CN108220369B

CN108220369B - Method for producing recombinant hirudin

Info

Publication number: CN108220369B
Application number: CN201711262810.4A
Authority: CN
Inventors: 陈杰鹏; 段丽丽; 洪琳; 陈煜藩; 陈鸿锐; 邱雪莲; 蔡桂珠; 胡留松; 许志锴; 叶红林; 黄晓莹; 纪烨瑜
Original assignee: Sungen Bioscience Co ltd
Current assignee: Sungen Bioscience Co ltd
Priority date: 2017-12-04
Filing date: 2017-12-04
Publication date: 2021-03-12
Anticipated expiration: 2037-12-04
Also published as: CN108220369A

Abstract

The invention discloses a method for producing recombinant hirudin, which comprises the following steps: the pBH-2 engineering bacteria (preservation number: CGMCC No.0908) is inoculated into a fermentation medium containing carbon source substances and nitrogen source substances, the culture is firstly carried out at the temperature of 25-35 ℃ until the logarithmic growth phase is finished, then the temperature is raised to 35-45 ℃ for continuous culture, and the temperature raising process is completed within 1 hour, optionally within 10 minutes, and optionally within 5 minutes.

Description

Method for producing recombinant hirudin

Technical Field

The present invention relates to bioengineering technology, especially fermentation engineering, and is especially process of producing recombinant hirudin.

Background

The hirudin is a thrombin specific inhibitor, can effectively prevent and treat disseminated intravascular coagulation, and has significant effects on cardiovascular and cerebral thrombosis diseases. Because the natural hirudin has limited yield, and each leech only contains 20 mu g of hirudin, the requirement of clinical application cannot be met, therefore, the research of producing the recombinant hirudin by adopting the biological engineering technology, in particular to a method for producing the recombinant hirudin with high yield, is particularly important.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for producing recombinant hirudin, which adopts pBH-2 engineering bacteria (Escherichia coli), China general microbiological culture Collection center, Beijing, Haitai Ministry of Hokko, No. 13, institute of microbiology of Chinese academy of sciences, preservation date: 03 and 19 days 2003, preservation number: CGMCC No.0908) to produce the recombinant hirudin by a high-density and high-expression fermentation process, and can greatly improve the yield of the recombinant hirudin.

In order to achieve the object of the present invention, the present invention provides a method for producing recombinant hirudin, the method comprising: the pBH-2 engineering bacteria (preservation number: CGMCC No.0908) is inoculated into a fermentation medium containing carbon source substances and nitrogen source substances, the culture is carried out under aerobic conditions at the temperature of 25-35 ℃ until the logarithmic phase is finished, and then the temperature is raised to 35-45 ℃ for continuous culture until the hirudin yield reaches the peak value.

In the above or other embodiments, the cultivation may be continued for 3-16 hours or more, so that the production of hirudin reaches a peak.

In the above or other embodiments, the culture may be conducted at a temperature of 30 ℃ until the end of the logarithmic growth phase.

In the above or other embodiments, the incubation may be continued for 3-16 hours or more at an elevated temperature of 42 ℃ to peak the production of hirudin.

In the above or other embodiments, the warming process may be completed within 1 hour.

In the above or other embodiments, the warming process may be completed within 10 minutes.

In the above or other embodiments, the warming process may be completed in 5 minutes.

In the above or other embodiments, the carbon source material may be selected from one or more of glucose, sucrose, maltose, starch, dextrin, lactose, fructose and glycerol. Wherein the addition amount of the carbon source material in the fermentation medium can be determined by those skilled in the art as needed. For example, in some cases, the amount of the carbon source material in the fermentation medium is 5-200g/L based on the total volume of the medium solution.

In the above or other embodiments, the carbon source material may be sucrose.

In the above or other embodiments, the carbon source material may be a combination of sucrose and one or more selected from glucose, maltose, starch, dextrin, lactose, fructose, and glycerol.

In the above or other embodiments, wherein the nitrogen source material may be selected from one or more of corn steep liquor, yeast powder, peptone, beef extract, yeast extract, ammonium nitrate, ammonium chloride and ammonium sulfate. The amount of the nitrogen source substance added to the fermentation medium can be determined as desired by a person skilled in the art. For example, in some cases, the amount of the nitrogen source material in the fermentation medium is 10-120g/L based on the total volume of the medium solution.

In the above or other embodiments, the nitrogen source substance may be yeast powder, peptone, or both yeast powder and peptone.

In the above or other embodiments, the nitrogen source substance may be yeast powder, peptone, or a combination of both yeast powder and peptone with one or more selected from corn steep liquor, beef extract, yeast extract, ammonium nitrate, ammonium chloride, and ammonium sulfate.

In the above or other embodiments, the nitrogen source may be selected from angel peptone FP318, angel yeast powder FM818, or both.

In the above or other embodiments, wherein the weight ratio of the carbon source material to the nitrogen source material in the fermentation medium may be from 0.2 to 40: 1.

in the above or other embodiments, wherein the weight ratio of the carbon source material to the nitrogen source material in the fermentation medium may be 1.1: 1.

In the above or other embodiments, other inorganic substances may also be added to the culture medium to promote the growth of the microorganism and to increase the yield of recombinant hirudin. For example, in the above or other embodiments, the fermentation medium may further comprise a phosphate salt, a magnesium salt, a sodium salt, or a combination thereof, optionally, the phosphate salt is selected from one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

In the above or other embodiments, the phosphate may be added in an amount of 0.2 to 20g/L based on the total volume of the medium solution.

In the above or other embodiments, the magnesium salt may be magnesium sulfate or magnesium chloride.

In the above or other embodiments, the magnesium salt may be added in an amount of 0.1 to 1.5g/L based on the total volume of the medium solution.

In the above or other embodiments, the sodium salt may be selected from one or more of sodium chloride, sodium citrate, sodium sulfate, sodium acetate.

In the above or other embodiments, the sodium salt may be added in an amount of 0.1 to 25g/L based on the total volume of the medium solution. The inventor of the application finds that the addition of the sodium salt is beneficial to promoting the pBH-2 engineering bacteria to express more recombinant hirudin.

In the above or other embodiments, the medium may further comprise one or more of iron salts, manganese salts, aluminum salts, cobalt salts, zinc salts, copper salts, nickel salts, boric acid, vitamin B1. The skilled person can select the above components and determine their addition according to the actual needs.

In the above or other embodiments, the fermentation medium may further comprise an antibiotic substance.

In the above or other embodiments, the antibiotic substance may be Ampicillin (AMP).

In the above or other embodiments, the antibiotic substance is added in an amount of 0.01 to 0.2g/L based on the total volume of the culture medium solution.

In the above or other embodiments, the antibiotic substance may be added to the fermentation medium in two portions, where the first addition is at the time of inoculation and the second addition is before the end of the log phase of growth of the biomass.

In the above or other embodiments, the antibiotic substance is added in an amount of 0.01-0.2g/L based on the total volume of the medium solution at the time of inoculation for the first time, and the antibiotic substance is added in an amount of 0.01-0.2g/L based on the total volume of the medium solution before the cells are cultured until the end of the logarithmic growth phase for the second time.

In the above or other embodiments, the second addition of antibiotic substance may be added as a stream. Through a great deal of experimental research, the inventor of the application finds that when the antibiotic substance such as ampicillin AMP is added for the second time, the feeding of the antibiotic substance into the fermentation liquid is more favorable for promoting the pBH-2 engineering bacteria to express more recombinant hirudin than the feeding of the antibiotic substance into the fermentation liquid once. For example, the feeding may be performed by controlling the flow rate to be 0.017mg/L to 0.33mg/L of the antibiotic substance per minute based on the total volume of the culture solution.

In the above or other embodiments, the constituent substances of the fermentation medium, such as the nitrogen source substance and the carbon source substance and other optional substances, may be added in two portions, where the first addition is added before inoculation and the second addition is added in a stream after inoculation and completed within 13 hours.

In the above or other embodiments, the pBH-2 engineered bacterium may be inoculated to the fermentation medium in an inoculum size of 1% to 20%.

In the above or other embodiments, the pBH-2 engineered bacterium may be inoculated to the fermentation medium at an inoculum size of 15%.

In the above or other embodiments, the strain is cultured at an initial pH of 5.5 to 11, wherein the initial pH is adjusted with 0.5N to 1N sodium hydroxide after the preparation of the culture medium.

In the above or other embodiments, the strain is cultured at an initial pH of 6.5 to 8.0, alternatively 7.0.

In the above or other embodiments, the pH is adjusted to 4-9.5, optionally 6.5-8.0, during the culturing process with 0.5N-1N HCl and 0.5N-1N NaOH or 10 wt% -20 wt% ammonia.

In the above or other embodiments, the strain culture of the present application is performed under aerobic conditions.

In the above or other embodiments, the dissolved oxygen value is controlled to be 40% or more during the culture of the strain of the present application.

In the above or other embodiments, during the culture of the strain of the present application, the culture medium is filled in a flask with 10-20ml/100ml and rotated at 250-300rpm, and then inoculated in a fermenter with a larger aeration rate (for example, dissolved oxygen is controlled to be more than 40%), and the regulation and control are achieved by adjusting the rotation rate, aeration rate and aeration rate.

In the above or other embodiments, the method further comprises inactivating the strain fermentation broth at a temperature of 60 ℃ to 80 ℃, optionally 75 ℃; the inactivation time is 5min-20 min.

In the above or other embodiments, the method further comprises: performing solid-liquid separation on the inactivated strain fermentation liquor by using a ceramic membrane with the aperture of 0.1-1 mu m, washing with purified water (for example, washing for three times), collecting a ceramic membrane dialysate, concentrating the ceramic membrane dialysate by using an ultrafiltration membrane with the aperture of less than 3 ten thousand molecular weights, adding 5-30 wt% of salt into the concentrated solution, and performing spray drying

In the above or other embodiments, the inactivation temperature is 75 ℃.

In the above or other embodiments, the inactivation time is 10 min.

In the above or other embodiments, the fermentation culture process may be carried out under apparatus and conditions conventional or known in the art, e.g., may be carried out using shake flasks at rotational speeds conventional or known in the art; it can also be carried out in conventional fermenters, for example in 5L fermenters.

When a 5L fermentation tank is required in the culture process, an inoculation mode of feeding liquid seeds into the tank can be adopted. Carbon source supplementing substances, nitrogen source supplementing substances, inorganic substances and antibiotic substances can be fed in the fermentation process. The pH value can be adjusted to 6.5-8.0 by 0.5N-1N HCl and 0.5N-1N NaOH or 10% -20% ammonia water in the whole fermentation process. Larger aeration volumes (e.g., dissolved oxygen values controlled to above 40%) were used after inoculation.

Compared with the prior art, the invention comprises a method for preparing recombinant hirudin, which comprises the steps of inoculating pBH-2 engineering bacteria (preservation number: CGMCC No.0908) into a fermentation medium containing carbon source substances and nitrogen source substances, culturing at the temperature of 25-35 ℃ until the logarithmic growth phase is finished, and then heating to 35-45 ℃ for continuous culture for 3-16 hours.

The method of the application can ensure that the strain specifically expresses and secretes the recombinant hirudin with high yield by using pBH-2 engineering bacteria (preservation number: CGMCC No.0908) and continuously culturing after the logarithmic phase is finished and heating to 35-45 ℃.

Further, the method of the present application further improves the expression and secretion of recombinant hirudins by specifically selecting carbon source substances such as sucrose and/or nitrogen source substances such as yeast powder and peptone and/or controlling the warming process within 10 minutes (optionally within 5 minutes) and/or performing the second addition of antibiotic substances and/or adding sodium salts in the form of a stream, in particular by controlling the warming process within 10 minutes (optionally within 5 minutes) and/or performing the second addition of antibiotic substances in the form of a stream.

In this application, temperature rise is a necessary induction process, and the principle is as follows: the expression plasmid of the strain used in the present application contains lambda P_LA promoter which promotes the expression of hirudin by bacteria, and a repressor cI which can prevent lambda P_LPromoter, when the temperature is increased, the repressor protein cI is inactivated, and the lambda P is released_LRepression of the promoter begins expression of hirudin. At 30 ℃ in culture: the regulatory gene generates active cI repressor protein which can be combined with the operator gene to prevent the transcription of exogenous gene and the mass growth of bacteria; when the temperature is rapidly increased to 42 ℃: the cI repressor protein is inactivated and is not combined with the operator gene, and the promoter starts the high-efficiency transcription of the exogenous gene to synthesize a large amount of recombinant hirudin. If the temperature is not raised, hirudin is not expressed basically.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.

FIGS. 1A-D are graphs showing the cells after crystal violet staining during the incubation of the present application, wherein FIG. 1A is a graph showing the cells after crystal violet staining for 3.5 hours of culture and the measured hirudin yield is 0, FIG. 1B is a graph showing the cells after crystal violet staining for 12 hours of culture and the measured hirudin yield is 9000ATU/ml, FIG. 1C is a graph showing the cells after crystal violet staining for 14 hours and the measured hirudin yield is 12000ATU/ml, FIG. 1D is a graph showing the cells after crystal violet staining for 20 hours of culture and the measured hirudin yield is 6000 ATU/ml. As can be seen from FIGS. 1A-D, the bacterial staining became bright with increasing hirudin production, and almost peaked when the bacterial staining became bright to some extent and began to lighten, while the hirudin production decreased as the color continued to lighten.

FIG. 2 is an electrophoretic picture of example 2 of the present application, wherein the first sample from the left is a protein molecular weight marker, the second sample is a hirudin sample after purification according to the present application, and the third sample is a hirudin sample without purification of the fermentation broth according to the present application.

FIG. 3 is an HPLC chart of example 2 of the present application, wherein FIG. 3A is an HPLC chart of a hirudin control and FIG. 3B is an HPLC chart of the present application after purification.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.

In addition, it should be noted that, for the yields calculated in the following examples, it is known to those skilled in the art that factors affecting the yield include the amount of inoculation and the carbon to nitrogen ratio in addition to the species in the formulation. For example, in example 5, the inoculation amount reaches 20%, so that the nutrition may be insufficient in the later period; example 7, the inoculum size was 8%, so that the late overnutrition caused nutrient depression.

Example 1 preparation of the bacterial species to be inoculated

Inoculating pBH-2 engineering bacteria (preservation number: CGMCC No.0908) glycerol preservation bacteria into a 100mL triangular flask filled with 10mL of seed culture medium (shown in table 1), and performing shake culture at 30 ℃ and 270rpm for 2 hours to obtain the strain to be inoculated.

Table 1: seed culture medium

Wherein, in the present example, L in g/L is the volume of the final medium.

Wherein, the sucrose is purchased from Guangzhou chemical reagent factory, and the specification is AR grade; peptone was purchased from OXOID and specified AR grade; yeast powder was purchased from OXOID and specified as AR grade; sodium chloride was purchased from Guangzhou chemical reagent works and its specification was AR grade; AMP was purchased from Shiyao pharmaceutical Co., Ltd and specified in pharmaceutical grade.

EXAMPLE 2 Shake flask fermentation

The strain obtained in example 1 was inoculated into a fermentation medium by a method of directly inoculating a seed culture, wherein the strain aged 2h was inoculated in an amount of 5% in terms of an inoculum size of OD600 of 0.7. The loading amount is 10ml/100ml triangular flask, the culture temperature is 30 ℃, and the rotation speed is 270 rpm. Under aerobic conditions, fermenting after inoculation until the measured OD value of the bacterial liquid tends to be stable, namely the logarithmic growth phase is finished, the time for use is 3.5 hours, then raising the temperature to 40 ℃ within 5 minutes, and continuing culturing until the bacterial body is lightened in color after crystal violet staining in microscopic examination, and the time for use is 11.5 hours. The fermentation period was 15 hours. Finally, inactivating the strain fermentation liquor at 75 ℃ for 10 min.

Wherein the fermentation medium consists of and has a pH of 7.2: 41g/L of sucrose, 20g/L of peptone, 13g/L of yeast powder, 6.5g/L of ammonium chloride, 0.25g/L of magnesium sulfate, 0.6g/L of disodium hydrogen phosphate, 0.6g/L of monopotassium phosphate, 1.2g/L of sodium sulfate, 0.17g/L of sodium citrate, 8g/L of sodium chloride, 10.028g/L of vitamin B, 40g/L of ferrous sulfate as a trace element, 28g/L of aluminum sulfate, 6.1g/L of manganese sulfate, 4g/L of cobalt chloride, 0.95g/L of zinc chloride, 2.16g/L of sodium molybdate, 0.5mg/L of boric acid, 2.93g/L of copper sulfate, 30g/L of nickel nitrate and 7.2 of pH value). 0.1g/L of AMP as an antibiotic substance was added at the time of inoculation. In this and the following examples, L in g/L is the volume of the final fermentation medium.

Further, the manufacturer and specification information of each component in the medium are shown in Table 2 below.

TABLE 2

And (3) performing solid-liquid separation on the inactivated fermentation liquor by using a ceramic membrane (a manufacturer: Xiamen Sanda Membrane science and technology Limited, equipment model: MFM-C-770) with the pore diameter of 0.1-1 mu m, washing the fermentation liquor by using purified water for three times, collecting a ceramic membrane dialysate, concentrating the ceramic membrane dialysate by using an ultrafiltration membrane (the manufacturer: Xiamen Sanda membrane science and technology Limited, equipment model: UFM-84S-2) with the pore diameter of less than 3 ten thousand, and adding 10 wt% of sodium chloride salt into the concentrated solution to perform spray drying to obtain a hirudin crude product.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to check the purity during purification. Pure proteins should generally have only one band on SDS electrophoresis.

In addition to SDS-PAGE, simultaneous HPLC detection of the hirudin control (purchased from SIGMA, cat # 377853-2000U) and the purified hirudin samples of the present application was performed, and the results are shown in FIG. 3.

Wherein, the test conditions of the high performance liquid phase are as follows:

an experimental instrument: agilent1260

A chromatographic column: agilent Zorbax 300SB-C3 column

Mobile phase: water (with 0.1% TFA): 90% acetonitrile (containing 0.1% TFA)

And (3) an elution mode: gradient elution

Flow rate: 1.2ml/min

UV：214nm

Column temperature: 35 ℃ is carried out.

In addition, the fermentation yield of hirudin measured and calculated according to the following method is 10000ATU/ml, and the purity of hirudin obtained by the purification method is more than 50% of electrophoresis purity:

1 experimental principle:

the reaction speed of the hirudin and the thrombin is high, the reaction is in a direct proportion, and the combination ratio of the hirudin to the thrombin is 1: 1. an ATU hirudin is the amount of hirudin required to inactivate one unit of thrombin at 37 ℃.

2 experimental reagent and equipment:

2.1 Thrombin (from Zhongzhou)

2.2 bovine fibrinogen (from sigma)

2.3 physiological saline

2.40.05 mol/L Tris-HCl: 25ml of 0.05mol/L trihydroxymethyl aminomethane solution, about 40ml of 0.1mol/L hydrochloric acid and 0.8766g of sodium chloride are mixed, dissolved, the pH value is adjusted to 7.5, and then distilled water is added to the mixture to reach the constant volume of 100 ml.

2.5 constant temperature water bath

3, experimental method:

3.10.5% bovine fibrinogen solution: bovine fibrinogen was dissolved in 0.05mol/L Tris-HCl buffer to prepare a 0.5% bovine fibrinogen solution.

3.2 preparation of thrombin solution: the solution with the appropriate thrombin concentration is prepared by using normal saline.

3.3 taking 100ul of sample, adding 200ul of 0.5% bovine fibrinogen solution into a 1.5ml test tube, shaking up, placing in a 37 ℃ water bath, leaching for 5 minutes, and dripping 5ul of thrombin solution (dripping 1 time every 1 minute, 5ul each time, and gently shaking up while dripping) until coagulation.

4, calculating the result:

the activity of hirudin is as follows: (C1 XV 1)/(V2 Xdilution factor).

C1 concentration of Thrombin solution, NIH/ml

V1 volume of thrombin solution consumed in. mu.l

V2, the addition amount of the test solution is unit of mul.

The fermentation yields of hirudin mentioned in the following examples were measured and calculated according to the method of this example.

EXAMPLE 3 Shake flask fermentation

The strain obtained in example 1 was inoculated into a fermentation medium by a method of directly inoculating a seed culture, wherein the strain aged 2h was inoculated in an amount of 10% in terms of the inoculum size of OD600 of 0.7. The loading amount is 10ml/100ml triangular flask, the culture temperature is 30 ℃, and the rotation speed is 270 rpm. Under aerobic conditions, fermenting after inoculation until the measured OD value of the bacterial liquid tends to be stable, namely the logarithmic growth phase is finished, the time for use is 3.5 hours, then, heating to 37 ℃ within 10 minutes, and continuing culturing until the bacterial body is lighter in color after crystal violet staining in microscopic examination, and the time for use is 11.5 hours. The fermentation period was 15 hours. Finally, inactivating the strain fermentation liquor at 80 ℃ for 5 min.

Wherein the fermentation medium consists of and has a pH of 7.2: 10g/L of glycerol, 10g/L of glucose, 20g/L of yeast powder, 0.5g/L of ammonium nitrate, 1.0g/L of magnesium sulfate, 5g/L of disodium hydrogen phosphate, 3.0g/L of sodium sulfate, 0.3g/L of sodium citrate, 20g/L of sodium chloride, 11 mg/L of vitamin B, 40g/L of ferrous sulfate as a trace element, 28mg/L of aluminum sulfate, 6.1mg/L of manganese sulfate, 4mg/L of cobalt chloride, 0.95g/L of zinc chloride, 2.16g/L of sodium molybdate, 0.5mg/L of boric acid, 2.93g/L of copper sulfate and 32g/L of nickel nitrate. 0.5g/L of AMP as an antibiotic substance was added at the time of inoculation.

Wherein, the manufacturers and specification information of glycerol, glucose and ammonium nitrate are as follows, and the information of other substances is the same as the above example:

glycerol: food grade, Guangzhou City Baojili chemical Co., Ltd

Glucose: food grade, Jiancheng Fine chemical Co., Ltd, Wujiang City

Ammonium nitrate: guangzhou chemical reagent works, AR grade

Purification was performed according to the purification method of example 2.

The calculated fermentation yield of the hirudin is 11000ATU/ml, and the purity of the hirudin of the purification method is 50 percent of electrophoresis purity.

Example 4 Shake flask fermentation

The strain obtained in example 1 was inoculated into a fermentation medium by direct inoculation of a seed culture, wherein the strain aged 2h was inoculated in an amount of 15% in terms of the inoculum size of OD600 of 0.7. The loading amount is 20ml/100ml triangular flask, the culture temperature is 28 ℃, and the rotation speed is 250 rpm. Under aerobic conditions, fermenting after inoculation until the measured OD value of the bacterial liquid tends to be stable, namely the logarithmic growth phase is finished, the time for use is 3.5 hours, then raising the temperature to 37 ℃ within 10 minutes, and continuing culturing until the bacterial body is lighter in color after crystal violet staining in microscopic examination, and the time for use is 11.5 hours. The fermentation period was 15 hours. Finally, inactivating the strain fermentation liquor at 70 ℃ for 20 min.

Wherein the fermentation medium consists of and has a pH of 7.2: 10g/L glucose, 10g/L sucrose, 10g/L yeast powder, 10g/L tryptone, 0.5g/L ammonium chloride, 0.9g/L magnesium sulfate, 1g/L dipotassium phosphate, 5.0g/L sodium sulfate, 0.87g/L sodium citrate, 16g/L chloride, 10.05mg/L vitamin B, 40g/L trace elements of sulfuric acid, 28mg/L aluminum sulfate, 6.1mg/L manganese sulfate, 4mg/L cobalt chloride, 0.95g/L zinc chloride, 2.16g/L sodium molybdate, 0.5mg/L boric acid, 2.93g/L copper sulfate and 32g/L nickel nitrate. And adding 0.5g/L of antibiotic substance AMP0 during inoculation. The information on the manufacturers and specifications of the components is the same as in the above example.

Purification was performed according to the purification method of example 2.

The calculated fermentation yield of the hirudin is 12000ATU/ml, and the purity of the hirudin of the purification method is 50% of electrophoresis purity.

EXAMPLE 5 Shake flask fermentation

The strain obtained in example 1 was inoculated into a fermentation medium by a method of directly inoculating a seed culture, wherein the strain aged 2h was inoculated in an amount of 20% in terms of the inoculum size of OD600 of 0.7. The loading amount is 20ml/100ml triangular flask, the culture temperature is 35 ℃, and the rotation speed is 250 rpm. Under aerobic conditions, fermenting after inoculation until the measured OD value of the bacterial liquid tends to be stable, namely the logarithmic growth phase is finished, the time for use is 3.5 hours, then raising the temperature to 45 ℃ within 60 minutes, and continuing culturing until the bacterial body is lightened in color after crystal violet staining in microscopic examination, and the time for use is 11.5 hours. The fermentation period was 15 hours. Finally, inactivating the strain fermentation liquor at 60 ℃ for 20 min.

Wherein the fermentation medium consists of and has a pH of 7.2: 10g/L glucose, 10g/L sucrose, 10g/L yeast powder, 10g/L soybean peptone, 0.5g/L ammonium chloride, 0.9g/L magnesium sulfate, 1g/L dipotassium phosphate, 5.0g/L sodium sulfate, 0.87g/L sodium citrate, 16g/L sodium chloride, 10.05mg/L vitamin B, 40g/L ferrous sulfate, 28mg/L aluminum sulfate, 4mg/L cobalt chloride, 0.95g/L zinc chloride, 2.16g/L sodium molybdate, 0.5mg/L boric acid, 2.93g/L copper sulfate and 32g/L nickel nitrate. 0.5g/L of AMP as an antibiotic substance was added at the time of inoculation. The information on the manufacturers and specifications of the components is the same as in the previous examples.

Purification was performed according to the purification method of example 2.

The calculated fermentation yield of the hirudin is 9000ATU/mL, and the hirudin purity of the purification method is 50% of electrophoresis purity.

Example 65L tank fermentation

The procedure of example 1 was followed except that the cells were cultured at 30 ℃ and 270rpm for 2 hours to obtain first-stage seed cells, which were inoculated into a fermentation medium in a 5-L fermentor by a liquid seed-in-tank method in an amount of 15% in terms of the inoculum size of OD600 of 0.7.

Wherein the fermentation medium consists of and has a pH of 7.2: 41g/L of sucrose, 20g/L of peptone FP318 (Angel Yeast Co., Ltd.), 13g/L of yeast powder FM818 (Angel Yeast Co., Ltd.), 6.5g/L of ammonium chloride, 0.25g/L of magnesium sulfate, 0.4g/L of disodium hydrogen phosphate, 0.5g/L of potassium dihydrogen phosphate, 1.3g/L of sodium sulfate, 0.12g/L of sodium citrate, 8.2g/L of sodium chloride, 10.02g/L of vitamin B, 3mg/L of microelement-ferrous sulfate, 2.3mg/L of aluminum sulfate, 0.5mg/L of manganese sulfate, 0.3mg/L of cobalt chloride, 0.07mg/L of zinc chloride, 0.2mg/L of sodium molybdate, 0.04mg/L of boric acid, 0.24mg/L of copper sulfate and 2.5mg/L of nickel nitrate. Ampicillin AMP0.04g/L was added during inoculation. The information on the manufacturers and specifications of the components is the same as in the above example.

And controlling the pH value to be 6.5-8.0 by using 20 weight percent of ammonia water and 0.5N HCl in the fermentation process. After inoculation, the dissolved oxygen value is controlled to be more than 40 percent. An additional 35g/L of sucrose, 16g/L of peptone, 10g/L of yeast powder and 7g/L of the inorganic substance disodium hydrogen phosphate 0.1g/L, potassium dihydrogen phosphate 0.1g/L and sodium chloride 1g/L (total 2L of feed solution) were fed in at a rate of 2.6 mL/min by the feeding method 1 hour after inoculation. Ampicillin AMP, 0.02g/L in medium, was added in a stream at a rate of 0.033mg/L per minute based on the total volume of the medium solution prior to the end of the logarithmic growth phase. The fermentation period was 15 hours. Wherein, in the process of adding additional substances in a flowing manner, the temperature is kept at 30 ℃ for fermentation for 11 hours (the OD value of the bacteria liquid measured at the moment tends to be stable, namely the logarithmic growth phase is finished), then the temperature is raised to 42 ℃ within 5 minutes, and the culture is continued to be controlled until the bacterial body is lightened after crystal violet staining in microscopic examination and used for 4 hours. And (3) inactivating the fermentation liquor at 75 ℃ for 10 min.

Purification was performed according to the purification method of example 2.

The calculated fermentation yield of the hirudin is 12500ATU/mL, and the purity of the hirudin of the purification method is 50% of electrophoresis purity.

Example 75L tank fermentation

The procedure of example 1 was followed except that the cells were cultured at 30 ℃ and 270rpm for 2 hours to obtain first-stage seed cells, which were inoculated into a fermentation medium in a 5-L fermentor by a liquid seed-in-tank method at an inoculum size of 8% in terms of OD600 of 0.7.

Wherein the fermentation medium consists of and has a pH of 7.2: 80g/L of sucrose, 0.7g/L of peptone FP31865g/L-OXOID, 12g/L of ammonium chloride, 0.7g/L of magnesium sulfate, 0.6g/L of disodium hydrogen phosphate, 0.6g/L of monopotassium phosphate, 1.7g/L of sodium sulfate, 0.17g/L of sodium citrate, 14g/L of sodium chloride, 0.028g/L of vitamin B, 3mg/L of microelement ferrous sulfate, 2.3mg/L of aluminum sulfate, 0.5mg/L of manganese sulfate, 0.3mg/L of cobalt chloride, 0.07mg/L of zinc chloride, 0.2mg/L of sodium molybdate, 0.04mg/L of boric acid, 0.24mg/L of copper sulfate and 2.5mg/L of nickel nitrate. Ampicillin AMP0.1g/L was added during inoculation. The information on the manufacturers and specifications of the components is the same as in the previous examples.

And controlling the pH value to be 6.5-8.0 by using 1N NaOH and 1N HCl in the fermentation process. After inoculation, the dissolved oxygen value is controlled to be more than 60 percent. An additional 10g/L of sucrose and 30g/L of peptone (2L of make-up liquid in total) were added at a rate of 2.6ml per minute by fed-batch method 40 minutes after inoculation. Ampicillin AMP, 0.1g/L of medium, was added in one portion after 5 hours of fermentation. The fermentation period was 15 hours. Wherein, in the process of adding additional substances in a flowing manner, the temperature is kept at 35 ℃ for fermentation for 11 hours (the OD value of the bacteria liquid measured at the moment tends to be stable, namely the logarithmic growth phase is finished), then the temperature is controlled to be increased from 35 ℃ to 40 ℃ within 30min, and the fermentation is continued until the bacterial body is lightened after crystal violet staining in microscopic examination and the bacterial body is used for 4 hours. And (3) inactivating the fermentation liquor at 80 ℃ for 5 min.

Purification was performed according to the purification method of example 2.

The calculated fermentation yield of the hirudin is 8000ATU/ml, and the purity of the hirudin of the purification method is 50% of electrophoresis purity.

Example 85L tank fermentation

The first-stage seed cells were obtained by culturing at 30 ℃ and 270rpm for 2 hours according to the method of example 1, inoculated with 10% of the inoculum size in terms of OD600 of 0.7 by the liquid seed tank method, and inoculated into the fermentation medium in a 5-L fermentor.

Wherein the fermentation medium consists of and has a pH of 7.2: 80g/L of sucrose, 65g/L of yeast powder, 12g/L of ammonium nitrate, 0.7g/L of magnesium sulfate, 0.6g/L of disodium hydrogen phosphate, 0.6g/L of monopotassium phosphate, 1.7g/L of sodium sulfate, 0.17g/L of sodium citrate, 14g/L of sodium chloride, 10.028g/L of vitamin B, 3mg/L of trace element ferrous sulfate, 2.3mg/L of aluminum sulfate, 0.5mg/L of manganese sulfate, 0.3mg/L of cobalt chloride, 0.07mg/L of zinc chloride, 0.2mg/L of sodium molybdate, 0.04mg/L of boric acid, 0.24mg/L of copper sulfate and 2.5mg/L of nickel nitrate. Ampicillin AMP0.04g/L was added at the time of inoculation. The information on the manufacturers and specifications of the components is the same as in the previous examples.

And controlling the pH value to be 6.5-8.0 by using 0.5N NaOH and 0.5N HCl in the fermentation process. Controlling the dissolved oxygen value to be more than 40% after inoculation. After inoculation for 1 hour by feeding, an additional 15g/L of sucrose and 30g/L of peptone were added at a rate of 2.6ml per minute. After 8 hours of fermentation, ampicillin AMP was added in an amount of 0.05g/L based on the culture medium. The fermentation period was 15 hours. Wherein, in the process of adding additional substances in a flowing manner, the temperature is kept at 30 ℃ for fermentation for 11 hours (the OD value of the bacteria liquid measured at the moment tends to be stable, namely the logarithmic growth phase is finished), the temperature is controlled to be increased from 35 ℃ to 45 ℃ within 10 minutes, the fermentation is continued until the bacterial body is lightened after crystal violet staining in microscopic examination, and the time is 4 hours. And (3) inactivating the fermentation liquor at the inactivation temperature of 60 ℃ for 20 min.

Purification was performed according to the purification method of example 2.

The calculated fermentation yield of the hirudin is 8500ATU/ml, and the purity of the hirudin of the purification method is 50% of electrophoresis purity.

Example 9

Recombinant hirudin was prepared and purified as in example 2, except that the fermentation medium was changed to a medium consisting of 41g/L sucrose, 20g/L peptone and 1L water, pH7.0 and inoculum size 1%.

The calculated fermentation yield of the hirudin is 6000ATU/ml, and the purity of the hirudin of the purification method is 50% of electrophoresis purity.

Example 10

Recombinant hirudin was prepared and purified by the method of example 9, except that sucrose was replaced with fructose 41g/L and peptone was replaced with ammonium chloride 20g/L, the initial culture temperature was adjusted to 25 ℃ and the sterilization conditions were adjusted to 60 ℃ for 20 minutes.

The calculated fermentation yield of the hirudin is 5500ATU/ml, and the purity of the hirudin obtained by the purification method is 50% of electrophoresis purity.

Example 11

Recombinant hirudin was prepared and purified as in example 6, except that the fermentation medium contained no disodium hydrogen phosphate 0.4g/L, sodium sulfate 1.2g/L, sodium citrate 0.17g/L, sodium chloride 8 g/L.

The calculated fermentation yield of hirudin is as follows: 11500ATU/ml, and the purity of hirudin obtained by the purification method is more than 50% of electrophoresis purity.

Example 12

Recombinant hirudin was prepared and purified as in example 6, except that the fermentation medium contained only sucrose 41g/L, peptone 20g/L, yeast powder 13g/L, ammonium chloride 6.5 g/L.

The calculated fermentation yield of the hirudin is 10500ATU/ml, and the purity of the hirudin obtained by the purification method is more than 50% of electrophoresis purity.

Example 13

Recombinant hirudin was prepared and purified as in example 6, except that the second addition of AMP was a one-shot addition after 1 hour fermentation.

The calculated fermentation yield of the hirudin is 10600ATU/ml, and the purity of the hirudin obtained by the purification method is 50% of electrophoresis purity.

Example 14

Recombinant hirudin was prepared and purified as in example 7, except that an additional 10g/L of sucrose and 30g/L of peptone added in one portion during the culture had been added to the fermentation medium at the same time as inoculation.

The calculated fermentation yield of the hirudin is 6500ATU/ml, and the purity of the hirudin obtained by the purification method is 50% of electrophoresis purity.

Example 15

Recombinant hirudin was prepared and purified according to the method of example 6, except that the inoculum size was 15%, sucrose was 41g/L, peptone FP318 (Qi Yeast Co., Ltd.) was 22g/L, and yeast powder FM818 (Angel Yeast Co., Ltd.) was 15.3 g/L.

The calculated fermentation yield of the hirudin is 13500ATU/ml, and the purity of the hirudin obtained by the purification method is 50% of electrophoresis purity.

Comparative example 1

Recombinant hirudin was prepared and purified as in example 2, except that it was cultured at 25 ℃ to the end of the logarithmic growth phase and then warmed to 32 ℃.

The calculated fermentation yield of the hirudin is 4500ATU/ml, and the purity of the hirudin obtained by the purification method is 50% of electrophoresis purity.

Comparative example 2

Recombinant hirudin was prepared and purified as in example 2, except that it was cultured at 20 ℃ to the end of the logarithmic growth phase and then warmed to 50 ℃.

The calculated fermentation yield of the hirudin is 500ATU/ml, and the purity of the hirudin obtained by the purification method is 50% of electrophoresis purity.

Comparative example 3

According to the method of patent No. ZL 03109244.6: inoculating the strain into 1L liquid seed culture medium at a ratio of 2%, and culturing at 37 deg.C and 190r/min for 12 hr to obtain seed liquid. Seed culture medium: peptone 1%, yeast extract 0.5%, sodium chloride 1%, ampicillin 100ug/m1, pH 7.0; fermentation medium: peptone 1%, yeast extract 0.5%, sodium glutamate 4%, malt extract 12%, ampicillin 100ug/m1, pH6.5, culturing at 37 deg.C, ventilation 4.48L/min, dissolved oxygen controlled at 12%, and cascade control of dissolved oxygen and rotation speed. And when the pH value of the fermentation liquor naturally falls below 6, adding acid and alkali to control the pH value to be 5.7-5.9, and when the pH value of the fermentation liquor rises above 8, finishing the fermentation culture.

The hirudin yields tested and calculated in the above examples are summarized in table 1 below:

TABLE 1

Although the embodiments of the present invention have been described above, the above description is only for the convenience of understanding the present invention, and is not intended to limit the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for producing recombinant hirudin, comprising: the pBH-2 engineering bacteria are preserved as follows: CGMCC No0908 is inoculated into a fermentation medium containing carbon source substances and nitrogen source substances, the fermentation medium is cultured at the temperature of 28-35 ℃ under aerobic conditions until the logarithmic growth phase is finished, and then the temperature is raised to 37-45 ℃ within 1 hour, the culture is continued until the hirudin yield reaches the peak value, wherein the fermentation medium further contains phosphate, magnesium salt, sodium salt or a combination thereof, and the fermentation medium further contains one or more of iron salt, manganese salt, aluminum salt, cobalt salt, zinc salt, copper salt, nickel salt, boric acid and vitamin B1;

wherein the fermentation medium further contains an antibiotic substance, and the antibiotic substance is ampicillin; adding the antibiotic substance into a fermentation medium by two times, wherein the first addition is 0.04g/L during inoculation, and the second addition is before the end of the logarithmic phase of the thallus culture; the second addition of antibiotic substance was made in the form of a feed addition of 0.033mg/L antibiotic substance per minute in relation to the total volume of the medium solution, at a rate of 0.02g/L medium.

2. The method according to claim 1, wherein the cultivation is continued for 3-16 hours or more so that the production of hirudin reaches a peak.

3. The method according to claim 2, wherein the culture is continued at a temperature of 30 ℃ to the end of the logarithmic growth phase, and then the temperature is raised to 42 ℃ for 3 to 16 hours or more.

4. The method of claim 1, wherein the warming process is completed in 10 minutes.

5. The method of claim 4, wherein the warming process is completed in 5 minutes.

6. The method of claim 1, wherein the carbon source material is selected from one or more of glucose, sucrose, maltose, starch, dextrin, lactose, fructose, and glycerol.

7. The method of claim 6, wherein the carbon source material is sucrose, or the carbon source material is sucrose in combination with one or more selected from the group consisting of glucose, maltose, starch, dextrin, lactose, fructose, and glycerol.

8. The method of claim 1, wherein the nitrogen source material is selected from one or more of corn steep liquor, yeast powder, peptone, beef extract, yeast extract, ammonium nitrate, ammonium chloride, and ammonium sulfate.

9. The method of claim 8, wherein the nitrogen source substance is yeast powder, peptone, or both yeast powder and peptone, or the nitrogen source substance is yeast powder, peptone, or a combination of both yeast powder and peptone and one or more selected from corn steep liquor, beef extract, yeast extract, ammonium nitrate, ammonium chloride, and ammonium sulfate.

10. The method of claim 1, wherein the weight ratio of the carbon source material to the nitrogen source material in the fermentation medium is 0.2-40: 1.

11. The method of claim 10, wherein the weight ratio of the carbon source material to the nitrogen source material in the fermentation medium is 1.1: 1.

12. The method of claim 1, wherein the phosphate is selected from one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate; the magnesium salt is magnesium sulfate or magnesium chloride; the sodium salt is selected from one or more of sodium chloride, sodium citrate, sodium sulfate and sodium acetate.

13. The method of claim 1, wherein the pBH-2 engineered bacterial deposit number is between 1% and 20%: CGMCC No0908 is inoculated to the fermentation medium.

14. The method of claim 13, wherein the inoculation is at 15%.

15. The method according to claim 1, wherein the strain is cultured at an initial pH of 5.5 to 11.

16. The method according to claim 15, wherein the strain culture initial pH is 6.5-8.0.

17. The method of claim 1, further comprising the steps of:

inactivating the strain fermentation liquor at the temperature of 60-80 ℃; inactivating for 5min-20 min;

and (3) performing solid-liquid separation on the inactivated strain fermentation liquor by using a ceramic membrane with the aperture of 0.1-1 mu m, washing with purified water, collecting a ceramic membrane dialysate, concentrating the ceramic membrane dialysate by using an ultrafiltration membrane with the aperture of less than 3 ten thousand molecular weights, and adding 5-30 wt% of salt into the concentrated solution for spray drying.

18. The method of claim 17, further comprising the steps of: the inactivation temperature is 75 ℃; inactivating for 10 min;

19. The process according to claim 1, wherein the components of the fermentation medium are added in two portions, wherein the first portion is added before inoculation and the second portion is added in a fed-batch manner after inoculation and is completed within 11 hours.