A method for producing recombinant human interferon beta 1b protein by large-scale fermentation

A recombinant human interferon and fermenter technology, which is applied in the field of large-scale fermentation and production of recombinant human interferon β1b protein, can solve the problems of low yield of fermentation products and the inability to apply β interferon industrial production, etc., to increase fermentation yield, Improve the effect of cell harvest and high-efficiency expression

Active Publication Date: 2021-05-07
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 by large-scale fermentation. The method comprises: preparing 2YT medium in a fermenter, and cooling down to 35-40°C after sterilization; inoculating a plasmid containing recombinant human interferon beta 1b gene Escherichia coli engineering bacteria fermentation culture, the fermentation temperature is controlled at 35-40°C, the rotation speed is 200-500r / min, the ventilation rate is 200-500L / min, and the pH is 5.0-8.0; to be cultivated until the cell density OD600 reaches about 2-8 Afterwards, add isopropylthiogalactoside (IPTG) to a final concentration of 0.2-1.5mM, or add lactose in batches to a final concentration of 2-10g / L for induction; after induction, supplement the volume of the original culture medium by 1-1. Fed-batch feeding was carried out at a rate of 10%, and the culture was continued for 2-8 hours to harvest the bacteria. The method of the invention is a large-scale fermentation method for stably and efficiently expressing 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 Patents(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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