CN107099569B - Method for large-scale fermentation production of recombinant human interferon beta 1b protein - Google Patents
Method for large-scale fermentation production of recombinant human interferon beta 1b protein Download PDFInfo
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Abstract
The invention provides a method for producing recombinant human interferon beta 1b by large-scale fermentation, which comprises the following steps: preparing a 2YT culture medium in a fermentation tank, sterilizing, and cooling to 35-40 ℃; inoculating an engineering bacterium of escherichia coli containing recombinant human interferon beta 1b gene plasmid, fermenting and culturing, controlling the fermentation temperature to be 35-40 ℃, the rotation speed to be 200-500 r/min, the ventilation volume to be 200-500L/min and the pH to be 5.0-8.0; after the culture is carried out until the thallus density OD600 reaches about 2-8, adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.2-1.5 mM, or adding lactose in batches to the final concentration of 2-10 g/L for induction; and after induction, feeding at a rate of 1-10% of the volume of the original culture medium per hour for feeding, and continuously culturing for 2-8 hours to obtain the thalli. The method is a large-scale fermentation method for stably and efficiently expressing the recombinant human interferon beta 1b protein.
Description
Technical Field
The invention relates to a method for producing recombinant human interferon beta 1b protein by large-scale fermentation, in particular to a large-scale fermentation method for stably and efficiently expressing recombinant human interferon beta 1b protein.
Background
The human beta interferon has antiviral and immunoregulatory effects, and its gene is located on chromosome 9 of human, and natural human beta interferon is a glycoprotein composed of 166 amino acids and with molecular weight of 20 KD. Genetically engineered interferon-beta has been approved by the FDA for use in the treatment of Multiple Sclerosis (MS), a neurological disease associated with an immune response upon which interferon-beta therapy is based. In addition, the beta interferon also has strong antiviral infection and antitumor effects, and has obvious clinical curative effects on hepatitis B, hepatitis C and other viral diseases.
The beta interferon medicine is used as a gene engineering protein medicine, the industrialization of the beta interferon medicine needs to solve the problem of large-scale fermentation production firstly, but the fermentation research on the beta interferon protein in the prior art only reaches the pilot-scale level to the maximum extent, the scale is basically to use a 50L fermentation tank to complete the fermentation of 30L culture, the yield of the fermentation product is low, and the beta interferon medicine cannot be applied to the industrialized production of the beta interferon.
Disclosure of Invention
One objective of the invention is to provide a method for producing recombinant human interferon beta 1b protein by large-scale fermentation.
To achieve the above object, the present invention provides a method for large-scale fermentation production of recombinant human interferon beta 1b protein, which comprises:
preparing a 2YT culture medium in a fermentation tank, sterilizing, and cooling to 35-40 ℃;
inoculating escherichia coli engineering bacteria containing recombinant human interferon beta 1b gene plasmids to a fermentation tank for fermentation culture, wherein the fermentation temperature is controlled to be 35-40 ℃, the rotation speed is controlled to be 200-500 r/min, the ventilation volume is controlled to be 200-500L/min, and the pH value is controlled to be 5.0-8.0;
monitoring the thallus density OD600 value of the bacteria liquid in the fermentation culture process; after the culture is carried out until the thallus density OD600 reaches about 2-8, adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.2-1.5 mM, or adding lactose in batches to the final concentration of 1-10 g/L for induction;
and after induction, feeding and supplementing materials (supplementing the original culture medium) at the rate of 1-10% of the volume of the supplemented original culture medium every hour, and continuously culturing for 2-8 hours to stop fermentation and harvest the thalli.
The method for producing the recombinant human interferon beta 1b protein can be beneficial to improving the expression level of the recombinant beta interferon.
According to the specific embodiment of the invention, the method can be scaled, and the volume of the fermentation tank is more than or equal to 200L and can reach more than 1000L.
According to a particular embodiment of the invention, in the method of the invention, the sterilization conditions of the 2YT medium are: sterilizing at 121 deg.C.
According to the specific embodiment of the invention, in the method, the inoculation amount of the escherichia coli engineering bacteria containing the recombinant human interferon beta 1b gene plasmid is 5-15%.
According to the specific embodiment of the invention, in the method, the content of the beta interferon protein in the harvested fermentation thallus accounts for more than 20% of the total protein of the thallus, the plasmid retention rate is 100%, and the yield of the inclusion bodies after the fermentation product is crushed reaches 250-705 g or even higher.
The method of the invention is a large-scale fermentation method for stably and efficiently expressing recombinant human interferon beta 1b protein, wherein a fed-batch fermentation mode is adopted, and a fed-batch culture medium with a certain concentration is added to ensure that the interferon beta 1b protein is efficiently expressed in engineering bacteria, so that the yield of bacteria and the expression quantity of the bacteria of the interferon beta protein are improved, the large-scale fermentation culture of escherichia coli engineering bacteria containing human interferon beta gene plasmids in a fermentation tank of more than 200L is realized, the fermentation process of the interferon beta is amplified, the fermentation yield is improved, and the foundation is laid for the industrialization of interferon beta medicines.
Detailed Description
The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure. The experimental procedures not specified in detail in the examples were carried out according to the routine procedures in the art. In the embodiment, the Escherichia coli containing recombinant human interferon beta 1b gene plasmid is constructed by Beijing Bioresearch biological products Limited according to the prior art, and the culture of mature recombinant Escherichia coli seed liquid is carried out according to the prior art.
Example 1
1. 300L of 2YT medium was prepared in a 500L fermenter, sterilized at 121 ℃ and then cooled to 35 ℃.
2. Inoculating 15L of mature recombinant Escherichia coli seed liquid for culturing, and culturing at the whole fermentation culture stage under the conditions of temperature of 35 + -1 deg.C, rotation speed of 200r/min, ventilation volume of 200L/min and pH of 5.0.
3. Sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the density OD600 of the cells reaches 2, and adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.2mM for induction.
4. After induction, feeding was started, the feed medium was likewise 2YT medium. The feed rate was fed at a rate of 5% of the volume of the original medium per hour. The fermentation was completed after further 4 hours of cultivation.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the beta interferon protein in the finally harvested fermentation thalli is 30%, the plasmid retention rate is 100%, and the yield of the inclusion body after the fermentation product is crushed is 385 g.
Example 2
1. 300L of 2YT medium was prepared in a 500L fermenter, sterilized at 121 ℃ and then cooled to 37 ℃.
2. Inoculating 30L of mature recombinant Escherichia coli seed liquid for culturing, and culturing at 37 + -1 deg.C, 300r/min rotation speed, 300L/min ventilation rate and 8.0 pH value in the whole fermentation culture stage.
3. Sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the density OD600 of the cells reaches 8, and adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.8mM for induction.
4. After induction, feeding was started, the feed medium was likewise 2YT medium. The feed rate was fed at a rate of 10% of the volume of the original medium per hour. The fermentation was completed by continuing the cultivation for 2 hours.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the beta interferon protein in the finally harvested fermentation thalli is 35 percent, the plasmid retention rate is 100 percent, and the yield of the inclusion body after the fermentation product is crushed is 525g
Example 3
1. Preparing 300L 2YT culture medium in 500L fermentation tank, sterilizing at 121 deg.C, and cooling to 40 deg.C;
2. inoculating 45L of mature recombinant Escherichia coli seed liquid for culturing, and culturing at 40 + -1 deg.C, 500r/min rotation speed, 500L/min ventilation rate and pH of 7.0 in the whole fermentation culture stage;
3. sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the density OD600 of the cells reaches 5, and adding isopropyl thiogalactoside (IPTG) to the final concentration of 1.5mM for induction.
4. After induction, feeding was started, the feed medium was likewise 2YT medium. The feed rate was fed at a rate of 1% of the volume of the original medium per hour. The fermentation is finished after 8 hours of continuous culture.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the beta interferon protein in the finally harvested fermentation thalli is 30%, the plasmid retention rate is 100%, and the yield of the inclusion bodies after the fermentation products are crushed is 475 g.
Example 4
1. Preparing 300L 2YT culture medium in 500L fermentation tank, sterilizing at 121 deg.C, and cooling to 35 deg.C;
2. inoculating 30L of mature recombinant Escherichia coli seed liquid for culture, and culturing at the whole fermentation culture stage under the conditions of temperature of 35 + -1 deg.C, rotation speed of 250r/min, ventilation capacity of 250L/min and pH of 7.0;
3. sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the thallus density OD600 reaches 4, and adding lactose in batches according to hours until the final concentration is 2g/L for induction.
4. After induction, feeding was started, the feed medium was likewise 2YT medium. The feed rate was fed at a rate of 2% of the volume of the original medium per hour. The fermentation was completed after further 6 hours of cultivation.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the beta interferon protein in the finally harvested fermentation thalli is 22%, the plasmid retention rate is 100%, and the yield of the inclusion body after the fermentation product is crushed is 258 g.
Example 5
1. Preparing 300L 2YT culture medium in 500L fermentation tank, sterilizing at 121 deg.C, and cooling to 38 deg.C;
2. inoculating 15L of mature recombinant escherichia coli seed liquid for culture, and culturing under the conditions that the temperature is maintained at 38 +/-1 ℃, the rotating speed is 350r/min, the ventilation volume is 350L/min and the pH value is 7.5 in the whole fermentation culture stage;
3. sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the thallus density OD600 reaches 4, and adding lactose to 10g/L for induction.
4. After induction, feeding was started, the feed medium was likewise 2YT medium. The feed rate was fed at a rate of 5% of the volume of the original medium per hour. The fermentation was completed after further 4 hours of cultivation.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the beta interferon protein in the finally harvested fermentation thalli is 30%, the plasmid retention rate is 100%, and the yield of the inclusion body after the fermentation product is crushed is 325 g.
Example 6
1. 600L of 2YT medium was prepared in a 1000L fermenter, sterilized at 121 ℃ and then cooled to 37 ℃.
2. Inoculating 50L of mature recombinant Escherichia coli seed liquid for culturing, and culturing at 37 + -1 deg.C, rotation speed of 270r/min, ventilation capacity of 270L/min and pH of 7.4 in the whole fermentation culture stage.
3. Sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the density OD600 of the cells reaches 4, and adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.8mM for induction.
4. After induction, feeding was started, the feed medium was likewise 2YT medium. The feed rate was fed at a rate of 3% of the volume of the original medium per hour. The fermentation was completed after further 4 hours of cultivation.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the interferon-beta protein in the finally harvested fermentation thalli is 32%, the plasmid retention rate is 100%, and the yield of the inclusion body after the fermentation product is crushed is 705 g.
Comparative example 1
1. Preparing 300L 2YT culture medium in 500L fermentation tank, sterilizing at 121 deg.C, and cooling to 37 deg.C;
2. inoculating 20L of mature recombinant Escherichia coli seed liquid for culturing, and culturing at 37 + -1 deg.C, 250r/min rotation speed, 250L/min ventilation rate and pH of 7.5 in the whole fermentation culture stage;
3. sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the density OD600 of the cells reaches 4, and adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.8mM for induction. The fermentation was terminated by continuing the culture for 5 hours.
4. After induction, 20% glucose solution is supplemented, the glucose solution with the volume of 5% of the original culture medium is supplemented every hour, and the glucose solution is supplemented until the fermentation is finished.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the beta interferon protein in the finally harvested fermentation thalli is 15%, the plasmid retention rate is 85%, and the yield of the inclusion body after the fermentation product is crushed is 135 g.
Comparative example 2
1. Preparing 300L 2YT culture medium in 500L fermentation tank, sterilizing at 121 deg.C, and cooling to 37 deg.C;
2. inoculating 10L of mature recombinant Escherichia coli seed liquid for culturing, and culturing at 37 + -1 deg.C, rotation speed of 270r/min, ventilation capacity of 270L/min and pH of 7.5 in the whole fermentation culture stage;
3. and (3) beginning to supplement 20% of glucose solution 1h after inoculation, wherein the supplement rate is 5% of the volume of the original culture medium per hour until the fermentation is finished.
4. Sampling at regular time during the fermentation culture process to determine the thallus density OD600 value of the bacteria liquid; culturing until the density OD600 of the cells reaches 8, and adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.5mM for induction. The fermentation was completed after further 3 hours of cultivation.
5. The samples were subjected to SDS-PAGE electrophoresis to determine the expression level of the cells, and the loss rate of the plasmid was determined by referring to 2015, pharmacopoeia, III, general rule 3406. The expression quantity of the beta interferon protein in the finally harvested fermentation thalli is 10%, the plasmid retention rate is 50%, and the yield of the inclusion body after the fermentation product is crushed is 105 g.
Finally, the description is as follows: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover any modifications or equivalents as may fall within the scope of the invention.
Claims (3)
1. A method for producing recombinant human interferon beta 1b by fermentation, which comprises the following steps:
preparing a 2YT culture medium in a fermentation tank with the volume of 200L-1000L, sterilizing, and cooling to 35-40 ℃;
inoculating escherichia coli engineering bacteria containing recombinant human interferon beta 1b gene plasmids to a fermentation tank for fermentation culture, wherein the fermentation temperature is controlled to be 35-40 ℃, the rotation speed is controlled to be 200-500 r/min, the ventilation volume is controlled to be 200-500L/min, and the pH value is controlled to be 5.0-8.0; wherein, the inoculation amount of the escherichia coli engineering bacteria containing recombinant human interferon beta 1b gene plasmids is 5-15%;
monitoring the thallus density OD600 value of the bacteria liquid in the fermentation culture process; after culturing until the thallus density OD600 reaches 2-8, adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.2-1.5 mM, or adding lactose in batches to the final concentration of 1-10 g/L for induction;
and after induction, feeding and supplementing materials at a rate of 1-10% of the volume of the original culture medium per hour, wherein the fed-back culture medium is a 2YT culture medium, and continuously culturing for 2-8 hours to stop fermentation and harvest the thalli.
2. The method of claim 1, wherein the 2YT medium is sterilized under the conditions: sterilizing at 121 deg.C.
3. The method according to claim 1, wherein the content of interferon-beta protein in the harvested fermented thallus accounts for more than 20% of the total thallus protein, the plasmid retention rate is 100%, and the yield of the inclusion bodies after the fermentation product is crushed reaches 250-550 g.
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