CN114350588B - Fermentation medium of rhG-CSF and fermentation method thereof - Google Patents

Fermentation medium of rhG-CSF and fermentation method thereof Download PDF

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CN114350588B
CN114350588B CN202111671633.1A CN202111671633A CN114350588B CN 114350588 B CN114350588 B CN 114350588B CN 202111671633 A CN202111671633 A CN 202111671633A CN 114350588 B CN114350588 B CN 114350588B
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CN114350588A (en
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张贵民
冀成法
马鲁南
刘忠
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Shandong New Time Pharmaceutical Co Ltd
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Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation medium of rhG-CSF and a fermentation method. According to the invention, the control range of fermentation temperature and the control range of fermentation pH value in different periods are optimized, the adding time of a basic culture medium carbon source is changed, the mixture ratio and the feeding amount of the culture medium are optimized, the feeding is carried out in a limited flow mode, the low-concentration IPTG and lactose are induced in a synergistic manner, and the high-density high-expression of rhG-CSF fermentation is facilitated. After fermentation, the OD600 value of the fermentation density reaches more than 100, the expression quantity of the target protein of the fermentation reaches more than 77%, the loss rate of plasmids is less than 4%, and the rhG-CSF high-density fermentation improves the production efficiency and has good application prospect.

Description

Fermentation medium of rhG-CSF and fermentation method thereof
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation medium of rhG-CSF and a fermentation method thereof.
Background
Granulocyte colony stimulating factor (granulocyte colony-stimulating factor, G-CSF) has the function of promoting proliferation and differentiation of granulocyte hematopoietic stem cells and enhancing mature granulocyte function, and has obvious curative effects on neutrophil deficiency caused by increase of neutrophil during bone marrow transplantation, cancer chemotherapy and neutrophil deficiency accompanied by aplastic anemia. The early clinical indications are cancer chemotherapy and neutropenia after bone marrow transplantation, and the early clinical indications are gradually expanded to neutropenia caused by various causes.
The high-density fermentation technology can improve the fermentation density of thalli and the specific productivity of products, not only can correspondingly reduce the fermentation scale and production batch, but also can shorten the production period and reduce the equipment investment so as to reduce the production cost, thereby greatly improving the competitiveness of the products. The genetic background research of the escherichia coli is thorough, and the escherichia coli is easy to culture, high in growth speed, mature in large-scale culture process, high in expression level and the like, so that the escherichia coli becomes a prokaryotic expression system for synthesizing heterologous proteins by using the most widely used genetic engineering.
However, the high-level expression of the exogenous gene not only relates to the interrelation among the host, the vector and the cloned gene, but also is closely related to the environmental conditions in which the exogenous gene is positioned, so that the production is far from sufficient only by the traditional fermentation process, the factors influencing the expression of the exogenous gene need to be analyzed, and a set of fermentation process suitable for the efficient expression of the exogenous gene is explored.
Two of the most important problems in the fermentation of genetically engineered bacteria are the high-density fermentation of the bacterial cells and the determination of the induction conditions. The high density growth of the strain will result in insufficient oxygen supply and the production of large amounts of acetic acid in the medium, which will greatly affect the growth of the cells; in addition, there is no correlation between the cell density and the expression level of the foreign protein, and the binding point between them is the determination of the induction condition.
The recombinant plasmid of the genetically engineered bacterium has certain instability in the fermentation process, which leads to the failure to obtain the expected target gene product and yield. The stability of a plasmid is affected by various genetic and environmental factors such as the host and plasmid genotypes, host and plasmid interactions, the degree of gene expression, culture temperature, nutrient limitations, and reactor operating modes. Genetic engineering fermentation generally requires high-density bacterial culture to obtain more target products, however, too high bacterial density can affect plasmid stability of engineering bacteria, and at present, few documents for investigating the plasmid stability in the high-density culture process are available.
The selection of the proper inducer type and concentration is the key to successful expression of the genetically engineered bacteria. isopropyl-beta-D-thiogalactoside (IPTG) has high toxicity and high cost, and lactose is used as disaccharide, is nontoxic and low in cost, can be used as a carbon source, and is a better IPTG substitution. Compared with other lactose analogue inducers such as lactose, IPTG is the most efficient inducer, so that experimental verification is needed for selecting the inducer.
Acetic acid is a main harmful byproduct in the fermentation process of escherichia coli, and accumulation of the acetic acid has obvious inhibition effect on the growth of thalli and the expression of products. The ratio of NADH/NAD+ and the distribution of carbon metabolism flow in the microbial cells can be obviously influenced by using carbon sources with different oxidation-reduction states. In microbial fermentation, when glucose, sorbitol and gluconate are used as carbon sources respectively, sorbitol can generate more NADH than glucose, and half of gluconate is directly converted into pyruvic acid without generating NADH. Xia it was found that when glucose or xylose was used as the carbon source, more AcCoA flowed to acetic acid than when sorbitol was used as the carbon source, enhancing the competing utilization of Ac CoA for acetic acid synthesis and PSA synthesis; and meanwhile, when glucose or xylose is used as a carbon source, the growth of thalli is influenced, and the toxic effect of acetic acid on engineering bacteria is added, so that the synthesis amount of PSA is lower. Meanwhile, when sorbitol is used as a carbon source, the inhibition effect of energy charge on pyruvate kinase is possibly reduced, and the bacterial growth and PSA synthesis are facilitated. Xu Zhina and the like, when escherichia coli is cultured to express human epidermal growth factor (hEGF), glucose is used for replacing acid liquor, and when the pH value is increased, acid is not added any more, but glucose liquor is automatically added for adjustment, but the adjustment has a certain lag, so that fluctuation of sugar concentration and accumulation of metabolic byproducts cannot be avoided. The rhG-CSF engineering bacteria have the same problems in the fermentation process, and excessive byproducts such as acetic acid are produced, so that bacterial growth can be inhibited, and finally, the fermentation density is low, and the fermentation yield is low. At present, glucose is usually added into a basic culture medium initially in recombinant escherichia coli fermentation, and the feeding method is generally used for controlling the feeding speed and reducing the production of acetic acid. In the actual fermentation process, the method still has poor effect, the initial growth rate of engineering bacteria is too fast and uncontrolled, and the fermentation level cannot be obviously improved.
Disclosure of Invention
In view of the current situation that the efficiency of the current fermentation production of rhG-CSF is low, the invention provides a method for improving the yield of the fermentation production of rhG-CSF by taking sorbitol or mannitol as a carbon source and utilizing a supplemented culture medium mode.
In a first aspect the invention provides a basal medium for the production of rhG-CSF comprising a carbon source, yeast extract, tryptone, disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, ferrous sulphate, magnesium sulphate and an antifoaming agent, wherein the carbon source is required to be formulated separately and sterilized.
Preferably, the carbon source is one or a combination of sorbitol or mannitol.
In a preferred embodiment, the basal medium comprises 100ml/L of a carbon source, 1-5g/L of yeast extract, 2-10g/L of tryptone, 20-40g/L of disodium hydrogen phosphate dodecahydrate, 2-10g/L of potassium dihydrogen phosphate, 2-10g/L of ammonium chloride, 0.5-3g/L of sodium chloride, 0.01-0.03g/L of ferrous sulfate, 0.5-2g/L of magnesium sulfate, and 0.2-1ml/L of an antifoaming agent, wherein the concentration of the carbon source is 200-600g/L, and is separately formulated and sterilized.
In a preferred embodiment, the basal medium comprises 100ml/L of a carbon source, 2-3g/L of yeast extract, 4-7g/L of tryptone, 25-35g/L of disodium hydrogen phosphate dodecahydrate, 4-7g/L of potassium dihydrogen phosphate, 4-7g/L of ammonium chloride, 1-2g/L of sodium chloride, 0.01-0.02g/L of ferrous sulfate, 0.5-1g/L of magnesium sulfate, and 0.3-0.5ml/L of an antifoaming agent, wherein the concentration of the carbon source is 300-500g/L, and is formulated and sterilized separately. .
In a second aspect the invention provides a feed medium for the production of rhG-CSF, the feed medium comprising one or a combination of sorbitol, mannitol, lactose, yeast extract, tryptone, magnesium sulphate.
In a preferred embodiment, the feed medium comprises feed medium 1 and feed medium 2.
In a preferred embodiment, the feed medium 1 comprises 400-600g/L sorbitol or mannitol, 35-55g/L yeast extract, 45-75g/L tryptone, 0.5-3g/L magnesium sulfate. The feed medium 2 comprises 200-300g/L of sorbitol or mannitol, 200-300g/L of lactose, 45-75g/L of tryptone and 0.5-3g/L of magnesium sulfate.
The third aspect of the invention provides a fermentation method for producing rhG-CSF, which comprises the following specific technical scheme:
I. amplifying engineering bacteria strains in a seed culture medium step by step to obtain seed liquid;
and II, inoculating the seed solution into a basic culture medium of a fermentation tank for high-density culture, feeding a carbon source, and feeding a feed supplement culture medium in batches until the culture is finished.
The method of the invention is suitable for engineering bacteria strain for producing rhG-CSF, in a preferred embodiment, the engineering bacteria strain for producing rhG-CSF is p ET-G-CSF /BL 21 (DE 3 ) PlysS, which was constructed and stored by the present laboratory, was the same method as CN102154189.
Preferably, the seed solution is inoculated into the basic culture medium of the fermentation tank in an inoculation volume of 5-10% of the volume of the basic culture medium.
Preferably, the carbon source in the fermentation base medium is fed to the fermentation system after 2-4 hours of fermentation, and the feeding is controlled to be completed for 2-4 hours.
Preferably, the fed-batch culture medium is fed-batch culture medium divided into two times, wherein the fed-batch culture medium for the first time is fed-batch culture medium 1 started when dissolved oxygen and pH of a fermentation system synchronously rise; the second feeding medium is fed with the feeding medium 2 after the inducer is added.
Preferably, the inducer is one or a combination of IPTG and lactose.
In a preferred embodiment, the fermentation process of rhG-CSF comprises the steps of:
a. the rhG-CSF engineering bacteria are shake cultured in a seed culture medium and used as seed liquid for rhG-CSF fermentation.
b. Inoculating the seed liquid for rhG-CSF fermentation into a fermentation tank containing a basic culture medium according to the inoculum size of 5% -10% for fermentation.
c. Controlling the temperature, dissolved oxygen and pH value of the fermentation liquid in the fermentation process, and ensuring the normal growth of engineering bacteria; after fermentation for 2-4h, slowly feeding separately prepared and sterilized sorbitol or mannitol solution into a fermentation tank, and continuing culturing.
d. When the nutrients are exhausted, the first feeding of the feed medium is carried out when the dissolved oxygen and the pH of the fermentation system synchronously rise, and the fed medium is the feed medium 1.
e. Inducing, and feeding a feeding culture medium for the second time, wherein the fed culture medium is a feeding culture medium 2 until the fermentation is finished.
Preferably, in the high-density culture process, the dissolved oxygen content of the fermentation liquid is controlled to be not lower than 30%, the fermentation temperature is 32-37 ℃, and the pH is 6.8+/-0.2.
Preferably, when the OD of the fermentation broth is 600 When the value is increased to 30-40And performing induction expression.
Acetic acid is produced during fermentation, the production of a large amount of acetic acid can reduce the pH value and inhibit the growth of thalli, and in order to reduce the influence of acetic acid on fermentation, the pH value of a fermentation system needs to be controlled, preferably ammonia water is used for controlling the pH value of the fermentation system, and the consumption of the ammonia water reflects the amount of acetic acid produced during fermentation.
In a preferred embodiment, the fermentation process for producing rhG-CSF comprises the steps of:
a. seed liquid culture: the rhG-CSF engineering bacterium seed liquid is a culture liquid for culturing the preserved engineering bacterium in a liquid seed culture medium. Shake culturing rhG-CSF engineering bacteria in liquid seed culture medium until the OD of bacterial liquid 600 Culturing to 2.0-2.5, and using as seed for rhG-CSF fermentation.
b. Pouring the prepared basic culture medium into a fermentation tank, and sterilizing at 121deg.C for 30min. After cooling, controlling the temperature of the fermentation tank, the air inlet amount, the initial rotating speed and the tank pressure, and calibrating the dissolved oxygen by 100% after stabilizing. The pH value of the culture medium is regulated to 6.8+/-0.2.
Seed liquid is inoculated to a fermentation tank under the protection of flame. After inoculation, controlling dissolved oxygen of the fermentation broth to be not lower than 30%, and controlling factors such as pH value, material supplementing and the like to ferment.
c. In the early stage of fermentation, the fermentation temperature is controlled, the pH value of the fermentation liquid can be gradually increased, and after 2-4 hours of fermentation, the separately prepared and sterilized sorbitol or mannitol solution is fed in a flowing way, and after the fed in the flowing way, the culture is continued. At this time, the pH value gradually decreases, and the pH value is adjusted to be 6.8+/-0.2 by ammonia water.
d. When the dissolved oxygen and the pH value of the fermentation liquid synchronously rise, the first feeding of the feed medium 1 is started, and the flow speed and the feed time are controlled.
e. Detection of the cell Density OD of the fermentation broth 600 Judging the growth condition of rhG-CSF engineering bacteria in the fermentation liquor. When the OD of the fermentation broth 600 When the value is increased to 30-40, induced expression is performed. After addition of the inducer, the reaction was allowed to stand for 20min and then fed with medium 2 a second time. Controlling flow rate and feeding time, fermenting temperature, dissolved oxygen and pH, and inducing Culturing is continued until the end.
In a preferred embodiment, step a seed solution culture step is as follows:
inoculating engineering bacteria strain onto the slant of solid seed culture medium, culturing overnight at 37deg.C, picking single colony from plate, inoculating into liquid seed culture medium, culturing at 37deg.C, 180r/min to OD 600 1.0-1.5, adding IPTG to a concentration of 0.1mmol/L, performing induced fermentation, centrifugally collecting thalli, performing SDS-PAGE electrophoresis analysis, screening to obtain rhG-CSF single colonies, selecting an optimal colony streaking plate for culturing overnight at 37 ℃, and preserving at 4 ℃ after culturing; picking single colony from the plate for re-streaking culture, inoculating into liquid seed culture medium, culturing at 30-37deg.C for 150r/min to OD 600 0.5-1.0 as primary seed liquid; inoculating the first seed solution into liquid seed culture medium, and culturing at 30-37deg.C and 150r/min to OD 600 And (3) microscopic examination and no bacteria are carried out until the temperature reaches 1.5-2.0, and the rhG-CSF fermentation seed is obtained.
The invention is not limited to the seed liquid culturing step, and any existing solid medium, liquid medium and culturing method for seed liquid culturing can be used in the invention. In one embodiment the solid seed medium consists of: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose; the liquid seed culture medium consists of: 10g/L peptone, 5g/L yeast extract, and 10g/L sodium chloride.
Preferably, feeding the feed medium for the first time, controlling the flow speed to be 2-3ml/min, and feeding for 3-4 hours; feeding the culture medium for the second time, controlling the flow speed to be 2-2.2ml/min, feeding for 3-6h, feeding for 2.2-2.5ml/min, feeding for 2-3h, feeding for 2.4-3ml/min, and feeding for 3-5h.
Preferably, the total feed medium time is 12-16h.
Specifically, in a preferred embodiment, the fermentation process for producing rhG-CSF comprises the steps of:
a. seed liquid culture:
the solid seed culture medium consists of: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose;
the liquid seed culture medium consists of: 10g/L peptone, 5g/L yeast extract, and 10g/L sodium chloride.
(1) And (3) strain screening: inoculating engineering bacteria strain onto the slant of solid seed culture medium, culturing overnight at 37deg.C, picking single colony from plate, inoculating into liquid seed culture medium, culturing at 37deg.C, 180r/min to OD 600 1.0-1.5, adding IPTG to a concentration of 0.2mmol/L, centrifuging to collect thalli after 4h of induction, screening to obtain rhG-CSF single colonies through SDS-PAGE electrophoresis analysis, selecting an optimal colony streaking plate for culturing overnight at 37 ℃, and preserving at 4 ℃ after culturing;
(2) Culturing primary seed liquid: single colonies were picked from the plates of the re-streaked culture and inoculated into liquid tubes containing 8mL of seed medium, incubated at 30-37℃at 150r/min to OD 600 0.5-1.0 as primary seed liquid;
(3) Culturing a secondary seed solution: inoculating the primary seed solution into 500mL triangular flask containing 250mL seed culture medium at 1% inoculating ratio, culturing at 30-37deg.C for 150r/min to OD 600 And (3) microscopic examination and no bacteria are carried out until the temperature reaches 1.5-2.0, and the rhG-CSF fermentation seed is obtained.
b. Pouring the prepared basal medium without carbon source into a fermentation tank, and sterilizing at 121deg.C for 30min. After cooling, controlling the temperature of the fermentation tank to be 37 ℃, the ventilation ratio of ventilation air to be 1:0.5, the initial rotating speed to be 200rpm, the tank pressure to be 0.05MPa, and calibrating dissolved oxygen to be 100% after stabilizing. The pH value of the culture medium is regulated to 6.8+/-0.2 by phosphoric acid or ammonia water, and the pH value is regulated to 6.8+/-0.2 by ammonia water in the fermentation process.
Inoculating the seed liquid into the fermentation tank with 5-10% of inoculation amount under the protection of flame. After inoculation, controlling dissolved oxygen of the fermentation broth to be not lower than 30%, and fermenting by pH value, feeding and other factors.
Wherein, the basic culture medium comprises 100ml/L of carbon source, 1-5g/L of yeast extract, 2-10g/L of tryptone, 20-40g/L of disodium hydrogen phosphate dodecahydrate, 2-10g/L of potassium dihydrogen phosphate, 2-10g/L of ammonium chloride, 0.5-3g/L of sodium chloride, 0.01-0.03g/L of ferrous sulfate, 0.5-2g/L of magnesium sulfate and 0.3-0.5ml/L of defoamer, wherein, the concentration of the carbon source is 200-600g/L, and the components are independently prepared and sterilized.
c. In the early stage of fermentation, the fermentation temperature is controlled to be 35-37 ℃, the pH value of fermentation liquid can be gradually increased, and after fermentation for 2-4 hours, 200-600g/L of sorbitol or mannitol solution which is independently prepared and sterilized is fed in, and the feeding time is 2-4 hours, and then culture is continued. At this time, the pH value gradually decreases, and the pH value is adjusted to be 6.8+/-0.2 by ammonia water.
d. After sorbitol or mannitol solution is fed, feeding the feed supplement culture medium 1, controlling the flow speed to be 2-3ml/min, and feeding for 3-4h when dissolved oxygen and pH value of the fermentation liquid synchronously rise.
Wherein, the 1 component of the feed medium is sorbitol or mannitol 400-600g/L, yeast extract 35-55g/L, tryptone 45-75g/L, and magnesium sulfate 0.5-3g/L.
e. Detection of the cell Density OD of the fermentation broth 600 Judging the growth condition of rhG-CSF engineering bacteria in the fermentation liquor. When the OD of the fermentation broth 600 When the value is increased to 30-40, induced expression is performed. Adding inducer IPTG to induce, adding inducer with final concentration of 0.06-0.1mM, waiting for 20min, feeding culture medium 2, controlling flow rate to 2-2.2ml/min, feeding for 3-6 hr, feeding for 2.2-2.5ml/min, feeding for 2-3 hr, feeding for 2.4-3ml/min, and feeding for 3-5 hr. The total feeding time is 12-16h. Controlling the fermentation temperature to be 32-35 ℃, controlling the dissolved oxygen to be 30-50%, controlling the pH to be 6.8+/-0.2, and finishing the fermentation after the material supplementing is finished.
Wherein the feed medium 2 comprises lactose 200-300g/L, sorbitol or mannitol 200-300g/L, tryptone 45-75g/L, and magnesium sulfate 0.5-3g/L.
Compared with the prior art, the invention has the following advantages:
the culture method of the invention ferments the rhG-CSF engineering bacteria, and can greatly improve the fermentation level of the escherichia coli. At the end of fermentation, fermentation Density OD 600 The value reaches more than 100, the expression quantity of the fermentation target protein reaches more than 77%, and the fermentation level of the escherichia coli is greatly improved. The control range of fermentation temperature and the control range of fermentation pH value in different periods are optimized, the adding time of a basic culture medium carbon source is changed, the mixture ratio and the feeding amount of the culture medium are optimized, the feeding is carried out in a limited flow mode, the synergistic induction of low-concentration IPTG and lactose is carried out, and the high-density high-expression of rhG-CSF fermentation is facilitated. Plasmid loss after fermentationThe rate is within 5%, and the rhG-CSF high-density fermentation improves the production efficiency and has good application prospect.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The materials used in the present invention are commercially available without any particular explanation.
Example 1
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
The solid seed culture medium consists of: 5g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
The liquid seed culture medium consists of: 10g/L peptone, 5g/L yeast extract, and 10g/L sodium chloride.
(1) And (3) strain activation and screening: culturing strain pET-rhG-CSF/BL21 (DE 3) PlysS to solid seed culture medium group plate for streaking activation at 33-37deg.C overnight, selecting single colony from plate, inoculating into 8ml liquid seed culture medium group, culturing at 37deg.C, 180r/min to OD 600 1.0-1.5, adding IPTG to a concentration of 0.2mmol/L, centrifuging to collect thalli after 4h of induction, screening to obtain rhG-CSF single colonies through SDS-PAGE electrophoresis analysis, selecting an optimal colony streaking plate for culturing overnight at 33-37 ℃, and preserving at 4 ℃ after culturing;
(2) Culturing primary seed liquid: single colonies were picked from the plates of the re-streaked culture and inoculated into tubes containing 8mL of liquid seed medium, cultured at 33-37℃at 150r/min to OD 600 0.5-1.0 as primary seed liquid;
(3) Culturing a secondary seed solution: inoculating the primary seed solution into 500mL triangular flask containing 250mL liquid seed culture medium at 1% inoculating ratio, and culturing at 33-37deg.C at 150r/min to OD 600 And (3) performing microscopic examination to 1.5-2.0, and performing sterile sterilization to obtain the seed liquid for rhG-CSF fermentation.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.4ml/L of defoamer; wherein, the concentration of sorbitol is 400g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of sorbitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, sorbitol 230g/L, magnesium sulfate 1g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then 3 hours of feeding the separately prepared and sterilized sorbitol solution (400 g/L, 1L); after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding the feed supplement culture medium 2 after adding IPTG for 20min, wherein the feeding acceleration is 2ml/min, 3h is fed by 2.2ml/min, 3h is fed by 2.4ml/min, 5h is fed by gradient feeding, the total feeding time is 15h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, the pH value is 6.80+/-0.2, and the fermentation is finished after the feeding is finished. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 2
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 3g/L of yeast extract, 7g/L of tryptone, 25g/L of disodium hydrogen phosphate dodecahydrate, 7g/L of potassium dihydrogen phosphate, 7g/L of ammonium chloride, 2g/L of sodium chloride, 0.02g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.5ml/L of defoamer; wherein, the concentration of sorbitol is 500g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 50g/L of yeast extract, 70g/L of tryptone, 460g/L of sorbitol and 2g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, sorbitol 250g/L, tryptone 50g/L, magnesium sulfate 2g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then 3 hours of feeding the separately prepared and sterilized sorbitol solution (500 g/L, 1L); after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. After adding IPTG for 20min, feeding a feeding culture medium 2, feeding 3h at a feeding speed of 2ml/min, feeding 3h at a feeding speed of 2.2ml/min, feeding 5h at a feeding speed of 2.4ml/min, feeding by gradient feeding, and maintaining dissolved oxygen at 30% -70% at a pH value of 6.80+ -0.2 for 15h at a total feeding time of 32-35 ℃. And after the material supplementing is finished, finishing the fermentation. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 3
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
The basic culture medium formula comprises: 100ml/L of sorbitol, 1g/L of yeast extract, 2g/L of tryptone, 20g/L of disodium hydrogen phosphate dodecahydrate, 2g/L of potassium dihydrogen phosphate, 2g/L of ammonium chloride, 2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 0.5g/L of magnesium sulfate and 0.3ml/L of defoamer. Wherein, the concentration of sorbitol is 200g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 55g/L of yeast extract, 75g/L of tryptone, 600g/L of sorbitol and 3g/L of magnesium sulfate.
Feed medium 2: lactose 300g/L, tryptone 75g/L, sorbitol 300g/L, magnesium sulfate 3g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then 3h of feeding a separately prepared and sterilized sorbitol solution (200 g/L, 1L); after the feeding is finished, feeding the feed supplement culture medium at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted and the dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding a feeding culture medium after adding IPTG for 20min, wherein the feeding acceleration is 2ml/min for 3h, 2.2ml/min for 3h, 2.4ml/min for 5h, feeding is carried out in a gradient mode, the total feeding time is 15h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, and the pH value is 6.80+/-0.2. And after the material supplementing is finished, finishing the fermentation. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 4
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
The basic culture medium formula comprises: 100ml/L of sorbitol, 5g/L of yeast extract, 10g/L of tryptone, 40g/L of disodium hydrogen phosphate dodecahydrate, 10g/L of potassium dihydrogen phosphate, 10g/L of ammonium chloride, 3g/L of sodium chloride, 0.03g/L of ferrous sulfate, 2g/L of magnesium sulfate and 0.5ml/L of defoamer. Wherein, the concentration of sorbitol is 600g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 33g/L of yeast extract, 45g/L of tryptone, 400g/L of sorbitol and 05g/L of magnesium sulfate.
Feed medium 2: lactose 200g/L, tryptone 45g/L, sorbitol 200g/L, magnesium sulfate 0.5g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then 3h of separately prepared and sterilized sorbitol solution (600 g/L, 1L) was fed in; after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. After adding IPTG for 20min, feeding a feeding culture medium 2, feeding 3h at a feeding speed of 2ml/min, feeding 3h at a feeding speed of 2.2ml/min, feeding 5h at a feeding speed of 2.4ml/min, feeding by gradient feeding, and maintaining dissolved oxygen at 30% -70% at a pH value of 6.80+ -0.2 for 15h at a total feeding time of 32-35 ℃. And after the material supplementing is finished, finishing the fermentation. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 5
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.4ml/L of defoamer; wherein, the concentration of sorbitol is 400g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of sorbitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, sorbitol 230g/L, magnesium sulfate 1g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 8% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by using phosphoric acid or ammonia water, and culturing for 4 hours; then 4h of separately prepared and sterilized sorbitol solution (400 g/L, 1L) was fed in; after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. After adding IPTG for 20min, feeding a feeding culture medium 2, feeding 3h at a feeding acceleration of 2ml/min, feeding 3h at a feeding speed of 2.2ml/min, feeding 5h at a feeding speed of 2.4ml/min, feeding by gradient feeding, and maintaining dissolved oxygen at 30% -70% at a pH value of 6.80+ -0.2 for 15h at a total feeding time of 37 ℃. And after the material supplementing is finished, finishing the fermentation. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 6
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: mannitol 100ml/L, yeast extract 2g/L, tryptone 5g/L, disodium hydrogen phosphate dodecahydrate 30g/L, potassium dihydrogen phosphate 5g/L, ammonium chloride 4g/L, sodium chloride 1.2g/L, ferrous sulfate 0.01g/L, magnesium sulfate 1g/L, and defoamer 0.4 ml/L; wherein, the mannitol concentration is 400g/L, and the mannitol is independently prepared and sterilized.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of mannitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, mannitol 230g/L, and magnesium sulfate 1g/L.
10L of basal medium without mannitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by using phosphoric acid or ammonia water, and culturing for 3 hours; then feeding separately prepared and sterilized mannitol solution (400 g/L, 1L) for 2.5 h; after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.6ml/min for 3 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. After adding IPTG for 20min, feeding a feeding culture medium 2, feeding for 4h at a feeding speed of 2ml/min, feeding for 4h at a feeding speed of 2.2ml/min, feeding for 3.5h at a feeding speed of 2.4ml/min, feeding by a gradient feeding, and keeping dissolved oxygen at 30% -70% at a temperature of 32-35 ℃ for 14.5h at a pH value of 6.80+ -0.2. And after the material supplementing is finished, finishing the fermentation. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 7
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.4ml/L of defoamer; wherein, the concentration of sorbitol is 400g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of sorbitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, sorbitol 230g/L, magnesium sulfate 1g/L. 10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by using phosphoric acid or ammonia water, and culturing for 2 hours; then feeding separately prepared and sterilized sorbitol solution (400 g/L, 1L) for 2h; after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 3ml/min for 3 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. After adding IPTG for 20min, feeding a feeding culture medium 2, feeding 3h at a feeding acceleration of 2.2ml/min, feeding 2h at a feeding speed of 2.5ml/min, feeding 4h at a feeding speed of 3ml/min, feeding by gradient feeding, wherein the total feeding time is 12h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, and the pH value is 6.80+/-0.2. And after the material supplementing is finished, finishing the fermentation. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 8
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.4ml/L of defoamer; wherein, the concentration of sorbitol is 400g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of sorbitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, sorbitol 230g/L, magnesium sulfate 1g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then 4h of separately prepared and sterilized sorbitol solution (400 g/L, 1L) was fed in; after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding the feed supplement culture medium 2 after adding IPTG for 20min, wherein the feeding acceleration is 2.0ml/min, the feeding speed is 2.2ml/min, the feeding speed is 3h, the feeding speed is 2.4ml/min, the feeding speed is 3h, the total feeding time is 16h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, the pH value is 6.80+/-0.2, and the fermentation is finished after the feeding is completed. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 9
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.04% of defoamer; wherein, the concentration of sorbitol is 100g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 300g/L of sorbitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 150g/L, tryptone 65g/L, sorbitol 100g/L, magnesium sulfate 1g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then 3 hours of feeding the separately prepared and sterilized sorbitol solution (100 g/L, 1L); after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding the feed supplement culture medium 2 after adding IPTG for 20min, wherein the feeding acceleration is 2ml/min, 3h is fed by 2.2ml/min, 3h is fed by 2.4ml/min, 5h is fed by gradient feeding, the total feeding time is 15h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, the pH value is 6.80+/-0.2, and the fermentation is finished after the feeding is finished. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 10
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.4ml/L of defoamer; wherein, the concentration of sorbitol is 400g/L, and the sorbitol is independently prepared and sterilized.
Feed medium: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of sorbitol and 1g/L of magnesium sulfate.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then 3 hours of feeding the separately prepared and sterilized sorbitol solution (400 g/L, 1L); after the feeding is finished, feeding the feed supplement culture medium at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted and the dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding a feeding culture medium after adding IPTG for 20min, wherein the feeding acceleration is 2ml/min, 3h is fed by 2.2ml/min, 3h is fed by 2.4ml/min, 5h is fed by gradient feeding, the total feeding time is 15h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, the pH value is 6.80+/-0.2, and the fermentation is finished after the feeding is finished. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 11
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
Basal medium: 100ml/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.4ml/L of defoamer; wherein, the concentration of sorbitol is 400g/L, and the sorbitol is independently prepared and sterilized.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of sorbitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, sorbitol 230g/L, magnesium sulfate 1g/L.
10L of basal medium without sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80+/-0.2 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then the sorbitol solution (400 g/L, 1L) was added instantaneously; when the basic culture medium is used up and dissolved oxygen and pH value synchronously rise, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And after adding IPTG for 20min, feeding a feeding culture medium 2, feeding the feed at the constant speed for 14h at the flow acceleration of 2.2ml/min, feeding the feed at the constant speed for 18h, wherein the total feeding time is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, and the pH value is 6.80+/-0.2. And after the material supplementing is finished, finishing the fermentation. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Example 12
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
The basic culture medium formula comprises: 40g/L of sorbitol, 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.04% of defoamer.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of sorbitol and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, sorbitol 230g/L, magnesium sulfate 1g/L.
10L of basal medium containing sorbitol is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seeds into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to be 6.80 by phosphoric acid or ammonia water, and when the basic culture medium is exhausted, the dissolved oxygen and the pH value rise synchronously, starting feeding the culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min, and the total feeding time is 4h. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding the feed supplement culture medium 2 after adding IPTG for 20min, wherein the feeding acceleration is 2ml/min, 3h is fed by 2.2ml/min, 3h is fed by 2.4ml/min, 5h is fed by gradient feeding, the total feeding time is 15h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, the pH value is 6.80, and the fermentation is finished after the feeding is completed. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Comparative example 1
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
The basic culture medium formula comprises: 2g/L of yeast extract, 5g/L of tryptone, 30g/L of disodium hydrogen phosphate dodecahydrate, 5g/L of potassium dihydrogen phosphate, 4g/L of ammonium chloride, 1.2g/L of sodium chloride, 0.01g/L of ferrous sulfate, 1g/L of magnesium sulfate and 0.04 percent of defoamer; wherein the defoaming agent is polyether or organic silicon defoaming agent.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone and 1g/L of magnesium sulfate.
Feed medium 2: lactose 300g/L, tryptone 65g/L, magnesium sulfate 1g/L.
10L of basal medium is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seeds into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to be 6.80 by phosphoric acid or ammonia water, and when the basic culture medium is exhausted, the dissolved oxygen and the pH value rise synchronously, starting feeding the culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min, and the total feeding time is 4h. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding the feed supplement culture medium 2 after adding IPTG for 20min, wherein the feeding acceleration is 2ml/min, 3h is fed by 2.2ml/min, 3h is fed by 2.4ml/min, 5h is fed by gradient feeding, the total feeding time is 15h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, the pH value is 6.80, and the fermentation is finished after the feeding is completed. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Comparative example 2
1. Step-by-step amplifying culture of engineering bacteria strain in seed culture medium
Seed liquid preparation parameters were as shown in example 1, and seed liquid for rhG-CSF fermentation was obtained.
2. High density fermentation culture
The basic culture medium formula comprises: glucose 100ml/L,1L (separately formulated and sterilized), yeast extract 2g/L, tryptone 5g/L, disodium hydrogen phosphate dodecahydrate 30g/L, potassium dihydrogen phosphate 5g/L, ammonium chloride 4g/L, sodium chloride 1.2g/L, ferrous sulfate 0.01g/L, magnesium sulfate 1g/L,0.04% defoamer; wherein the glucose concentration is 400g/L, and the preparation and sterilization are carried out independently.
Feed medium 1: 40g/L of yeast extract, 65g/L of tryptone, 530g/L of glucose and 1g/L of magnesium sulfate.
Feed medium 2: lactose 250g/L, tryptone 65g/L, glucose 230g/L, magnesium sulfate 1g/L.
10L of basal medium without glucose is added into a 20L fermentation tank, and sterilization is carried out for 30 minutes at 121 ℃; after sterilization, when the temperature of the basic culture medium is reduced to 35-37 ℃, inoculating the rhG-CSF fermentation seed into a fermentation tank according to the volume ratio of 10% of the inoculum size, culturing at 37 ℃ and 200rpm, gradually adjusting the rotation speed to 200-800rpm along with the growth of thalli, maintaining dissolved oxygen to be more than 35%, adjusting the pH value to 6.80 by phosphoric acid or ammonia water, and culturing for 3.5 hours; then glucose solution (400 g/L, 1L) was fed over 3 h; after the feeding is finished, feeding the feed supplement culture medium 1 at a constant speed with the flow acceleration of 2.2ml/min for 4 hours when the nutrient of the basic culture medium is exhausted, dissolved oxygen and the pH value synchronously rise. When the nutrient of the culture medium is exhausted and dissolved oxygen and the pH value synchronously rise, adding IPTG with the final concentration of 0.20mmol/L into the fermentation tank for induction. And (3) feeding the feed supplement culture medium 2 after adding IPTG for 20min, wherein the feeding acceleration is 2ml/min, 3h is fed by 2.2ml/min, 3h is fed by 2.4ml/min, 5h is fed by gradient feeding, the total feeding time is 9h, the temperature is 32-35 ℃, the dissolved oxygen is maintained at 30-70%, the pH value is 6.80, and the fermentation is finished after the feeding is completed. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
Comparative example 3
1. Culturing seeds: seed culture medium 210mL was placed in a 500mL Erlenmeyer flask, sterilized by heat and humidity at 121℃for 15min, and cooled to room temperature. pET-rhG-CSF/BL21 (DE 3) PlysS glycerol strain is inoculated into a seed culture medium with an inoculum size of 0.1%, and is subjected to shaking culture in a shaker at 37 ℃ and a rotation speed of 180rpm for 9 hours, and OD600 is measured to be 1.0, so as to obtain a fermentation seed culture solution. Wherein the seed culture medium comprises the following components in percentage by weight: 20g/L of tryptone, 10g/L of yeast powder and 10g/L of sodium chloride are prepared by deionized water, and the pH value is adjusted to 7.1 after sterilization.
2. Preparation before inoculation: firstly, cleaning a fermentation tank, dissolving a fermentation culture medium in purified water, uniformly stirring, pouring the mixture into the fermentation tank, performing off-line sterilization for 20min at 121 ℃ by steam and damp heat, and cooling to 37 ℃ for standby. Wherein the composition of the fermentation medium is: tryptone 20g/L, yeast extract 10g/L, naCl10g/L, lactose 1g/L.
Dissolving the feed supplement culture medium in purified water, uniformly stirring, sterilizing for 20min in an off-line mode by steam wet heat at 121 ℃, and cooling for later use. Wherein the composition of the feed medium is: 100g/L of tryptone, 50g/L of yeast extract, 360g/L of glycerol and 2g/L of lactose.
3. Inoculating: under the protection of flame, inoculating 210mL of qualified fermentation seed culture solution into a 10L fermentation tank according to the inoculation amount of 3% of the volume of the fermentation medium, filling 7L of the fermentation seed culture solution, and starting fermentation culture at the culture temperature of 37 ℃ and the stirring rotation speed of 375 rpm. The initial value of dissolved oxygen is corrected to 100% by adjusting the rotating speed, the air flow and the tank pressure after the fermentation seed liquid is inoculated to the fermentation culture medium, and the dissolved oxygen value is not controlled in the fermentation process.
4. Fermentation: the fermentation process adopts the following intermediate control method:
4.1 fermenting for 5h, and feeding the feed medium prepared in the step 2, wherein the feed rate is 200mL/h, and the volume of the feed medium is 2L;
4.2 fermenting for 8h, wherein the rotating speed is adjusted to 250rpm from 375 rpm;
4.3 the total feed supplement in the fermentation process of this example was 2L;
4.4 ph=7.0±0.1 during the fermentation in this example, 16.0% naoh solution, 25% hcl solution was automatically fed through the reactor to adjust;
4.5, fermenting for 20 hours to obtain fermentation bacteria liquid. And (3) taking fermentation liquor SDS-PAGE electrophoresis analysis to determine the expression quantity of the rhG-CSF protein and the loss rate of the plasmid.
The results of fermentation broth measurements for examples 1-12 and comparative examples 1-3 are shown in Table 1.
TABLE 1 fermentation broth detection results

Claims (2)

1. A fermentation method for producing rhG-CSF, which is characterized by comprising the following specific technical scheme:
I. amplifying engineering bacteria strains in a seed culture medium step by step to obtain seed liquid;
II, inoculating the seed liquid into a basic culture medium without a carbon source for high-density culture, feeding the carbon source into a fermentation system after 2-4h of culture, and controlling the fed-batch culture medium to be fed in batches after 2-4h of fed-batch culture is finished;
wherein the batch fed-batch feed medium is fed-batch medium divided into two times, and the fed-batch medium 1 is fed-batch for the first time when the dissolved oxygen and the pH of the fermentation system rise synchronously; after the first feeding is finished, when the OD600 value of the fermentation broth is increased to 30-40, adding an inducer for induction culture; feeding the feed medium 2 for the second time after adding the inducer;
The basal medium comprises the following components:
wherein the carbon source is sorbitol or mannitol, the concentration of the carbon source is 200-600g/L, and the carbon source needs to be prepared independently;
the feed medium 1 comprises the following components: 400-600g/L of sorbitol or mannitol, 35-55 g/L of yeast extract, 45-75 g/L of tryptone and 0.5-3 g/L of magnesium sulfate; feed medium 2 contained the following components: 200-300g/L of sorbitol or mannitol, 200-300g/L of lactose, 45-75 g/L of tryptone and 0.5-3 g/L of magnesium sulfate; the inducer is IPTG; the engineering bacteria strain is pET-G-CSF/BL21 (DE 3) PlysS.
2. The fermentation method according to claim 1, wherein the dissolved oxygen content of the fermentation broth is controlled to be not lower than 30% in the high-density culture process, the fermentation temperature is 32-37 ℃, and the pH is 6.8+ -0.2.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101410410A (en) * 2006-03-06 2009-04-15 卡迪拉保健有限公司 Process for preparing human G-CSF
WO2011086447A2 (en) * 2010-01-12 2011-07-21 Lupin Limited Fermentation process for the preparation of recombinant heterologous proteins
CN102154189A (en) * 2010-12-31 2011-08-17 山东新时代药业有限公司 Fermentation culture method of rhG-CSF (recombinant human granulocyte colony-stimulating factor) recombination engineering bacteria
CN104152515A (en) * 2014-08-14 2014-11-19 江苏奥赛康药业股份有限公司 Medium used for preparation of recombinant human granulocyte colony stimulating factor and fermentation method
CN110066331A (en) * 2018-01-23 2019-07-30 江苏恒瑞医药股份有限公司 A kind of preparation method of recombinant human granulocyte colony stimulating factor
CN112029810A (en) * 2019-06-03 2020-12-04 鲁南制药集团股份有限公司 Culture medium for producing insulin glargine and fermentation method

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101410410A (en) * 2006-03-06 2009-04-15 卡迪拉保健有限公司 Process for preparing human G-CSF
WO2011086447A2 (en) * 2010-01-12 2011-07-21 Lupin Limited Fermentation process for the preparation of recombinant heterologous proteins
CN102154189A (en) * 2010-12-31 2011-08-17 山东新时代药业有限公司 Fermentation culture method of rhG-CSF (recombinant human granulocyte colony-stimulating factor) recombination engineering bacteria
CN104152515A (en) * 2014-08-14 2014-11-19 江苏奥赛康药业股份有限公司 Medium used for preparation of recombinant human granulocyte colony stimulating factor and fermentation method
CN110066331A (en) * 2018-01-23 2019-07-30 江苏恒瑞医药股份有限公司 A kind of preparation method of recombinant human granulocyte colony stimulating factor
CN112029810A (en) * 2019-06-03 2020-12-04 鲁南制药集团股份有限公司 Culture medium for producing insulin glargine and fermentation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Production of recombinant human G-CSF from non-classical inclusion bodies in Escherichia coli;Trinh NTM等;Brazilian Journal of Microbiology(第52期);第541-546页，参见全文 *

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