WO2020210965A1 - 水蛭素突变体的提取纯化方法及其应用 - Google Patents

水蛭素突变体的提取纯化方法及其应用 Download PDF

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WO2020210965A1
WO2020210965A1 PCT/CN2019/082785 CN2019082785W WO2020210965A1 WO 2020210965 A1 WO2020210965 A1 WO 2020210965A1 CN 2019082785 W CN2019082785 W CN 2019082785W WO 2020210965 A1 WO2020210965 A1 WO 2020210965A1
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lys47
hirudin mutant
hirudin
mutant
blood collection
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PCT/CN2019/082785
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English (en)
French (fr)
Inventor
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
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广东双骏生物科技有限公司
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Priority to PCT/CN2019/082785 priority Critical patent/WO2020210965A1/zh
Priority to EP19925351.9A priority patent/EP3838911A4/en
Priority to JP2021525292A priority patent/JP7375012B2/ja
Priority to US17/282,303 priority patent/US20210317162A1/en
Publication of WO2020210965A1 publication Critical patent/WO2020210965A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin

Definitions

  • This application includes, but is not limited to, bioengineering technology, especially a method for extracting and purifying the hirudin mutant HV2-Lys47 from the fermentation broth of E. coli fermentation to produce the hirudin mutant HV2-Lys47 (SEQ ID NO.:1), and The purified hirudin mutant HV2-Lys47 thus obtained.
  • This application also relates to biomedical devices, especially the application of the hirudin mutant HV2-Lys47 purified by the above method in anticoagulation blood collection tubes.
  • Hirudin is the most active and most studied ingredient among the various active ingredients extracted from Leech and its salivary glands. It is a small molecule protein composed of 65-66 amino acids ( Polypeptide). Leech is rich in hirudin. Hirudin has a strong inhibitory effect on thrombin and is the strongest natural specific inhibitor of thrombin found so far. It can be used to treat various thrombotic diseases, especially the treatment of venous thrombosis and diffuse vascular coagulation; it can also be used to prevent the formation of arterial thrombosis after surgery, prevent the formation of thrombus after thrombolysis or revascularization; improve extracorporeal blood circulation and Hemodialysis process.
  • hirudin can promote wound healing. Studies have also shown that hirudin can also play a role in tumor treatment. It can prevent the metastasis of tumor cells, and has proven effective tumors such as fibrosarcoma, osteosarcoma, angiosarcoma, melanoma and leukemia. Hirudin can also be combined with chemotherapy and radiotherapy, due to its ability to promote blood flow in the tumor and enhance its efficacy.
  • the anticoagulant activity of the hirudin mutant currently used in the market is 16000 ATU/mg, and the purity is about 93%.
  • heparin anticoagulated plasma which can be centrifuged to separate the plasma for machine testing immediately after the blood is taken, but whether heparin sodium or lithium heparin anticoagulated plasma will affect certain biochemical indicators.
  • Natural hirudin is a polypeptide extracted and isolated from the salivary glands of blood-sucking leeches (commonly known as leeches). It is an important active ingredient in leeches, but its output is extremely limited and cannot meet the requirements of clinical applications.
  • This application provides a method for extracting and purifying hirudin mutant HV2-Lys47, which has a simple process, less waste water, and is suitable for industrial scale-up.
  • the present application provides a method for extracting and purifying the hirudin mutant HV2-Lys47 from the fermentation broth of E. coli for the production of the hirudin mutant HV2-Lys47 (SEQ ID NO.: 1), The method includes:
  • the fermentation broth that has passed through a ceramic membrane or a centrifuge is processed by an ultrafiltration membrane to remove impurities;
  • the filtrate was passed through molecular sieve column chromatography only once, eluted with water, and the collected liquid was concentrated to obtain the crude product of the hirudin mutant HV2-Lys47;
  • the crude hirudin mutant HV2-Lys47 is dissolved in water, and then salt or organic solvent is added to it to precipitate out the hirudin mutant HV2-Lys47, which is dried to obtain the pure hirudin mutant HV2-Lys47.
  • the elevated temperature may be a high temperature capable of sterilization, such as a temperature of 65°C, 75°C, and the like.
  • the drying may be vacuum drying.
  • the drying may be freeze vacuum drying.
  • the sterilized fermentation broth is processed by a centrifuge.
  • the centrifuge may be a tube centrifuge or a disc centrifuge.
  • the purpose of ceramic membrane treatment or centrifuge treatment is to remove bacteria
  • the purpose of ultrafiltration membrane treatment is to remove impurities
  • the purpose of nanofiltration membrane treatment is to concentrate.
  • the high temperature treatment may be maintained at 65°C-67°C for 5-20 minutes, and optionally for 10 minutes.
  • the salt may be selected from one or more of sodium chloride, potassium chloride, ammonium sulfate, sodium sulfate, or potassium sulfate.
  • the molecular sieve column chromatography can be selected from the group consisting of sephadex G25, sephadex G50, sephadex G75 and sephadex G100.
  • the above-mentioned molecular sieve column chromatography are all commercially available products.
  • the main purpose of using molecular sieve column chromatography is to remove pigments, salts, and polypeptides and polysaccharides whose molecular weight is farther from hirudin.
  • the concentration of the collected liquid obtained by molecular sieve column chromatography can be carried out by vacuum concentration under reduced pressure, concentration by nanofiltration membrane or concentration by reverse osmosis membrane.
  • the organic solvent may be selected from one or more of ethanol, methanol, acetone, isopropanol, or acetonitrile.
  • the weight in grams of the salt added as an auxiliary material is 5% or more, optionally 10% or more, based on the volume in milliliters of the crudely separated concentrate.
  • the inventor of the present application found that adding salt in an amount of 5% or more by weight and volume fraction as above can achieve the purification purpose of the present application. Adding salt less than 5% of hirudin will cause a large loss, and adding too much salt will cost too much.
  • the volume in milliliters of the water used to dissolve the dry powder is more than 10 times the weight in grams of the dry powder.
  • the purpose of choosing a certain amount of water to dissolve the dry powder is to keep the weight of the salt in grams at 5% or less of the volume in milliliters based on the aqueous solution, so that part of the water-insoluble protein can pass through Remove by filtration.
  • the weight in grams of the salt added is 20% of the volume in milliliters of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved. %-30%, the volume of the added organic solvent is 5-9 times the volume of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved.
  • the step of re-precipitating the recombinant hirudin is to remove polysaccharides and polypeptides with a molecular weight closer to hirudin.
  • the E. coli fermentation broth may be produced by culturing the strain Escherichia coli pBH2 CGMCC No0908 with pBH-2 as an expression vector.
  • the cultivation includes culturing under aerobic conditions at a temperature of 25°C-35°C until the end of the logarithmic growth phase, and then raising the temperature to 35°C-45°C to continue culturing until the hirudin mutant The production of HV2-Lys47 reached its peak.
  • the fermentation broth of the hirudin mutant HV2-Lys47 can be obtained with reference to the production method of the patent application with the application number 201711262810.4, and the patent application with the application number 201711262810.4 is hereby incorporated by reference in its entirety.
  • the applied hirudin mutant HV2-Lys47 is called “recombinant hirudin” in the patent application with application number 201711262810.4. Therefore, in this article, "hirudin mutant HV2-Lys47" and “recombinant hirudin” represent the same substance and can be used interchangeably.
  • this application adopts membrane technology and one-time column chromatography technology, and only uses water elution, the process is simple, and the waste water is small, which is suitable for industrial scale-up production of hirudin mutant HV2-Lys47.
  • the present application provides a hirudin mutant HV2-Lys47 produced according to any of the methods described above.
  • 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 .
  • anticoagulant activity is different from purity. Anticoagulant activity refers to the biological activity unit per milligram of protein. With high purity, anticoagulant activity is not necessarily high, because the active protein may decrease activity due to subtle changes in conformation. .
  • the application provides the application of the hirudin mutant HV2-Lys47 in anticoagulation blood collection tubes.
  • the anticoagulant activity of the hirudin mutant HV2-Lys47 may be greater than or equal to 18000 ATU/mg.
  • the hirudin mutant HV2-Lys47 may be cultured and produced by the strain Escherichia coli pBH2 CGMCC No0908 with pBH-2 as an expression vector, and the hirudin mutant
  • the anticoagulant activity of HV2-Lys47 can be greater than or equal to 18000 ATU/mg.
  • the hirudin mutant HV2-Lys47 can be directly added to the anticoagulation blood collection tube.
  • the hirudin mutant HV2-Lys47 may be applied to the anticoagulation blood collection tube in the form of being contained in a coating.
  • the dosage of the hirudin mutant HV2-Lys47 may be 8.5ug/ml blood.
  • the hirudin mutant HV2-Lys47 may be applied to the anticoagulation blood collection tube in a form contained in the coating, and then the coating may be dried by freeze-vacuum drying .
  • the inventor of the present application found in experiments that the activity of hirudin is not lost by freeze-vacuum drying.
  • the anticoagulation blood collection tube can be used to contain blood, and the blood can be used for one of the five items of blood routine, blood biochemistry, electrolytes, tumor markers, homocysteine, and hepatitis B.
  • One or more detections due to the high purity of the hirudin mutant HV2-Lys47 of the present application, will not interfere with the detection results.
  • the present application provides an anticoagulation blood collection tube, which may have directly added hirudin mutant HV2-Lys47 or coated with a coating containing hirudin mutant HV2-Lys47.
  • the hirudin mutant HV2-Lys47 may be cultured and produced by the strain Escherichia coli pBH2 CGMCC No0908 with pBH-2 as an expression vector, and the hirudin mutant
  • the anticoagulant activity of HV2-Lys47 can be greater than or equal to 18000 ATU/mg. Among them, every 1mg is greater than or equal to 18000ATU, and the purity is greater than 95%.
  • the required addition of the hirudin mutant HV2-Lys47 is correspondingly reduced, from the original 0.01mg/ml blood to 8.5ug/ml blood.
  • the process usually used is high-temperature drying, and the loss of hirudin activity is about 10%.
  • the freeze-vacuum drying method is adopted in this application, and the activity of hirudin is not lost.
  • the hirudin mutant HV2-Lys47 used in the present application has almost no interference in the detection of blood indicators due to its high purity.
  • an anticoagulant blood collection tube containing hirudin mutant HV2-Lys47 can be used for blood routine, blood biochemistry, electrolytes, tumor markers, homocysteine And testing for various items such as five items of hepatitis B.
  • Hirudin mutant HV2-Lys47 as an anticoagulant for blood collection tubes is characterized by a small amount of anticoagulant, a fast reaction speed, and the anticoagulated plasma has fast separation of plasma and no interference with the test items, and it can maintain the blood
  • the original character, nature and shape can interfere with blood less than other anticoagulants, no hemolysis, no false thrombocytopenia, no heavy metals, impurity interference, no need to modify the value of calcium.
  • amino acid sequence of the hirudin mutant HV2-Lys47 of the present application is as follows:
  • Disulfide bond positions Cys6-Cys14; Cys16-Cys28; Cys22-Cys39.
  • the hirudin mutant HV2-Lys47 or recombinant hirudin of this application replaces the 47-position asparagine of natural hirudin with lysine.
  • Figure 1 is an electrophoresis diagram of the hirudin mutant HV2-Lys47 prepared in Example 1;
  • Fig. 2 is an HPLC chart of the hirudin mutant HV2-Lys47 prepared in Example 1, wherein the peak with a retention time of 18.991 min is the corresponding peak of the hirudin mutant HV2-Lys47.
  • Figure 3 is a sequence diagram of amino acid residues of hirudin mutant HV2-Lys47;
  • Figure 4 shows the three-dimensional structure diagram of the simulated hirudin mutant HV2-Lys47 in the biomolecule database webpage (https://www.rcsb.org/).
  • the pBH-2 engineered bacteria (preservation number: CGMCC No. 0908) glycerol preservation bacteria were inoculated into the fermentation medium at 5% of the inoculation amount.
  • the filling volume is 10ml/100ml Erlenmeyer flask, the culture temperature is 30°C, and the rotation speed is 270rpm.
  • the measured OD value of the bacteria liquid becomes stable after inoculation, that is, the logarithmic growth phase is over. It takes 3.5 hours, and then the temperature is raised to 40°C within 5 minutes and the culture is continued until it crystallizes in the microscope. After purple staining, the color of the bacteria becomes lighter, and it takes 11.5 hours.
  • the fermentation period is 15 hours.
  • the medium consists of the following composition and its pH is 7.2: glucose 10g/L, sucrose 10g/L, yeast powder 10g/L, tryptone 10g/L, ammonium chloride 0.5g/L, magnesium sulfate 0.9g/L, Dipotassium hydrogen phosphate 1g/L, sodium sulfate 5.0g/L, sodium citrate 0.87g/L, chloride 16g/L, vitamin B1 0.05mg/L, trace elements-sulfurous acid 40g/L, aluminum sulfate 28mg/L , Manganese sulfate 6.1mg/L, cobalt chloride 4mg/L, zinc chloride 0.95g/L, sodium molybdate 2.16g/L, boric acid 0.5mg/L, copper sulfate 2.93g/L, nickel nitrate 32g/L.
  • the antibiotic substance AMP 0.5g/L was added during inoculation. Among them, in this embodiment and the following embodiments
  • the hirudin mutant HV2-Lys47 was extracted and purified from the fermentation broth as follows:
  • step 2) Centrifuge the sterilized fermentation broth in step 1) through a tube centrifuge (Liaoyang Zhenxing Vacuum Equipment Factory GQB-770) to remove E. coli;
  • step 3 Pass the centrifugal supernatant of step 2) through an ultrafiltration membrane (Xiamen Sanda Membrane Technology Co., Ltd., model: NFM-84S-6/3) to remove impurities;
  • step 4) Concentrate the ultrafiltration dialysate in step 3) through a nanofiltration membrane (Xiamen Sanda Membrane Technology Co., Ltd., model: UFM-84S-2) to obtain a crude separation concentrate;
  • step 4) The crude separation concentrate obtained in step 4) is added with sodium chloride as an auxiliary material and spray-dried to obtain a dry powder, wherein the weight of the salt added as an auxiliary material in grams is the volume of the crude separation concentrate in milliliters 10%;
  • step 6) Dissolve the dry powder obtained in step 5) in water and filter out impurities to obtain a filtrate, wherein the volume of water used to dissolve the dry powder in milliliters is 10 times the weight of the dry powder in grams;
  • step 6) The filtrate in step 6) is purified by molecular sieve column chromatography (sephadex G50, manufacturer: General Electric Company (GE)) once, eluted with pure water, and the collected liquid is concentrated under reduced pressure and vacuum;
  • molecular sieve column chromatography sephadex G50, manufacturer: General Electric Company (GE)
  • step 8) Add 9 times the volume of ethanol to the concentrated solution obtained in step 7) to precipitate the hirudin mutant HV2-Lys47, and dry the precipitate under reduced pressure and vacuum.
  • Electrophoresis analysis of the above-prepared hirudin mutant HV2-Lys47 is shown in Figure 1, and the obtained electrophoresis pattern shows a single band (see the figure corresponding to hirudin 1 in Figure 1, where hirudin 1 represents It is the hirudin mutant HV2-Lys47 prepared in this application, and the molecular weight of the obtained pure recombinant hirudin is proved to be consistent with the theoretical value by electrophoresis.
  • the electrophoresis operating conditions are: concentrated gel 3% separation gel 15% loading volume 30uL (10,000 ATU/ml), first run the gel at a voltage of 100V for 1 hour, and then run the gel at a voltage of 200V for 2.2 hours.
  • the anticoagulant activity of the prepared hirudin mutant HV2-Lys47 was calculated to be 17000ATU/mg by biochemical methods.
  • specific anticoagulant activity determination please refer to the 2005 edition of "Chinese Pharmacopoeia" on page 84 Left column.
  • the purity of the hirudin mutant HV2-Lys47 calculated based on the ratio of color bands in gel electrophoresis has reached 95%.
  • Example 1 The method of Example 1 was used to prepare and purify the hirudin mutant HV2-Lys47. The difference is that in step 2), a dish centrifuge (Alfa laval stainless products skogstop: 881095-05-S/01) was used to remove E. coli The weight in grams of the salt added as an auxiliary material in step 5) is 8% of the volume in milliliters of the crudely separated concentrate.
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 15800ATU/mg.
  • the purity is about 93%.
  • Example 1 The method of Example 1 was used to prepare and purify the hirudin mutant HV2-Lys47. The difference is that in step 2), a ceramic membrane (Sanda Membrane Technology (Xiamen) Co., Ltd.) was used to remove E. coli.
  • a ceramic membrane Sanda Membrane Technology (Xiamen) Co., Ltd.
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 16800ATU/mg, and the purity reached more than 93%.
  • Example 1 The method of Example 1 was used to purify the recombinant hirudin, except that in step 2), a hollow fiber membrane (Sanda Membrane Technology (Xiamen) Co., Ltd.) was used to remove E. coli.
  • a hollow fiber membrane Sanda Membrane Technology (Xiamen) Co., Ltd.
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 16000 ATU/mg, and the purity reached more than 93%.
  • the recombinant hirudin was purified by the method of Example 1, except that in step 8), 8 times methanol was added for precipitation
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 15800ATU/mg.
  • the purity is about 93%.
  • the recombinant hirudin was purified by the method of Example 1. In step 8), 5 times acetone was added for precipitation.
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 17000 ATU/mg, and the purity reached 95%.
  • the recombinant hirudin was purified by the method of Example 1. The difference is that in step 8), ammonium sulfate with a saturation of 75% is used for precipitation, wherein the weight of ammonium sulfate in grams is the dissolved hirudin mutation 20% of the volume in milliliters of the aqueous solution of crude HV2-Lys47.
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 15000 ATU/mg.
  • the recombinant hirudin was purified by the method of Example 1, except that the sterilized fermentation broth was directly passed through the ceramic membrane for bacterial liquid separation, and the dialysate of the ceramic membrane was treated with nanofiltration membrane once, and in step 8)
  • sodium chloride is used for precipitation, wherein the weight of sodium chloride in grams is 30% of the volume in milliliters of the aqueous solution in which the crude hirudin mutant HV2-Lys47 is dissolved.
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 14500ATU/mg.
  • the recombinant hirudin was purified by the method of Example 1, except that in step 8), 9 times the volume of isopropanol was used to precipitate the hirudin mutant HV2-Lys47.
  • the obtained hirudin mutant HV2-Lys47 was analyzed by electrophoresis, and the result was the same as that in Fig. 1, and the electrophoresis showed a single band.
  • the anticoagulant activity of the obtained hirudin mutant HV2-Lys47 was 18000ATU/mg. The purity is 98%.
  • the hirudin prepared in Example 9 was dissolved in pure water, and the dissolved concentration was 3.4 g/100 ml sterile pure water. Using a 2 ml blood collection tube, spray each of the above-prepared hirudin mutant HV2-Lys47 solution with a needle spray, and then dry.
  • the drying process commonly used in this field is high-temperature drying: drying at 45°C, 50°C, and 70°C three times, each for 60 seconds;
  • the drying method of the present application the sprayed blood collection tube is dried in a freeze vacuum dryer, the temperature is -50 degrees Celsius, and the vacuum degree is -4Mpa.
  • the anticoagulant effect of the hirudin mutant HV2-Lys47 prepared in Example 9 can be achieved when the addition amount of the blood collection tube is above 0.005mg.
  • hirudin The effect of the hirudin mutant HV2-Lys47 (hereinafter referred to as hirudin) produced in Example 9 on the blood routine and blood biochemical indexes of the anticoagulant blood collection tube, wherein the hirudin mutant HV2-Lys47 was applied to the vacuum by spraying In the blood collection tube.
  • the blood routine test indexes of hirudin anticoagulation and K2 ⁇ EDTA anticoagulation vacuum blood collection tube are shown in Table 3 below. Among them, the 20 indicators of the 2 kinds of vacuum blood collection tubes were highly correlated (r value>0.8), and 4 items were moderately correlated (0.5 ⁇ r value ⁇ 0.8).
  • the five hepatitis B items are qualitative detection indicators, and the results are shown in Table 5. From the results, the serum and plasma test indicators are very consistent. The serum negative index plasma test result is also negative, and the serum positive index plasma test result is also positive.
  • results show that the results of the hirudin anticoagulation routine and the K2 ⁇ EDTA blood routine are consistent, and the detection indicators are highly or moderately correlated with the results of Menssen H (see “Measurement of Hematological, Clinical Chemistry, and Infection Parameters from Hirudinized Blood Collected in Universal Blood Sampling Tubes", Hans D. Menssen, etc., SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 27, NUMBER 4 2001, pages 349-356).
  • serum samples are commonly used in the detection of clinical biochemical indicators. In theory, compared with serum, plasma samples are closer to the physiological state.
  • K2 ⁇ EDTA and hirudin anticoagulant have similar effects, but K2 ⁇ EDTA can only measure blood routine, while hirudin blood collection tube can also measure serum and plasma biochemistry, electrolytes, tumor markers and homocysteamine Acid and other indicators.
  • the anticoagulant activity of hirudin currently on the market is: 40 ATU of hirudin is required per milliliter of blood.
  • the purity of the hirudin in this application is high, so the addition amount of hirudin is lower than that of low purity hirudin when preparing blood collection tubes.
  • the hirudin of the present application has significantly better stability.
  • the specific data are as follows:
  • Hirudin blood collection tube (2ml): (accelerated experiment temperature 40°C ⁇ 2 humidity 75% ⁇ 5)
  • the blood collection tube of the present application is the blood collection tube with the coating of the dried hirudin mutant HV2-Lys47 prepared by freeze-drying in Example 9.
  • the purchased blood collection tube is Sarstedt, Germany, which is also a hirudin blood collection tube.

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Abstract

提供了一种从大肠杆菌发酵生产水蛭素突变体HV2-Lys47的发酵液中提取纯化水蛭素突变体HV2-Lys47的方法、水蛭素突变体HV2-Lys47在抗凝采血管中的应用及包含水蛭素突变体HV2-Lys47的抗凝采血管。该纯化方法采用膜技术及一次柱层析技术，从而获得水蛭素突变体HV2-Lys47。

Description

水蛭素突变体的提取纯化方法及其应用 技术领域
本申请包括但不限于涉及生物工程技术，尤指从大肠杆菌发酵生产水蛭素突变体HV2-Lys47(SEQ ID NO.:1)的发酵液中提取纯化水蛭素突变体HV2-Lys47的方法，及由此得到的纯化的水蛭素突变体HV2-Lys47。
本申请还涉及生物医疗器械，尤指由上述方法纯化制得的水蛭素突变体HV2-Lys47在抗凝采血管中的应用。
背景技术
天然水蛭素(Hirudin)是水蛭(Leech)及其唾液腺中已提取出的多种活性成分中活性最显著并且研究得最多的一种成分，它是由65-66个氨基酸组成的小分子蛋白质(多肽)。水蛭含有丰富的水蛭素，水蛭素对凝血酶有极强的抑制作用，是迄今为止所发现最强的凝血酶天然特异抑制剂。它可用于治疗各种血栓疾病，尤其是静脉血栓和弥漫性血管凝血的治疗；也可用于外科手术后预防动脉血栓的形成，预防溶解血栓后或血管再造后血栓的形成；改善体外血液循环和血液透析过程。在显微外科手术中常因为吻合处血管栓塞而导致失败，采用水蛭素可促进伤口愈合。研究还表明，水蛭素在肿瘤治疗中也能发挥作用。它能阻止肿瘤细胞的转移，已证明有疗效的肿瘤如纤维肉瘤、骨肉瘤、血管肉瘤、黑素瘤和白血病等。水蛭素还可配合化学治疗和放射治疗，归因于可促进肿瘤中的血流而使疗效增强。
由于天然水蛭素产能较低，临床应该量大，国内外开展了大量重组水蛭素研究，自上个世纪八十年代末研制出与天然水蛭素结构相似、作用相同的重组水蛭素，产能大。
然而，现有蛋白和肽类的纯化基本都需要两种或三种以上的多次层析分离才能拿到纯品，并且洗脱剂包含大量的水和盐，对环境污染大，污水处理工程压力大。
目前市场所用的水蛭素突变体的抗凝活性为16000ATU/mg，纯度为93％左右。
比如，陈华友等(安徽农业科学，2009年，37(34)；第16757-16759页及第16768页)公开了发酵液经离心、三氯乙酸处理、超滤浓缩脱盐，再上阴离子交换柱，S100分子筛柱，最后得到95％的纯品；韦利军等人(2004年全国生物技术学术研讨会论文集，第104-112页)公开了使用大孔树脂层析、DEAE层析、反相色谱层析的三次层析法分离得到95％纯品。其中，层析步骤越多，成本越高且越费时，环境污染也越高。
因此，存在对工艺简洁、废水少、适合工业放大的水蛭素突变体HV2-Lys47的提取纯化方法的需求。
此外，血液标本的采集和分离在临床生化检测中至关重要，是保证质量的一个重要环节。传统的标本采集从开始采血到血清析出上机检测，往往需要较长时间，使全自动生化分析仪的高效性受到限制。部分生化实验室使用肝素抗凝血浆，取血后可以立即离心分离出血浆上机检测，但无论肝素钠还是肝素锂抗凝血浆，对某些生化指标都会产生影响。
1997年我国开始推广真空采血技术，是对传统采血方式的一次重大改进。由于是在全封闭系统下完成采血程序，因而从根本上排除了血液污染和交叉感染的可能性，采血更安全、更准确、更规范，因而易于普及和推广。在真空采血管内可加入分离胶、促凝剂，以及各种抗凝剂，以管帽的颜色来区分不同用途的采血管。
近年来，含促凝剂和分离胶的真空采血管在生化检验被广泛使用，它大大缩短了血液凝固的时间，一般放置20min后就可进行离心分离出血清，但有一定比例的采血管离心效果不佳，血清中存在蛋白凝丝或凝块。分离胶是一种由数种化合物组成的半固体惰性胶，不影响血清各成份的含量，但促凝管因促凝剂的原料产地、性质、制作工艺不同，可能对生化检测造成不同的影响。
天然水蛭素是从吸血水蛭(俗称蚂蟥)的唾液腺中提取、分离得到的一种多肽，是水蛭体内的重要活性成分，但产量极其有限，不能满足临床应用的要求。
因此，存在抗凝活性更佳的、来源更稳定的抗凝剂需求。
发明概述
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本申请提供了一种工艺简洁、废水少、适合工业放大的水蛭素突变体HV2-Lys47的提取纯化方法。
在本申请的实施方案中，本申请提供了一种从大肠杆菌发酵生产水蛭素突变体HV2-Lys47(SEQ ID NO.:1)的发酵液中提取纯化水蛭素突变体HV2-Lys47的方法，所述方法包括：
在升高的温度下处理大肠杆菌发酵液，以灭菌且去除杂蛋白；
通过陶瓷膜或通过离心机来处理灭菌的发酵液，以去除菌体；
可选地，通过超滤膜来处理通过陶瓷膜或离心机的发酵液，以去除杂蛋白；
通过纳滤膜来处理通过陶瓷膜或离心机的发酵液、或通过超滤膜的发酵液，以获得粗分离的浓缩液；
将盐添加到所述粗分离的浓缩液中作为辅料，喷雾干燥，得到干粉；
将所述干粉溶解在水中并滤除杂质，可选地通过滤纸、砂芯漏斗或膜设备来进行过滤；
使滤液仅通过分子筛柱层析1次，用水洗脱，浓缩收集液，得到水蛭素突变体HV2-Lys47粗品；
将所述水蛭素突变体HV2-Lys47粗品溶解到水中，然后向其中添加盐或有机溶剂，以沉淀出水蛭素突变体HV2-Lys47，干燥即得水蛭素突变体HV2-Lys47纯品。
在以上或其他实施方式中，升高的温度可以是能够灭菌的高温，比如65℃、75℃的温度等。
在以上或其他实施方式中，所述干燥可以为真空干燥。
在以上或其他实施方式中，所述干燥可以为冷冻真空干燥。
在以上或其他实施方式中，通过离心机来处理灭菌的发酵液。
在以上或其他实施方式中，离心机可以为管式离心机或碟式离心机。
其中，陶瓷膜处理或离心机处理的目的是为了去除菌体，超滤膜处理的目的是去除杂蛋白，且纳滤膜处理的目的是进行浓缩。
在以上或其他实施方式中，所述高温处理可以是在65℃-67℃下保持5-20分钟，可选地保持10分钟。
在以上或其他实施方式中，所述盐可以选自氯化钠、氯化钾、硫酸铵、硫酸钠或硫酸钾中的一种或多种。
在以上或其他实施方式中，所述分子筛柱层析可以选自由sephadex G25、sephadex G50、sephadex G75和sephadex G100组成的组。本领域技术人员将理解的是，上述这些分子筛柱层析均为市售产品。其中，使用分子筛柱层析的目的主要是除去色素、盐及分子量离水蛭素较远的多肽和多糖。
在以上或其他实施方式中，经由分子筛柱层析得到的收集液的浓缩可以通过减压真空浓缩、纳滤膜浓缩或反渗透膜浓缩来进行。
在以上或其他实施方式中，所述有机溶剂可以选自乙醇、甲醇、丙酮、异丙醇或乙腈中的一种或更多种。
在以上或其他实施方式中，作为辅料添加的盐的以克计的重量为基于粗分离的浓缩液的以毫升计的体积的5％或更大，可选地10％或更大。本申请的发明人发现，如上以重量体积分数5％或更大的量添加盐就可以达到本申请的纯化目的，加盐少于5％水蛭素损失大，而加盐过多成本过高。
在以上或其他实施方式中，用于溶解所述干粉的水的以毫升计的体积为所述干粉的以克计的重量的10倍以上。其中，选择用一定量的水来溶解干粉的目的是为了保持盐的以克计的重量为基于水溶液的以毫升计的体积的5％或更小，从而使得不溶于水的部分杂蛋白可以通过过滤除去。
在以上或其他实施方式中，为了使水蛭素突变体HV2-Lys47沉淀，所添加的盐的以克计的重量为溶解有水蛭素突变体HV2-Lys47粗品的水溶液的以毫升计的体积的20％-30％，所添加的有机溶剂的体积为溶解有水蛭素突变体HV2-Lys47粗品的水溶液的体积的5-9倍。
其中，使重组水蛭素重新沉淀出的步骤是为了除去分子量离水蛭素较近 的多糖和多肽。
在以上或其他实施方式中，所述大肠杆菌发酵液可以是通过以pBH-2为表达载体的菌株大肠埃希氏菌(Escherichia coli)pBH2 CGMCC No0908培养生产的。
在以上或其他实施方式中，所述培养包括在需氧条件下，在25℃-35℃的温度下培养至对数生长期结束，然后升温至35℃-45℃继续培养至水蛭素突变体HV2-Lys47的产量达到峰值。
在本申请中，水蛭素突变体HV2-Lys47的发酵液的获得可参考申请号为201711262810.4的专利申请的生产方法，其中申请号为201711262810.4的专利申请在此以引用的方式整体并入，其中本申请的水蛭素突变体HV2-Lys47在申请号为201711262810.4的专利申请中被称为“重组水蛭素”。因此，在本文中，“水蛭素突变体HV2-Lys47”与“重组水蛭素”代表的是同一物质，可以互换使用。
与现有技术相比，本申请采用膜技术及一次柱层析技术，且只采用水洗脱，工艺简洁，废水少，适合工业放大生产水蛭素突变体HV2-Lys47。
在另一方面，本申请提供一种根据以上所述的任一方法生产的水蛭素突变体HV2-Lys47。
在另外的方面，本申请提供一种根据以上所述的任一方法生产的水蛭素突变体HV2-Lys47，其中所述水蛭素突变体HV2-Lys47的抗凝活性可以为大于或等于18000ATU/mg。本领域已知的是，抗凝活性跟纯度不同，抗凝活性是指单位毫克蛋白质的生物学活性单位，纯度高，抗凝活性不一定高，因为活性蛋白可能由于构象的细微变化引起活性降低。
在又一方面，本申请提供水蛭素突变体HV2-Lys47在抗凝采血管中的应用。
在以上或其他实施方式中，所述水蛭素突变体HV2-Lys47的抗凝活性可以为大于或等于18000ATU/mg。
在以上或其他实施方式中，所述水蛭素突变体HV2-Lys47可以是以pBH-2为表达载体的菌株大肠埃希氏菌(Escherichia coli)pBH2 CGMCC  No0908培养生产的，所述水蛭素突变体HV2-Lys47的抗凝活性可以为大于或等于18000ATU/mg。
在以上或其他实施方式中，所述水蛭素突变体HV2-Lys47可以被直接添加到所述抗凝采血管中。
在以上或其他实施方式中，所述水蛭素突变体HV2-Lys47可以以包含在涂层中的形式被应用到所述抗凝采血管中。
在以上或其他实施方式中，所述水蛭素突变体HV2-Lys47的用量可以为8.5ug/ml血液。
在以上或其他实施方式中，可以将水蛭素突变体HV2-Lys47以包含在涂层中的形式被涂覆到所述抗凝采血管中，然后采用冷冻真空干燥的方式来干燥所述涂层。本申请的发明人在实验中发现，采用冷冻真空干燥的方式，水蛭素活性不损失。
在以上或其他实施方式中，所述抗凝采血管可以用于盛装血液，所述血液可以用于血常规、血液生化、电解质、肿瘤标志物、同型半胱氨酸以及乙肝五项中的一种或更多种的检测，由于本申请的水蛭素突变体HV2-Lys47的高纯度，因此对检测结果无干扰。
在另外的方面，本申请提供一种抗凝采血管，所述抗凝采血管可以具有直接添加的水蛭素突变体HV2-Lys47或涂覆有包含水蛭素突变体HV2-Lys47的涂层。
在以上或其他实施方式中，所述水蛭素突变体HV2-Lys47可以是以pBH-2为表达载体的菌株大肠埃希氏菌(Escherichia coli)pBH2 CGMCC No0908培养生产的，所述水蛭素突变体HV2-Lys47的抗凝活性可以为大于或等于18000ATU/mg。其中，每1mg大于等于18000ATU，纯度大于95％。
在本申请的抗凝采血管中，所需添加的水蛭素突变体HV2-Lys47相应减少，由原来的0.01mg/ml血液，减少到8.5ug/ml血液。在采血管生产加工过程中，通常所用工艺为高温干燥，水蛭素活性损失在10％左右，本申请采用冷冻真空干燥方法，水蛭素活性不损失。并且，本申请所用的水蛭素突变体HV2-Lys47由于其高纯度，所以在血液指标检测的时候几乎无干扰。
本申请的发明人通过大量的临床实验研究发现，可以采用一种含有水蛭素突变体HV2-Lys47的抗凝采血管，用于血常规、血液生化、电解质、肿瘤标志物、同型半胱氨酸以及乙肝五项等多种项目的检测。用水蛭素突变体HV2-Lys47作为采血管抗凝剂的特点是抗凝剂用量少、反应速度快、所抗凝的血浆具有分离的快血浆并对检测项目无干扰，更能够保持血液的原始性状、性质跟形态、能够比其他抗凝剂对血液的干扰更小、无溶血现象、无假性血小板减少现象、无重金属、杂质干扰、不需修正钙质的数值。
并且，实验证明，采用水蛭素突变体HV2-Lys47作为抗凝剂，可以用一个管来取代几种颜色的真空采血管，彻底减轻医护人员每次需要分辨采血管的颜色来确定该采血样品的预期用途的负担，从而减轻医护人员的劳动强度和误差。而且还可以减少患者的采血量以及减少医用垃圾的产生，具有重大的社会经济效益。
其中，本申请的水蛭素突变体HV2-Lys47的氨基酸序列如下所示：
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)
二硫键位置：Cys6-Cys14；Cys16-Cys28；Cys22–Cys39。
与天然水蛭素相比，本申请的水蛭素突变体HV2-Lys47或重组水蛭素是把天然水蛭素的47位天冬酰胺用赖氨酸取代。
在阅读并理解了附图和详细描述后，可以明白其他方面。
附图概述
附图用来提供对本申请技术方案的进一步理解，并且构成说明书的一部分，与本申请的实施例一起用于解释本申请的技术方案，并不构成对本申请技术方案的限制。
图1为实施例1制得的水蛭素突变体HV2-Lys47的电泳图；
图2为实施例1制得的水蛭素突变体HV2-Lys47的HPLC图，其中保留时间为18.991min的峰为水蛭素突变体HV2-Lys47的对应峰。
图3为水蛭素突变体HV2-Lys47的氨基酸残基序列图；
图4为在生物分子数据库网页(https://www.rcsb.org/)里模拟水蛭素突变体HV2-Lys47的三维结构图。
详述
为使本申请的目的、技术方案和优点更加清楚明白，下文中将结合附图对本申请的实施例进行详细说明。需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互任意组合。
实施例1
制备发酵液：
将pBH-2工程菌(保藏号：CGMCC No.0908)甘油保藏菌按接种量5％接种到发酵培养基中。装量为10ml/100ml三角瓶、培养温度为30℃、转速270rpm。在需氧条件下，接种后发酵至测得的菌液的OD值趋于稳定，即对数生长期结束，用时3.5小时，然后在5分钟内升温至40℃继续培养至显微镜镜检中结晶紫染色后菌体着色变浅，用时11.5小时。发酵周期为15小时。
具体操作请参见申请号为201711262810.4的实施例2。
其中，培养基由如下组成且其pH为7.2：葡萄糖10g/L、蔗糖10g/L、酵母粉10g/L、胰蛋白胨10g/L、氯化铵0.5g/L、硫酸镁0.9g/L、磷酸氢二钾1g/L、硫酸钠5.0g/L、柠檬酸钠0.87g/L、氯化16g/L、维生素B1 0.05mg/L，微量元素-硫酸亚40g/L、硫酸铝28mg/L、硫酸锰6.1mg/L、氯化钴4mg/L、氯化锌0.95g/L、钼酸钠2.16g/L、硼酸0.5mg/L、硫酸铜2.93g/L、硝酸镍32g/L。接种时加入抗生素物质AMP0.5g/L。其中，在本实施例及以下的实施例中，g/L中的L是最终发酵培养基的体积。
其中物料的来源如下表所示：
Figure PCTCN2019082785-appb-000001
Figure PCTCN2019082785-appb-000002
通过以下的步骤从上述发酵液中提取纯化水蛭素突变体HV2-Lys47：
1)发酵液经65℃、5min高温灭菌除杂；
2)将步骤1)中的灭菌发酵液经管式离心机(辽阳振兴真空设备厂GQB-770)离心去除大肠杆菌；
3)将步骤2)的离心清液通过超滤膜(厦门三达膜科技有限公司，型号：NFM-84S-6/3)除杂蛋白；
4)将步骤3)中的超滤透析液经纳滤膜(厦门三达膜科技有限公司，型号：UFM-84S-2)浓缩得到粗分离浓缩液；
5)将步骤4)所得粗分离浓缩液，加入氯化钠作为辅料，喷雾干燥，得到干粉，其中作为辅料添加的盐的以克计的重量为粗分离的浓缩液的以毫升计的体积的10％；
6)将步骤5)所得干粉溶解在水中并滤除杂质，得滤液，其中用于溶解所述干粉的水的以毫升计的体积为所述干粉的以克计的重量的10倍；
7)使步骤6)中的滤液经过分子筛柱层析(sephadex G50，生产厂家：General Electric Company(GE))纯化1次，用纯水洗脱，收集液减压真空浓缩；
8)将步骤7)得到的浓缩液，加入9倍体积乙醇，使水蛭素突变体HV2-Lys47沉淀，使沉淀物经过减压真空干燥即得。
对上述制得的水蛭素突变体HV2-Lys47进行电泳分析，如图1所示，得到的电泳图中呈现出单一条带(参见图1中水蛭素1所对应的图形，其中水蛭素1代表的是本申请制得的水蛭素突变体HV2-Lys47)，且通过电泳法证明所得的重组水蛭素纯品的分子量与理论值相一致。
其中，电泳操作条件为：浓缩胶3％分离胶15％上样量30uL(1万ATU/ml)，先电压100v跑胶1h，再电压200V跑胶2.2h。
电泳具体操作如下所示：
1、安装预制胶在min电泳仪中，加入SDS-PAGE电泳缓冲液，没过胶顶部，拔出样品槽梳齿。
2、取样品40uL，加入10uL的5×蛋白上样缓冲液，混匀后100℃水浴5min，取40uL加入胶上的样品槽中。
3、上样完毕后，连接电泳仪电源，设置电压150V恒压，时间50min左右，溴酚蓝线至胶底部则电泳结束。
4、固定20min，染色20min，脱色2h后可以观察结果。
经过抗凝活性分析，制得的水蛭素突变体HV2-Lys47的抗凝活性通过生物化学方法测定计算为17000ATU/mg，具体的抗凝活性的测定请参见2005年版的《中国药典》第84页左栏。并且，根据凝胶电泳中色带的比例算出来的水蛭素突变体HV2-Lys47的纯度已经达到95％。
此外，经过HPLC测定，证明所制得的水蛭素突变体HV2-Lys47中确实存在显著较大的水蛭素突变体HV2-Lys47的峰，如图2所示。
实施例2
采用实施例1的方法制备且纯化水蛭素突变体HV2-Lys47，不同的是，在步骤2)中，采用碟式离心机(Alfa laval stainless products skogstop：881095-05-S/01)去除大肠杆菌，步骤5)中作为辅料添加的盐的以克计的重量为粗分离的浓缩液的以毫升计的体积的8％。
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为15800ATU/mg。纯度约93％。
实施例3
采用实施例1的方法制备且纯化水蛭素突变体HV2-Lys47，不同的是，在步骤2)中，采用陶瓷膜(三达膜科技(厦门)有限公司)去除大肠杆菌。
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为16800ATU/mg，纯度达到93％以上。
实施例4
采用实施例1的方法进行提纯重组水蛭素，不同的是，在步骤2)中，采用中空纤维膜(三达膜科技(厦门)有限公司)去除大肠杆菌。
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为16000ATU/mg，纯度达到93％以上。
实施例5
采用实施例1的方法进行提纯重组水蛭素，不同的是，在步骤8)中，加入8倍甲醇进行沉淀
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为15800ATU/mg。纯度约93％。
实施例6
采用实施例1的方法进行提纯重组水蛭素，在步骤8)中，加入5倍丙酮进行沉淀。
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为17000ATU/mg，纯度达到95％。
实施例7
采用实施例1的方法进行提纯重组水蛭素，不同的是，在步骤8)中，采用饱和度为75％的硫酸铵盐进行沉淀，其中硫酸铵的以克计的重量为溶解有水蛭素突变体HV2-Lys47粗品的水溶液的以毫升计的体积的20％。
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为15000ATU/mg。
实施例8
采用实施例1的方法进行提纯重组水蛭素，不同的是，直接将灭菌的发酵液通过陶瓷膜进行菌液分离，陶瓷膜的透析液再用纳滤膜处理1次，而且在步骤8)中，采用氯化钠进行沉淀，其中氯化钠的以克计的重量为溶解有 水蛭素突变体HV2-Lys47粗品的水溶液的以毫升计的体积的30％。
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为14500ATU/mg。
实施例9
采用实施例1的方法进行提纯重组水蛭素，不同的是，步骤8)中，采用9倍体积异丙醇，使水蛭素突变体HV2-Lys47沉淀。
对得到的水蛭素突变体HV2-Lys47进行电泳分析，结果与图1相同，其中电泳单显示一条带。
经测试，得到的水蛭素突变体HV2-Lys47的抗凝活性为18000ATU/mg。纯度为98％。
实施例10
干燥工艺对在抗凝采血管中的水蛭素突变体HV2-Lys47活性的影响：
将实施例9所制得的水蛭素溶解在纯水中，溶解浓度为3.4g/100ml无菌纯水。采用2ml的采血管，采用针头喷雾将每只喷涂上述制得的水蛭素突变体HV2-Lys47的溶液，然后干燥。
本领域常用的干燥工艺为高温干燥：分别在45℃、50℃、70℃干燥三次，每次60秒；
本申请的干燥方法：喷涂后的采血管于冷冻真空干燥机中干燥，温度为-50摄氏度、真空度为-4Mpa。
高温干燥与冷冻真空干燥方法对水蛭素突变体HV2-Lys47活性影响如下表1所示。
表1
Figure PCTCN2019082785-appb-000003
Figure PCTCN2019082785-appb-000004
从表中可知，真空冷冻干燥时，涂覆的水蛭素突变体HV2-Lys47的活性损失最小。
实施例11
实施例9所制得的通过真空冷冻干燥的水蛭素突变体HV2-Lys47的用量对血液凝固的影响如下表2所示的：
表2
水蛭素突变体HV2-Lys47用量(mg) 采血量(ml) 凝固现象
0.001 2 很快凝固
0.005 2 无凝固
0.008 2 无凝固
0.02 2 无凝固
0.08 2 无凝固
0.16 2 无凝固
0.5 2 无凝固
1 2 无凝固
5 2 无凝固
10 2 无凝固
15 2 无凝固
20 2 无凝固
根据上表可知，实施例9所制得的水蛭素突变体HV2-Lys47在采血管中的添加量在0.005mg以上就可以达到抗凝血效果。
实施例12
实施例9所制得的水蛭素突变体HV2-Lys47(以下简称水蛭素)抗凝采血管对血常规和血液生化指标的影响，其中水蛭素突变体HV2-Lys47是采用喷涂的方式应用至真空采血管中。
(一)血常规
水蛭素抗凝和K2·EDTA抗凝真空采血管对血常规检测指标如下表3所示。其中，2种真空采血管的20项指标间呈高度相关性(r值>0.8)，4项呈中度相关(0.5<r值<0.8)。
表3.水蛭素抗凝血和K2·EDTA抗凝血血常规比较
Figure PCTCN2019082785-appb-000005
Figure PCTCN2019082785-appb-000006
(二)血液生化、肿瘤标志物及同型半胱氨酸
血清(分离胶/二氧化硅真空采血管)和血浆(水蛭素真空采血管)生化、肿瘤标志物及同型半胱氨酸检测结果见表4。2种真空采血管22项指标间呈高度相关性(r值>0.8)，2项呈中度相关(0.5<r值<0.8)。
表4.水蛭素抗凝血浆和常规血清生化、肿瘤标志物及同型半胱氨酸比较
Figure PCTCN2019082785-appb-000007
Figure PCTCN2019082785-appb-000008
(三)乙肝五项
乙肝五项为定性检测指标，结果如表5所示。从结果来看血清和血浆检测指标非常一致，血清阴性指标血浆检测结果亦为阴性，血清阳性指标血浆检测结果亦为阳性。
表5
Figure PCTCN2019082785-appb-000009
Figure PCTCN2019082785-appb-000010
结果表明水蛭素抗凝血血常规和K2·EDTA血常规结果一致，检测指标呈高度或者中度相关性，与Menssen H的结果(参见“Measurement of Hematological,Clinical Chemistry,and Infection Parameters from Hirudinized Blood Collected in Universal Blood Sampling Tubes”,Hans D.Menssen等，SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 27,NUMBER 4 2001，第349-356页)一致。目前临床生化指标的检测常用血清样品，从理论上讲与血清相比，血浆样品更接近于生理状态。结果表明除了总蛋白、 白蛋白和球蛋白的相关性较差((0.5<r值<0.8))外，血清和血浆生化、电解质、肿瘤标志物及同型半胱氨酸高度相关。(r值>0.9)。由于血浆中含有纤维蛋白原使得血浆中蛋白浓度(75.07±4.31)高于血清(70.89±3.48)。
从如上可知，K2·EDTA与水蛭素抗凝剂的效果相近，但是K2·EDTA只能测定血常规，而水蛭素采血管还可以测定血清和血浆生化、电解质、肿瘤标志物及同型半胱氨酸等指标。
此外，目前市场上的水蛭素的抗凝血活性为：每毫升血液需要40ATU的水蛭素，本申请的水蛭素纯度高，因此在制备采血管时添加量比纯度低的水蛭素添加量低，同时，因为纯度高，因此避免了因为杂质的存在对血液凝固以及血液指标检测的影响。并且，由于纯度高，本申请的水蛭素具有显著更好的稳定性，具体数据如下：
水蛭素采血管(2ml)：(加速实验温度40℃±2 湿度75％±5)
检测周期 0周 2周 1个月 2个月 3个月 6个月
本申请采血管 2625ATU 2650ATU 2625ATU 2580ATU 2600ATU 2575ATU
外购采血管 3020ATU 2800ATU 2548ATU 1890ATU 1600ATU 1500ATU
其中本申请采血管为实施例9中具有通过冷冻干燥所制得的干燥的水蛭素突变体HV2-Lys47的涂层的采血管。外购采血管为：德国Sarstedt，也是水蛭素采血管。
本领域的普通技术人员应当理解，可以对本申请的技术方案进行修改或者等同替换，而不脱离本申请技术方案的精神和范围，均应涵盖在本申请的权利要求范围当中。

Claims (24)

  1. 一种从大肠杆菌发酵生产水蛭素突变体HV2-Lys47(SEQ ID NO.:1)的发酵液中提取纯化水蛭素突变体HV2-Lys47的方法，包括以下步骤：
    在升高的温度下处理大肠杆菌发酵液，以灭菌且去除杂蛋白；
    通过陶瓷膜或通过离心机来处理灭菌的发酵液，以去除菌体；
    可选地，通过超滤膜来处理通过陶瓷膜或离心机的发酵液，以去除杂蛋白；
    通过纳滤膜来处理通过陶瓷膜或离心机的发酵液、或通过超滤膜的发酵液，以获得粗分离的浓缩液；
    将盐添加到所述粗分离的浓缩液中作为辅料，喷雾干燥，得到干粉；
    将所述干粉溶解在水中并过滤除杂，可选地通过滤纸、砂芯漏斗或膜设备来进行过滤；
    使滤液仅通过分子筛柱层析1次，用水洗脱，浓缩收集液，得到水蛭素突变体HV2-Lys47粗品；
    将所述水蛭素突变体HV2-Lys47粗品溶解到水中，然后向其中添加盐或有机溶剂，以沉淀出水蛭素突变体HV2-Lys47，干燥即得水蛭素突变体HV2-Lys47纯品，可选地进行真空干燥，更可选地进行冷冻真空干燥。
  2. 根据权利要求1所述的方法，其中，通过离心机来处理高温灭菌的发酵液，可选地所述离心机为管式离心机或碟式离心机。
  3. 根据权利要求1所述的方法，其中，所述高温处理是在65℃-67℃下保持5-20分钟，可选地10分钟。
  4. 根据权利要求1所述的方法，其中，所述盐选自氯化钠、氯化钾、硫酸铵、硫酸钠、硫酸钾中的一种或更多种。
  5. 根据权利要求1所述的方法，其中，所述分子筛柱层析选自由sephadex G25、sephadex G50、sephadex G75和sephadex G100组成的组。
  6. 根据权利要求1所述的方法，其中，浓缩经由分子筛柱层析得到的收集液是通过减压真空浓缩、纳滤膜浓缩或反渗透膜浓缩来进行的。
  7. 根据权利要求1所述的方法，其中，所述有机溶剂选自乙醇、甲醇、丙酮、异丙醇或乙腈中的一种或更多种。
  8. 根据权利要求1-7中任一项所述的方法，其中，作为辅料添加的盐的以克计的重量为粗分离的浓缩液的以毫升计的体积的5％或更大。
  9. 根据权利要求1-7中任一项所述的方法，其中，用于溶解所述干粉的水的以毫升计的体积为所述干粉的以克计的重量的10倍以上。
  10. 根据权利要求1-7中任一项所述的方法，其中，为了使水蛭素突变体HV2-Lys47沉淀，所添加的盐的以克计的重量为溶解有水蛭素突变体HV2-Lys47粗品的水溶液的以毫升计的体积的20％-30％，所添加的有机溶剂的体积为溶解有水蛭素突变体HV2-Lys47粗品的水溶液的体积的5-9倍。
  11. 根据权利要求1-7中任一项所述的方法，其中所述大肠杆菌发酵液是通过以pBH-2为表达载体的菌株大肠埃希氏菌(Escherichia coli)pBH2CGMCC No0908培养生产的，可选地，所述培养包括在需氧条件下，在25℃-35℃的温度下培养至对数生长期结束，然后升温至35℃-45℃继续培养至水蛭素突变体HV2-Lys47的产量达到峰值。
  12. 一种水蛭素突变体HV2-Lys47，所述水蛭素突变体HV2-Lys47根据权利要求1-11中任一项所述的方法来生产。
  13. 如权利要求12所述的水蛭素突变体HV2-Lys47，所述水蛭素突变体HV2-Lys47的抗凝活性为大于或等于18000ATU/mg。
  14. 水蛭素突变体HV2-Lys47(SEQ ID NO.:1)在抗凝采血管中的应用。
  15. 根据权利要求14所述的应用，所述水蛭素突变体HV2-Lys47的抗凝活性为大于或等于18000ATU/mg。
  16. 根据权利要求14所述的应用，其中所述水蛭素突变体HV2-Lys47被直接添加到所述抗凝采血管中。
  17. 根据权利要求14所述的应用，其中所述水蛭素突变体HV2-Lys47以包含在涂层中的形式被应用到所述抗凝采血管中。
  18. 根据权利要求16或17所述的应用，其中所述水蛭素突变体HV2-Lys47的用量为8.5μg/ml血液。
  19. 根据权利要求17所述的应用，其中将水蛭素突变体HV2-Lys47以包含在涂层中的形式被涂覆到所述抗凝采血管中，然后采用冷冻真空干燥的方式来干燥所述涂层。
  20. 根据权利要求14所述的应用，其中所述抗凝采血管用于盛装血液，所述血液用于血常规、血液生化、电解质、肿瘤标志物、同型半胱氨酸以及乙肝五项中的一种或更多种的检测。
  21. 根据权利要求14-20中任一项所述的应用，其中所述水蛭素突变体HV2-Lys47是通过权利要求1-11中任一项所述的方法来制备的，或所述水蛭素突变体HV2-Lys47是如权利要求13所述的水蛭素突变体HV2-Lys47。
  22. 一种抗凝采血管，所述抗凝采血管具有直接添加的水蛭素突变体HV2-Lys47(SEQ ID NO.:1)或涂覆有包含水蛭素突变体HV2-Lys47(SEQ ID NO.:1)的涂层。
  23. 根据权利要求22所述的抗凝采血管，所述水蛭素突变体HV2-Lys47的抗凝活性为大于或等于18000ATU/mg。
  24. 根据权利要求22或23所述的抗凝采血管，其中所述水蛭素突变体HV2-Lys47是根据权利要求1-11中任一项所述的方法生产的水蛭素突变体HV2-Lys47，或所述水蛭素突变体HV2-Lys47是如权利要求13所述的水蛭素突变体HV2-Lys47。
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