阅读说明：本技术 一种重组水蛭素的制备方法 (Preparation method of recombinant hirudin ) 是由 杨爱岗 吴扬 于 2019-11-15 设计创作，主要内容包括：本发明涉及发酵的技术领域,更具体地,本发明提供了一种重组水蛭素的制备方法。本发明的第一方面提供一种重组水蛭素的制备方法,包括：将种子液置于初始培养基中,当DO值大于55％时,于初始培养基中滴加补料培养基；其中,初始培养基的制备过程包括步骤：将糖溶液进行灭菌处理,并添加于发酵罐中；将微量金属溶液过滤除菌,并添加于发酵罐中；将维生素溶液过滤除菌,并添加于发酵罐中,即得初始培养基。(The invention relates to the technical field of fermentation, and particularly provides a preparation method of recombinant hirudin. In a first aspect, the present invention provides a method for preparing recombinant hirudin, comprising: placing the seed solution in an initial culture medium, and dropwise adding a feed culture medium into the initial culture medium when the DO value is greater than 55%; wherein the preparation process of the initial culture medium comprises the following steps: sterilizing the sugar solution, and adding into a fermentation tank; filtering the trace metal solution for sterilization, and adding the solution into a fermentation tank; filtering the vitamin solution for sterilization, and adding into a fermentation tank to obtain the initial culture medium.)

1. A preparation method of recombinant hirudin is characterized in that the preparation process of the recombinant hirudin comprises the following steps: placing the seed solution in an initial culture medium, and dropwise adding a feed supplement culture medium when the DO value is more than 55%; wherein, the preparation raw materials of the initial culture medium comprise sugar solution, trace metal solution and vitamin solution.

2. The method for preparing recombinant hirudin according to claim 1, wherein the sugar solution comprises an amino-free yeast nitrogen source, a carbon source and an amino acid solution.

3. The method for producing recombinant hirudins according to claim 2, wherein the amino acid solution comprises at least one of L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

4. The method for preparing recombinant hirudin according to claim 1, wherein the trace metal solution comprises at least one of zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt, copper salt.

5. The method for preparing recombinant hirudin according to claim 1, wherein the vitamin solution comprises at least one of biotin, nicotinic acid, thiamine hydrochloride, vitamin B2.

6. The process for preparing recombinant hirudin according to claim 5, wherein the vitamin solution further comprises inositol and pyridoxal hydrochloride.

7. The process for preparing recombinant hirudin according to any one of claims 1 to 6, wherein the pH of the initial medium is controlled to 4.5 to 5.5 by adding dropwise ammonia water during the preparation of the recombinant hirudin.

8. The process for preparing recombinant hirudins according to any one of claims 1 to 6, wherein the inoculation amount of the seed liquid is 10 to 15%.

9. The process for the preparation of recombinant hirudins according to any one of claims 1 to 6, wherein the feed medium comprises a solution of trace metals, a solution of vitamins and a carbon source.

10. A recombinant hirudin produced by the method for producing a recombinant hirudin according to any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of fermentation, and particularly provides a preparation method of recombinant hirudin.

Background

The hirudin has relatively independent inhibition effect on thrombin, does not need other coagulation factors, has selectivity and specificity on thrombin, and can directly inhibit the thrombin from blocking the coagulation function. Hirudin is a natural inhibitor of thrombin, has a single target, and is relatively safe and effective in the existing known anticoagulant drugs. Hirudin can prevent severe blood stasis caused by thrombin-catalyzed coagulation factor activation and platelet reaction. Hirudin is a promising drug and has significant therapeutic effects in many forms of thrombotic diseases, such as venous thrombosis, disseminated blood vessel coagulation, prevention of arterial thrombosis after surgical operations, prevention of thrombosis after thrombolysis or vascular remodeling, and the like.

However, the natural hirudin has a limited yield, and each leech only contains 20 mu g of hirudin, which cannot meet the requirements of clinical application, so the research of producing recombinant hirudin by adopting the biological engineering technology, in particular to a method for producing high-yield recombinant hirudin, is very important.

The expression level of the recombinant hirudin prepared at present is low, and the problem of preparation of high-activity hirudin cannot be solved well, so that the invention provides a novel preparation process of the recombinant hirudin.

Disclosure of Invention

In order to solve the above technical problems, a first aspect of the present invention provides a method for preparing recombinant hirudin, wherein the preparation process of the recombinant hirudin comprises: placing the seed solution in an initial culture medium, and dropwise adding a feed culture medium into the initial culture medium when the DO value is greater than 55%; wherein, the preparation raw materials of the initial culture medium comprise sugar solution, trace metal solution and vitamin solution.

In a preferred embodiment of the present invention, the sugar solution comprises an amino-free yeast nitrogen source, a carbon source and an amino acid solution.

In a preferred embodiment of the present invention, the amino acid solution comprises at least one of L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

As a preferable technical solution of the present invention, the trace metal solution includes at least one of zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt, and copper salt.

In a preferred embodiment of the present invention, the vitamin solution includes at least one of biotin, nicotinic acid, thiamine hydrochloride, and vitamin B2.

As a preferred technical scheme of the invention, in the preparation process of the recombinant hirudin, the pH of an initial culture medium is controlled to be 4.5-5.5 by dropwise adding ammonia water.

As a preferred technical scheme of the invention, the temperature of the initial culture medium is 25-40 ℃.

As a preferable technical scheme of the invention, the inoculation amount of the seed liquid is 10-15%.

As a preferable technical scheme of the invention, the feed medium comprises a trace metal solution, a vitamin solution and a carbon source.

In a second aspect, the invention provides a recombinant hirudin prepared according to the recombinant hirudin preparation method.

Compared with the prior art, the preparation method of the recombinant hirudin provided by the invention can obtain a large amount of secreted thalli and improve the expression of active products, thereby improving the production efficiency of the hirudin.

Detailed Description

Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

The technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, and the scope of protection is not limited thereto.

The words "preferred", "preferably", "more preferred", and the like, in the present invention, refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention. The sources of components not mentioned in the present invention are all commercially available.

In a first aspect, the present invention provides a method for preparing recombinant hirudin, comprising: placing the seed solution in an initial culture medium, and dropwise adding a feed culture medium into the initial culture medium when the DO value is greater than 55%; wherein, the preparation raw materials of the initial culture medium comprise sugar solution, trace metal solution and vitamin solution.

The applicant has found that, surprisingly, the addition of a feed medium when the DO value in the system is greater than 55% is beneficial to improving the expression activity of the obtained hirudin, while the addition of the feed medium too early or too late has negative effects, which can reduce the growth of the thalli or the expression amount of the product, and may affect the expression effect of the final effective hirudin due to different absorption capacities of the thalli on the feed medium under different DO values.

In a preferred embodiment, the recombinant hirudin is prepared by a process comprising: placing the seed liquid in an initial culture medium, introducing sterile air, and keeping the DO value to be more than 40%; when the DO value is more than 55%, the feeding medium is added dropwise in the initial medium in a feeding mode.

In one embodiment, the ventilation amount of the sterile air is 3-7L/min; preferably, the ventilation of sterile air is 5L/min.

In one embodiment, the temperature of the initial medium is 25 to 40 ℃; preferably, the temperature of the initial culture medium is 27-35 ℃; more preferably, the temperature of the initial medium is 30 ℃.

In one embodiment, the recombinant hirudin is prepared in a manner and under conditions conventional or well known in the art.

In one embodiment, the recombinant hirudin is prepared in a shake flask at a rotation speed of 300-1000 r/min; preferably, the rotation speed is 650 r/min.

In one embodiment, the recombinant hirudin is prepared in a 5L fermenter.

In one embodiment, the pH of the initial culture medium is controlled to be 4.5-5.5 by dropwise adding ammonia water; preferably, the pH of the initial medium is controlled to 5.0 by dropwise addition of ammonia water.

In one embodiment, the concentration of the ammonia water is 20-30 wt%; preferably, the concentration of ammonia is 25 wt%.

In one embodiment, the inoculation amount of the seed solution is 10-15%; preferably, the inoculation amount of the seed liquid is 11-13%.

In one embodiment, the seed liquid is prepared by: inoculating engineering bacteria glycerol preservation bacteria into a bottle filled with a seed culture medium, and culturing to obtain a seed solution to be inoculated.

Preferably, the preparation method of the seed solution comprises the steps of inoculating ① engineering bacteria glycerol preservation bacteria into a 100mL triangular flask filled with 5mL seed culture medium, carrying out shake culture at 30 ℃ and 220rpm for 24 hours, transferring ② engineering bacteria glycerol preservation bacteria into a 250mL triangular flask filled with 50mL seed culture medium, carrying out shake culture at 30 ℃ and 220rpm for 24 hours, transferring ③ engineering bacteria glycerol preservation bacteria into a 1000mL triangular flask filled with 250mL seed culture medium (two flasks in total), and carrying out shake culture at 30 ℃ and 220rpm for 12 hours to obtain the seed solution to be inoculated.

In one embodiment, the seed medium comprises glucose, YNB, amino acids, and water.

In one embodiment, the glucose content in the seed culture medium is 10-30 g/L of the total volume of the solution of the initial culture medium; preferably, the sucrose content in the seed medium is 20g/L of the total volume of the solution of the initial medium.

In one embodiment, the weight ratio of glucose, YNB and amino acid is (10-30): (5-9): (1-2); preferably, the weight ratio of glucose, YNB and amino acid is 20: 7: 1.5.

in one embodiment, the water content in the seed culture medium is 400-550 mL; preferably, the water content in the seed medium is 480 mL.

In one embodiment, the amino acid is selected from the group consisting of any one or more of L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine; preferably, the amino acids are L-arginine, L-histidine and L-tryptophan; more preferably, the weight ratio of the amino acid to the L-arginine, the L-histidine and the L-tryptophan is 1: (0.8-1.2): (0.8 to 1.2); more preferably, the weight ratio of L-arginine, L-histidine and L-tryptophan is 1: 1: 1.

in one embodiment, the inoculated engineered bacteria is a strain of INVSC1 Saccharomyces cerevisiae.

In one embodiment, the sugar solution comprises an amino-free yeast nitrogen source (YNB), a carbon source, and an amino acid solution; preferably, the sugar solution further comprises at least one of ammonium salt, phosphate, magnesium salt, zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt and copper salt; more preferably, the sugar solution further comprises an antifoaming agent.

In one embodiment, the sugar solution comprises an amino-free yeast nitrogen source (YNB), a carbon source, an amino acid solution, an ammonium salt, a phosphate salt, a magnesium salt, and an antifoaming agent.

In one embodiment, the content of the nitrogen source (YNB) without amino yeast is 4 to 10g/L of the total volume of the solution of the initial medium; preferably, the content of the nitrogen source (YNB) without amino yeast is 6-8 g/L of the total volume of the solution of the initial culture medium; more preferably, the content of nitrogen source (YNB) without amino yeast is 7g/L of the total volume of the solution of the initial medium.

In one embodiment, the carbon source content is 20-50 g/L of the total volume of the solution of the initial culture medium; preferably, the content of the carbon source is 30-40 g/L of the total volume of the solution of the initial culture medium; more preferably, the carbon source content is 35g/L of the total volume of the solution of the initial medium.

In one embodiment, the amino acid solution is 10-40 mL/L of the total volume of the solution of the initial culture medium; preferably, the content of the amino acid solution is 15-30 mL/L of the total volume of the solution of the initial culture medium; more preferably, the amino acid solution content is 20mL/L of the total volume of the solution of the starting medium.

In one embodiment, the weight ratio of ammonium salt, phosphate salt and magnesium salt in the sugar solution is (1-1.5): (1.5-3): 1; preferably, the weight ratio of ammonium salt, phosphate salt and magnesium salt in the sugar solution is 1.3: 2.5: 1.

in one embodiment, the content of ammonium salt in the sugar solution is 10-30 g/L of the total volume of the solution of the initial culture medium; preferably, the ammonium salt content of the sugar solution is 15g/L of the total volume of the solution of the starting medium.

In one embodiment, the content of the antifoaming agent is 0.1-1 mL/L of the total volume of the solution of the initial medium; preferably, the antifoaming agent content is 0.5mL/L of the total volume of the solution of the starting medium.

In one embodiment, the defoamer is a silicone-based defoamer and/or a polyether-based defoamer; preferably, the antifoaming agent is tween 80.

In one embodiment, the amino acid solution comprises at least one of L-arginine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine; preferably, the amino acid solution comprises L-histidine, L-leucine, L-lysine and L-methionine; further preferably, the weight ratio of L-histidine, L-leucine, L-lysine and L-methionine is 1: (2-4): (1-3): (0.5 to 1.5); more preferably, the weight ratio of L-histidine, L-leucine, L-lysine and L-methionine is 1: 3: 1.5: 1.

in one embodiment, the L-histidine content is 1-3 g/L of a 100X amino acid solution; preferably, the L-histidine content is 2g/L of the amino acid solution.

In one embodiment, the content of the trace metal solution in the preparation raw material of the initial culture medium is 5-20 mL/L of the total volume of the solution of the initial culture medium; preferably, the content of the trace metal solution in the starting material for the preparation of the starting medium is 10mL/L of the total volume of the solution of the starting medium.

In one embodiment, the content of the vitamin solution in the raw material for preparing the initial culture medium is 5-20 mL/L of the total volume of the solution of the initial culture medium; preferably, the content of the vitamin solution in the raw materials for preparing the initial culture medium is 10-15 mL/L of the total volume of the solution of the initial culture medium; the content of the vitamin solution in the starting material for the preparation of the starting medium was 12mL/L of the total volume of the solution of the starting medium.

In one embodiment, the feed medium comprises a trace metal solution, a vitamin solution, and a carbon source; preferably, the feed medium further comprises at least one of phosphate, ammonium salt, phosphate, magnesium salt, zinc salt, manganese salt, cobalt salt, iron salt, calcium salt, sodium salt, copper salt and potassium salt.

In one embodiment, the feed medium comprises a trace metal solution, a vitamin solution, a carbon source, phosphate, magnesium salt, and potassium salt.

The applicant has found that, surprisingly, the hirudin obtained by the preparation method of the invention has a high activity under the synergistic effect of a specific initial culture medium and a supplemented culture medium, and the yield of the hirudin which is expressed effectively finally is high, probably because the hirudin is beneficial to the sufficiency of nutrient components in different stages of the thallus fermentation process and the high yield of active substances, and the decomposition of the newly-generated hirudin is avoided.

In one embodiment, the content of the carbon source in the feed medium is 300-500 g/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the carbon source in the feed culture medium is 350-450 g/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the carbon source content in the feed medium is 400g/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the content of the vitamin solution in the feed medium is 5-20 mL/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the vitamin solution in the feed culture medium is 10-15 mL/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the content of the vitamin solution in the feed medium is 12mL/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the content of the trace metal solution in the feed medium is 3-15 mL/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the trace metal solution in the feed culture medium is 8-12 mL/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the content of the trace metal solution in the feed medium is 10mL/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the content of potassium salt in the feed medium is 1.5-6 g/L of the total volume of the solution of the initial medium and the solution of the feed medium; preferably, the content of the potassium salt in the feed culture medium is 3-5 g/L of the total volume of the solution of the initial culture medium and the solution of the feed culture medium; more preferably, the potassium salt content in the feed medium is 3.5g/L of the total volume of the solution of the initial medium and the solution of the feed medium.

In one embodiment, the potassium salt, magnesium salt, and phosphate are present in the feed medium in a weight ratio of 1: (1-3): (2-5); preferably, the weight ratio of potassium salt, magnesium salt and phosphate in the feed medium is 1: 1.5: 3.5.

in one embodiment, the carbon source is selected from any one or a combination of more of glucose, sucrose, galactose, starch, maltose, lactose.

In one embodiment, the trace metal solution comprises at least one of a zinc salt, a manganese salt, a cobalt salt, an iron salt, a calcium salt, a sodium salt, a copper salt, and a potassium salt; preferably, the trace metal solution includes sodium, zinc, calcium and manganese salts; further preferably, the weight ratio of the sodium salt, the zinc salt, the calcium salt and the manganese salt is 1: (50-90): (20-40): (3-10); more preferably, the weight ratio of the sodium salt, zinc salt, calcium salt and manganese salt is 1: 80: 30: 5.

in one embodiment, the content of sodium salt in the trace metal solution is 0.05-0.2 g/L of the trace metal solution; preferably, the content of the sodium salt in the trace metal solution is 0.1g/L of the trace metal solution.

In one embodiment, the trace metal solution further comprises 0.5M EDTA; further preferably, the content of EDTA is 70-90 mL/L of the trace metal solution; more preferably, the EDTA content is 80mL/L of the trace metal solution.

In one embodiment, zinc salts that may be mentioned include, but are not limited to: zinc sulfate, zinc chloride; manganese salts that may be mentioned include, but are not limited to: manganese sulfate, manganese dichloride and manganese glycinate; cobalt salts that may be mentioned include, but are not limited to: cobalt dichloride; iron salts that may be mentioned include, but are not limited to: ferrous sulfate, ferrous chloride; calcium salts that may be mentioned include, but are not limited to: calcium chloride, calcium glycinate; sodium salts that may be mentioned include, but are not limited to: sodium chloride, sodium molybdate; copper salts that may be mentioned include, but are not limited to: anhydrous copper sulfate, copper glycinate; illustrative potassium salts include, but are not limited to: potassium sulfate, potassium chloride, potassium molybdate; ammonium salts that may be mentioned include, but are not limited to: ammonium nitrate, ammonium sulfate, ammonium chloride; exemplary phosphates include, but are not limited to: potassium dihydrogen phosphate, sodium dihydrogen phosphate, and calcium dihydrogen phosphate.

In one embodiment, the vitamin solution comprises at least one of biotin, niacin, thiamine hydrochloride, vitamin B2; preferably, the vitamin solution comprises biotin, niacin, and thiamine hydrochloride; further preferably, the weight ratio of biotin, nicotinic acid and thiamine hydrochloride is 1: (15-30): (10-30); more preferably, the weight ratio of biotin, nicotinic acid and thiamine hydrochloride is 1: 22: 20.

in one embodiment, the content of biotin is 0.03-0.1 g/L of the vitamin solution; preferably, the biotin content is 0.05g/L of the vitamin solution.

In one embodiment, the vitamin solution further comprises inositol and pyridoxal hydrochloride, preferably, the weight ratio of niacin, inositol and pyridoxal hydrochloride is 1: (0.5-1.5): (0.5 to 1.5); more preferably, the weight ratio of nicotinic acid, inositol and pyridoxal hydrochloride is 1: 1: 1.

the activity of the obtained hirudin can be further optimized by adding inositol and pyridoxal hydrochloride into the system in the preparation process of the applicant, and the inositol and the pyridoxal hydrochloride can promote the growth of thalli and avoid the decomposition of newly generated thalli, so that the expression activity of the obtained hirudin is improved.

In one embodiment, the initial medium is prepared by a process comprising the steps of:

(1) sterilizing the sugar solution, and adding into a fermentation tank;

(2) filtering the trace metal solution for sterilization, and adding the solution into a fermentation tank;

(3) filtering the vitamin solution for sterilization, and adding into a fermentation tank to obtain the initial culture medium.

In a preferred embodiment, the preparation of the initial medium comprises the following steps:

(1) sterilizing the sugar solution, and adding into a fermentation tank by feeding;

(2) filtering and sterilizing the trace metal solution by a sterile filter of 0.22 mu m, and adding the solution into a fermentation tank;

(3) filtering vitamin solution with 0.22 μm sterile filter, sterilizing, and adding into fermentation tank to obtain initial culture medium.

In a second aspect, the invention provides a recombinant hirudin prepared according to the recombinant hirudin preparation method.