.0 during the entire fermentation period;

[0032] 3. During the fermentation and cultivation process, take samples regularly to measure the OD600 value of the cell density of the bacterial liquid; cultivate until the cell density OD600 reaches 5, and then add isopropylthiogalactopyranoside (IPTG) to a final concentration of 1.5mM for induction.

[0033] 4. Start feeding after induction, and the feeding medium is also 2YT medium. The feeding rate was fed by feeding at a rate of 1% of the original medium volume per hour. Continue to cultivate for 8 hours and the fermentation ends.

[0034] 5. Sampling was used to determine the expression level of the bacteria by SDS-PAGE electrop...

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Abstract

The invention provides a method for producing recombinant human interferon beta 1b protein through large-scale fermentation. The method includes: preparing a 2YT culture medium in a fermentation tank, and sterilizing before cooling to 35-40 DEG C; inoculating an Escherichia coli engineering strain containing recombinant human interferon beta 1b gene plasmid for fermentation culture, and controlling fermentation temperature to 35-40 DEG C, rotating speed to 200-500r/min, ventilation quantity to 200-500L/min and pH to 5.0-8.0; after thallus density OD600 reaches about 2-8 through culture, adding isopropyl thiogalactoside (IPTG) until final concentration is 0.2-1.5mM, or adding lactose in batches until the final concentration is 2-10g/L for induction; after induction, performing flow feeding at a rate of supplementing 1-10% of original volume of the culture medium per hour, and continuing culture for 2-8h to obtain thalli. The method is a large-scale fermentation method which is stable and efficient in expressing the recombinant human interferon beta 1b protein.

Description

technical field [0001] The invention relates to a large-scale fermentation method for producing recombinant human interferon β1b protein, more specifically, a large-scale fermentation method for stably and efficiently expressing recombinant human interferon β1b protein. Background technique [0002] Human beta interferon has antiviral and immunoregulatory effects, and its gene is located on human chromosome 9. Natural human beta interferon is a glycoprotein with a molecular weight of 20KD composed of 166 amino acids. Genetically engineered interferon-beta has been approved by the FDA for the treatment of multiple sclerosis (MS), a neurological disease related to immune response, and the treatment of interferon-beta is based on its role in immune response. In addition, beta interferon also has strong antiviral infection and antitumor effects, and has obvious curative effects on hepatitis B, hepatitis C and other viral diseases clinically. [0003] Interferon-beta drugs are g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12R1/19
CPCC07K14/565
Inventor 杨云凯马昱朱鑫硕韩凝刘明张雅博周玉
Owner BEIJING BIOLOGICAL PROD INST CO LTD
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