CN103146787A - Fermentation medium and fermentation method for preparing recombinant human interferon alpha2b - Google Patents
Fermentation medium and fermentation method for preparing recombinant human interferon alpha2b Download PDFInfo
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Abstract
The invention discloses a fermentation medium and a fermentation method for preparing recombinant human interferon alpha2b, including a fermentation medium formula which is suitable for constitutive expression recombinant human interferon alpha2b engineering bacteria and a fermentation method thereof; the fermentation medium formula comprises the following components in parts by weight: 25-35 parts of tryptone, 15-25 pats of yeast extract, 4-8 parts of disodium hydrogen phosphate, 2-4 parts of mono-potassium phosphate, 0.4-0.6 parts of ammonium chloride, 0.4-0.6 parts of sodium chloride, 0.2-0.4 parts of magnesium sulfate, 0.05-0.15 parts of calcium chloride and 10-20 parts of glycerol; the glycerol is used as the only carbon source during a culture process of the fermentation method, and the expression rate of target protein is regulated by using proper phosphate concentration, therefore the excellent growing status of the engineering bacteria is ensured, and the high-density culture and high-efficiency expression are achieved.
Description
Technical field
The present invention relates to field of medicaments, relate in particular to a kind of fermention medium and fermentation process for preparing recombinant human interferon alpha 2 b.
Background technology
Development along with genetic engineering technique, the zymotechnique of engineering bacteria obtains using very widely, the purpose of bioengineered strain fermentation is to obtain a large amount of ectogenic gene products, reduce as far as possible the pollution of host cell and foeign element, so the cultivation of high-density, high yield high expression level has become target and the direction of fermentation industry.aspect High Cell Density And High Expression, intestinal bacteria are representative and the most most widely used Host Strains, in intestinal bacteria, the expression of recombinant proteins mode has two kinds, a kind of is constitutive expression, a kind of is the inducible expression, at present, in the high density fermentation of recombinant protein engineering bacteria is cultivated, mostly be greatly inducible expression's bacterial classification, its advantage is by temperature-induced or chemical induction, can make engineering bacteria ramp at short notice, reach high specific growth rate, reach simultaneously maximum expression amount, shortcoming is that the meta-bolites of bacterium in the mushroom process of bacterium also can produce rapidly, as organic acid, the growth of the run-up meeting anti-bacteria of the objectionable impuritiess such as carbonic acid gas and the expression of product, and the generation of maximum by-product acetic acid, it is generally acknowledged that bacterium is under low specific growth rate condition, be enough to satisfy the demand of synthetic and dissimilation in energy by oxidative metabolism effect generation, can not produce acetic acid, and when high specific growth rate, intestinal bacteria only depend on oxidative metabolism that enough energy can not be provided, must be by acetic acid generation approach deposit ATP and NADH, will too much produce acetic acid like this, and the increase meeting of acid is to bacterial reproduction and Product Expression restraining effect, simultaneously also can bring very large burden to subsequent purification.
In intestinal bacteria, the constitutive expression mode of recombinant protein is a kind of mode of slow expression, need not to induce, continue growth and express in the process of bacterial growth, its growth cycle internal cause is not induced mushroom process, the speed of growth is even, in meta-bolites, acetic acid content is low, can obtain desirable Product Expression rate, but is difficult to realize high-density culture.
Therefore, prior art awaits further improve and develop.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of fermention medium and fermentation process for preparing recombinant human interferon alpha 2 b, by the control of a kind of defined medium and fermenting process, realize high-density culture and the high efficient expression of the recombinant human interferon alpha 2 b engineering bacteria of constitutive expression.
Technical scheme of the present invention is as follows:
A kind of fermention medium that is applicable to the recombinant human interferon alpha 2 b engineering bacteria of constitutive expression, it comprises following component according to weight part for it:
Tryptones 25-35 part, yeast extract 15-25 part, Sodium phosphate dibasic 4-8 part, potassium primary phosphate 2-4 part, ammonium chloride 0.4-0.6 part, sodium-chlor 0.4-0.6 part, sal epsom 0. 2-0.4 part, calcium chloride 0.05-0.15 part and glycerol 10-20 part.
Described fermention medium, wherein, Tryptones concentration is 25-35 g/L, the concentration of yeast extract is 15-25 g/L, and Sodium phosphate dibasic concentration is 4-8 g/L, and the biphosphate potassium concn is 2-4 g/L, ammonium chloride concentration is 0.4-0.6g/L, sodium chloride concentration is 0.4-0.6 g/L, and magnesium sulfate concentration is 0. 2-0.4g/L, and calcium chloride concentration is that 0.05-0.15 g/L and glycerol concentration are 10-20g/L.
A kind of fermentation process for preparing recombinant human interferon alpha 2 b, it comprises the following steps:
A, according to weight part with Tryptones 25-35 part, yeast extract 15-25 part, Sodium phosphate dibasic 4-8 part, potassium primary phosphate 2-4 part, ammonium chloride 0.4-0.6 part, sodium-chlor 0.4-0.6 part, sal epsom 0. 2-0.4 part, calcium chloride 0.05-0.15 part and glycerol 10-20 part add in fermentor tank, and add water for injection fully to be mixed to get substratum in described fermentor tank;
B, add again the sodium hydroxide solution of phosphoric acid and 50% in the described fermentor tank, regulate the pH value to 7.0 of described substratum ± 0.2, and then the engineering bacteria bacterial classification is inoculated into described substratum cultivates, in described fermentor tank, culture temperature is 37 ℃, obtains tunning;
C, adopt the low-temperature centrifugation method to obtain thalline described tunning, and described thalline is carried out cracking and a series of purification, obtain recombinant human interferon alpha 2 b albumen.
Described fermentation culture method, wherein, described step B also comprises:
B1, add described phosphoric acid in described fermentor tank, the pH value of described substratum is adjusted to 4.2 ± 0.2, and sterilization 15 minutes under 121 ℃ of conditions;
B2, the described substratum temperature after sterilizing are controlled at 37 ℃, then add 50% sodium hydroxide solution in the described fermentor tank, and the pH value of described substratum is adjusted to 7.0 ± 0.2;
Add the sodium hydroxide solution of phosphoric acid and 50% in B3, fermentation culture process in the described fermentor tank, the pH value of controlling described substratum is 7.0 ± 0.2 in good time; Along with the dissolved oxygen of bacterial growth media is reduced to zero gradually from 90%.
the invention provides a kind of fermention medium and fermentation process for preparing recombinant human interferon alpha 2 b, take glycerol as sole carbon source, adjust the expression speed of target protein with appropriate phosphate concn, and will cultivate based under acidic conditions and sterilize, reduce sterilization time to reduce the loss of medium nutrient content, phosphoric acid and 50% sodium hydroxide with 50% in culturing process are regulated the pH value of fermented liquid as acid-base modifier, guaranteed the growth conditions that engineering bacteria is good, high-density culture and high efficient expression have been realized, technique of the present invention is simple, metabolic by-prods is few, the subsequent purification that is conducive to expression product, reduced the manufacturing cost of recombinant human interferon alpha 2 b.
Description of drawings
Fig. 1 is the schematic flow sheet of fermentation process in the present invention;
Fig. 2 is the Dynamic Graph that in the present invention, engineering bacteria growth curve and target protein are expressed;
Fig. 3 is the schematic diagram of the electrophoretogram of tunning in the present invention.
Embodiment
The invention provides a kind of fermention medium and fermentation process for preparing recombinant human interferon alpha 2 b, clearer, clear and definite for making purpose of the present invention, technical scheme and effect, below the present invention is described in more detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
The invention provides a kind of fermentative medium formula that is applicable to the recombinant human interferon alpha 2 b engineering bacteria of constitutive expression, it comprises following component according to weight part for it: Tryptones 25-35 part, yeast extract 15-25 part, Sodium phosphate dibasic 4-8 part, potassium primary phosphate 2-4 part, ammonium chloride 0.4-0.6 part, sodium-chlor 0.4-0.6 part, sal epsom 0. 2-0.4 part, calcium chloride 0.05-0.15 part and glycerol 10-20 part.
Further, Tryptones concentration is 25-35 g/L, the concentration of yeast extract is 15-25 g/L, Sodium phosphate dibasic concentration is 4-8 g/L, the biphosphate potassium concn is 2-4 g/L, and ammonium chloride concentration is 0.4-0.6g/L, and sodium chloride concentration is 0.4-0.6 g/L, magnesium sulfate concentration is 0. 2-0.4g/L, and calcium chloride concentration is that 0.05-0.15 g/L and glycerol concentration are 10-20g/L.Take glycerol as sole carbon source, adjust the expression speed of target protein with appropriate phosphate concn, and will cultivate based under acidic conditions and sterilize, reduce sterilization time to reduce the loss of medium nutrient content, phosphoric acid and 50% sodium hydroxide with 50% in culturing process are regulated the pH value of fermented liquid as acid-base modifier, guarantee the growth conditions that engineering bacteria is good, realized high-density culture and high efficient expression.
The invention provides a kind of fermentation process for preparing recombinant human interferon alpha 2 b, as shown in Figure 1, it comprises the following steps:
Step 101: according to weight part with Tryptones 25-35 part, yeast extract 15-25 part, Sodium phosphate dibasic 4-8 part, potassium primary phosphate 2-4 part, ammonium chloride 0.4-0.6 part, sodium-chlor 0.4-0.6 part, sal epsom 0. 2-0.4 part, calcium chloride 0.05-0.15 part and glycerol 10-20 part add in fermentor tank, and add water for injection fully to be mixed to get substratum in described fermentor tank;
Step 102: the sodium hydroxide solution that adds again phosphoric acid and 50% in the described fermentor tank, regulate the pH value to 7.0 of described substratum ± 0.2, and then the engineering bacteria bacterial classification is inoculated into described substratum cultivates, in described fermentor tank, culture temperature is 37 ℃, obtains tunning;
Step 103: adopt the low-temperature centrifugation method to obtain thalline described tunning, and described thalline is carried out cracking purify, obtain recombinantinterferonα 2b albumen.
It is more specifically:
First according to weight part with Tryptones 25-35 part, yeast extract 15-25 part, Sodium phosphate dibasic 4-8 part, potassium primary phosphate 2-4 part, ammonium chloride 0.4-0.6 part, sodium-chlor 0.4-0.6 part, sal epsom 0. 2-0.4 part, calcium chloride 0.05-0.15 part and glycerol 10-20 part add in fermentor tank, and add the above-mentioned substratum of the abundant mixed dissolution of water for injection of specified quantity in the described fermentor tank.
Then adjust PH to 4.2 ± 0.2,121 ℃ sterilization 15 minutes with 50 % phosphoric acid;
Above-mentioned substratum is being cooled to 37 ℃, is adjusting PH to 7.0 ± 0.2 with 50% sodium hydroxide solution, inoculating suitable engineering bacteria seed liquid and cultivate;
Wherein in fermenting process, culture parameters is: 37 ℃ of culture temperature, pH value are 7.0 ± 0.2, and dissolved oxygen 90 % stir revolution 200rpm;
Add the sodium hydroxide adjustment pH value of 50 % phosphoric acid and 50 %. in culturing process according to the needs of environment of bacteria growth, foam is controlled with defoamer, and initial dissolved oxygen is set as 90 %, along with the bacterial growth dissolved oxygen drops to zero gradually;
And when continuing to be cultured to 12～14 hours, when the growth rate of bacterium and the resultant quantity of target protein peak, stop cultivating;
At last, adopt the low-temperature centrifugation method to collect thalline, detect Interferon, rabbit purity and content with SDS-PAGE electrophoretic method and ELISA method, detect and to carry out cracking and purification to thalline after qualified, obtain recombinantinterferonα 2b albumen.
Further, described step 102 also comprises:
Add described phosphoric acid in described fermentor tank, the pH value of described substratum is adjusted to 7.0 ± 0.2, and sterilization 15 minutes under 121 ℃ of conditions;
Described substratum temperature after sterilization is controlled at 37 ℃, then adds 50% sodium hydroxide solution in the described fermentor tank, the pH value of described substratum is adjusted to 7.0 ± 0.2;
Add the sodium hydroxide solution of phosphoric acid and 50% in the fermentation culture process in the described fermentor tank, the pH value of controlling described substratum is 7.0 ± 0.2 in good time; Along with the dissolved oxygen of bacterial growth media is reduced to zero gradually from 90%.Technique of the present invention is simple, and metabolic by-prods is few, is conducive to the subsequent purification of expression product, has reduced the manufacturing cost of recombinantinterferonα 2b.
In order further to describe the present invention, below enumerating more specifically, embodiment describes.
Embodiment 1
The component of fermention medium is: Tryptones 6000g, yeast extract 4000g, Sodium phosphate dibasic 1200g, potassium primary phosphate 600g, ammonium chloride 100g, sodium-chlor 100g, sal epsom 60g, calcium chloride 20g and glycerol 3000g.
The fermentation culture method of preparation recombinant human interferon alpha 2 b comprises the following steps:
According to weight, said components is made into substratum;
With the described substratum of dissolving in appropriate water for injection, cumulative volume is settled to 200L in the 300L fermentor tank;
Adjust the pH value to 4.2 of described substratum ± 0.2 with phosphoric acid, and sterilization 15 minutes under 121 ℃ of conditions;
And then described substratum is cooled to 37 ℃, and adjust described substratum pH value to 7.0 ± 0.2 with 50% sodium hydroxide solution, inoculation recombinant human interferon alpha 2 engineering bacteria seed liquid (pADUA-4/ K-12LE392 engineering bacteria) is cultivated;
Then with postvaccinal described substratum 37 ± 0.5 ℃ of culture temperature, the pH value is controlled at 7.0 ± 0.2, stirring velocity 200+/-5 r.p.m, air flow 0.5 vvm., working pressure 0.5 atm., the condition bottom fermentation of dissolved oxygen 90 % min is cultivated; And the sodium hydroxide of regularly adding the phosphoric acid of 50 % and 50 % according to the needs of environment of bacteria growth is adjusted the pH value 7.0 ± 0.2; Control with defoamer when producing foam during the fermentation; As shown in Figure 2, adopt the inventive method to realize the high-density culture of engineering bacteria of combined expression and the high efficient expression of target protein;
Above-mentioned substratum is continued continuously be cultured to 12～14 hours, detection OD
600Be 24.00, stop cultivating;
Described substratum after stopping at last cultivating adopts the low-temperature centrifugation method to collect thalline, obtain thalline 4370g, detect with the SDS-PAGE electrophoretic method, expressing quantity is 30.9%, the ELISA method detects Interferon, rabbit content 200.44g/L, after thalline is carried out cracking and purification, can obtain recombinantinterferonα 2b albumen.
Embodiment 2
The component of fermention medium is: Tryptones 6000g, yeast extract 4000g, Sodium phosphate dibasic 1200g, potassium primary phosphate 600g, ammonium chloride 100g, sodium-chlor 100g, sal epsom 60g, calcium chloride 20g and glycerol 3000g.
The fermentation culture method of preparation recombinant human interferon alpha 2 b comprises the following steps:
According to weight, said components is made into substratum;
With the described substratum of dissolving in appropriate water for injection, cumulative volume is settled to 200L in the 300L fermentor tank;
Adjust the pH value to 4.2 of described substratum ± 0.2 with phosphoric acid, and sterilization 15 minutes under 121 ℃ of conditions;
And then described substratum is cooled to 37 ℃, and adjust described substratum pH value to 7.0 ± 0.2 with 50% sodium hydroxide solution, inoculation recombinant human interferon alpha 2 engineering bacteria seed liquid (pADUA-4/ K-12LE392 engineering bacteria) is cultivated;
Then with postvaccinal described substratum 37 ± 0.5 ℃ of culture temperature, the pH value is controlled at 7.0 ± 0.2, stirring velocity 200+/-5 r.p.m, air flow 0.5 vvm., working pressure 0.5 atm., the condition bottom fermentation of dissolved oxygen 90 % min is cultivated; And the sodium hydroxide of regularly adding the phosphoric acid of 50 % and 50 % according to the needs of environment of bacteria growth is adjusted the pH value 7.0 ± 0.2; Control with defoamer when producing foam during the fermentation; As shown in Figure 3, adopt the inventive method to realize the high-density culture of engineering bacteria of combined expression and the high efficient expression of target protein;
Above-mentioned substratum is continued continuously be cultured to 12～14 hours, detection OD
600Be 25.20, stop cultivating;
Described substratum after stopping at last cultivating adopts the low-temperature centrifugation method to collect thalline, obtain thalline 5200g, detect with the SDS-PAGE electrophoretic method, expressing quantity is 31.6%, the ELISA method detects Interferon, rabbit content 253.38g/L, after thalline is carried out cracking and purification, can obtain recombinantinterferonα 2b albumen.
Embodiment 3
The component of fermention medium is: Tryptones 6000g, yeast extract 4000g, Sodium phosphate dibasic 1200g, potassium primary phosphate 600g, ammonium chloride 100g, sodium-chlor 100g, sal epsom 60g, calcium chloride 20g and glycerol 3000g.
The fermentation culture method of preparation recombinant human interferon alpha 2 b comprises the following steps:
According to weight, said components is made into substratum;
With the described substratum of dissolving in appropriate water for injection, cumulative volume is settled to 200L in the 300L fermentor tank;
Adjust the pH value to 4.2 of described substratum ± 0.2 with phosphoric acid, and sterilization 15 minutes under 121 ℃ of conditions;
And then described substratum is cooled to 37 ℃, and adjust described substratum pH value to 7.0 ± 0.2 with 50% sodium hydroxide solution, inoculation recombinant human interferon alpha 2 engineering bacteria seed liquid (pADUA-4/ K-12LE392 engineering bacteria) is cultivated;
Then with postvaccinal described substratum 37 ± 0.5 ℃ of culture temperature, the pH value is controlled at 7.0 ± 0.2, stirring velocity 200+/-5 r.p.m, air flow 0.5 vvm., working pressure 0.5 atm., the condition bottom fermentation of dissolved oxygen 90 % min is cultivated; And the sodium hydroxide of regularly adding the phosphoric acid of 50 % and 50 % according to the needs of environment of bacteria growth is adjusted the pH value 7.0 ± 0.2; Control with defoamer when producing foam during the fermentation; As Fig. 2 and shown in Figure 3, adopt the inventive method to realize the high-density culture of engineering bacteria of combined expression and the high efficient expression of target protein;
Above-mentioned substratum is continued continuously be cultured to 12～14 hours, detection OD
600Be 24.30, stop cultivating;
Described substratum after stopping at last cultivating adopts the low-temperature centrifugation method to collect thalline, obtain thalline 4830g, detect with the SDS-PAGE electrophoretic method, expressing quantity is 39.8%, the ELISA method detects Interferon, rabbit content 445.67g/L, after thalline is carried out cracking and purification, can obtain recombinantinterferonα 2b albumen.
In order further to highlight advance of the present invention, below enumerate the fermentation culture method of part preparation recombinant human interferon alpha 2 b of the prior art, compare.
Embodiment 4
In prior art, the component of recombinantinterferonα 2b recombinant base is: Tryptones 20g/L, yeast extract 12g/L, glucose 13.7g/L, sodium-chlor 10g/L, dipotassium hydrogen phosphate 2.3g/L, potassium primary phosphate 1.5g/L and sal epsom 0.25g/L.
In prior art, the fermentation culture method of preparation recombinant human interferon alpha 2 b comprises the following steps:
According to weight, said components is made into substratum;
The above-mentioned substratum of preparation 200L in the 300L fermentor tank;
Adjust described Medium's PH Value as 7.0 ± 0.2 take 4 molar sodium hydroxides, and sterilized 30 minutes under 121 ℃ of conditions;
Described substratum is cooled to 37 ℃, and inoculation recombinant human interferon alpha 2 engineering bacteria seed liquid (pADUA-4/ K-12LE392 engineering bacteria) is cultivated;
Then be 37 ± 0.5 oC with postvaccinal described substratum in culture temperature, the pH value is controlled at 7.0 ± 0.2, stirring velocity 250+/-5 r.p.m, air flow 0.5 vvm, working pressure 0.5 atm., the condition bottom fermentation of dissolved oxygen 30-75 % min. is cultivated; And the sodium hydroxide of regularly adding 50 % according to the needs of environment of bacteria growth is adjusted the pH value 7.0 ± 0.2; Control with defoamer when producing foam during the fermentation;
Above-mentioned substratum is continued continuously be cultured to 14 hours, detection OD
600Be 18.2 o'clock, stop cultivating;
Described substratum after stopping at last cultivating adopts the low-temperature centrifugation method to collect thalline, obtain thalline 2500g, detect with the SDS-PAGE electrophoretic method, expressing quantity is 18.5%, the ELISA method detects Interferon, rabbit content 145.22 mg/L, after thalline is carried out cracking and purification, can obtain recombinantinterferonα 2b albumen in prior art.
Recombinantinterferonα 2b albumen in the prior art of the recombinantinterferonα 2b albumen of embodiment 1, embodiment 2, embodiment 3 acquisitions and embodiment 4 acquisitions is detected, detected result is listed in table 1, cultivation average and the thalline weight that can find out embodiment 1, embodiment 2, embodiment 3 from table 1, Fig. 2 and Fig. 3 can reach 4.8kg, the expression amount of Interferon, rabbit can reach 34.1%, it is 299.83 that ELISA detects the interferon protein amount, obviously is better than the result of control group embodiment 4.
| Lot number | Thalline weight (kg) | Electrophoresis expression amount (%) | IFN content (mg/L) | Microbiological manipulation |
| Embodiment 1 | 4.37 | 30.9 | 200.44 | Qualified |
| Embodiment 2 | 5.20 | 31.6 | 253.38 | Qualified |
| Embodiment 3 | 4.83 | 39.8 | 445.67 | Qualified |
| Embodiment 1,2 and 3 average | 4.8 | 34.1 | 299.83 | Qualified |
| Embodiment 4 | 2.5 | 18.5 | 145.22 | Qualified |
Should be understood that, application of the present invention is not limited to above-mentioned giving an example, and for those of ordinary skills, can be improved according to the above description or conversion, and all these improve and conversion all should belong to the protection domain of claims of the present invention.
Claims (4)
1. fermention medium for preparing recombinant human interferon alpha 2 b, it comprises following component according to weight part for it:
Tryptones 25-35 part, yeast extract 15-25 part, Sodium phosphate dibasic 4-8 part, potassium primary phosphate 2-4 part, ammonium chloride 0.4-0.6 part, sodium-chlor 0.4-0.6 part, sal epsom 0. 2-0.4 part, calcium chloride 0.05-0.15 part and glycerol 10-20 part.
2. fermention medium according to claim 1, it is characterized in that, Tryptones concentration is 25-35 g/L, the concentration of yeast extract is 15-25 g/L, and Sodium phosphate dibasic concentration is 4-8 g/L, and the biphosphate potassium concn is 2-4 g/L, ammonium chloride concentration is 0.4-0.6g/L, sodium chloride concentration is 0.4-0.6 g/L, and magnesium sulfate concentration is 0. 2-0.4g/L, and calcium chloride concentration is that 0.05-0.15 g/L and glycerol concentration are 10-20g/L.
3. fermentation process for preparing recombinant human interferon alpha 2 b, it comprises the following steps:
A, according to weight part with Tryptones 25-35 part, yeast extract 15-25 part, Sodium phosphate dibasic 4-8 part, potassium primary phosphate 2-4 part, ammonium chloride 0.4-0.6 part, sodium-chlor 0.4-0.6 part, sal epsom 0. 2-0.4 part, calcium chloride 0.05-0.15 part and glycerol 10-20 part add in fermentor tank, and add water for injection fully to be mixed to get substratum in described fermentor tank;
B, add again the sodium hydroxide solution of phosphoric acid and 50% in the described fermentor tank, regulate the pH value to 7.0 of described substratum ± 0.2, and then the engineering bacteria bacterial classification is inoculated into described substratum cultivates, in described fermentor tank, culture temperature is 37 ℃, obtains tunning;
C, adopt the low-temperature centrifugation method to obtain thalline described fermented product, and described thalline is carried out cracking purify, obtain recombinantinterferonα 2b albumen.
4. fermentation process according to claim 3, is characterized in that, described step B also comprises:
B1, add described phosphoric acid in described fermentor tank, the pH value of described substratum is adjusted to 4.2 ± 0.2, and sterilization 15 minutes under 121 ℃ of conditions;
B2, the described substratum temperature after sterilizing are controlled at 37 ℃, then add 50% sodium hydroxide solution in the described fermentor tank, and the pH value of described substratum is adjusted to 7.0 ± 0.2;
Add the sodium hydroxide solution of phosphoric acid and 50% in B3, fermentation culture process in the described fermentor tank, the pH value of controlling described substratum is 7.0 ± 0.2 in good time; Along with the dissolved oxygen of bacterial growth media is reduced to zero gradually from 90%.
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| CN106636263A (en) * | 2017-01-24 | 2017-05-10 | 广州余仁谷生物科技有限公司 | Preparation method of recombinant human interferon |
| CN111996225A (en) * | 2020-09-18 | 2020-11-27 | 深圳科兴药业有限公司 | Fermentation method for producing high-activity recombinant human interferon alpha 2b by using microorganisms |
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| CN106636263A (en) * | 2017-01-24 | 2017-05-10 | 广州余仁谷生物科技有限公司 | Preparation method of recombinant human interferon |
| CN111996225A (en) * | 2020-09-18 | 2020-11-27 | 深圳科兴药业有限公司 | Fermentation method for producing high-activity recombinant human interferon alpha 2b by using microorganisms |
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