CN106282227B - High-density fermentation method of naked plasmid of recombinant human hepatocyte growth factor - Google Patents

Abstract

The invention relates to the technical field of biological pharmacy, and particularly provides a high-density fermentation method of a naked plasmid of a recombinant human hepatocyte growth factor. The high-density fermentation method of the naked plasmid of the recombinant human hepatocyte growth factor comprises the following steps: 1) culturing working seeds; 2) inoculating into a fermentation tank for fermentation. The fermentation method can realize high expression of the naked plasmid of the recombinant human hepatocyte growth factor in 50L scale fermentation, the final thallus concentration OD600 can reach more than 55, and the content of the naked plasmid can reach more than 420mg/L, thereby solving the technical problems that the content of the naked plasmid of the recombinant human hepatocyte growth factor is low and the yield can not reach the scale requirement, and laying a solid foundation for the industrialization of the naked plasmid of the recombinant human hepatocyte growth factor.

Description

High-density fermentation method of naked plasmid of recombinant human hepatocyte growth factor

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a high-density fermentation method of naked plasmid of a recombinant human hepatocyte growth factor.

Background

The recombinant human hepatocyte growth factor naked plasmid is a gene therapeutic drug for treating vascular ischemic diseases, is injected into muscles of ischemic positions to transfect striated muscle cells, expresses and secretes Hepatocyte Growth Factor (HGF) with the function of vascular growth, locally forms collateral circulation, establishes a 'molecular bypass' mechanism, increases the blood supply of the ischemic positions, and achieves the purpose of treating the arterial ischemic diseases.

The preparation method of the recombinant human hepatocyte growth factor naked plasmid adopts a gene engineering method, namely a gene transfer system is constructed by utilizing a biological engineering technology, and the recombinant human hepatocyte growth factor naked plasmid is mainly prepared by replication expression in escherichia coli and separation and purification; at present, the recombinant human hepatocyte growth factor naked plasmid products are still in the research stage, and no recombinant human hepatocyte growth factor naked plasmid products are on the market at home and abroad.

This company transformed naked plasmid (pCK-HGF) of recombinant human hepatocyte growth factor into Escherichia coli DH5 alpha for proliferationAnd (4) producing. Although there is no report on fermentation process directly related to naked plasmid of recombinant human hepatocyte growth factor, there are many reports on the research on fermentation process related to plasmid DNA, for example, Zheng Shuzhen studies the fermentation process of recombinant Escherichia coli producing gene therapy plasmid DNA in 2L fermentation tank (fermentation scale 1.5L), and as a result, under optimized culture conditions, the yield of pUK21CMV beta 1.2 plasmid can reach 601.9 mg/L (Zheng Shuzhen, academic paper of Xiamen university, 2007). Wang Yongxin study on the fermentation conditions of pcD-awte candidate malaria DNA vaccine in a 5L fermentation tank (fermentation scale 3L) resulted in the optimal bacterial concentration OD₆₀₀Can reach 43-45, and the yield of pcD-awte plasmid can reach 125-130 mg/L (Wan Yongxin et al, journal of biology 2007;24(5): 16-19). The fermentation conditions for producing the nucleic acid vaccine by Escherichia coli DH5A were investigated in a 15L fermentor (10L fermentation scale), and the OD of the cell concentration under the optimized culture conditions was found₆₀₀The yield of the pVAX244 plasmid can reach over 22.6, and the yield of the pVAX244 plasmid can reach 75 mg/L (Kyowa et al, university of eastern China school, 2006; 31(1) 38-42). Pilot plant preparation and quality study of multi-epitope HBV DNA vaccine are carried out in 100L Guoza fermenter (fermentation scale 50L), and the result shows that the plasmid yield can reach 60 mg/L (Guoza et al, university of southern medical science, 2009; 29(1) 118-. In summary, it is known from the reported literature on plasmid DNA fermentation processes that, due to the influence of factors such as the characteristics of strains and plasmids, most of the existing fermentation processes can obtain higher bacterial concentration and plasmid content in smaller-scale fermentation, but can only obtain lower bacterial concentration and plasmid content in larger-scale fermentation, and therefore, the problem in many of the reported plasmid fermentation processes is that the fermentation scale, bacterial concentration and plasmid content cannot be simultaneously presented at higher levels, and the total yield cannot meet the large-scale requirement.

In addition, with the rapid development of gene therapy, the research on non-viral vectors is more and more emphasized by people, the number and the proportion of the non-viral vectors are gradually increased, and particularly, plasmid DNA has the advantages of easy preparation, weak host reaction, no integration into host DNA and the like, and is generally accepted as safe and convenient to be introduced into target cells as a gene vector. However, plasmid DNA has the defects of low cell transfection efficiency, short expression duration, low expression amount, large dosage of medicine and the like, so whether the preparation of the plasmid DNA in a large scale can be realized becomes one of the key steps of applying the plasmid to gene therapy.

Based on the reasons, a set of fermentation process capable of realizing high yield and large scale of naked plasmid of the recombinant human hepatocyte growth factor is developed, and has great significance for the development of the industrialization research thereof.

Disclosure of Invention

The invention aims to provide a high-density fermentation method of a recombinant human hepatocyte growth factor naked plasmid, thereby achieving the purpose of efficiently producing the recombinant human hepatocyte growth factor naked plasmid.

The high-density fermentation method of the naked plasmid of the recombinant human hepatocyte growth factor comprises the following steps:

1) and (3) culturing working seeds:

inoculating strains in the working seed bank into a primary seed culture medium, placing the primary seed culture medium in a shaking table for culturing, controlling the temperature to be 35-38 ℃, the rotating speed to be 200-230rpm, and culturing for 7-9h to obtain a primary seed solution; inoculating the primary seed liquid into a secondary seed culture medium in an inoculation amount of 2-8%, placing the secondary seed culture medium in a shaking table for culturing for 15-17h at the controlled temperature of 35-38 ℃ and the rotation speed of 200-;

2) inoculating in a fermentation tank for fermentation:

inoculating the prepared fermentation working seed liquid into a fermentation tank containing a fermentation culture medium for fermentation according to the inoculation amount of 2-5%, wherein the mechanical stirring rotation speed is 150-80 rpm, the ventilation volume is 10-80L/min, the tank pressure is 0.2-0.4par, the dissolved oxygen level is maintained at 20-60%, the fermentation temperature is 36-38 ℃, and 25-28% (v/v) ammonia water is added to control the fermentation pH to be 6.9-7.2;

measuring the thallus concentration OD with ultraviolet spectrophotometer₆₀₀When the concentration is 1-2, starting to supplement the carbon source supplementing culture medium at the flow rate of 4 mL/L.h, performing off-line detection on the glucose concentration every 1h in the fermentation process, feeding back according to the glucose concentration, and controlling the glucose concentration to be 0.2-0.8 g/L; determination of thallus concentration OD by ultraviolet spectrophotometer₆₀₀When the flow rate is 4-6, the organic nitrogen source feed medium is started to be supplemented at the flow rate of 5-7 mL/L.h; culturing until the growth speed of the thalli is reduced, and stopping fermentation and collecting the thalli. Wherein the carbon source supplementing culture medium is a 50% glucose solution, and the organic nitrogen source supplementing culture medium is a mixed solution of 20% tryptone and yeast powder (1: 3).

The method has the advantages that according to the growth rule of the thalli, the substrate concentration feedback material feeding is combined, the flow acceleration of the carbon source and the organic nitrogen material feeding is adjusted, the glucose concentration is maintained at 0.2-0.8g/L, and the C: N ratio is controlled, so that the growth speed of the thalli and the synthesis efficiency of a target product are adjusted. The fermentation method can realize high-efficiency fermentation of naked plasmid of recombinant human hepatocyte growth factor, and the final thallus concentration OD obtained by fermentation in 100L pilot-scale fermentation tank (fermentation scale 50L)₆₀₀Can reach more than 55, and the plasmid content can reach more than 420mg/L, so that the fermentation scale, the final thallus concentration and the plasmid content can be presented at a higher level at the same time, the fermentation process has stronger practicability and higher efficiency, and a solid foundation is laid for the industrialization of the naked plasmid of the recombinant human hepatocyte growth factor.

Drawings

FIG. 1 is a graph showing the growth of cells in a 30L fermenter during high-density fermentation; in the figure, the horizontal axis represents fermentation time, and the vertical axis represents cell concentration OD₆₀₀。

FIG. 2 is a schematic diagram showing the change of wet weight of cells in a 30L fermenter during high-density fermentation; in the figure, the horizontal axis represents fermentation time, and the vertical axis represents wet weight of the cells.

FIG. 3 is a 30L fermentor test high density fermentation expression agarose gel electrophoresis chart of naked plasmid of recombinant human hepatocyte growth factor; in the figure, M is Marker, and lanes 1 and 2 are samples after fermentation.

FIG. 4 is a graph showing the growth of cells in a 100L fermenter during high-density fermentation; in the figure, the horizontal axis represents fermentation time, and the vertical axis represents cell concentration OD₆₀₀。

FIG. 5 is a schematic diagram showing the change of wet weight of cells in a 100L fermenter during high-density fermentation; in the figure, the horizontal axis represents fermentation time, and the vertical axis represents wet weight of the cells.

FIG. 6 is a diagram of agarose gel electrophoresis of naked plasmid recombinant human hepatocyte growth factor in 100L fermenter through high density fermentation; in the figure, M is Marker, and lanes 1 and 2 are samples after fermentation.

Detailed Description

The invention is further illustrated by the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any way.

The materials used in the examples are as follows:

working strain: DH5 alpha/pCK-HGF engineering bacteria self-constructed by the company are adopted as working strains.

The main equipment is as follows: 100L fermentors (Hanil, LIFLUS SP 100L), 30L fermentors (Shanghai Baoching, BIOTECH-30JS), autoclave (Jiangyin Binjiang, LS-B100L-1), gas bath constant temperature shaker (Jiangsu Taicang, THZ-22), electronic balance (Sartorius, TE4101-L, TE212-L), UV spectrophotometer (Agilent Technologies, Cary60), super clean bench (Suzhou Anta, SW-CJ-1F), pH meter (Mettler Toledo, FE20), biochemical analyzer (Shandong academy of sciences institute of sciences, SBA-40C), gel imager (Tanon, 1600), bench centrifuge (Sigma, 1-13), continuous flow high speed freezing centrifuge (GITACHI, CR21 II).

Seed culture medium: 10g/L of tryptone, 5g/L of yeast powder and 1g/L of sodium chloride.

Fermentation medium: 13.3g/L of monopotassium phosphate, 4g/L of diammonium phosphate, 1.7g/L of citric acid monohydrate, 1g/L of tryptone, 1g/L of yeast powder, 1.145g/L of magnesium sulfate heptahydrate, 1g/L of glucose, 4mL/L of trace element solution and 0.03g/L of kanamycin sulfate.

Carbon source feeding medium: 50% glucose solution.

Organic nitrogen source feed medium: 20% organic nitrogen source mixed solution (tryptone: yeast powder =1: 3).

Example 1

Pilot high density fermentation in 1.30L fermenter:

1) working seed culture

Taking a strain from a working seed library stored at-80 ℃, taking 100 mu L of bacterial liquid after the strain is completely melted, inoculating the strain into a primary seed culture medium (50 mL in a 250mL triangular bottle containing 50 mu g/mL kanamycin), placing the primary seed culture medium in a shaking table for culturing, controlling the temperature at 37 ℃, and the rotating speed at 230rpm, and culturing for 8 hours to obtain a primary seed liquid; inoculating the first-stage seed solution into a second-stage seed culture medium (400 mL in 2L triangular flasks containing 50 μ g/mL kanamycin), culturing in a shaking table at 37 deg.C and 230rpm for 15-17h to obtain the fermentation seed solution.

2) Inoculating into 30L fermentation tank for fermentation

Inoculating the prepared fermentation working seed solution into a 30L fermentation tank containing 14L fermentation medium in an inoculation amount of 5% for fermentation; the mechanical stirring speed is 150-500rpm, the ventilation amount is 10-80L/min, the tank pressure is 0.2-0.4par, the dissolved oxygen level is maintained at 20-50%, the fermentation temperature is controlled at 37 ℃, and 25-28% (v/v) ammonia water is added to control the fermentation pH to be 6.9-7.1.

Measuring the thallus concentration OD with ultraviolet spectrophotometer₆₀₀When the concentration is about 1.5, the carbon source supplementing culture medium is started to be supplemented at the flow rate of 4 mL/L.h, the glucose concentration is detected off-line every 1h in the fermentation process, and the glucose concentration is controlled to be 0.2-0.5g/L by feeding back the supplementing culture medium according to the glucose concentration; determination of thallus concentration OD by ultraviolet spectrophotometer₆₀₀At about 5, the organic nitrogen source feed medium was fed at a flow rate of 5 mL/L.h.

3) Collecting thallus in lower tank

And after 4 hours of induced expression, stopping feeding the carbon source supplementing culture medium and the organic nitrogen source supplementing culture medium, closing all control parameters, ending the fermentation process, and then discharging the zymocyte liquid to a 100L collecting tank by using the tank pressure. Centrifuging the collected zymophyte liquid by adopting a continuous flow centrifuge (9500 rpm, 4 ℃ and the sampling flow rate of 100 mL/min), discarding the supernatant, collecting the precipitate, and weighing the total weight of the precipitate to obtain the wet weight of the total thalli.

2. The main index determination method comprises the following steps:

2.1 measurement of cell concentration

1) Sampling: sampling is carried out from the fermentation tank every 1h, the sampling amount is 100mL each time, the first 50mL of zymogen liquid is discarded during sampling, and the last 50mL of zymogen liquid is collected.

2) Sample treatment: blowing and uniformly mixing the collected zymophyte liquid, taking a proper amount of zymophyte liquid, and diluting with pure water to ensure that the light absorption value OD of the diluted zymophyte liquid is₆₀₀The readings ranged from 0.2 to 0.8.

3) And (3) determination: pure water is used as a blank control, and an ultraviolet spectrophotometer is adopted to measure the light absorption value of the diluent at the wavelength of 600 nm.

4) Data processing: the bacterial concentration OD is calculated according to a formula (bacterial concentration = absorbance of the diluent x dilution multiple)₆₀₀And the fermentation time is used as the abscissa and the cell concentration OD is used₆₀₀And drawing a thallus growth curve chart in the fermentation process as a vertical coordinate.

2.2 measurement of Wet weight of cells

1) Sampling: sampling from the fermentation tank every 1h, wherein the sampling amount is 100mL each time, discarding the first 50mL of zymogen liquid during sampling, and collecting the last 50mL of zymogen liquid.

2) And (3) determination: and blowing and uniformly mixing the collected zymophyte liquid, respectively filling 1 mL of the bacteria liquid into 4 weighed 1.5mL centrifuge tubes, centrifuging at 12000rpm for 2min, discarding the supernatant, and finally weighing the 4 centrifuge tubes by using an electronic balance to obtain the total weight.

3) Data processing: according to the formula { wet weight of thallus = (total weight-total tare)/4 × 10^-3And (4) calculating to obtain the wet weight of the thallus in unit volume, and drawing a thallus wet weight change schematic diagram in the fermentation process by taking the fermentation time as a horizontal coordinate and the wet weight of the thallus as a vertical coordinate.

2.3 determination of plasmid content:

the method for determining the plasmid content by adopting an agarose gel electrophoresis method and an ultraviolet spectrophotometry comprises the following specific operations:

1) sampling: and (3) when the fermentation is finished, taking a proper amount of zymocyte liquid (100-500 mu L), centrifuging 2 pieces at 12000rpm for 2min, discarding the supernatant, and collecting the thalli.

2) Sample treatment: extracting the plasmids in the sample according to the operation instructions of the column type plasmid extraction kit.

3) Determination of plasmid content by agarose gel electrophoresis

a.1% agarose gel preparation: weighing 0.467g of agarose, pouring into a triangular flask, adding 50mL of 1 XTAE working solution, heating in a microwave oven for 110 seconds, taking out, cooling to a temperature of 50-60 ℃, adding 3 μ l of Goldview dye, mixing, pouring into a glue-making mold, coating uniformly, placing a comb on one side of the mold, standing, and solidifying for 30 min. .

b. Loading and running electrophoresis: after plasmid extraction, 5. mu.l of sample was mixed with 1. mu.l of 6 Xloading buffer (two wells), 5. mu.l of control was mixed with 1. mu.l of 6 Xloading buffer, 6. mu.l of standard molecular weight was taken, preheated at 65 ℃ for 5min and added to the wells, respectively, and electrophoresis was carried out at a constant voltage of 72v for about 1h, at which time the electrophoresis indicator should be about 1cm from the edge of the glass plate. .

c. Data processing: after electrophoresis, the electrophoresis Gel is transferred to a white plate, a Tanon Gel imager is used for shooting the electrophoresis Gel, and the electrophoresis picture is analyzed by utilizing Tanon Gel Image System1D analysis software to obtain the plasmid content.

4) Ultraviolet spectrophotometry method for determining plasmid content

a. Sample dilution: diluting the sample with water for injection to the measured absorbance OD₂₆₀The value is in the range of 0.2 to 1.0.

b. And (3) detection: the diluted plasmid solution 500 u L, put into the sample volume of 500 u L cuvette, using water for injection as blank control, using ultraviolet spectrophotometer determination 260nm and 280nm wavelength OD value.

c. And (4) calculating a result:

sample plasmid content (μ g) = OD₂₆₀X 50. mu.g/mL x dilution factor x 0.1 mL;

unit cell plasmid content (μ g/g) = sample plasmid content (μ g) × sample volume (L) ÷ retention volume (L) ÷ cell mass (g) ÷ 70%.

Remarking: the plasmid extraction efficiency of the column type plasmid extraction kit is about 70 percent.

3. The experimental results are as follows:

example 1 the final cell concentration OD was determined by measuring the cell concentration₆₀₀Up to 61 or more (see fig. 1); by the determination of the wet weight of the thalli,shows that the final wet weight of the thallus can reach more than 153g/L (see figure 2); the plasmid content can be up to 428mg/L (see figure 3).

Example 2

1.100L fermenter pilot scale high density fermentation:

1) working seed culture

Taking a strain from a working seed library at-80 ℃, taking 200 mu L of bacterial liquid after the strain is completely melted, inoculating the strain into a primary seed culture medium (100 mL in a 500mL triangular bottle containing 50 mu g/mL kanamycin), placing the strain in a shaking table for culturing, controlling the temperature at 37 ℃, rotating speed at 230rpm, and culturing for 8 hours to obtain a primary seed liquid; inoculating the primary seed solution into a secondary seed culture medium (650 mL in 4 2L triangular flasks containing 50 ug/mL kanamycin respectively) at an inoculation amount of 4%, placing in a shaking table, culturing at 37 deg.C and 230rpm for 15-17h to obtain the fermentation seed solution.

2) Inoculating into 100L fermentation tank for fermentation

Inoculating the prepared fermentation working seed liquid into a 100L fermentation tank containing 50L of fermentation medium for fermentation at the inoculation amount of 5%, wherein the rotation speed of mechanical stirring is 150-.

Measuring the thallus concentration OD with ultraviolet spectrophotometer₆₀₀When the concentration is about 1, starting to supplement the carbon source supplementing culture medium at the flow rate of 4 mL/L.h, performing off-line detection on the glucose concentration every 1h in the fermentation process, feeding back according to the glucose concentration, and controlling the glucose concentration to be 0.2-0.5 g/L; determination of thallus concentration OD by ultraviolet spectrophotometer₆₀₀At about 5, the organic nitrogen source feed medium was fed at a flow rate of 5 mL/L.h.

3) Collecting thallus in lower tank

And after 4 hours of induced expression, stopping feeding the carbon source supplementing culture medium and the organic nitrogen source supplementing culture medium, closing all control parameters, ending the fermentation process, and then discharging the zymocyte liquid to a 100L collecting tank by using the tank pressure. Centrifuging the collected zymophyte liquid by adopting a continuous flow centrifuge (9500 rpm, 4 ℃ and the sampling flow rate of 100 mL/min), discarding the supernatant, collecting the precipitate, and weighing the total weight of the precipitate to obtain the wet weight of the total thalli.

2. The main index determination method comprises the following steps:

2.1 measurement of cell concentration

1) Sampling: sampling is carried out from the fermentation tank every 1h, the sampling amount is 100mL each time, the first 50mL of zymogen liquid is discarded during sampling, and the last 50mL of zymogen liquid is collected.

2) Sample treatment: and blowing and uniformly mixing the collected zymophyte liquid, taking a proper amount of zymophyte liquid, and diluting with pure water to ensure that the light absorption value OD600 of the diluted liquid is read within the range of 0.2-0.8.

3) And (3) determination: pure water is used as a blank control, and an ultraviolet spectrophotometer is adopted to measure the light absorption value of the diluent at the wavelength of 600 nm.

4) Data processing: the bacterial concentration OD is calculated according to a formula (bacterial concentration = absorbance of the diluent x dilution multiple)₆₀₀And the fermentation time is used as the abscissa and the cell concentration OD is used₆₀₀And drawing a thallus growth curve chart in the fermentation process as a vertical coordinate.

2.2 measurement of Wet weight of cells

1) Sampling: sampling from the fermentation tank every 1h, wherein the sampling amount is 100mL each time, discarding the first 50mL of zymogen liquid during sampling, and collecting the last 50mL of zymogen liquid.

2) And (3) determination: and blowing and uniformly mixing the collected zymophyte liquid, respectively filling 1 mL of the bacteria liquid into 4 weighed 1.5mL centrifuge tubes, centrifuging at 12000rpm for 2min, discarding the supernatant, and finally weighing the 4 centrifuge tubes by using an electronic balance to obtain the total weight.

3) Data processing: according to the formula { wet weight of thallus = (total weight-total tare)/4 × 10^-3And (4) calculating to obtain the wet weight of the thallus in unit volume, and drawing a thallus wet weight change schematic diagram in the fermentation process by taking the fermentation time as a horizontal coordinate and the wet weight of the thallus as a vertical coordinate.

2.3 determination of plasmid content:

the method for determining the plasmid content by adopting an agarose gel electrophoresis method and an ultraviolet spectrophotometry comprises the following specific operations:

1) sampling: and (3) when the fermentation is finished, taking a proper amount of zymocyte liquid (100-500 mu L), centrifuging 2 pieces at 12000rpm for 2min, discarding the supernatant, and collecting the thalli.

2) Sample treatment: extracting the plasmids in the sample according to the operation instructions of the column type plasmid extraction kit.

3) Determination of plasmid content by agarose gel electrophoresis

a.1% agarose gel preparation: weighing 0.467g of agarose, pouring into a triangular flask, adding 50mL of 1 XTAE working solution, heating in a microwave oven for 110 seconds, taking out, cooling to a temperature of 50-60 ℃, adding 3 μ l of Goldview dye, mixing, pouring into a glue-making mold, coating uniformly, placing a comb on one side of the mold, standing, and solidifying for 30 min. .

b. Loading and running electrophoresis: after plasmid extraction, 5. mu.l of sample was mixed with 1. mu.l of 6 Xloading buffer (two wells), 5. mu.l of control was mixed with 1. mu.l of 6 Xloading buffer, 6. mu.l of standard molecular weight was taken, preheated at 65 ℃ for 5min and added to the wells, respectively, and electrophoresis was carried out at a constant voltage of 72v for about 1h, at which time the electrophoresis indicator should be about 1cm from the edge of the glass plate. .

c. Data processing: after electrophoresis, the electrophoresis Gel is transferred to a white plate, a Tanon Gel imager is used for shooting the electrophoresis Gel, and the electrophoresis picture is analyzed by utilizing Tanon Gel Image System1D analysis software to obtain the plasmid content.

4) Ultraviolet spectrophotometry method for determining plasmid content

a. Sample dilution: diluting the sample with water for injection to the measured absorbance OD₂₆₀The value is in the range of 0.2 to 1.0.

b. And (3) detection: the diluted plasmid solution 500 u L, put into the sample volume of 500 u L cuvette, using water for injection as blank control, using ultraviolet spectrophotometer determination 260nm and 280nm wavelength OD value.

c. And (4) calculating a result:

sample plasmid content (μ g) = OD₂₆₀X 50. mu.g/mL x dilution factor x 0.1 mL;

unit cell plasmid content (μ g/g) = sample plasmid content (μ g) × sample volume (L) ÷ retention volume (L) ÷ cell mass (g) ÷ 70%.

Remarking: the plasmid extraction efficiency of the column type plasmid extraction kit is about 70 percent.

3. The experimental results are as follows:

example 2 the final cell concentration OD was determined by measuring the cell concentration₆₀₀Up to 55 or more (see fig. 4); the wet weight of the bacteria is measured, and the final unit wet weight of the bacteria can be up to more than 141g/L (see figure 5); the plasmid content can be up to 423mg/L or more through plasmid content determination (see figure 6).

Claims (4)

1. A high-density fermentation method of naked plasmid of recombinant human Hepatocyte Growth Factor (HGF), which comprises the following steps:

1) and (3) culturing working seeds:

inoculating strains in the working seed bank into a primary seed culture medium, placing the primary seed culture medium in a shaking table for culturing, controlling the temperature at 37 ℃, and culturing for 7-9h at the rotation speed of 200 plus 230rpm to obtain a primary seed solution; inoculating the primary seed liquid into a secondary seed culture medium in an inoculation amount of 4-6%, placing the secondary seed culture medium in a shaking table for culturing for 15-17h at the controlled temperature of 37 ℃ and the rotation speed of 200-; the primary seed culture medium and the secondary seed culture medium comprise tryptone 10g/L, yeast powder 5g/L, sodium chloride 1g/L and kanamycin 50 mu g/mL; the strain is obtained by transforming the recombinant human hepatocyte growth factor naked plasmid pCK-HGF into escherichia coli DH5 alpha;

2) inoculating in a fermentation tank for fermentation:

inoculating the prepared fermentation working seed liquid into a fermentation tank containing a fermentation culture medium by an inoculation amount of 5% for fermentation, wherein the mechanical stirring rotation speed is 150-80 rpm, the ventilation volume is 10-80L/min, the tank pressure is 0.2-0.4par, the dissolved oxygen level is maintained at 20-50%, the fermentation temperature is 37 ℃, and ammonia water with the volume of 25-28% is added to control the fermentation pH to be 6.9-7.1; the fermentation medium comprises 13.3g/L of monopotassium phosphate, 4g/L of diammonium phosphate, 1.7g/L of citric acid monohydrate, 1g/L of tryptone, 1g/L of yeast powder, 1.145g/L of magnesium sulfate heptahydrate, 1g/L of glucose, 4mL/L of trace element solution and 0.03g/L of kanamycin sulfate;

measuring the thallus concentration OD with ultraviolet spectrophotometer₆₀₀When the concentration is 1-2, starting to supplement the carbon source supplementing culture medium at the flow rate of 4 mL/L.h, performing off-line detection on the glucose concentration every 1h in the fermentation process, feeding back according to the glucose concentration, and controlling the glucose concentration to be 0.2-0.5 g/L; determination of thallus concentration OD by ultraviolet spectrophotometer₆₀₀When the flow rate is 4-6, the organic nitrogen source feed medium is started to be supplemented at the flow rate of 5 mL/L.h; culturing until the growth speed of the thalli is reduced, stopping fermentation and collecting the thalli; the carbon source supplementing culture medium is a 50% glucose solution, the organic nitrogen source supplementing culture medium is a mixed solution of 20% tryptone and yeast powder, and the volume ratio of the tryptone to the yeast powder in the mixed solution is 1: 3.

2. the method of claim 1, wherein the high-density fermentation is performed on a 50L scale.

3. The method according to claim 1, wherein the final bacterial concentration OD₆₀₀Can reach more than 55.

4. The method of claim 1, wherein the amount of naked plasmid is up to 420mg/L or more.

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