CN109957008B

CN109957008B - Extraction and purification method of hirudin mutant and application thereof

Info

Publication number: CN109957008B
Application number: CN201910301614.6A
Authority: CN
Inventors: 陈杰鹏; 段丽丽; 陈鸿锐; 胡留松; 陈煜藩; 洪琳; 许志锴; 叶红林; 纪烨瑜; 蔡春丽
Original assignee: Sungen Bioscience Co ltd
Current assignee: Sungen Bioscience Co ltd
Priority date: 2019-04-16
Filing date: 2019-04-16
Publication date: 2021-12-24
Anticipated expiration: 2039-04-16
Also published as: CN109957008A

Abstract

The application discloses a method for extracting and purifying a hirudin mutant HV2-Lys47 from fermentation liquor of a hirudin mutant HV2-Lys47 produced by fermenting escherichia coli, wherein the method adopts a membrane technology and a primary column chromatography technology, so that the high-purity hirudin mutant HV2-Lys47 is obtained. The application also discloses application of the hirudin mutant HV2-Lys47 prepared in an anticoagulated blood collection tube and the anticoagulated blood collection tube containing the hirudin mutant HV2-Lys 47.

Description

Extraction and purification method of hirudin mutant and application thereof

Technical Field

The application relates to a biological engineering technology, in particular to a method for extracting and purifying a hirudin mutant HV2-Lys47 from fermentation liquor of a hirudin mutant HV2-Lys47(SEQ ID NO.:1) produced by fermenting escherichia coli, and the purified hirudin mutant HV2-Lys47 obtained by the method.

The application also relates to a biomedical device, in particular to application of the hirudin mutant HV2-Lys47 purified by the method in an anticoagulant blood collection tube.

Background

Natural Hirudin (Hirudin) is the most active and most studied of the many active ingredients extracted from leeches (Leech) and their salivary glands, and is a small molecular protein (polypeptide) consisting of 65-66 amino acids. The leech contains abundant hirudin which has extremely strong inhibition effect on thrombin and is the strongest natural specific thrombin inhibitor discovered so far. It can be used for treating various thrombotic diseases, especially venous thrombosis and disseminated blood vessel coagulation; can also be used for preventing the formation of arterial thrombus after surgical operation, and preventing the formation of thrombus after dissolving thrombus or revascularization; improving extracorporeal blood circulation and hemodialysis procedures. In microsurgery, failure is often caused by vascular embolism at the anastomosis site, and hirudin is used to promote wound healing. Studies have also shown that hirudin can also play a role in the treatment of tumors. It can prevent tumor cell metastasis, and has proven curative effect on tumors such as fibrosarcoma, osteosarcoma, angiosarcoma, melanoma, leukemia, etc. Hirudin can also be used in combination with chemotherapy and radiotherapy to enhance the therapeutic effect due to the promotion of blood flow in tumors.

Because the natural hirudin has low productivity and large clinical requirement, a large amount of recombinant hirudin researches are carried out at home and abroad, and the recombinant hirudin which has a similar structure and the same function as the natural hirudin is developed at the end of the last eighties of the century, so that the productivity is high.

However, the purification of the existing proteins and peptides basically requires two or more than three times of chromatographic separation to obtain pure products, and the eluent contains a large amount of water and salt, so that the environmental pollution is great and the pressure of sewage treatment engineering is great.

The anticoagulant activity of the hirudin mutant used in the current market is 16000ATU/mg, and the purity is about 93%.

For example, Chenhuayou et al (Anhui agricultural science, 2009, 37 (34); pages 16757, 16759 and 16768) disclose that fermentation broth is subjected to centrifugation, trichloroacetic acid treatment, ultrafiltration concentration and desalination, then is subjected to anion exchange column and S100 molecular sieve column, and finally 95% pure product is obtained; weley et al (2004. the Collection of the national institute of Biotechnology and academic, symposium, pp. 104-112) disclose the use of three chromatographic separations including macroporous resin chromatography, DEAE chromatography, and reversed-phase chromatography to obtain 95% pure product. Wherein, the more chromatography steps, the higher the cost and the more time-consuming, and the higher the environmental pollution.

Therefore, there is a need for a method for extracting and purifying hirudin mutant HV2-Lys47, which is simple in process, less in waste water and suitable for industrial scale-up.

In addition, the collection and separation of blood samples are of great importance in clinical biochemical detection, and are an important link for ensuring quality. The traditional sample collection usually needs a long time from the beginning of blood collection to the blood serum separation on-machine detection, so that the high efficiency of the full-automatic biochemical analyzer is limited. Heparin anticoagulant plasma is used in some biochemical laboratories, and after blood is taken, the blood can be immediately centrifugally separated for on-board detection, but heparin sodium anticoagulant plasma and heparin lithium anticoagulant plasma have influence on certain biochemical indexes.

China began to popularize the vacuum blood sampling technology in 1997, which is a major improvement on the traditional blood sampling mode. Because the blood sampling process is completed under a totally closed system, the possibility of blood pollution and cross infection is fundamentally eliminated, and the blood sampling is safer, more accurate and more standard, thereby being easy to popularize and popularize. Separating gel, coagulant and various anticoagulants can be added into the vacuum blood collection tube, and the color of the tube cap is used for distinguishing the blood collection tubes with different purposes.

In recent years, the containing coagulant and separation gel vacuum blood collection tube in biochemical test is widely used, it has greatly shortened blood coagulation time, generally placed 20 minutes after centrifugal separation of serum, but there is a certain proportion of blood collection tube centrifugal effect is not good, in the serum exists protein coagulation silk or clot. The separating gel is a semi-solid inert gel composed of a plurality of compounds, does not affect the content of each component of serum, but the procoagulant tube may have different influences on biochemical detection due to different raw material producing areas, properties and manufacturing processes of the procoagulant.

The natural hirudin is a polypeptide extracted and separated from salivary glands of blood sucking leeches (commonly called leeches), is an important active ingredient in leeches, but has extremely limited yield and can not meet the requirements of clinical application.

Therefore, there is a need for anticoagulants with better anticoagulant activity and more stable sources.

Disclosure of Invention

The application provides an extraction and purification method of hirudin mutant HV2-Lys47, which has the advantages of simple process, less waste water and suitability for industrial amplification.

To achieve the object of the present application, the present application provides a process for the extraction of a purified hirudin mutant HV2-Lys47 from a fermentation broth of Escherichia coli for the fermentative production of the hirudin mutant HV2-Lys47(SEQ ID No.:1), said process comprising:

treating the escherichia coli fermentation broth at an elevated temperature to sterilize and remove contaminating proteins;

treating the sterilized fermentation broth with a ceramic membrane or a centrifuge to remove the biomass;

optionally, treating the fermentation broth passed through a ceramic membrane or centrifuge by ultrafiltration membrane to remove contaminating proteins;

treating the fermentation liquor passing through a ceramic membrane or a centrifugal machine or the fermentation liquor passing through an ultrafiltration membrane by using a nanofiltration membrane to obtain a roughly separated concentrated solution;

adding salt into the roughly separated concentrated solution as an auxiliary material, and performing spray drying to obtain dry powder;

dissolving the dry powder in water and filtering out impurities, optionally filtering through filter paper, a sand core funnel or membrane equipment;

making filtrate pass through molecular sieve column chromatography for 1 time, eluting with water, concentrating the collected solution to obtain hirudin mutant HV2-Lys47 crude product;

dissolving the crude hirudin mutant HV2-Lys47 in water, adding salt or organic solvent to precipitate hirudin mutant HV2-Lys47, and drying to obtain pure hirudin mutant HV2-Lys 47.

In the above or other embodiments, the elevated temperature may be an elevated temperature capable of sterilization, such as a temperature of 65 ℃ or the like.

In the above or other embodiments, the drying may be vacuum drying.

In the above or other embodiments, the drying may be freeze vacuum drying.

In the above or other embodiments, the sterilized fermentation broth is processed by a centrifuge.

In the above or other embodiments, the centrifuge may be a tube centrifuge or a disk centrifuge.

Among them, the ceramic membrane treatment or the centrifuge treatment is intended to remove cells, the ultrafiltration membrane treatment is intended to remove foreign proteins, and the nanofiltration membrane treatment is intended to concentrate.

In the above or other embodiments, the high temperature treatment may be at 65 ℃ to 67 ℃ for 5 to 20 minutes, optionally 10 minutes.

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

In the above or other embodiments, the molecular sieve column chromatography may be selected from the group consisting of sephadex G25, sephadex G50, sephadex G75, and sephadex G100. It will be appreciated by those skilled in the art that these molecular sieve column chromatographs are all commercially available products. Wherein, the purpose of molecular sieve column chromatography is mainly to remove pigments, salts, and polypeptides and polysaccharides with molecular weights far away from hirudin.

In the above or other embodiments, the concentration of the collected liquid obtained via molecular sieve column chromatography may be performed by vacuum concentration under reduced pressure, nanofiltration membrane concentration, or reverse osmosis membrane concentration.

In the above or other embodiments, the organic solvent may be selected from one or more of ethanol, methanol, acetone, isopropanol, or acetonitrile.

In the above or other embodiments, the weight in grams of salt added as an adjunct is 5% or more, alternatively 10% or more, of the volume in milliliters of the crude separated concentrate. The inventors of the present application have found that the purification object of the present application can be achieved by adding salt in an amount of 5% by weight or more by volume as above, the loss of hirudin is large when less than 5% of salt is added, and the cost is too high when too much salt is added.

In the above or other embodiments, the volume in milliliters of water used to dissolve the dry powder is more than 10 times the weight in grams of the dry powder. Wherein the amount of water is selected to dissolve the dry powder in order to maintain the weight of the salt in grams at 5% or less of the volume of the aqueous solution in milliliters so that a portion of the contaminating proteins that are not soluble in water can be removed by filtration.

In the above or other embodiments, the salt is added in a weight in grams of 20-30% of the volume in milliliters of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved, and the volume of the organic solvent added is 5-9 times the volume of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved, in order to precipitate the hirudin mutant HV2-Lys 47.

Wherein the step of re-precipitating the recombinant hirudin is performed in order to remove polysaccharides and polypeptides having a molecular weight closer to that of hirudin.

In the above or other embodiments, the E.coli fermentation broth may be produced by culturing the strain Escherichia coli (Escherichia coli) pBH2 CGMCC No0908 with pBH-2 as an expression vector.

In the above or other embodiments, the culturing comprises culturing under aerobic conditions at a temperature of 25 ℃ to 35 ℃ to the end of the logarithmic growth phase, and then increasing the temperature to 35 ℃ to 45 ℃ to continue culturing until the production of the hirudin mutant HV2-Lys47 reaches a peak.

In the present application, reference is made to the production of fermentation broths of hirudin mutant HV2-Lys47 by reference to the patent application with application number 201711262810.4, wherein application number 201711262810.4 is hereby incorporated by reference in its entirety. Wherein the hirudin mutant HV2-Lys47 of the present application is referred to as "recombinant hirudin" in the patent application with application number 201711262810.4. Thus, in this context, the expression "hirudin mutant HV2-Lys 47" and "recombinant hirudin" represent the same substance and are used interchangeably.

Compared with the prior art, the method adopts a membrane technology and a column chromatography technology, only adopts water for elution, has simple process and less waste water, and is suitable for industrial amplification production of the hirudin mutant HV2-Lys 47.

In a further aspect, the present application provides a hirudin mutant HV2-Lys47 produced according to any of the methods described above.

In a further aspect, the present application provides a hirudin mutant HV2-Lys47 produced according to any of the methods described above, wherein the anticoagulant activity of the hirudin mutant HV2-Lys47 may be greater than or equal to 18000 ATU/mg. It is known in the art that anticoagulant activity, which means units of biological activity per milligram of protein, is different from purity, which is high, and anticoagulant activity is not necessarily high, because the active protein may cause a decrease in activity due to a slight change in conformation.

In yet another aspect, the application provides the use of hirudin mutant HV2-Lys47 in an anticoagulated blood collection tube.

In the above or other embodiments, wherein the anti-coagulant activity of said hirudin mutant HV2-Lys47 may be greater than or equal to 18000 ATU/mg.

In the above or other embodiments, the hirudin mutant HV2-Lys47 may be produced by culturing the strain Escherichia coli (Escherichia coli) pBH2 CGMCC No0908 with pBH-2 as an expression vector, and the hirudin mutant HV2-Lys47 may have an anticoagulant activity of 18000ATU/mg or more.

In the above or other embodiments, wherein the hirudin mutant HV2-Lys47 may be added directly to the anticoagulated blood collection tube.

In the above or other embodiments, wherein the hirudin mutant HV2-Lys47 may be applied to the anticoagulated blood collection tube in a form contained in a coating.

In the above or other embodiments, wherein the hirudin mutant HV2-Lys47 may be used in an amount of 8.5ug/ml blood.

In the above or other embodiments, the hirudin mutant HV2-Lys47 may be coated in a form contained in a coating layer into the anticoagulation blood collection tube, and then the coating layer is dried by freeze vacuum drying. In experiments, the inventor of the application finds that the hirudin activity is not lost by adopting a freeze vacuum drying mode.

In the above or other embodiments, the anticoagulated blood collection tube can be used for holding blood, and the blood can be used for detecting one or more of blood routine, blood biochemistry, electrolyte, tumor marker, homocysteine and hepatitis B, and has no interference on detection results due to the high purity of the hirudin mutant HV2-Lys 47.

In a further aspect, the present application provides an anticoagulated blood collection tube which may have a hirudin mutant HV2-Lys47 added directly or coated with a coating comprising the hirudin mutant HV2-Lys 47.

In the above or other embodiments, the hirudin mutant HV2-Lys47 may be produced by culturing the strain Escherichia coli (Escherichia coli) pBH2 CGMCC No0908 with pBH-2 as an expression vector, and the hirudin mutant HV2-Lys47 may have an anticoagulant activity of 18000ATU/mg or more. Wherein, every 1mg is more than or equal to 18000ATU, and the purity is more than 95 percent.

In the anticoagulant blood collection tube, the hirudin mutant HV2-Lys47 required to be added is correspondingly reduced from 0.01mg/ml of original blood to 8.5ug/ml of original blood. In the production and processing process of the blood collection tube, the process is high-temperature drying generally, the loss of the activity of the hirudin is about 10 percent, and the freeze vacuum drying method is adopted, so that the activity of the hirudin is not lost. Furthermore, the hirudin mutant HV2-Lys47 used in the present application is almost non-interfering in the detection of blood markers due to its high purity.

Through a large number of clinical experimental researches, the inventor of the application finds that an anticoagulant blood collection tube containing hirudin mutant HV2-Lys47 can be used for detecting various items such as blood routine, blood biochemistry, electrolytes, tumor markers, homocysteine, hepatitis B and the like. The hirudin mutant HV2-Lys47 as the anticoagulant of the blood collection tube has the characteristics of small dosage of the anticoagulant, high reaction speed, capability of separating plasma from the anticoagulated plasma without interference on detection items, capability of keeping original properties and properties of the blood, capability of keeping the original properties and the forms of the blood, capability of reducing the interference on the blood compared with other anticoagulants, no hemolysis phenomenon, no pseudo-platelet reduction phenomenon, no interference of heavy metal and impurities and no need of correcting the numerical value of calcium.

Moreover, experiments prove that the hirudin mutant HV2-Lys47 is used as an anticoagulant, one tube can be used for replacing vacuum blood collection tubes with a plurality of colors, the burden that medical staff need to distinguish the color of the blood collection tube every time to determine the expected application of the blood collection sample is thoroughly reduced, and the labor intensity and the error of the medical staff are reduced. But also can reduce the blood sampling amount of patients and the generation of medical waste, and has great social and economic benefits.

Wherein the amino acid sequence of the hirudin mutant HV2-Lys47 of the application is shown as follows:

Ile Thr Tyr Thr Asp CysThrGluSerGlyGlnAsnLeuCysLeuCysGluGlySerAsn Val CysGlyLys GlyAsn Lys Cys Ile LeuGlySerAsnGly Lys GlyAsnGlnCys Val ThrGlyGluGlyThr Pro Lys Pro GluSerHisAsnAsnGly Asp PheGluGlu Ile ProGluGlu Tyr LeuGln(SEQ ID NO.:1)

disulfide bond position: cys6-Cys 14; cys16-Cys 28; cys 22-Cys 39

Compared with natural hirudin, the hirudin mutant HV2-Lys47 or recombinant hirudin of the present application has the asparagine at position 47 of the natural hirudin replaced by lysine.

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

Drawings

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

FIG. 1 shows the electrophoretogram of hirudin mutant HV2-Lys47 prepared in example 1;

FIG. 2 is an HPLC picture of the hirudin mutant HV2-Lys47 prepared in example 1, in which the peak with retention time of 18.991min is the corresponding peak of the hirudin mutant HV2-Lys 47.

FIG. 3 is a diagram showing the amino acid residue sequence of hirudin mutant HV2-Lys 47;

FIG. 4 is a three-dimensional structure diagram of the mutant hirudin HV2-Lys47 in the webpage of the biomolecule database (https:// www.rcsb.org /).

Detailed Description

To make the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application will be described in detail below with reference to the accompanying drawings. 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.

Example 1

Preparing a fermentation liquid:

the glycerol preserved strain of pBH-2 engineering bacteria (preservation number: CGMCC No.0908) is inoculated into a fermentation medium according to the inoculation amount of 5%. 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.

For a detailed operation, see example 2, application No. 201711262810.4.

Wherein the culture medium consists of the following components and the pH value is 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. In this and the following examples, L in g/L is the volume of the final fermentation medium.

Wherein the sources of the materials are shown in the following table:

the purified hirudin mutant HV2-Lys47 was extracted from the fermentation broth by the following steps:

1) sterilizing the fermentation liquor at 65 deg.C for 5 min;

2) centrifuging the sterilized fermentation liquor obtained in the step 1) by a tube centrifuge (GQB-770 of Liaoyang Shaxing vacuum equipment factory) to remove escherichia coli;

3) passing the centrifuged clear solution of step 2) through an ultrafiltration membrane (Xiamen Sanda Membrane science and technology Co., Ltd., model: NFM-84S-6/3) trash protein;

4) subjecting the ultrafiltration dialysate obtained in step 3) to nanofiltration membrane (Xiamen Sanda Membrane science and technology Co., Ltd., type: UFM-84S-2) to obtain a crude separation concentrated solution;

5) adding sodium chloride into the crude separation concentrated solution obtained in the step 4) as an auxiliary material, and performing spray drying to obtain dry powder, wherein the weight of salt added as the auxiliary material in grams is 10% of the volume of the crude separation concentrated solution in milliliters;

6) dissolving the dry powder obtained in the step 5) in water and filtering out impurities to obtain a filtrate, wherein the volume of the water used for dissolving the dry powder in milliliters is 10 times of the weight of the dry powder in grams;

7) subjecting the filtrate from step 6) to molecular sieve column chromatography (sephadex G50, manufacturer: general Electric Company (GE)) was purified 1 time, eluted with pure water, and the collected solution was concentrated under reduced pressure in vacuo;

8) adding 9 times volume of ethanol into the concentrated solution obtained in the step 7) to precipitate hirudin mutant HV2-Lys47, and drying the precipitate under reduced pressure and vacuum to obtain the hirudin mutant hirudin.

The hirudin mutant HV2-Lys47 prepared above was analyzed by electrophoresis, as shown in FIG. 1, and the obtained electrophoretogram showed a single band (see the figure corresponding to hirudin 1 in FIG. 1), and the molecular weight of the pure recombinant hirudin was confirmed to be consistent with the theoretical value by electrophoresis.

Wherein, the electrophoresis operation conditions are as follows: the gel loading amount of the concentrated gel 3% separation gel 15% is 30uL (1 ten thousand ATU/ml), the gel is run for 1h under the voltage of 100V, and then the gel is run for 2.2h under the voltage of 200V.

The electrophoresis was performed as follows:

1. and (3) mounting the prefabricated gel in a min electrophoresis apparatus, adding SDS-PAGE electrophoresis buffer solution, submerging the top of the gel, and pulling out the comb teeth of the sample tank.

2. 40uL of sample is taken, 10uL of 5 Xprotein loading buffer solution is added, after uniform mixing, water bath at 100 ℃ is carried out for 5min, and 40uL of sample is taken and added into a sample groove on the gel.

3. And after the sample loading is finished, connecting an electrophoresis apparatus power supply, setting the voltage to be 150V constant voltage for about 50min, and ending the electrophoresis when the bromophenol blue line reaches the bottom of the gel.

4. Fixing for 20min, dyeing for 20min, and decolorizing for 2 h.

After the analysis of anticoagulation activity, the anticoagulation activity of the prepared hirudin mutant HV2-Lys47 is calculated as 17000ATU/mg by biochemical method determination, and the specific anticoagulation activity is determined as shown in the left column of page 84 of Chinese pharmacopoeia 2005 edition. Furthermore, the purity of the hirudin mutant HV2-Lys47, calculated on the basis of the ratio of the color bands in gel electrophoresis, has reached 95%.

In addition, HPLC determination proves that the prepared hirudin mutant HV2-Lys47 indeed has a significantly larger peak of the hirudin mutant HV2-Lys47, as shown in FIG. 2.

Example 2

Hirudin mutant HV2-Lys47 was prepared and purified by the method of example 1, except that, in step 2), a disk centrifuge (Alfa laval stain products skogstop: 881095-05-S/01) to remove E.coli, the weight in grams of the salt added as adjuvant in step 5) being 8% of the volume in milliliters of the crude separated concentrate.

The obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, which showed the same result as that in FIG. 1, wherein a single band was shown by electrophoresis.

The hirudin mutant HV2-Lys47 was tested to have an anticoagulant activity of 15800 ATU/mg. The purity was about 93%.

Example 3

The hirudin mutant HV2-Lys47 was prepared and purified as described in example 1, except that in step 2) a ceramic membrane (Sanda Membrane technology (Xiamen) Co., Ltd.) was used for the removal of E.coli.

Tests show that the obtained hirudin mutant HV2-Lys47 has the anticoagulation activity of 16800ATU/mg and the purity of more than 93 percent.

Example 4

The recombinant hirudin was purified by the method of example 1, except that in step 2), the E.coli was removed by using a hollow fiber membrane (Sanda Membrane technology (Xiamen, Ltd.).

Tests show that the obtained hirudin mutant HV2-Lys47 has the anticoagulation activity of 16000ATU/mg and the purity of more than 93 percent.

Example 5

Purification of recombinant hirudin was carried out by the method of example 1, except that in step 8) 8 times methanol was added for precipitation

Example 6

The recombinant hirudin was purified by the method of example 1 and, in step 8), precipitated by adding 5 times the amount of acetone.

Tests show that the obtained hirudin mutant HV2-Lys47 has the anticoagulation activity of 17000ATU/mg and the purity of 95%.

Example 7

Purification of recombinant hirudin was carried out by the method of example 1, except that in step 8) a precipitation with an ammonium sulfate salt with a saturation of 75% was carried out, wherein the weight in grams of ammonium sulfate was 20% of the volume in ml of the aqueous solution in which the crude hirudin mutant HV2-Lys47 was dissolved.

The hirudin mutant HV2-Lys47 was tested for its anticoagulant activity at 15000 ATU/mg.

Example 8

The recombinant hirudin was purified by the method of example 1, except that the sterilized fermentation broth was directly subjected to a bacterial liquid separation by means of a ceramic membrane, the dialysate from the ceramic membrane was treated 1 time with a nanofiltration membrane and in step 8) a precipitation was carried out with sodium chloride, the weight of which in grams was 30% of the volume in ml of the aqueous solution in which the crude hirudin mutant HV2-Lys47 was dissolved.

The hirudin mutant HV2-Lys47 was tested to have an anticoagulant activity of 14500 ATU/mg.

Example 9

The purification of recombinant hirudin was carried out as described in example 1, except that in step 8) the hirudin mutant HV2-Lys47 was precipitated using 9 volumes of isopropanol.

The hirudin mutant HV2-Lys47 was tested to have an anticoagulant activity of 18000 ATU/mg. The purity was 98%.

Example 10

Effect of drying process on the Activity of hirudin mutant HV2-Lys47 in anticoagulated blood collection tubes:

the hirudin prepared in example 9 was dissolved in pure water at a concentration of 3.4g/100ml of sterile pure water. Each of the above-prepared solutions of hirudin mutant HV2-Lys47 was sprayed with a 2ml blood collection tube using a needle spray and then dried.

Drying processes commonly used in the art are high temperature drying: drying at 45 deg.C, 50 deg.C, 70 deg.C for three times, 60 s each time;

the drying method of the present application: drying the sprayed blood collection tube in a freeze vacuum drier at-50 deg.C and-4 Mpa.

The effect of the high temperature drying and freeze vacuum drying method on hirudin activity is shown in table 1 below.

TABLE 1

As can be seen from the table, the loss of activity of the coated hirudin mutant HV2-Lys47 is minimal upon vacuum freeze-drying.

Example 11

The effect of the amount of the hirudin mutant HV2-Lys47 prepared in example 9 by vacuum freeze-drying on blood coagulation is shown in Table 2 below:

TABLE 2

Hirudin mutant HV2-Lys47 dosage (mg)	Blood volume (ml)	Coagulation phenomenon
			0.001	2	Quickly solidify
0.005	2	Without coagulation
			0.008	2	Without coagulation
0.02	2	Without coagulation
			0.08	2	Without coagulation
0.16	2	Without coagulation
			0.5	2	Without coagulation
1	2	Without coagulation
			5	2	Without coagulation
10	2	Without coagulation
			15	2	Without coagulation
20	2	Without coagulation

As can be seen from the above table, the hirudin mutant HV2-Lys47 prepared in example 9 can achieve an anticoagulant effect when the amount of the mutant is more than 0.005 mg.

Example 12

The hirudin mutant HV2-Lys47 (hereinafter referred to as hirudin) anticoagulant blood collection tube prepared in example 9 has influence on the conventional blood and biochemical indexes of blood, wherein the hirudin mutant HV2-Lys47 is applied to the vacuum blood collection tube by spraying.

Blood routine

The conventional blood test indexes of hirudin anticoagulation and K2 EDTA anticoagulation vacuum blood collection tubes are shown in the following table 3. Among them, 2 kinds of vacuum blood collection tube 20 indexes in high correlation (r value >0.8), 4 items in moderate correlation (0.5< r value < 0.8).

TABLE 3 comparison of hirudin anticoagulation with K2 EDTA anticoagulated blood

(II) blood biochemistry, tumor marker and homocysteine

The results of biochemical and tumor markers and homocysteine detection of serum (separation gel/silica vacuum blood collection tube) and plasma (hirudin vacuum blood collection tube) are shown in Table 4. The 22 indexes of 2 vacuum blood collection tubes are highly correlated (r value >0.8), and 2 indexes are moderately correlated (0.5< r value < 0.8).

TABLE 4 comparison of hirudin anticoagulated plasma with conventional serum biochemical, tumor markers and homocysteine

The third five items of hepatitis B

The five hepatitis B items are qualitative detection indexes, and the results are shown in Table 5. From the results, the serum and plasma detection indexes are very consistent, the serum negative index plasma detection result is negative, and the serum positive index plasma detection result is positive.

TABLE 5

The results showed that the conventional hirudin anticoagulated Blood AND the conventional K2 EDTA Blood were consistent, AND the detection index was highly or moderately correlated with the results of Menssen H (see "Measurement of hematology, Clinical Chemistry, AND Infection Parameters from fragmented Blood Collected in Universal Blood Sampling Tubes", Hans D.Menssen et al, SEMINARS IN THROMBOSIS AND HEMOSSTATIS/VOLUME 27, NUMBER 42001, page 349 AND 356). At present, a serum sample is commonly used for detecting clinical biochemical indexes, and compared with serum, a plasma sample is closer to a physiological state in theory. The results indicate that serum and plasma biochemistry, electrolytes, tumor markers and homocysteine are highly correlated, except for poor correlation of total protein, albumin and globulin ((0.5< r value < 0.8)). (r value > 0.9). The plasma contains fibrinogen, so that the protein concentration (75.07 +/-4.31) in the plasma is higher than that in the serum (70.89 +/-3.48).

As can be seen from the above, K2. EDTA and hirudin anticoagulant effect is similar, but K2. EDTA can only determine blood routine, and hirudin blood collection tube can also determine serum and plasma biochemical, electrolyte, tumor marker and homocysteine index.

In addition, the anticoagulant activity of hirudin currently on the market is: the hirudin of 40ATU is needed for each milliliter of blood, the hirudin has high purity, so the addition amount of the hirudin is lower than that of the hirudin with low purity when the blood collection tube is prepared, and simultaneously, the influence on blood coagulation and blood index detection due to the existence of impurities is avoided because of high purity. Moreover, due to the high purity, the hirudin of the application has significantly better stability, and the specific data are as follows:

hirudin blood collection tube (2 ml): (accelerated test temperature 40 ℃. + -. 2 humidity 75%. + -. 5)

Detection period

0 week

2 weeks

1 month

2 months old

3 months old

6 months old

Blood collection tube

2625ATU

2650ATU

2625ATU

2580ATU

2600ATU

2575ATU

External blood sampling tube

3020ATU

2800ATU

2548ATU

1890ATU

1600ATU

1500ATU

Wherein the blood collection tube of the present application is the blood collection tube of example 9 having a coating of the dried hirudin mutant HV2-Lys47 prepared by freeze-drying. The externally purchased blood collection tube comprises: sarstedt, germany, is also a hirudin blood collection tube.

Although the embodiments disclosed in the present application are described above, the descriptions are only for the convenience of understanding the present application, and are not intended to limit the present application. 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 disclosure as defined by the appended claims.

Claims

1. A method for extracting and purifying a hirudin mutant HV2-Lys47 from a fermentation broth of Escherichia coli for producing the hirudin mutant HV2-Lys47 by fermentation, wherein the sequence of the hirudin mutant HV2-Lys47 is shown in SEQ ID NO. 1, and the Escherichia coli fermentation broth is produced by culturing a strain Escherichia coli (Escherichia coli) pBH2 CGMCC No0908 with pBH-2 as an expression vector, and the method comprises the following steps:

treating an escherichia coli broth at an elevated temperature for 5-20 minutes at 65-67 ℃ to sterilize and remove contaminating proteins;

treating the fermentation broth passing through the ceramic membrane or centrifuge by a nanofiltration membrane to obtain a coarsely separated concentrated solution;

dissolving the dry powder in water and filtering to remove impurities;

2. The method according to claim 1, wherein the method comprises the steps of:

treating the fermentation broth passing through the ceramic membrane or centrifuge with an ultrafiltration membrane to remove the foreign proteins;

treating the fermentation liquor passing through the ultrafiltration membrane by a nanofiltration membrane to obtain a roughly separated concentrated solution;

dissolving the dry powder in water and filtering to remove impurities;

3. The method according to claim 1 or 2, wherein in dissolving the dry powder in water and filtering out impurities, the filtering is performed by filter paper, sand core funnel or membrane device.

4. The process according to claim 1 or 2, wherein the crude hirudin mutant HV2-Lys47 is dissolved in water, followed by addition of a salt or an organic solvent to precipitate the hirudin mutant HV2-Lys47, and vacuum drying to obtain the pure hirudin mutant HV2-Lys 47.

5. The process of claim 4, wherein the crude hirudin mutant HV2-Lys47 is dissolved in water, followed by addition of a salt or an organic solvent to precipitate the hirudin mutant HV2-Lys47, followed by lyophilization to obtain pure hirudin mutant HV2-Lys 47.

6. The process according to claim 1 or 2, wherein the autoclaved fermentation broth is treated by a centrifuge.

7. The method of claim 6, wherein the centrifuge is a tube centrifuge or a disk centrifuge.

8. The method of claim 1 or 2, wherein the treating the E.coli fermentation broth at elevated temperature is at 65-67 ℃ for 10 minutes.

9. The method of claim 1 or 2, wherein the salt is selected from one or more of sodium chloride, potassium chloride, ammonium sulfate, sodium sulfate, potassium sulfate.

10. The method of claim 1 or 2, wherein the molecular sieve column chromatography is selected from the group consisting of sephadex G25, sephadex G50, sephadex G75 and sephadex G100.

11. The method according to claim 1 or 2, wherein the concentration of the collected liquid obtained by the molecular sieve column chromatography is performed by vacuum concentration under reduced pressure, nanofiltration membrane concentration or reverse osmosis membrane concentration.

12. The process according to claim 1 or 2, wherein the organic solvent is selected from one or more of ethanol, methanol, acetone, isopropanol or acetonitrile.

13. A process according to claim 1 or 2, wherein the weight in grams of salt added as an adjunct is 5% or more of the volume in milliliters of the crude separated concentrate.

14. The method according to claim 1 or 2, wherein the volume in milliliters of water used to dissolve the dry powder is more than 10 times the weight in grams of the dry powder.

15. The process according to claim 1 or 2, wherein for the precipitation of the hirudin mutant HV2-Lys47, the salt is added in a weight in grams of 20-30% of the volume in milliliters of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved, and the volume of organic solvent added is 5-9 times the volume of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved.

16. The process according to claim 1, wherein the cultivation comprises cultivation under aerobic conditions at a temperature of 25-35 ℃ to the end of the logarithmic growth phase, followed by warming to 35-45 ℃ and continuing the cultivation until the production of hirudin mutant HV2-Lys47 reaches a peak.