CN104152515B - Culture medium for preparing recombinant human granulocyte colony stimulating factor and fermentation method - Google Patents
Culture medium for preparing recombinant human granulocyte colony stimulating factor and fermentation method Download PDFInfo
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Abstract
The invention discloses a culture medium for preparing a recombinant human granulocyte colony stimulating factor and a fermentation method, belonging to the technical field of fermentation engineering; the method comprises the steps of culturing recombinant strain seed liquid; inoculating the seed liquid into a fermentation culture medium; supplementing a supplemented medium during fermentation; obtaining fermentation liquor after fermentation; the fermentation medium and the feeding medium both contain inducer lactose; the composition of the fermentation medium is (g/L): 15-20 parts of tryptone, 10-20 parts of yeast extract, 10-20 parts of sodium chloride, 1-5 parts of lactose and water; the composition of the feed medium was (g/L): 50-100 parts of tryptone, 25-50 parts of yeast extract, 300 parts of glycerol, 2-6 parts of lactose and water. The invention simplifies the process, has low cost, is safe and nontoxic, improves the yield of the thalli and the expression quantity of the protein, and is beneficial to realizing industrial production.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a culture medium for preparing a recombinant human granulocyte colony-stimulating factor and a fermentation method.
Background
Human granulocyte colony-stimulating factors (hG-CSF) are a glycoprotein hematopoietic growth factor that specifically acts on granulocyte progenitor cells to promote proliferation and differentiation of the progenitor cells into mature neutrophils and maintain their functions and survival. In 1986, hG-CSF gene was isolated from the human squamous cell carcinoma cell line CHU-II by Nagata et al, and its nucleotide sequence was first determined and expressed in COS cells (Nagata S et al, Nature, 1986, 319: 415-418). There are two different kinds of human G-CSF cDNAs, which encode precursor proteins containing 207 and 204 amino acids, respectively, and each have 30-amino acid signal peptides, and the mature protein molecules are 177 and 174 amino acids, the former having the same sequence as the 174 amino acid molecule except that 3 amino acids are inserted at the 35-position at the N-terminus of the mature molecule. Human G-CSF has a molecular weight of 19.6kD, PI of 6.1, O-glycosylation, and is relatively stable against acids and bases (pH 2-10), heat, denaturants, and the like. The hG-CSF has 5 cysteine residues, wherein 4 cysteine residues form two pairs of disulfide bonds between Cys36 and Cys42, Cys74 and Cys 64; cys17 is an unpaired cysteine, and disulfide bond formation is important for maintaining G-CSF biological function.
The G-CSF has important significance in clinical application, and can promote the recovery of neutrophils during bone marrow transplantation; improving neutropenia accompanied by aplastic anemia; the severe neutrophilic granulocyte deficiency caused by cancer chemotherapy is obviously improved, the tumor treatment strength is enhanced, and the treatment effect is enhanced; can be widely used for treating other diseases accompanied with neutropenia, resisting infection, etc. The natural G-CSF product has very limited source and far from clinical requirement, and the recombinant G-CSF with similar biological activity to natural one may be produced in large scale.
In 1985, Welte K successfully purified and refined hG-CSF from the culture supernatant of human bladder cancer cell strain 5637, then Welte K and Souza L M further determined the N-segment amino acid sequence of this hG-CSF, cloned hG-CSF gene from 5637 cell strain, and inserted the gene into Escherichia coli by gene engineering technology to successfully prepare hG-CSF, thereby developing recombinant human granulocyte colony stimulating factor rhG-CSF, which is commercially known as Filgratin (Whitblood). The drug was approved by the U.S. FDA for marketing in 1991. In 1993, Dianthus superbus Quiquus et al firstly cloned human G-CSF cDNA in China and obtained expression in Escherichia coli. Since then, many researchers have been devoted to research on this aspect, and it is very important to optimize the fermentation process, improve the product quality, and reduce the production cost because G-CSF has the disadvantages of low expression level and inferior biological activity to natural products in prokaryotes.
At present, the research on the fermentation production process of the recombinant granulocyte colony stimulating factor mainly focuses on the aspects of engineering strains, high-density fermentation culture, inclusion body renaturation, purification and the like. Inducible expression is currently the most common and efficient strategy for the clonal expression of recombinant proteins in E.coli. There are three main ways of induction: IPTG (isopropyl-beta-D-thiogalactoside), heat induction and lactose induction. Recombinant granulocyte colony stimulating factor fermentation processes currently employ either IPTG induction or thermal induction (e.g., 42 ℃). IPTG is a very efficient lactose promoter inducer, the induction conditions of IPTG are simple, the effect is durable and stable, but IPTG has potential toxicity, the IPTG is not advocated to be used by pharmacopoeias of various countries, and the cost is high, so that IPTG is not suitable for large-scale production of gene engineering products in fermentation tanks. In addition, heat induction tends to make inclusion body renaturation difficult and reduce the renaturation rate. Lactose is disaccharide, has no toxicity, has the function of inducing lactose operon naturally, is cheap and easy to obtain, can be used as an inducer for replacing IPTG, and has very important practical significance and application value for large-scale production of recombinant protein by using a prokaryotic expression system. However, lactose can be metabolized and utilized by cells as a carbon source while being used as an inducer, the concentration of lactose is dynamically changed, multiple enzymes are involved in transportation and transformation, the induction mechanism is more complicated than IPTG, the induction conditions are not easy to master, and the growth and the induction conditions of the thalli need to be more finely researched and optimized to stably and efficiently induce the recombinant thalli to ferment and express hG-CSF.
Disclosure of Invention
One of the purposes of the invention is to provide a high-efficiency expression culture medium for preparing recombinant human granulocyte colony stimulating factor, which comprises lactose as an inducer and a carbon source, and solves the problems of complex process and low recombinant protein expression amount of the lactose as the carbon source and the inducer in the prior art by researching and breaking through the types and contents of the carbon source, the nitrogen source, the inducer and other nutrient components in the culture medium and the proportion of each culture medium; another object of the present invention is to provide a fermentation method for preparing recombinant human granulocyte colony stimulating factor by using the above high-efficiency expression culture medium, wherein the fermentation method comprises an inducer in the culture medium, omits the common steps of IPTG induction and thermal induction, simplifies the process, and has the advantages of no toxicity of lactose, low cost, suitability for large-scale industrial production of recombinant medical protein and extremely important value.
The technical scheme of the invention is as follows: a culture medium for preparing a recombinant human granulocyte colony stimulating factor comprises a seed culture medium, a fermentation culture medium and a supplemented culture medium, wherein the fermentation culture medium comprises the following components in percentage by weight: 15-20 g/L of tryptone, 10-20 g/L of yeast extract, 10-20 g/L of sodium chloride, 1-5 g/L of lactose and the balance of water; the feed culture medium comprises the following components in percentage by weight: 50-100 g/L of tryptone, 25-50 g/L of yeast extract, 300-500 g/L of glycerol, 2-6 g/L of lactose and the balance of water; the volume of the feed medium is 20-30% of the volume of the fermentation medium.
Further, the fermentation medium comprises the following components in percentage by weight: 18-20 g/L of tryptone, 10-20 g/L of yeast extract, 10-20 g/L of sodium chloride, 1-5 g/L of lactose and the balance of water; the feed culture medium comprises the following components in percentage by weight: 70-100 g/L of tryptone, 40-50 g/L of yeast extract, 360-500 g/L of glycerol, 2-6 g/L of lactose and the balance of water.
Preferably, the components and the content of the seed culture medium are 15-25 g/L of tryptone, preferably 20-25 g/L, 7-14 g/L of yeast powder, preferably 10-14 g/L, 6-15 g/L of sodium chloride, preferably 10-15 g/L, the balance of water, and the pH value is adjusted to 7.0-7.2; the volume of the seed culture medium is 1-3% of the volume of the fermentation culture medium.
The invention also provides a fermentation method for preparing the recombinant human granulocyte colony stimulating factor by using the culture medium, which comprises the following steps:
inoculating host bacteria transformed with recombinant vector plasmid constructed by pET-9a vector and human G-CSF gene to seed culture medium and culturing to OD6000.8-1.2, obtaining a fermented seed liquid, wherein the human G-CSF gene sequence is shown as SEQ ID No. 1;
secondly, inoculating the fermentation seed liquid into a sterilized fermentation culture medium according to the proportion of 1-3% of the volume of the fermentation culture medium, and culturing for 3-6 hours;
supplementing the sterilized supplemented medium into the fermentation system according to the proportion of 20-30% of the volume of the fermentation medium, continuing fermentation, and finishing fermentation 18-22 hours later to obtain recombinant bacterium fermentation liquor, wherein the fermentation medium comprises the following components in percentage by volume: 15-20 g/L of tryptone, 10-20 g/L of yeast extract, 10-20 g/L of sodium chloride, 1-5 g/L of lactose and the balance of water; the feed culture medium comprises the following components in percentage by weight: 50-100 g/L of tryptone, 25-50 g/L of yeast extract, 300-500 g/L of glycerol, 2-6 g/L of lactose and the balance of water.
Further, the fermentation medium comprises the following components in percentage by weight: 18-20 g/L of tryptone, 10-20 g/L of yeast extract, 10-20 g/L of sodium chloride, 1-5 g/L of lactose and the balance of water; the feed culture medium comprises the following components in percentage by weight: 70-100 g/L of tryptone, 40-50 g/L of yeast extract, 360-500 g/L of glycerol, 2-6 g/L of lactose and the balance of water.
Preferably, the host bacteria of the transformed recombinant vector plasmids are inoculated in a seed culture medium to obtain a fermented seed solution, the host bacteria of the transformed recombinant vector plasmids are selected from glycerol tube strains and inoculated in a culture bottle containing the seed culture medium, the inoculation amount is 0.1%, and the fermented seed solution is obtained by culturing for 8-10 hours at 35-39 ℃ and 160-180 rpm.
Preferably, the components and the content of the seed culture medium are 15-25 g/L of tryptone, preferably 20-25 g/L, 7-14 g/L of yeast powder, preferably 10-14 g/L, 6-15 g/L of sodium chloride, preferably 10-15 g/L, the balance of water, and the pH value is adjusted to 7.0-7.2.
The temperature is an important factor for fermentation and growth of engineering bacteria, and can affect the growth rate of bacteria, the biochemical reaction rate, the activity of an enzyme system, the absorption and utilization of a substrate and the like. pH has an effect on both normal cell growth and efficient expression of foreign proteins. Common acids and bases for controlling pH are HCl, H3PO4、H2SO4、NaOH、KOH、NH3·H2O, and the like. The rotating speed of the shaking table is also an important factor for controlling the fermentation growth of the engineering bacteria. In the process of fermentation growth, the rotating speed of the shaking table needs to be adjusted according to the growth and metabolism conditions of the engineering bacteria so as to optimize the expression quantity of the recombinant protein.
Preferably, in the fermentation process, the culture temperature of the fermentation is 35-39 ℃, and the pH of a fermentation system is controlled to be 6.0-7.5 by automatically feeding acid and alkali solution in a reactor; fermenting for 0-8 hours, wherein the rotating speed of the fermentation tank is 300-400 rpm, and after 8 hours, the rotating speed is adjusted to 200-400 rpm; after the fermentation seed liquid is inoculated to the fermentation culture medium, the rotating speed, the air flow and the tank pressure are adjusted to correct the dissolved oxygen initial value to be 100 percent, and the dissolved oxygen value is not controlled any more in the fermentation process.
Further, the acid and alkali solution used for controlling the pH of the fermentation system is hydrochloric acid and sodium hydroxide solution, preferably 25% hydrochloric acid and 16% sodium hydroxide solution.
The culture method of the genetic engineering bacteria comprises the following steps: batch culture, fed-batch culture, continuous culture, dialysis culture, immobilized culture, and the like. Batch culture and fed-batch culture are among the most commonly used culture methods. The batch culture is to add all nutrients into the fermentation tank at one time, and the product is obtained at one time after the fermentation is finished. The fed-batch culture is a culture method in which seeds are inoculated into a fermentation tank for culture, and after a period of time, fresh culture medium is fed in batch or continuously to further grow the thalli. The fed-batch culture method is also called fed-batch culture, and is based on batch culture, and is supplemented with carbon sources, nitrogen sources, inorganic salts and the like so as to solve the inhibition of the carbon sources and the nitrogen sources, eliminate the inhibition effect of high-concentration substrates on cell growth, and make up the defect that low-concentration substrates are not beneficial to cell growth, thereby effectively controlling the growth process of thalli, prolonging the logarithmic phase of the thalli and improving the cell density. Commonly used feeding strategies are: constant rate, stepwise increase and exponential addition.
Preferably, the sterilized feed medium is fed to the fermentation system in fed-batch mode, the feeding time is 10 hours, and the feeding rate is set to a value obtained by dividing the feeding volume by the feeding time. In a preferred embodiment of the invention, the total feeding volume is 2L, and the feeding rate is 2L/10 h-200 mL/h. In another preferred embodiment of the present invention, the total feeding volume is 5L, and the feeding rate is 5L/10 h-500 mL/h.
Preferably, the host bacterium is Escherichia coli (Escherichia coli) BL21(DE 3).
As is well known to those skilled in the art, the seed culture medium, the fermentation culture medium and the feed culture medium are sterilized at 115-121 ℃ for 15-20 min before use.
The invention has the advantages that:
(1) lactose is successfully applied to replace IPTG in high-density fermentation to induce an expression system constructed based on lactose manipulator mechanism to stably and efficiently express the recombinant human granulocyte colony stimulating factor;
(2) in the method, lactose serving as an inducer is contained in the culture medium, and culture and induction are carried out in the whole fermentation process, so that the method does not need additional steps like thermal induction or IPTG induction, simplifies the process, has fast thallus growth and good repeatability, and greatly shortens the fermentation period. In addition, no IPTG is present, and the post-treatment is simple.
(3) Compared with IPTG, lactose is much cheaper and has no toxicity, and the small amount of lactose residue in the product has little influence on human body. The method is favorable for establishing the preparation process of the recombinant granulocyte colony stimulating factor with low cost, safety and no toxicity.
(4) According to the technical scheme, the yield of escherichia coli BL21(DE3) is high, the wet weight of the escherichia coli is 20-40 g/L, the expression level of target protein is high, and the level of hG-CSF in total protein of the escherichia coli can reach 40-70%, and is equivalent to the level of IPTG induction.
Drawings
FIG. 1 is a plasmid map of pET-9a-G-CSF prepared in the first example;
FIG. 2 is a graph showing the growth of the engineered bacteria in a 10L fermenter;
FIG. 3 is a graph showing the growth of the engineered bacteria in a 50L fermentor.
Detailed Description
The present invention provides a culture medium for preparing recombinant human granulocyte colony stimulating factor and a fermentation method for preparing recombinant human granulocyte colony stimulating factor by using the culture medium, which are further described by the following embodiments.
Example one
Construction of prokaryotic expression strain pET-9a-G-CSF
The human G-CSF gene sequence is shown in SEQ ID No. 1. Synthesizing a human G-CSF gene with a sequence shown as SEQ ID No.1 according to a conventional molecular cloning method, and introducing a terminator TAA and NdeI and BamHI enzyme cutting sites at two ends of a target gene; the prokaryotic expression plasmid vector pET-9a and the human G-CSF gene fragment are respectively subjected to double enzyme digestion by NdeI and BamHI, the pET-9a subjected to double enzyme digestion and the human G-CSF gene fragment are connected to obtain a pET-9a-G-CSF recombinant plasmid (shown in figure 1), and then the pET-9a-G-CSF recombinant plasmid is transformed into an E.coli DH5 alpha strain. The work is completed by Nanjing Kingsrei.
E.coli DH 5. alpha. strain containing pET-9a-G-CSF recombinant plasmid was inoculated at 1% inoculum size into LB medium containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃ on a shaker (180rpm), purified plasmid was extracted using a plasmid endotoxin-free medium extraction kit (purchased from Biomega), purified plasmid was introduced into E.coli BL21(DE3) (purchased from Invitrogen) according to the transformation method in "molecular cloning"), and positive clones were selected using LB agar plate containing 50. mu.g/mL kanamycin. Single colonies of 10 positive clones were selected, labeled, inoculated into SuperBroth medium (containing 50. mu.g/mL kanamycin, 4.1% SuperBroth, 1g/L lactose), and cultured on a shaker (180rpm) at 37 ℃ for 20-24 hours. Taking 500 mu l of bacterial liquid, centrifuging at 3500rpm, discarding supernatant, collecting bacterial sludge, re-suspending the bacterial sludge by using 1mL of PBS, taking 15 mu l of the re-suspended bacterial suspension, adding 15 mu l of 2 Xloading Buffer solution (Loading Buffer), placing in a boiling water bath for 5min, centrifuging at 10000rpm for 5min, taking 15 mu l of supernatant, and carrying out SDS-PAGE whole bacterial electrophoresis under the electrophoresis conditions: 4.5% concentrated gel 100v electrophoresis for 10min, 15% separating gel 120v electrophoresis until bromophenol blue to the bottom of gel, stop electrophoresis. After the end, staining with Coomassie brilliant blue for 30min, decolorizing for 3h, and detecting the expression level of G-CSF on a BIO-RAD gel imaging system. The strain with the maximum expression amount of 40% is selected for PCR and sequencing identification, and is named as E.coli BL21(DE3) -pET-9 a-G-CSF.
2. Seed preservation
After the screening and identification, the E.coli BL21(DE3) -pET-9a-G-CSF single colony was inoculated into SuperBroth medium (containing 50. mu.g/mL kanamycin, 4.1% SuperBroth), and cultured at 37 ℃ for 6-8 hours in a shaker (180rpm) until OD is reached600When the culture reaches 1, the culture is stopped,adding sterilized glycerol to make the concentration of glycerol reach 15%, subpackaging in sterilized cryopreservation tubes, and preserving at-80 deg.C for later use.
EXAMPLE two (taking 10L fermenter as an example)
1. Culturing seeds: a500 mL Erlenmeyer flask was filled with 210mL of seed medium, heat-sterilized at 121 ℃ for 15min, and cooled to room temperature. The E.coli BL21(DE3) -pET-9a-G-CSF glycerol tube strain prepared in example one was inoculated in a seed medium in an amount of 0.1% and cultured with shaking in a shaker at a temperature of 35 ℃ and a rotation speed of 160rpm for 8 hours to determine OD600The yield was 0.8, and a fermentation seed culture was obtained. Wherein the seed culture medium comprises the following components in percentage by weight: 15g/L tryptone, 7g/L yeast powder and 6g/L sodium chloride are prepared by deionized water, and the pH value is adjusted to 7.0 after sterilization. The cultured strain has no contamination and good thallus morphology.
2. Preparation before inoculation: cleaning a fermentation tank, dissolving a fermentation culture medium in purified water, stirring uniformly, pouring into the fermentation tank, performing wet-heat off-line sterilization with steam at 121 ℃ for 20min, and cooling to 35 ℃ for later use. Wherein the fermentation medium comprises the following components: tryptone 15g/L, yeast extract 14g/L, NaCl15g/L, lactose 3 g/L.
Dissolving the supplemented medium in purified water, stirring, performing wet-heat off-line sterilization with 121 deg.C steam for 20min, and cooling. Wherein the composition of the feed medium is as follows: 50g/L of tryptone, 25g/L of yeast extract, 300g/L of glycerol and 4g/L of lactose.
3. Inoculation: under the protection of flame, 70mL of qualified fermentation seed culture solution is inoculated into a 10L fermentation tank according to the inoculation amount of 1% of the volume of the fermentation medium, the liquid loading amount is 7L, the culture temperature is 35 ℃, the stirring speed is 300rpm, and fermentation culture is started. The initial dissolved oxygen value is corrected to 100% by adjusting the rotating speed, air flow and tank pressure after the fermentation seed liquid is inoculated to the fermentation medium, and the dissolved oxygen value is not controlled any more in the fermentation process.
4. Fermentation: the fermentation process adopts the following intermediate control method:
feeding the supplemented medium prepared in the step 2 when the fermentation time is 4.1 hours to 3 hours, wherein the feeding rate is 140mL/h, and the volume of the supplemented medium is 1.4L;
4.2 fermenting for 8h, and adjusting the rotating speed from 300rpm to 200 rpm;
4.3 the total feed supplement amount in the fermentation process of this example was 1.4L;
4.4 during the fermentation process, the pH was 6.1 ± 0.1, and 16.0% NaOH solution and 25% HCl solution were automatically fed through the reactor;
4.5 fermenting for 18h to obtain zymophyte liquid.
5. The experimental results are as follows: through detection, the fermentation broth OD600The yield reaches 16.1, a tubular centrifuge is adopted to collect thalli to obtain 169G of wet bacterial sludge, the target protein G-CSF accounts for 40.2 percent of the total protein of the thalli, and the wet bacterial sludge is subjected to cell disruption to obtain 25.6G of inclusion bodies.
EXAMPLE three (taking 10L fermenter as an example)
1. Culturing seeds: a500 mL Erlenmeyer flask was filled with 210mL of seed medium, heat-sterilized at 121 ℃ for 15min, and cooled to room temperature. The E.coli BL21(DE3) -pET-9a-G-CSF glycerol tube strain prepared in example one was inoculated in a seed medium in an amount of 0.1% and cultured with shaking in a shaker at 37 ℃ and 180rpm for 9 hours to determine OD600The yield was 1.2, and a fermentation seed culture was obtained. Wherein the seed culture medium comprises the following components in percentage by weight: the tryptone is 20g/L, the yeast powder is 10g/L, the sodium chloride is 10g/L, the tryptone is prepared by deionized water, and the pH value is adjusted to 7.1 after sterilization. The cultured strain has no contamination and good thallus morphology.
2. Preparation before inoculation: cleaning a fermentation tank, dissolving a fermentation culture medium in purified water, stirring uniformly, pouring into the fermentation tank, performing wet-heat off-line sterilization with steam at 121 ℃ for 20min, and cooling to 37 ℃ for later use. Wherein the fermentation medium comprises the following components: tryptone 20g/L, yeast extract 10g/L, NaCl10g/L, lactose 1 g/L.
Dissolving the supplemented medium in purified water, stirring, performing wet-heat off-line sterilization with 121 deg.C steam for 20min, and cooling. Wherein the composition of the feed medium is as follows: 100g/L of tryptone, 50g/L of yeast extract, 360g/L of glycerol and 2g/L of lactose.
3. Inoculation: under the protection of flame, 210mL of qualified fermentation seed culture solution is inoculated into a 10L fermentation tank according to the inoculation amount of 3% of the volume of the fermentation medium, the liquid loading amount is 7L, the culture temperature is 37 ℃, the stirring speed is 375rpm, and the fermentation culture is started. The initial dissolved oxygen value is corrected to 100% by adjusting the rotating speed, air flow and tank pressure after the fermentation seed liquid is inoculated to the fermentation medium, and the dissolved oxygen value is not controlled any more in the fermentation process.
4. Fermentation: the fermentation process adopts the following intermediate control method:
feeding the supplemented medium prepared in the step 2 when the fermentation time is 4.1 to 5 hours, wherein the feeding rate is 200mL/h, and the volume of the supplemented medium is 2L;
4.2 fermenting for 8h, and adjusting the rotating speed from 375rpm to 250 rpm;
4.3 the total feed supplement amount in the fermentation process of the embodiment is 2L;
4.4 during the fermentation process, the pH was 7.0 ± 0.1, and 16.0% NaOH solution and 25% HCl solution were automatically fed through the reactor;
4.5, finishing the fermentation after 20 hours to obtain zymogen liquid.
5. The experimental results are as follows: through detection, the fermentation broth OD600And (3) reaching 22.4, collecting the thallus by using a tubular centrifuge to obtain 270.2G of wet bacterial sludge, wherein the target protein G-CSF accounts for 71 percent of the total protein of the thallus, and the wet bacterial sludge is subjected to cell disruption to obtain 45.6G of inclusion bodies.
EXAMPLE four (taking 10L fermenter as an example)
1. Culturing seeds: a500 mL Erlenmeyer flask was filled with 210mL of seed medium, heat-sterilized at 121 ℃ for 15min, and cooled to room temperature. The E.coli BL21(DE3) -pET-9a-G-CSF glycerol tube strain prepared in example one was inoculated in a seed medium in an amount of 0.1% and cultured with shaking in a shaker at a temperature of 39 ℃ and a rotation speed of 170rpm for 10 hours to determine OD600The yield was 1.0, and a fermentation seed culture was obtained. Wherein the seed culture medium comprises the following components in percentage by weight: 25g/L tryptone, 14g/L yeast powder and 15g/L sodium chloride are prepared by deionized water, and the pH value is adjusted to 7.2 after sterilization. The cultured strain has no contamination and good thallus morphology.
2. Preparation before inoculation: cleaning a fermentation tank, dissolving a fermentation culture medium in purified water, stirring uniformly, pouring into the fermentation tank, performing wet-heat off-line sterilization with steam at 121 ℃ for 20min, and cooling to 39 ℃ for later use. Wherein the fermentation medium comprises the following components: tryptone 18g/L, yeast extract 20g/L, NaCl20g/L, lactose 5 g/L.
Dissolving the supplemented medium in purified water, stirring, performing wet-heat off-line sterilization with 121 deg.C steam for 20min, and cooling. Wherein the composition of the feed medium is as follows: 70g/L of tryptone, 40g/L of yeast extract, 500g/L of glycerol and 6g/L of lactose.
3. Inoculation: under the protection of flame, 110mL of qualified fermentation seed culture solution is inoculated into a 10L fermentation tank according to the inoculation amount of 1.6% of the volume of the fermentation medium, the liquid loading amount is 7L, the culture temperature is 39 ℃, the stirring speed is 400rpm, and fermentation culture is started. The initial dissolved oxygen value is corrected to 100% by adjusting the rotating speed, air flow and tank pressure after the fermentation seed liquid is inoculated to the fermentation medium, and the dissolved oxygen value is not controlled any more in the fermentation process.
4. Fermentation: the fermentation process adopts the following intermediate control method:
feeding the supplemented medium prepared in the step 2 when the fermentation time is 4.1 h to 6h, wherein the feeding rate is 210mL/h, and the volume of the supplemented medium is 2.1L;
4.2 in the fermentation process, the stirring speed is kept at 400 rpm;
4.3 the total feed supplement amount in the fermentation process of this example was 2.1L;
4.4 during the fermentation process, the pH was 7.4 ± 0.1, and 16.0% NaOH solution and 25% HCl solution were automatically fed through the reactor;
4.5 fermenting for 22h to obtain zymophyte liquid.
5. The experimental results are as follows: through detection, the fermentation broth OD600And (3) collecting the thallus by using a tubular centrifuge to obtain 227.5G of wet bacterial sludge, wherein the target protein G-CSF accounts for 64 percent of the total protein of the thallus, and the wet bacterial sludge is subjected to cell disruption to obtain 36.4G of inclusion bodies. The growth curve of the engineering bacteria is shown in figure 2.
EXAMPLE five (taking 50L fermenter as an example)
1. Culturing seeds: the seeds of this example were grown as in the examples.
2. Preparation before inoculation: the preparation before inoculation in this example is the same as in the example.
3. Inoculation: under the protection of flame, 250mL of qualified fermentation seed culture solution is inoculated into a 50L fermentation tank according to the inoculation amount of 1% of the volume of the fermentation medium, the liquid loading amount is 25L, the culture temperature is 35 ℃, the stirring speed is 300rpm, and fermentation culture is started. The initial dissolved oxygen value is corrected to 100% by adjusting the rotating speed, air flow and tank pressure after the fermentation seed liquid is inoculated to the fermentation medium, and the dissolved oxygen value is not controlled any more in the fermentation process.
4. Fermentation: the fermentation process adopts the following intermediate control method:
feeding the fed-batch culture medium prepared in the step 2 when the fermentation time is 4.1 hours to 3 hours, wherein the feeding rate is 625 mL/hour, and the volume of the fed-batch culture medium is 6.25L;
4.2 fermenting for 8h, and adjusting the rotating speed from 300rpm to 200 rpm;
4.3 the total feed supplement amount in the fermentation process of this example was 6.25L;
4.4 during the fermentation process, the pH was 6.1 ± 0.1, and 16.0% NaOH solution and 25% HCl solution were automatically fed through the reactor;
4.5 fermenting for 18h to obtain zymophyte liquid.
5. The experimental results are as follows: through detection, the fermentation broth OD600And (3) collecting the thallus by using a tubular centrifuge to obtain 937.6G of wet bacterial sludge, wherein the target protein G-CSF accounts for 42% of the total protein of the thallus, and the wet bacterial sludge is subjected to cell disruption to obtain 93.8G of inclusion bodies.
EXAMPLE six (taking 50L fermenter as an example)
1. Culturing seeds: the seeds of this example were cultured as in the examples.
2. Preparation before inoculation: the preparation before inoculation in this example is the same as in the example.
3. Inoculation: under the protection of flame, 400mL of qualified fermentation seed culture solution is inoculated into a 50L fermentation tank according to the inoculation amount of 1.6% of the volume of the fermentation medium, the liquid loading amount is 25L, the culture temperature is 37 ℃, the stirring speed is 375rpm, and fermentation culture is started. The initial dissolved oxygen value is corrected to 100% by adjusting the rotating speed, air flow and tank pressure after the fermentation seed liquid is inoculated to the fermentation medium, and the dissolved oxygen value is not controlled any more in the fermentation process.
4. Fermentation: the fermentation process adopts the following intermediate control method:
feeding the supplemented medium prepared in the step 2 when the fermentation time is 4.1 to 5 hours, wherein the feeding rate is 500mL/h, and the volume of the supplemented medium is 5L;
4.2 fermenting for 8h, and adjusting the rotating speed from 375rpm to 250 rpm;
4.3 the total feed supplement amount in the fermentation process of the embodiment is 5L;
4.4 during the fermentation process, the pH was 7.0 ± 0.1, and 16.0% NaOH solution and 25% HCl solution were automatically fed through the reactor;
4.5, finishing the fermentation after 20 hours to obtain zymogen liquid.
5. The experimental results are as follows: through detection, the fermentation broth OD60031.5, collecting the thallus by using a tubular centrifuge to obtain 1203G of wet bacterial sludge, wherein the target protein G-CSF accounts for 71.3 percent of the total protein of the thallus, and the wet bacterial sludge is subjected to cell disruption to obtain 152G of inclusion bodies. The growth curve of the engineering bacteria is shown in figure 3.
EXAMPLE seven (taking a 50L fermenter as an example)
1. Culturing seeds: the seed culture of this example was the same as that of example four.
2. Preparation before inoculation: the preparation before inoculation in this example is the same as in the fourth example.
3. Inoculation: under the protection of flame, 750mL of qualified fermentation seed culture solution is inoculated into a 50L fermentation tank according to the inoculation amount of 3% of the volume of the fermentation medium, the liquid loading amount is 25L, the culture temperature is 39 ℃, the stirring speed is 400rpm, and fermentation culture is started. The initial dissolved oxygen value is corrected to 100% by adjusting the rotating speed, air flow and tank pressure after the fermentation seed liquid is inoculated to the fermentation medium, and the dissolved oxygen value is not controlled any more in the fermentation process.
4. Fermentation: the fermentation process adopts the following intermediate control method:
feeding the supplemented medium prepared in the step 2 when the fermentation time is 4.1 hours to 6 hours, wherein the feeding rate is 750 mL/hour, and the volume of the supplemented medium is 7.5L;
4.2 in the fermentation process, the stirring speed is kept at 400 rpm;
4.3 the total feed supplement amount in the fermentation process of this example was 7.5L;
4.4 during the fermentation process, the pH was 7.4 ± 0.1, and 16.0% NaOH solution and 25% HCl solution were automatically fed through the reactor;
4.5 fermenting for 22h to obtain zymophyte liquid.
5. The experimental results are as follows: through detection, the fermentation broth OD60027, collecting the thallus by using a tubular centrifuge to obtain 1137.2G of wet bacterial sludge, wherein the target protein G-CSF accounts for 65.5 percent of the total protein of the thallus, and the wet bacterial sludge is subjected to cell disruption to obtain 130.2G of inclusion bodies.
The invention is used for the production of the recombinant human granulocyte colony stimulating factor and has wide application prospect. The lactose-induced fermentation production process of the recombinant human granulocyte colony-stimulating factor engineering bacteria is researched and developed, so that the operation is simpler, the production cost is reduced, the safety and the non-toxicity are realized, the thallus yield and the target protein expression quantity are improved, and the industrial production is favorably realized.
Finally, it should be noted that the above-mentioned embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described by referring to the preferred embodiments of the present invention, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered in the claims of the present invention.
Claims (4)
1. A fermentation method for preparing recombinant human granulocyte colony stimulating factor comprises the following steps:
inoculating host bacteria transformed with recombinant vector plasmids constructed by using a pET-9a vector and a human G-CSF gene into a seed culture medium to culture until OD600 is 0.8-1.2, and obtaining fermented seed liquid, wherein the human G-CSF gene sequence is shown as SEQ ID No. 1;
secondly, inoculating the fermentation seed liquid into a sterilized fermentation culture medium according to the proportion of 1-3% of the volume of the fermentation culture medium, and culturing for 3-6 hours;
thirdly, supplementing the sterilized supplementary culture medium into the fermentation system according to the proportion of 20-30% of the volume of the fermentation culture medium for continuous fermentation, finishing the fermentation after 18-22 hours to obtain recombinant bacterium fermentation liquor,
the fermentation medium is characterized by comprising the following components in percentage by weight: tryptone 20g/L, yeast extract 10g/L, sodium chloride 10g/L, lactose 1g/L, and the balance of water, wherein the components and the contents of the supplementary culture medium are as follows: 100g/L of tryptone, 50g/L of yeast extract, 360g/L of glycerol, 2g/L of lactose and the balance of water; or the fermentation medium comprises the following components in percentage by weight: the feed medium comprises 18g/L of tryptone, 20g/L of yeast extract, 20g/L of sodium chloride, 5g/L of lactose and the balance of water, and comprises the following components in percentage by weight: 70g/L of tryptone, 40g/L of yeast extract, 500g/L of glycerol, 6g/L of lactose and the balance of water;
the seed culture medium comprises 15-25 g/L of tryptone, 7-14 g/L of yeast powder, 6-15 g/L of sodium chloride and the balance of water, and the pH value is adjusted to 7.0-7.2;
in the fermentation process, the culture temperature of the fermentation is 35-39 ℃, and the pH of a fermentation system is controlled to be 6.0-7.5 by adding acid and alkali solution; fermenting for 0-8 hours, wherein the rotating speed of the fermentation tank is 300-400 rpm, and after 8 hours, the rotating speed is adjusted to 200-400 rpm;
the sterilized feed medium is fed into the fermentation system in a fed-batch mode, the feed time is 10 hours, and the feed rate is set to be a numerical value obtained by dividing the feed volume by the feed time;
the host bacterium is Escherichia coli (Escherichia coli) BL21(DE 3).
2. The fermentation method for preparing recombinant human granulocyte colony stimulating factor as claimed in claim 1, wherein the host bacteria of the transformed recombinant vector plasmid is inoculated into a seed culture medium for culture to obtain a fermentation seed solution, the host bacteria of the transformed recombinant vector plasmid is selected from glycerol tube strains and inoculated into a culture flask containing the seed culture medium, the inoculation amount is 0.1%, and the fermentation seed solution is obtained by culturing the host bacteria at 35-39 ℃ and 160-180 rpm for 8-10 hours.
3. The fermentation method for preparing recombinant human granulocyte colony stimulating factor as claimed in claim 1, wherein the seed culture medium contains tryptone 20-25 g/L, yeast powder 10-14 g/L, and sodium chloride 10-15 g/L.
4. The fermentation method for preparing recombinant human granulocyte colony stimulating factor of claim 1, wherein the acid and alkali solution used for controlling the pH of the fermentation system is hydrochloric acid and sodium hydroxide solution.
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