CN107099569A - A kind of method of scale fermenting and producing recombinant human interferon beta 1b albumen - Google Patents
A kind of method of scale fermenting and producing recombinant human interferon beta 1b albumen Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/565—IFN-beta
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Abstract
The invention provides a kind of scale fermenting and producing recombinant human interferon beta 1b method, this method includes：2YT culture mediums are prepared in fermentation tank, 35~40 DEG C are cooled to after sterilizing；Be inoculated with the colibacillus engineering fermented and cultured containing recombinant human interferon beta 1b gene plasmids, control fermentation temperature be 35~40 DEG C, 200~500r/min of rotating speed, 200~500L/min of throughput, pH5.0~8.0；After culture reach 2~8 or so to cell density OD600 after, add isopropylthiogalactoside (IPTG) to 0.2~1.5mM of final concentration or be added portionwise lactose to 2~10g/ of final concentration rise induced；The speed for adding former culture volume 1~10% after induction per hour carries out flow feeding, continues to cultivate 2~8h harvest thalline.The method of the present invention is a kind of stabilization, the scale fermentation process of efficiently expressing recombinant human interferon beta 1b albumen.
Description
Technical field
The present invention is
On a kind of stabilization, the scale fermentation process of efficiently expressing recombinant human interferon beta 1b albumen.
Background technology
Human beta interferon has antiviral and immunoregulation effect, and its gene is located on people's Chromosome 9, and natural human β is done
It is the glycoprotein that a kind of molecular weight being made up of 166 amino acid is 20KD to disturb element.FDA is examined genetic engineering beta interferon,
For treating multiple sclerosis (MS), the disease is the nervous system disease related to immune response, and the treatment of beta interferon is based on
Its immune response is acted on.In addition beta interferon also has very strong viral infection resisting and antitumor action, clinically to hepatitis B,
Hepatitis C and other viral diseases have obvious curative effect.
Beta interferon class medicine is as engineered protein medicine, and its industrialization need to solve large scale fermentation production first and ask
Topic, but the fermentation research maximum to beta interferon albumen only reaches pilot scale level in the prior art, and scale is essentially sent out using 50L
Fermentation tank completes the fermentation of 30L cultures, and tunning harvest yield is low, it is impossible to the industrialization production applied to beta interferon.
The content of the invention
It is an object of the present invention to provide a kind of method that large scale fermentation produces recombinant human interferon beta 1b albumen.
For up to above-mentioned purpose, the invention provides a kind of method that large scale fermentation produces recombinant human interferon beta 1b albumen,
This method includes：
2YT culture mediums are prepared in fermentation tank, 35~40 DEG C are cooled to after sterilizing；
It is inoculated with the colibacillus engineering containing recombinant human interferon beta 1b gene plasmids to fermentation tank and carries out fermented and cultured,
Control fermentation temperature for 35~40 DEG C, 200~500r/min of rotating speed, 200~500L/min of throughput, pH5.0~8.0；
The cell density OD600 values of bacterium solution are monitored during fermented and cultured；Wait to cultivate to cell density OD600 and reach 2~8
Behind left and right, isopropylthiogalactoside (IPTG) is added to final concentration of 0.2~1.5mM, or lactose is added portionwise to dense eventually
Spend to rise for 1~10g/ and induced；
Each hour carries out flow feeding according to the speed for adding former culture volume 1~10% and (adds former training after induction
Support base), continue to cultivate 2~8h stopping fermentation harvest thalline.
The method of the production recombinant human interferon beta 1b albumen of the present invention, can be beneficial to improve recombinant beta interferon expression level.
According to specific embodiments of the present invention, method of the invention can scale, the fermenter volume is more than or equal to
200L, up to more than 1000L.
According to specific embodiments of the present invention, in method of the invention, the sterilising conditions of the 2YT culture mediums are：121
DEG C sterilizing.
According to specific embodiments of the present invention, in method of the invention, contain recombinant human interferon beta 1b gene plasmids
The inoculum concentration of colibacillus engineering is 5~15%.
According to specific embodiments of the present invention, in method of the invention, beta interferon protein content in harvest fermentation thalli
Account for more than the 20% of bacterial protein amount, plasmid retention is 100%, after tunning is broken inclusion body yield reach 250 to
705g even more highs.
The method of the present invention, is a kind of stabilization, the scale fermentation process of efficiently expressing recombinant human interferon beta 1b albumen,
The fermentation method of flow feeding is wherein used, by adding certain density supplemented medium, it is ensured that interferon beta 1b albumen is in work
High efficient expression in journey bacterium, improves the thalline expression quantity of thalline harvest yield and beta interferon albumen, realizes containing human beta interferon
Scale fermented and cultured of the colibacillus engineering of gene plasmid in more than 200L fermentation tank, work of being fermented to beta interferon
Skill is exaggerated, and improves fermentation yield, is that beta interferon class drug industries lay the first stone.
Embodiment
The beneficial effect for describing the implementation process of the present invention in detail below by way of specific embodiment and producing, it is intended to which help is read
Reader more fully understand the present invention essence and feature, not as to this case can practical range restriction.It is not detailed in each embodiment
Carefully dated experimental method, is carried out according to the routine operation of art.It is used in embodiment to contain recombinant human interferon beta 1b bases
Because the Escherichia coli of plasmid voluntarily build for Beijing North Sheng Yan biological products Co., Ltd according to prior art, maturation restructuring large intestine
The culture of bacillus seed liquor is carried out with reference to prior art.
Embodiment 1
1st, 300L 2YT culture mediums are prepared in 500L fermentation tanks, 35 DEG C are cooled to after 121 DEG C of sterilizings.
2nd, inoculation maturation recombination bacillus coli seed liquor 15L is cultivated, and whole fermentation culture stage maintains the temperature to be
35 ± 1 DEG C, rotating speed 200r/min, throughput 200L/min, pH be 5.0 under the conditions of cultivated.
3rd, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
2 are reached, isopropylthiogalactoside (IPTG) extremely final concentration of 0.2mM is added and is induced.
4th, feed supplement is started after inducing, supplemented medium is similarly 2YT culture mediums.Feed rate adds former culture on an hourly basis
5% speed of matrix product carries out flow feeding.Continue to cultivate 4 hours fermentation ends.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 30% in final harvest fermentation thalli, and plasmid retention is 100%, fermentation production
Inclusion body yield is 385g after thing is broken.
Embodiment 2
1st, 300L 2YT culture mediums are prepared in 500L fermentation tanks, 37 DEG C are cooled to after 121 DEG C of sterilizings.
2nd, inoculation maturation recombination bacillus coli seed liquor 30L is cultivated, and whole fermentation culture stage maintains the temperature to be
37 ± 1 DEG C, rotating speed 300r/min, throughput 300L/min, pH be 8.0 under the conditions of cultivated.
3rd, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
8 are reached, isopropylthiogalactoside (IPTG) extremely final concentration of 0.8mM is added and is induced.
4th, feed supplement is started after inducing, supplemented medium is similarly 2YT culture mediums.Feed rate adds former culture on an hourly basis
10% speed of matrix product carries out flow feeding.Continue to cultivate 2 hours fermentation ends.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 35% in final harvest fermentation thalli, and plasmid retention is 100%, fermentation production
Inclusion body yield is 525g after thing is broken
Embodiment 3
1st, 300L 2YT culture mediums are prepared in 500L fermentation tanks, 40 DEG C are cooled to after 121 DEG C of sterilizings；
2nd, inoculation maturation recombination bacillus coli seed liquor 45L is cultivated, and whole fermentation culture stage maintains the temperature to be
40 ± 1 DEG C, rotating speed 500r/min, throughput 500L/min, pH be 7.0 under the conditions of cultivated；
3rd, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
5 are reached, isopropylthiogalactoside (IPTG) extremely final concentration of 1.5mM is added and is induced.
4th, feed supplement is started after inducing, supplemented medium is similarly 2YT culture mediums.Feed rate adds former culture on an hourly basis
1% speed of matrix product carries out flow feeding.Continue to cultivate 8 hours fermentation ends.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 30% in final harvest fermentation thalli, and plasmid retention is 100%, fermentation production
Inclusion body yield is 475g after thing is broken.
Embodiment 4
1st, 300L 2YT culture mediums are prepared in 500L fermentation tanks, 35 DEG C are cooled to after 121 DEG C of sterilizings；
2nd, inoculation maturation recombination bacillus coli seed liquor 30L is cultivated, and whole fermentation culture stage maintains the temperature to be
35 ± 1 DEG C, rotating speed 250r/min, throughput 250L/min, pH be 7.0 under the conditions of cultivated；
3rd, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
4 are reached, lactose is added portionwise by the hour and is induced to final concentration of 2g/L.
4th, feed supplement is started after inducing, supplemented medium is similarly 2YT culture mediums.Feed rate adds former culture on an hourly basis
2% speed of matrix product carries out flow feeding.Continue to cultivate 6 hours fermentation ends.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 22% in final harvest fermentation thalli, and plasmid retention is 100%, fermentation production
Inclusion body yield is 258g after thing is broken.
Embodiment 5
1st, 300L 2YT culture mediums are prepared in 500L fermentation tanks, 38 DEG C are cooled to after 121 DEG C of sterilizings；
2nd, inoculation maturation recombination bacillus coli seed liquor 15L is cultivated, and whole fermentation culture stage maintains the temperature to be
38 ± 1 DEG C, rotating speed 350r/min, throughput 350L/min, pH be 7.5 under the conditions of cultivated；
3rd, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
4 are reached, lactose is added portionwise by the hour and is induced to final concentration of 10g/L.
4th, feed supplement is started after inducing, supplemented medium is similarly 2YT culture mediums.Feed rate adds former culture on an hourly basis
5% speed of matrix product carries out flow feeding.Continue to cultivate 4 hours fermentation ends.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 30% in final harvest fermentation thalli, and plasmid retention is 100%, fermentation production
Inclusion body yield is 325g after thing is broken.
Embodiment 6
1st, 600L 2YT culture mediums are prepared in 1000L fermentation tanks, 37 DEG C are cooled to after 121 DEG C of sterilizings.
2nd, inoculation maturation recombination bacillus coli seed liquor 50L is cultivated, and whole fermentation culture stage maintains the temperature to be
37 ± 1 DEG C, rotating speed 270r/min, throughput 270L/min, pH be 7.4 under the conditions of cultivated.
3rd, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
4 are reached, isopropylthiogalactoside (IPTG) extremely final concentration of 0.8mM is added and is induced.
4th, feed supplement is started after inducing, supplemented medium is similarly 2YT culture mediums.Feed rate adds former culture on an hourly basis
3% speed of matrix product carries out flow feeding.Continue to cultivate 4 hours fermentation ends.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 32% in final harvest fermentation thalli, and plasmid retention is 100%, fermentation production
Inclusion body yield is 705g after thing is broken.
Reference examples 1
1st, 300L 2YT culture mediums are prepared in 500L fermentation tanks, 37 DEG C are cooled to after 121 DEG C of sterilizings；
2nd, inoculation maturation recombination bacillus coli seed liquor 20L is cultivated, and whole fermentation culture stage maintains the temperature to be
37 ± 1 DEG C, rotating speed 250r/min, throughput 250L/min, pH be 7.5 under the conditions of cultivated；
3rd, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
4 are reached, isopropylthiogalactoside (IPTG) extremely final concentration of 0.8mM is added and is induced.Continue to cultivate 5 hours fermentation knots
Beam.
4th, start to add 20% glucose solution after inducing, feed rate is to add former culture volume 5% per hour
Above-mentioned glucose solution, feed supplement to fermentation ends.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 15% in final harvest fermentation thalli, and plasmid retention is 85%, fermentation production
Inclusion body yield is 135g after thing is broken.
Reference examples 2
1st, 300L 2YT culture mediums are prepared in 500L fermentation tanks, 37 DEG C are cooled to after 121 DEG C of sterilizings；
2nd, inoculation maturation recombination bacillus coli seed liquor 10L is cultivated, and whole fermentation culture stage maintains the temperature to be
37 ± 1 DEG C, rotating speed 270r/min, throughput 270L/min, pH be 7.5 under the conditions of cultivated；
3rd, 1h starts to add 20% glucose solution after being inoculated with, and feed rate is to add former culture volume 5% per hour
Above-mentioned glucose solution, until fermentation ends.
4th, timing sampling determines the cell density OD600 values of bacterium solution during fermented and cultured；Cultivate to cell density OD600
8 are reached, isopropylthiogalactoside (IPTG) extremely final concentration of 0.5mM is added and is induced.Continue to cultivate 3 hours fermentation knots
Beam.
5th, sampling SDS-PAGE electrophoresis technique determining thalline expression quantity, with reference to 2015 editions《Pharmacopeia》III general rule 3406 is determined
Plasmid loss rate.Beta interferon expressing quantity is 10% in final harvest fermentation thalli, and plasmid retention is 50%, fermentation production
Inclusion body yield is 105g after thing is broken.
What is finally illustrated is：Above example is merely to illustrate the implementation process and feature of the present invention, and unrestricted is sent out
Bright technical scheme, although the present invention is described in detail with reference to above-described embodiment, one of ordinary skill in the art should
Work as understanding：The present invention can still be modified or equivalent substitution, without departing from the spirit and scope of the present invention any
Modification or local replacement, all should cover among protection scope of the present invention.
Claims (5)
1. a kind of fermenting and producing recombinant human interferon beta 1b method, this method includes：
2YT culture mediums are prepared in fermentation tank, 35~40 DEG C are cooled to after sterilizing；
It is inoculated with the colibacillus engineering containing recombinant human interferon beta 1b gene plasmids to fermentation tank and carries out fermented and cultured, control
Fermentation temperature be 35~40 DEG C, 200~500r/min of rotating speed, 200~500L/min of throughput, pH5.0~8.0；
The cell density OD600 values of bacterium solution are monitored during fermented and cultured；Wait to cultivate to cell density OD600 and reach 2~8 or so
Afterwards, isopropylthiogalactoside (IPTG) is added to final concentration of 0.2~1.5mM or lactose is added portionwise to final concentration of 1
~10g/ rises and induced；
The speed for adding former culture volume 1~10% according to each hour after induction carries out flow feeding, continues to cultivate 2~8h
Stop fermentation harvest thalline.
2. according to the method described in claim 1, wherein, the fermenter volume be 200L~1000L.
3. according to the method described in claim 1, wherein, the sterilising conditions of the 2YT culture mediums are：121 DEG C of sterilizings.
4. according to the method described in claim 1, wherein, the engineered E. coli containing recombinant human interferon beta 1b gene plasmids
The inoculum concentration of bacterium is 5~15%.
5. according to the method described in claim 1, wherein, harvest fermentation thalli in beta interferon protein content account for bacterial protein
More than the 20% of amount, plasmid retention is 100%, and inclusion body yield reaches 250 to 550g after tunning is broken.
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Cited By (1)
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| CN110862945A (en) * | 2019-11-29 | 2020-03-06 | 宁波酶赛生物工程有限公司 | Fermentation method for escherichia coli induced protein expression |
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