CN117736962A - Fermentation medium and application thereof in preparation of teriparatide - Google Patents

Abstract

The invention provides a fermentation medium and application thereof in preparation of teriparatide, and belongs to the technical field of biological pharmacy. The fermentation medium comprises the following components: yeast peptone, yeast extract powder, dipotassium phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, glycerol, microelement solution, defoamer and pure water as solvent. The microelement solution added in the fermentation culture medium is more beneficial to the expression of inclusion bodies by controlling the types and the addition amount of the microelement, and the strategy of starting and adjusting the feeding flow rate at a fixed time point is adopted in the fermentation process, so that the stability of fermentation production is improved.

Description

Fermentation medium and its application in the preparation of teriparatide

Technical field

The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a fermentation culture medium and its application in the preparation of teriparatide.

Background technique

Osteoporosis (OP) is a metabolic bone disease caused by bone loss and bone quality reduction caused by a variety of reasons, leading to increased bone fragility until fracture occurs. Osteoporosis can be divided into primary and secondary types. Type I primary osteoporosis occurs in postmenopausal women, and type II primary osteoporosis occurs in the elderly. The risk of fractures caused by osteoporosis seriously affects patients' physical and mental health and quality of life. Osteoporosis often has no obvious clinical symptoms in the early stages, so it is called the "silent killer". Therefore, early prevention and treatment are of great significance. Currently, there are three main types of drugs commonly used in clinical practice: those that promote bone mineralization, those that resist bone resorption, and those that enhance bone synthesis.

The molecular formula of teriparatide is C ₁₈₁ H ₂₉₁ N ₅₅ O ₅₁ S ₂ , the molecular weight is 4117.72, and the theoretical isoelectric point is 8.29. It is a derivative of parathyroid hormone (PTH) and contains 84 amino acids of human endogenous parathyroid gland. The N-terminal region of parathyroid hormone has a biologically active 1-34 amino acid fragment, and its immunological and biological properties are exactly the same as endogenous parathyroid hormone.

Teriparatide is an anti-osteoporosis drug that helps regulate calcium metabolism and promote bone formation. By increasing the activity and number of osteoblasts, it promotes new bone formation, improves bone strength, effectively reduces the risk of vertebral and non-vertebral fractures, improves back pain, and improves mobility. This drug promotes the growth of new bone, whereas other osteoporosis drugs increase bone density by inhibiting bone resorption or breakdown.

Teriparatide is the first new bone-forming agent approved by the U.S. Food and Drug Administration. It is suitable for the treatment of osteoporosis in postmenopausal women with a high risk of fractures. It can significantly reduce the number of vertebrae in postmenopausal women. and risk of nonvertebral fractures. Teriparatide injection was approved by the FDA in November 2002, then by the EMEA in June 2003, and by the PMDA in July 2010. In March 2011, teriparatide injection was approved to enter the Chinese drug market and was approved by the State Food and Drug Administration for the treatment of postmenopausal female patients with severe osteoporosis in China.

The preparation of teriparatide generally adopts the fermentation method of genetically engineered bacteria. How to improve the fermentation yield of teriparatide has gradually become a hot topic.

Recombinant expression of teriparatide using Escherichia coli has been reported in some literature and patents. For example, in the authorized patent CN110938151B, Feng Qiang et al. constructed pET28a-HSTP/BL21 (DE3) engineering bacteria, using TB medium containing 50 μg/ml kanamycin, cultured in a 60L fermentation volume for 5 hours and induced with IPTG for 3 hours. The average wet bacterial strains of the three batches of fermentation were 66.7, 65.4 and 66.7g/L respectively, and the average inclusion body yields were 11.6, 9.3 and 8.3g/L respectively.

By increasing oxygen supply to achieve high-density fermentation, there have also been attempts to improve the expression of recombinant protein products. In Chinese patent CN111378027B, Wen Liangzhu and others tried to use high-density fermentation to recombinantly express the semaglutide precursor (similar in size to teriparatide). The engineering strain also used the BL21 (DE3) host strain in the form of inclusion bodies. Express the polypeptide of interest. In this study, glucose 10g/L, yeast powder: 30g/L, potassium dihydrogen phosphate 4g/L, disodium hydrogenphosphate 4g/L, ammonium sulfate 6g/L, calcium chloride dihydrate 0.02g/L, sulfuric acid heptahydrate Magnesium 10g/L is used as the fermentation medium, yeast powder: 20g/L, glucose: 30g/L are used as the feed medium; control the temperature at 34°C, pH around 6.9, dissolved oxygen above 30%, and use IPTG for induction for 6 hours This expression method was later used to increase the wet bacterial weight of fermentation to 145g/L. In another Chinese patent CN 104592381 A, Wen Liangzhu and others tried to use high-density fermentation to recombinantly express liraglutide (similar in size to teriparatide). The engineering strain also used the BL21 (DE3) host strain. Express the target polypeptide in the form of inclusion bodies. In this study, glucose 10g/L; yeast powder: 30g/L; disodium hydrogen phosphate 4g/L; potassium dihydrogen phosphate 4g/L; ammonium sulfate 6g/L; magnesium sulfate heptahydrate 10g/L; calcium chloride dihydrate 0.02g/L; VB1 trace as fermentation medium, yeast powder: 20g/L, glucose: 30g/L as feeding medium; after feeding for 6 hours, control the temperature to 30°C, pH around 7.0, and dissolved oxygen to 30 %, using this expression method after IPTG induction for 6 hours, the wet bacterial weight of fermentation was increased to 178g/L, and the wet weight of inclusion bodies was 30g/L fermentation broth.

Through equipment modification, designs adapted to high-density fermentation of teriparatide have also been reported. For example, in Chinese patent CN219174485 U, Deng Quanyu and others improved the stirring effect during teriparatide fermentation by optimizing the stirring paddle. However, the patent did not disclose the specific process of high-density fermentation of teriparatide and the expression level of teriparatide inclusion bodies.

However, the yield of teriparatide prepared by existing methods in E. coli high-density fermentation inclusion bodies is low and cannot better meet the demand. Therefore, there is an urgent need to develop a method that can increase the yield of teriparatide in E. coli high-density fermentation. Fermentation medium and fermentation method.

Contents of the invention

Based on the deficiencies in the prior art, the present invention obtains a fermentation medium by optimizing the fermentation conditions of teriparatide, which can increase the production of inclusion bodies of teriparatide in high-density fermentation of E. coli.

The fermentation medium of the present invention can increase the inclusion body yield of high-density fermentation of E. coli teriparat to a level of 40-45g/L.

In order to achieve the above objects, the present invention adopts the following technical solutions:

On the one hand, the invention provides a fermentation medium, which includes the following components: yeast peptone 12-20g/L, yeast extract powder 4-10g/L, and dipotassium hydrogen phosphate trihydrate 3-5g. /L, potassium dihydrogen phosphate 0.5-2g/L, ammonium sulfate 0.5-3g/L, magnesium sulfate heptahydrate 0.5-2g/L, glycerin 3-10g/L, trace element solution 1-3ml/L, defoaming agent 0.1-0.5g/L, the solvent is pure water, use 2mol/L sodium hydroxide solution to adjust pH to 6.8-7.2;

The trace element solution contains nickel sulfate, titanium tetrachloride and ferrous chromite.

Preferably, the concentrations of nickel sulfate, titanium tetrachloride and ferrous chromite are 1-3g/L, 0.5-2g/L and 10-20g/L respectively.

Preferably, the trace element solution also includes the following components: disodium ethylenediaminetetraacetate dihydrate, manganese sulfate, sodium molybdate dihydrate, cobalt chloride, zinc sulfate, copper sulfate and boric acid, and the solvent is pure water.

Further preferably, the trace element solution also includes the following components: disodium ethylenediaminetetraacetate dihydrate 20-30g/L, manganese sulfate 8-10g/L, sodium molybdate dihydrate 0.5-1.5g/L , cobalt chloride 16-20g/L, zinc sulfate 6-10g/L, copper sulfate 4-6g/L and boric acid 2-4g/L, the solvent is pure water.

As some preferred embodiments, the trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 20-30g/L, manganese sulfate 8-10g/L, ferrous chromite 10-20g/L L, nickel sulfate 1-3g/L, cobalt chloride 16-20g/L, titanium tetrachloride 0.5-2g/L, zinc sulfate 6-10g/L, copper sulfate 4-6g/L, sodium molybdate dihydrate 0.5-1.5g/L, boric acid 2-4g/L, solvent is pure water.

Preferably, the fermentation medium includes the following components: yeast peptone 15-18g/L, yeast extract powder 5-8g/L, dipotassium hydrogen phosphate trihydrate 4-5g/L, potassium dihydrogen phosphate 1-1.5 g/L, ammonium sulfate 1-2g/L, magnesium sulfate heptahydrate 1-1.5g/L, glycerin 5-8g/L, trace element solution 1.5-2.5ml/L, defoaming agent 0.2-0.4g/L, The solvent is pure water, and the pH is adjusted to 6.8-7.2 with 2mol/L sodium hydroxide solution;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 22-28g/L, manganese sulfate 9-10g/L, ferrous chromite 12-18g/L, nickel sulfate 1.5-2.5 g/L, cobalt chloride 17-19g/L, titanium tetrachloride 1-1.5g/L, zinc sulfate 7-9g/L, copper sulfate 4.5-5.5g/L, sodium molybdate dihydrate 1-1.5g /L, boric acid 2.5-3.5g/L, solvent is pure water.

As a preferred embodiment, the fermentation medium includes the following components: yeast peptone 20g/L, yeast extract powder 10g/L, dipotassium hydrogen phosphate trihydrate 5g/L, potassium dihydrogen phosphate 2g/L, sulfuric acid Ammonium 3g/L, magnesium sulfate heptahydrate 2g/L, glycerin 10g/L, trace element solution 3ml/L, defoaming agent 0.5g/L, solvent is pure water, adjust pH to 7.2 with 2mol/L sodium hydroxide solution ;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, titanium tetrachloride 2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

As another preferred embodiment, the fermentation medium includes the following components: yeast peptone 12g/L, yeast extract powder 4g/L, dipotassium hydrogen phosphate trihydrate 3g/L, potassium dihydrogen phosphate 0.5g/L , ammonium sulfate 0.5g/L, magnesium sulfate heptahydrate 0.5g/L, glycerol 3g/L, trace element solution 1ml/L, defoaming agent 0.1g/L, the solvent is pure water, use 2mol/L sodium hydroxide solution Adjust pH6.8;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, titanium tetrachloride 2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The defoaming agent is selected from one or more of silicone defoaming agents, polymer defoaming agents, and biological defoaming agents (i.e., fermentation-specific defoaming agents), and is preferably a biological defoaming agent (i.e., fermentation-specific defoaming agents). defoaming agent).

On the other hand, the present invention also provides the use of the above fermentation medium in the preparation of teriparatide.

On the other hand, the present invention also provides a method for preparing teriparatide inclusion bodies using the above-mentioned fermentation medium, including the following steps:

(1) First configure other components except the trace element solution into culture medium A, then filter the trace element solution and add it to culture medium A to obtain a fermentation medium; then add the seed liquid to the culture medium for fermentation ;

(2) When the OD600 of the fermentation broth is ≥ 60, add the inducer and cool down. After 5-7 hours of induced expression, the fermentation is completed and the fermentation broth is obtained. First, centrifuge the fermentation broth to collect the bacterial precipitate. The bacterial precipitate is resuspended, broken, and centrifuged. Inclusion bodies were obtained.

As some preferred embodiments, the filtration described in step (1) is filtration using a 0.22 micron filter membrane.

The inoculum amount of the seed liquid described in step (1) is 5-10v/v%, the initial culture conditions are 37°C, pH 6.7-7.3, and dissolved oxygen ≥ 30%.

The amount of dissolved oxygen is controlled by the stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1-1.5vvm.

During the fermentation process described in step (1), feed needs to be fed, and as the density of the microbial cells increases, the flow rate of the feed needs to be gradually increased.

The specific variable speed feeding plan is as follows: start feeding 2.5ml/h·L ^-1 after 1 hour of culture, increase to 5ml/h·L ^-1 after 1.5 hours, and then increase the feeding speed by 5ml/h·L every 30 minutes. ^-1 , up to 35ml/h·L ^-1 , when OD600≥30, the feeding speed increases to 40ml/h·L ^-1 .

Wherein, the supplement includes the following components: yeast peptone 180-220g/L, yeast extract powder 80-120g/L, glycerol 350-400g/L, defoaming agent 1-2g/L, and the solvent is pure water;

As a preferred embodiment, the feed includes the following components: yeast peptone 220g/L, yeast extract powder 120g/L, glycerol 400g/L, defoaming agent 2g/L, and the solvent is pure water.

The defoaming agent is a biological defoaming agent.

The inducer described in step (2) is IPTG, the addition amount of the inducer is 0.1-0.5 mmol/L; the cooling temperature is 30°C.

The resuspension operation described in step (3) is: add sterilization buffer to the bacterial sediment and resuspend to obtain a resuspension; the mass-to-volume ratio of the bacterial sediment to the sterile buffer is 1kg: 5-20L.

The bacteriolysis buffer includes the following components: trishydroxymethylaminomethane 4-8g/L, disodium ethylenediaminetetraacetate dihydrate 0.2-0.5g/L, and the solvent is pure water.

As a preferred embodiment, the bacteriolysis buffer includes the following components: 8 g/L trishydroxymethylaminomethane, 0.5 g/L disodium ethylenediaminetetraacetate dihydrate, and the solvent is pure water.

As another preferred embodiment, the bacteriolysis buffer includes the following components: 4 g/L trishydroxymethylaminomethane, 0.2 g/L disodium ethylenediaminetetraacetate dihydrate, and the solvent is pure water.

Compared with the existing technology, the beneficial effects of this application are:

1. The teriparatide high-density fermentation medium of the present invention has clear composition and stable source, which is more conducive to ensuring product quality in the production of biological drugs;

2. The composition of the trace element solution added to the teriparatide high-density fermentation medium of the present invention is more conducive to the expression of inclusion bodies;

3. The teriparatide high-density fermentation method of the present invention adopts a strategy of starting at a fixed time point and adjusting the feeding flow rate, which is more conducive to the stability of fermentation production.

4. The high-density fermentation method of teriparatide of the present invention adopts a low-temperature induction method, which is more conducive to reducing the side effects of the inducer on bacterial growth in the fermentation environment and increasing the expression rate.

Detailed ways

The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents used is not indicated, they are all conventional products that can be purchased on the market. The defoaming agent used in the following examples was purchased from Yantai Hengxin Chemical Technology Co., Ltd., and the model is THIX-298 high-efficiency fermentation special disinfectant. Foaming agent.

Example 1 A fermentation medium and its application in the preparation of teriparatide

The fermentation medium includes the following components: yeast peptone 12g/L, yeast extract powder 4g/L, dipotassium hydrogen phosphate trihydrate 3g/L, potassium dihydrogen phosphate 0.5g/L, ammonium sulfate 0.5g/L, Magnesium sulfate heptahydrate 0.5g/L, glycerol 3g/L, trace element solution 1ml/L, defoaming agent 0.1g/L, the solvent is pure water, and 2mol/L sodium hydroxide solution is used to adjust pH to 6.95;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 20g/L, manganese sulfate 8g/L, ferrous chromite 10g/L, nickel sulfate 1g/L, cobalt chloride 16g/L. L, titanium tetrachloride 0.5g/L, zinc sulfate 6g/L, copper sulfate 4g/L, sodium molybdate dihydrate 0.5g/L, boric acid 2g/L, the solvent is pure water.

The method for preparing teriparatide inclusion bodies includes the following steps:

(1) First configure other components except the trace element solution into culture medium A. Filter the trace element solution through a 0.22 micron filter membrane and add it to culture medium A to obtain the fermentation culture medium. Then add the seed liquid (OD ₆₀₀ 4.5 ) is inserted into the culture medium for fermentation, the inoculum volume is 5v/v%, the initial culture conditions are 37°C, pH 7.0, and the dissolved oxygen is controlled to ≥ 30% through stirring speed, ventilation and oxygen, and the total ventilation of air and oxygen is 1vvm ;

The fermentation process requires variable speed feeding, specifically: starting to feed 2.5ml/h·L ^-1 after 1 hour of cultivation, increasing to 5ml/h·L ^-1 after 1.5 hours, and then increasing the feed every 30 minutes. The feeding speed is 5ml/h·L ^-1 , up to 35ml/h·L ^-1 . When OD ₆₀₀ ≥ 30, the feeding speed is increased to 40ml/h·L ^-1 ;

(2) When the OD ₆₀₀ of the fermentation broth is ≥60, add the inducer IPTG at an amount of 0.1 mmol/L. Reduce the fermentation control temperature to 30°C. After inducing expression for 5 hours, the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacteria. The cells are precipitated, and the cells are resuspended in sterilization buffer (1kg of cells is added to 10L of sterilization buffer) to obtain a resuspension. The resuspension is broken by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The feed material described in step (1) includes the following components: yeast peptone 180g/L, yeast extract powder 80g/L, glycerol 350g/L, defoaming agent 1g/L, and the solvent is pure water.

The bacteriolysis buffer described in step (2) includes the following components: trishydroxymethylaminomethane 4g/L, disodium ethylenediaminetetraacetate dihydrate 0.2g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.243g/g _{of bacterial cells} and 40.1g/L _{of fermentation broth} .

Example 2 A fermentation medium and its application in the preparation of teriparatide

The fermentation medium includes the following components: yeast peptone 20g/L, yeast extract powder 10g/L, dipotassium hydrogen phosphate trihydrate 5g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 3g/L, heptahydrate Magnesium sulfate 2g/L, glycerol 10g/L, trace element solution 3ml/L, defoaming agent 0.5g/L, solvent is pure water, adjust pH to 7.1 with 2mol/L sodium hydroxide solution;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 20g/L, manganese sulfate 8g/L, ferrous chromite 10g/L, nickel sulfate 1g/L, cobalt chloride 16g/L. L, titanium tetrachloride 0.5g/L, zinc sulfate 6g/L, copper sulfate 4g/L, sodium molybdate dihydrate 0.5g/L, boric acid 2g/L, the solvent is pure water.

The method for preparing teriparatide inclusion bodies includes the following steps:

(1) First configure other components except the trace element solution into culture medium A. Filter the trace element solution through a 0.22 micron filter membrane and add it to culture medium A to obtain the fermentation culture medium. Then add the seed liquid (OD ₆₀₀ 4.5 ) is inserted into the culture medium for fermentation, the inoculum volume is 6v/v%, the initial culture conditions are 37°C, pH 7.0, and the dissolved oxygen is controlled to ≥ 30% through stirring speed, ventilation and oxygen, and the total ventilation of air and oxygen is 1vvm ;

The fermentation process requires variable speed feeding, specifically: starting to feed 2.5ml/h·L ^-1 after 1 hour of cultivation, increasing to 5ml/h·L ^-1 after 1.5 hours, and then increasing the feed every 30 minutes. The feeding speed is 5ml/h·L ^-1 , up to 35ml/h·L ^-1 . When OD ₆₀₀ ≥ 30, the feeding speed is increased to 40ml/h·L ^-1 ;

(2) When the OD ₆₀₀ of the fermentation broth is ≥60, add the inducer IPTG at an amount of 0.2 mmol/L. Reduce the fermentation control temperature to 30°C. After inducing expression for 5 hours, the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacteria. The cells are precipitated, and the cells are resuspended in sterilization buffer (1kg of cells is added to 10L of sterilization buffer) to obtain a resuspension. The resuspension is broken by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The feed material described in step (1) includes the following components: yeast peptone 180g/L, yeast extract powder 80g/L, glycerol 350g/L, defoaming agent 1g/L, and the solvent is pure water.

The bacteriolysis buffer described in step (2) includes the following components: trishydroxymethylaminomethane 4g/L, disodium ethylenediaminetetraacetate dihydrate 0.2g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.241g/g _{of bacterial cells} and 40.5g/L _{of fermentation broth} .

Embodiment 3 A fermentation medium and its application in the preparation of teriparatide

The fermentation medium includes the following components: yeast peptone 12g/L, yeast extract powder 4g/L, dipotassium hydrogen phosphate trihydrate 3g/L, potassium dihydrogen phosphate 0.5g/L, ammonium sulfate 0.5g/L, Magnesium sulfate heptahydrate 0.5g/L, glycerol 3g/L, trace element solution 1ml/L, defoaming agent 0.1g/L, the solvent is pure water, and 2mol/L sodium hydroxide solution is used to adjust pH to 6.8;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, titanium tetrachloride 2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The method for preparing teriparatide inclusion bodies includes the following steps:

(1) First configure other components except the trace element solution into culture medium A. Filter the trace element solution through a 0.22 micron filter membrane and add it to culture medium A to obtain the fermentation culture medium. Then add the seed liquid (OD ₆₀₀ 4.5 ) is inserted into the culture medium for fermentation, the inoculum volume is 7v/v%, the initial culture conditions are 37°C, pH 7.0, and the dissolved oxygen is controlled to ≥ 30% through stirring speed, ventilation and oxygen, and the total ventilation of air and oxygen is 1vvm ;

The fermentation process requires variable speed feeding, specifically: starting to feed 2.5ml/h·L ^-1 after 1 hour of cultivation, increasing to 5ml/h·L ^-1 after 1.5 hours, and then increasing the feed every 30 minutes. The feeding speed is 5ml/h·L ^-1 , up to 35ml/h·L ^-1 . When OD ₆₀₀ ≥ 30, the feeding speed is increased to 40ml/h·L ^-1 ;

(2) When the OD ₆₀₀ of the fermentation broth is ≥60, add the inducer IPTG at an amount of 0.2 mmol/L. Reduce the fermentation control temperature to 30°C. After inducing expression for 5 hours, the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacteria. The cells are precipitated, and the cells are resuspended in sterilization buffer (1kg of cells is added to 10L of sterilization buffer) to obtain a resuspension. The resuspension is broken by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The feed material described in step (1) includes the following components: yeast peptone 220g/L, yeast extract powder 120g/L, glycerin 400g/L, defoaming agent 2g/L, and the solvent is pure water.

The bacteriolysis buffer described in step (2) includes the following components: trishydroxymethylaminomethane 4g/L, disodium ethylenediaminetetraacetate dihydrate 0.2g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.259g/g _{of bacterial cells} and 43.6g/L _{of fermentation broth} .

Embodiment 4 A fermentation medium and its application in the preparation of teriparatide

The fermentation medium comprises the following components: 20 g/L yeast peptone, 10 g/L yeast extract powder, 5 g/L potassium dihydrogen phosphate trihydrate, 2 g/L potassium dihydrogen phosphate, 3 g/L ammonium sulfate, 2 g/L magnesium sulfate heptahydrate, 10 g/L glycerol, 3 ml/L trace element solution, 0.5 g/L defoamer, the solvent is pure water, and the pH is adjusted to 7.2 with 2 mol/L sodium hydroxide solution;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, titanium tetrachloride 2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The method for preparing teriparatide inclusion bodies includes the following steps:

(1) First configure other components except the trace element solution into culture medium A. Filter the trace element solution through a 0.22 micron filter membrane and add it to culture medium A to obtain the fermentation culture medium. Then add the seed liquid (OD ₆₀₀ 4.5 ) is inserted into the culture medium for fermentation, the inoculum volume is 6v/v%, the initial culture conditions are 37°C, pH 7.0, and the dissolved oxygen is controlled to ≥ 30% through the stirring speed, ventilation volume and oxygen, of which the total ventilation volume of air and oxygen is 1.2 vvm;

The fermentation process requires variable speed feeding, specifically: starting to feed 2.5ml/h·L ^-1 after 1 hour of cultivation, increasing to 5ml/h·L ^-1 after 1.5 hours, and then increasing the feed every 30 minutes. The feeding speed is 5ml/h·L ^-1 , up to 35ml/h·L ^-1 . When OD ₆₀₀ ≥ 30, the feeding speed is increased to 40ml/h·L ^-1 ;

(2) When the OD ₆₀₀ of the fermentation broth is ≥60, add the inducer IPTG at an amount of 0.3 mmol/L. Reduce the fermentation control temperature to 30°C. After inducing expression for 5 hours, the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacteria. The cells are precipitated, and the cells are resuspended in sterilization buffer (1kg of cells is added to 5L of sterilization buffer) to obtain a resuspension. The resuspension is broken by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The feed material described in step (1) includes the following components: yeast peptone 220g/L, yeast extract powder 120g/L, glycerin 400g/L, defoaming agent 2g/L, and the solvent is pure water.

The bacteriolysis buffer described in step (2) includes the following components: trishydroxymethylaminomethane 8g/L, disodium ethylenediaminetetraacetate dihydrate 0.5g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.279g/g _{of bacterial cells} and 44.1g/L _{of fermentation broth} .

Example 5 A fermentation medium and its application in the preparation of teriparatide

The fermentation medium includes the following components: yeast peptone 12g/L, yeast extract powder 4g/L, dipotassium hydrogen phosphate trihydrate 3g/L, potassium dihydrogen phosphate 0.5g/L, ammonium sulfate 0.5g/L, Magnesium sulfate heptahydrate 0.5g/L, glycerol 3g/L, trace element solution 1ml/L, defoaming agent 0.1g/L, the solvent is pure water, and 2mol/L sodium hydroxide solution is used to adjust pH to 6.8;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 20g/L, manganese sulfate 8g/L, ferrous chromite 10g/L, nickel sulfate 1g/L, cobalt chloride 16g/L. L, titanium tetrachloride 0.5g/L, zinc sulfate 6g/L, copper sulfate 4g/L, sodium molybdate dihydrate 0.5g/L, boric acid 2g/L, the solvent is pure water.

The method for preparing teriparatide inclusion bodies includes the following steps:

(1) First configure other components except the trace element solution into culture medium A. Filter the trace element solution through a 0.22 micron filter membrane and add it to culture medium A to obtain the fermentation culture medium. Then add the seed liquid (OD ₆₀₀ 4.5 ) is inserted into the culture medium for fermentation, the inoculum volume is 8v/v%, the initial culture conditions are 37°C, pH 7.0, and the dissolved oxygen is controlled to ≥ 30% through the stirring speed, ventilation volume and oxygen, of which the total ventilation volume of air and oxygen is 1.5 vvm;

The fermentation process requires variable speed feeding, specifically: starting to feed 2.5ml/h·L ^-1 after 1 hour of cultivation, increasing to 5ml/h·L ^-1 after 1.5 hours, and then increasing the feed every 30 minutes. The feeding speed is 5ml/h·L ^-1 , up to 35ml/h·L ^-1 . When OD ₆₀₀ ≥ 30, the feeding speed is increased to 40ml/h·L ^-1 ;

(2) When the OD ₆₀₀ of the fermentation broth is ≥60, add the inducer IPTG at an amount of 0.4 mmol/L. Reduce the fermentation control temperature to 30°C. After inducing expression for 5 hours, the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacteria. The cells are precipitated, and the cells are resuspended in sterilization buffer (20L of sterilization buffer is added to 1kg of cells) to obtain a resuspension. The resuspension is broken by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The feed material described in step (1) includes the following components: yeast peptone 220g/L, yeast extract powder 120g/L, glycerin 400g/L, defoaming agent 2g/L, and the solvent is pure water.

The bacteriolysis buffer described in step (2) includes the following components: trishydroxymethylaminomethane 8g/L, disodium ethylenediaminetetraacetate dihydrate 0.5g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.252g/g _{of bacterial cells} and 42.9g/L _{of fermentation broth} .

Example 6 A fermentation medium and its application in the preparation of teriparatide

The fermentation medium includes the following components: yeast peptone 20g/L, yeast extract powder 10g/L, dipotassium hydrogen phosphate trihydrate 5g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 3g/L, heptahydrate Magnesium sulfate 2g/L, glycerol 10g/L, trace element solution 3ml/L, defoaming agent 0.5g/L, solvent is pure water, adjust pH to 7.2 with 2mol/L sodium hydroxide solution;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, titanium tetrachloride 2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The method for preparing teriparatide inclusion bodies includes the following steps:

(1) First configure other components except the trace element solution into culture medium A. Filter the trace element solution through a 0.22 micron filter membrane and add it to culture medium A to obtain the fermentation culture medium. Then add the seed liquid (OD ₆₀₀ 4.5 ) is inserted into the culture medium for fermentation, the inoculum volume is 5v/v%, the initial culture conditions are 37°C, pH 7.0, and the dissolved oxygen is controlled to ≥ 30% through stirring speed, ventilation and oxygen, and the total ventilation of air and oxygen is 1vvm ;

The fermentation process requires variable speed feeding, specifically: starting to feed 2.5ml/h·L ^-1 after 1 hour of cultivation, increasing to 5ml/h·L ^-1 after 1.5 hours, and then increasing the feed every 30 minutes. The feeding speed is 5ml/h·L ^-1 , up to 35ml/h·L ^-1 . When OD ₆₀₀ ≥ 30, the feeding speed is increased to 40ml/h·L ^-1 ;

(2) When the OD ₆₀₀ of the fermentation broth is ≥60, add the inducer IPTG at an amount of 0.5 mmol/L. Reduce the fermentation control temperature to 30°C. After inducing expression for 5 hours, the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacteria. The cells are precipitated, and the cells are resuspended in sterilization buffer (1kg of cells is added to 10L of sterilization buffer) to obtain a resuspension. The resuspension is broken by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The feed material described in step (1) includes the following components: yeast peptone 180g/L, yeast extract powder 80g/L, glycerol 350g/L, defoaming agent 1g/L, and the solvent is pure water.

The bacteriolysis buffer described in step (2) includes the following components: trishydroxymethylaminomethane 8g/L, disodium ethylenediaminetetraacetate dihydrate 0.5g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.251g/g _{of bacterial cells} and 41.6g/L _{of fermentation broth} .

Embodiment 7 A fermentation medium and its application in the preparation of teriparatide

The fermentation medium includes the following components: yeast peptone 16g/L, yeast extract powder 7g/L, dipotassium hydrogen phosphate trihydrate 4g/L, potassium dihydrogen phosphate 1.5g/L, ammonium sulfate 2g/L, seven Aqueous magnesium sulfate 1.5g/L, glycerol 7g/L, trace element solution 2ml/L, defoaming agent 0.3g/L, solvent is pure water, adjust pH to 7.2 with 2mol/L sodium hydroxide solution;

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 25g/L, manganese sulfate 9g/L, ferrous chromite 15g/L, nickel sulfate 2g/L, cobalt chloride 18g/L. L, titanium tetrachloride 1g/L, zinc sulfate 8g/L, copper sulfate 5g/L, sodium molybdate dihydrate 1g/L, boric acid 3g/L, the solvent is pure water.

The method for preparing teriparatide inclusion bodies includes the following steps:

(1) First configure other components except the trace element solution into culture medium A. Filter the trace element solution through a 0.22 micron filter membrane and add it to culture medium A to obtain the fermentation culture medium. Then add the seed liquid (OD ₆₀₀ 4.5 ) is inserted into the culture medium for fermentation, the inoculum volume is 5v/v%, the initial culture conditions are 37°C, pH 7.0, and the dissolved oxygen is controlled to ≥ 30% through stirring speed, ventilation and oxygen, and the total ventilation of air and oxygen is 1vvm ;

The fermentation process requires variable speed feeding, specifically: starting to feed 2.5ml/h·L ^-1 after 1 hour of cultivation, increasing to 5ml/h·L ^-1 after 1.5 hours, and then increasing the feed every 30 minutes. The feeding speed is 5ml/h·L ^-1 , up to 35ml/h·L ^-1 . When OD ₆₀₀ ≥ 30, the feeding speed is increased to 40ml/h·L ^-1 ;

(2) When the OD ₆₀₀ of the fermentation broth is ≥60, add the inducer IPTG at an amount of 0.5 mmol/L. Reduce the fermentation control temperature to 30°C. After inducing expression for 5 hours, the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacteria. The cells are precipitated, and the cells are resuspended in sterilization buffer (1kg of cells is added to 10L of sterilization buffer) to obtain a resuspension. The resuspension is broken by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The feed material described in step (1) includes the following components: yeast peptone 200g/L, yeast extract powder 100g/L, glycerin 375g/L, defoaming agent 1.5g/L, and the solvent is pure water.

The bacteriolysis buffer described in step (2) includes the following components: trishydroxymethylaminomethane 6g/L, disodium ethylenediaminetetraacetate dihydrate 0.4g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.268g/g _{of bacteria} and 42.8g/L _{of fermentation broth} .

Comparative example 1

The difference from Example 4 is that no trace element solution is added, and the contents and preparation methods of other components are the same as those in Example 4.

The inclusion body yield calculated by weighing was 0.152g/g _{of bacterial cells} and 21.1g/L _{of fermentation broth} .

Comparative Example 2 A fermentation medium and its application in the preparation of teriparatide

The difference from Example 4 is that the induction temperature is 37°C, and the contents and preparation methods of other components are the same as those in Example 4. Specifically:

When the OD600 of the fermentation broth is ≥ 60, add the inducer IPTG at a dosage of 0.3mmol/L, keep the fermentation temperature at 37°C, and induce expression for 5 hours. After 5 hours of fermentation, the fermentation is completed and the fermentation broth is obtained. The fermentation broth is centrifuged to collect the bacterial precipitate. Resuspend in sterilization buffer (add 10L of sterilization buffer to 1kg of bacteria) to obtain a resuspension. The resuspension is crushed by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The inclusion body yield calculated by weighing was 0.174g/g _{of bacterial cells} and 26.8g/L _{of fermentation broth} .

Comparative Example 3 A fermentation medium and its application in the preparation of teriparatide

The difference from Example 4 is that no trace element solution is added, materials are fed at a constant rate during the fermentation process, and the induction temperature is 37°C. The contents and preparation methods of other components are the same as in Example 4. Specifically:

The method for preparing teriparatide inclusion bodies comprises the following steps:

(1) Add the seed liquid (OD6004.5) to the culture medium for fermentation. The inoculum volume is 5v/v%. The initial culture conditions are 37°C and pH 7.0. The dissolved oxygen is controlled to ≥ 30% through stirring speed, ventilation volume and oxygen. , where the total ventilation volume of air and oxygen is 1vvm;

The fermentation process requires variable speed feeding, specifically: starting to feed 20ml/h·L-1 after 1 hour of cultivation, and feeding at this flow rate until the end of fermentation;

(2) When the OD600 of the fermentation broth is ≥ 60, add the inducer IPTG at a dosage of 0.1 mmol/L, keep the fermentation temperature at 37°C, and induce expression for 5 hours after the fermentation is completed to obtain the fermentation broth; centrifuge the fermentation broth to collect the bacterial sediment. Resuspend the bacteria in sterilization buffer (add 5L of sterilization buffer to 1kg of bacteria) to obtain a resuspension. The resuspension is crushed by a high-pressure homogenizer and centrifuged again. The centrifugal precipitate is collected as inclusion bodies.

The inclusion body yield calculated by weighing was 0.166g/g _{of bacterial cells} and 23.7g/L _{of fermentation broth} .

Comparative Example 4 A fermentation medium and its application in the preparation of teriparatide

The difference from Example 4 is that no trace element solution is added, materials are fed at a constant rate during the fermentation process, and the contents and preparation methods of other components are the same as in Example 4. Specifically:

(1) Add the seed liquid (OD6004.5) to the culture medium for fermentation. The inoculum volume is 10v/v%. The initial culture conditions are 37°C and pH 7.0. The dissolved oxygen is controlled to ≥30% through stirring speed, ventilation volume and oxygen. , where the total ventilation volume of air and oxygen is 1.5vvm;

The fermentation process requires variable speed feeding, specifically: starting feeding at 20 ml/h·L-1 after 1 hour of cultivation, and feeding at this flow rate until the end of fermentation.

The inclusion body yield calculated by weighing was 0.168g/g _{of bacterial cells} and 25.9g/L _{of fermentation broth} .

Comparative example 5

The difference from Example 4 is that it does not contain ferrous chromite, 32 g/L of ferrous sulfate heptahydrate is added, and the contents and preparation methods of other components are the same as those in Example 4. Right now:

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous sulfate heptahydrate 32g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, titanium tetrachloride 2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The inclusion body yield calculated by weighing was 0.231g/g _{of bacterial cells} and 36.2g/L _{of fermentation broth} .

Comparative example 6

The difference from Example 4 is that it does not contain titanium tetrachloride, and the contents and preparation methods of other components are the same as those in Example 4. Right now:

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The inclusion body yield calculated by weighing was 0.232g/g _{of bacterial cells} and 36.9g/L _{of fermentation broth} .

Comparative example 7

The difference from Example 4 is that it does not contain nickel sulfate, and the contents and preparation methods of other components are the same as those in Example 4. Right now:

The trace element solution includes the following components: the trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, Cobalt chloride 20g/L, titanium tetrachloride 2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The inclusion body yield calculated by weighing was 0.241g/g _{of bacterial cells} and 37.6g/L _{of fermentation broth} .

Comparative example 8

The difference from Example 4 is that it does not contain nickel sulfate and titanium tetrachloride, and the contents and preparation methods of other components are the same as those in Example 4. Right now:

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, cobalt chloride 20g/L, zinc sulfate 10g/L. L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, and the solvent is pure water.

The inclusion body yield calculated by weighing was 0.226g/g _{of bacterial cells} and 34.8g/L _{of fermentation broth} .

Comparative example 9

The difference from Example 4 is that 4.8 g/L of nickel sulfate and 0.2 g/L of titanium tetrachloride are added, and the contents and preparation methods of other components are the same as those in Example 4. Right now:

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 20g/L, nickel sulfate 4.8g/L, cobalt chloride 20g /L, titanium tetrachloride 0.2g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, the solvent is pure water.

The inclusion body yield calculated by weighing was 0.233g/g _{of bacterial cells} and 36.4g/L _{of fermentation broth} .

Comparative example 10

The difference from Example 4 is that 2 g/L of ferrous chromite, 3.3 g/L of titanium tetrachloride, and 30 g/L of ferrous sulfate heptahydrate are added. The contents and preparation methods of other components are the same as those of Example 4. Right now:

The trace element solution includes the following components: disodium ethylenediaminetetraacetate dihydrate 30g/L, manganese sulfate 10g/L, ferrous chromite 2g/L, nickel sulfate 3g/L, cobalt chloride 20g/L. L, titanium tetrachloride 3.3g/L, zinc sulfate 10g/L, copper sulfate 6g/L, sodium molybdate dihydrate 1.5g/L, boric acid 4g/L, ferrous sulfate heptahydrate 30g/L, the solvent is pure water.

The inclusion body yield calculated by weighing was 0.227g/g _{of bacterial cells} and 34.5g/L _{of fermentation broth} .

Comparative example 11

The difference from Example 4 is that the trace element solution includes the following components: ferrous sulfate 3.2g/L, manganous chloride 1g/L, cobalt nitrate 1.5g/L, calcium chloride 1g/L, chlorine Copper oxide 0.25g/L, zinc sulfate 0.4g/L, boric acid 0.3g/L and sodium molybdate 3g/L, the solvent is 1mol/L hydrochloric acid, and the content and preparation method of other components are the same as in Example 4.

The inclusion body yield calculated by weighing was 0.199g/g _{of bacterial cells} and 28.9g/L _{of fermentation broth} .

Performance data:

Compare the inclusion body yields of each embodiment and the comparative example, and the results are shown in Table 1.

Table 1

According to the test results in Table 1 above, it can be seen that the fermentation medium provided in Examples 1-5 of the present invention is used to prepare teriparatide inclusion bodies, and the yield of the inclusion bodies obtained is ≥0.241g/g _{bacterial cells} , ≥40.1g/ L _{fermentation broth} ; in the comparative example, changing the type and content of elements in the trace element solution or omitting the trace element solution, or changing the feeding method during the fermentation process will lead to a reduction in the production of inclusion bodies.

The above embodiments are only used to illustrate the technical solutions of the present invention but not to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art can still make modifications or equivalent substitutions to the specific implementations of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are within the scope of the claims of the present invention.

Claims (15)

1. A fermentation medium, characterized in that: it includes the following components: yeast peptone 12-20g/L, yeast extract powder 4-10g/L, dipotassium hydrogen phosphate trihydrate 3-5g/L, potassium dihydrogen phosphate 0.5-2g/L, ammonium sulfate 0.5-3g/L, magnesium sulfate heptahydrate 0.5-2g/L, glycerin 3-10g/L, trace element solution 1-3ml/L, defoaming agent 0.1-0.5g/L, The solvent is pure water, and the pH is adjusted to 6.8-7.2 with 2mol/L sodium hydroxide solution; The trace element solution contains nickel sulfate, titanium tetrachloride and ferrous chromite. 2. The fermentation medium according to claim 1, characterized in that: the concentrations of described nickel sulfate, titanium tetrachloride and ferrous chromite are 1-3g/L, 0.5-2g/L and 10 respectively. -20g/L. 3. The fermentation culture medium according to claim 2, characterized in that: the trace element solution further includes the following components: disodium ethylenediaminetetraacetate dihydrate, manganese sulfate, sodium molybdate dihydrate, chloride Cobalt, zinc sulfate, copper sulfate and boric acid, the solvent is pure water. 4. The fermentation culture medium according to claim 3, characterized in that: the trace element solution also includes components of the following concentration: disodium ethylenediaminetetraacetate dihydrate 20-30g/L, manganese sulfate 8- 10g/L, sodium molybdate dihydrate 0.5-1.5g/L, cobalt chloride 16-20g/L, zinc sulfate 6-10g/L, copper sulfate 4-6g/L and boric acid 2-4g/L, the solvent is Pure water. 5. Application of the fermentation medium according to claim 1 in the preparation of teriparatide. 6. The method for preparing teriparatide inclusion bodies from a fermentation medium according to claim 1, characterized in that it includes the following steps: (1) First configure other components except the trace element solution into culture medium A, then filter the trace element solution and add it to culture medium A to obtain a fermentation medium; then add the seed liquid to the culture medium for fermentation ; (2) When the OD600 of the fermentation broth is ≥ 60, add the inducer and cool down. After 5-7 hours of induced expression, the fermentation is completed and the fermentation broth is obtained. First, centrifuge the fermentation broth to collect the bacterial precipitate. The bacterial precipitate is resuspended, broken, and centrifuged. Crude inclusion bodies were obtained. 7. The method according to claim 6, characterized in that: the inoculum amount of the seed liquid described in step (1) is 5-10v/v%, the initial culture conditions are 37°C, pH 6.7-7.3, and dissolved oxygen ≥30%. 8. The method according to claim 6, characterized in that: the amount of dissolved oxygen is controlled by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1-1.5 vvm. 9. The method according to claim 6, characterized in that: during the fermentation process in step (1), feeding material needs to be fed, and as the density of the microbial cells increases, the flow rate of the feeding material needs to be gradually increased. 10. The method according to claim 9, characterized in that: the method of adding feed is variable speed feeding, and the variable speed feeding scheme is as follows: start feeding 2.5ml/h·L after 1 hour of cultivation. ^-1 , increase to 5ml/h·L ^-1 after 1.5 hours, then increase the feeding rate by 5ml/h·L ^-1 every 30 minutes, up to 35ml/h·L ^-1 , feed when OD600≥30 The speed is increased to 40ml/h·L ^-1 . 11. The method according to claim 9, characterized in that: the supplement includes the following components: yeast peptone 180-220g/L, yeast extract powder 80-120g/L, glycerin 350-400g/L, digestion The foaming agent is 1-2g/L, and the solvent is pure water. 12. The method according to claim 6, characterized in that: the inducer described in step (2) is IPTG, and the addition amount of the inducer is 0.1-0.5 mmol/L. 13. The method according to claim 6, characterized in that: the cooling temperature in step (2) is 30°C. 14. The method according to claim 6, characterized in that: the resuspension operation described in step (3) is: adding bacteriostasis buffer to the bacterial sediment and resuspension to obtain a resuspension; The mass-volume ratio of bacterial sediment and bacteriolysis buffer is 1kg:5-20L. 15. The method according to claim 14, characterized in that: the bacteriolysis buffer includes the following components: trishydroxymethylaminomethane 4-8g/L, disodium ethylenediaminetetraacetate dihydrate 0.2- 0.5g/L, the solvent is pure water.