Method for separating and purifying hirudin based on thrombin affinity magnetic microspheres and application thereof

Abstract

The present application belongs to the technical field of biological medicine separation and purification, and particularly relates to a leech-derived hirudin separation and purification method based on thrombin affinity magnetic microspheres and application thereof. The present application uses self-prepared leaching liquor to coarsely extract hirudin from leech powder, then selects carboxyl magnetic microspheres to prepare thrombin affinity magnetic microspheres, and further separates and purifies by using the thrombin affinity magnetic microspheres, so as to finally obtain natural hirudin with an activity of 17000 ATU / g or more. The self-prepared leaching liquor can destroy the cell wall of leech, so that the hirudin in the leech is fully dissolved, thereby providing higher efficiency for the coarse extraction of hirudin in leech powder. The prepared thrombin affinity magnetic microspheres can fully utilize the sample enrichment effect thereof, and compared with agarose microspheres and polymer microspheres, can separate and purify hirudin with higher activity and purity, has a long storage period and stable quality, can provide safe and high-quality raw materials for health food, medicine or cosmetics, and is beneficial to industrialized production and application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine separation and purification, and relates to extraction of animal effective components, in particular to a leech antithrombin separation and purification method based on thrombin affinity magnetic microspheres and application thereof. BACKGROUND

[0002] Leech antithrombin is a small molecule acid single-chain polypeptide composed of 65-66 amino acid residues and free of glycosylation, which is prepared by separation and purification from leech salivary glands. Leech antithrombin has highly specific antithrombin activity and can inhibit thrombin binding to substrates. It is the strongest thrombin-specific inhibitor discovered so far, can improve blood microcirculation, and has good anticoagulant and blood stasis effects. It has significant efficacy on human cardiovascular and cerebrovascular diseases, especially cerebral thrombosis and other thrombotic diseases. In addition, leech antithrombin also has strong antioxidant properties, can assist immune cells in recognizing, phagocytizing, encapsulating and eliminating free radicals, and prevent skin from being oxidized to produce fine lines, color spots, dryness and other signs of aging. Compared with natural leech antithrombin, recombinant leech antithrombin is not sulfated at the 63rd amino acid (tyrosine), and has slightly lower activity, with an inhibition effect on thrombin only 1 / 10 of that of natural leech antithrombin. Therefore, natural leech antithrombin has great application prospects in the fields of medicine, health products and cosmetics.

[0003] Currently, natural leech antithrombin is mostly extracted from live leeches. For example, the patent with publication number CN 102286108 A provides a production method of natural leech antithrombin, which first extracts natural leech antithrombin crude product from live blood-sucking leeches, and then obtains natural leech antithrombin powder through separation and purification. The patent with publication number CN 108727487 A provides a liquid membrane extraction method of leech antithrombin, which grinds frozen leeches to prepare leech antithrombin crude extract. The patent with publication number CN 102964446 A provides a method for multiple extraction of natural leech antithrombin from live blood-sucking leeches. However, most of the methods for extraction from live leeches are complicated, time-consuming and not conducive to industrial production. Meanwhile, the activity of the extracted leech antithrombin is not high, with a value of about 150-6000 ATU / g, and the impurity content is also high, which is not suitable for injection drugs. The patent with publication number CN 107936115 A provides a leech antithrombin extraction method, which extracts leech antithrombin from leech powder and is simple to operate, but the activity of the obtained leech antithrombin is still low. Therefore, it is of great significance to develop a leech antithrombin extraction method with simple steps and higher activity of leech antithrombin for further research and application of leech antithrombin. SUMMARY

[0004] In view of the technical problems in the background art, the present application provides a leech-derived hirudin separation and purification method based on thrombin affinity magnetic microspheres and an application thereof, which extracts high-quality natural hirudin with high activity, is simple to operate, low in cost, and can realize rapid industrialized production.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] The leech-derived hirudin separation and purification method based on thrombin affinity magnetic microspheres comprises the following steps:

[0007] (1) Coarsely extracting hirudin from leech powder: adding a leaching solution in an amount of 2-6 times the weight of the leech powder, soaking, ultrasonicating, centrifuging to obtain supernatant, repeatedly extracting the precipitate twice, collecting all the supernatant, and adjusting the pH to 4.5; using the difference in solubility of different proteins at different pH values to precipitate and remove part of the impure proteins, and then centrifuging to obtain supernatant, and adjusting the pH to 7.0 to prepare for the binding of hirudin and magnetic microspheres under neutral conditions.

[0008] (2) Preparing thrombin affinity magnetic microspheres: mixing thrombin and carboxyl magnetic microspheres, then adding ethanolamine for further reaction, separating and removing the liquid, and washing to obtain thrombin affinity magnetic microspheres; wherein, the ethanolamine is added to shield the carboxyl groups on the surface of the magnetic microspheres that have not been completely reacted, so as to prevent the adsorption of impure proteins in the leech crude extract and cause the purity of hirudin to decrease.

[0009] (3) Separating and purifying thrombin affinity magnetic microspheres: adding the thrombin affinity magnetic microspheres prepared in step (2) into the hirudin crude extract of step (1) and mixing uniformly, then standing, then removing the supernatant by magnetic separation, then sequentially washing and eluting, and collecting the eluate.

[0010] (4) Desalting and concentrating: dialyzing the eluate of step (3), changing the water 2-8 times in the middle, with an interval of not less than 2 h each time; concentrating the eluate after dialysis by rotary evaporation (40-70℃, 100-130r / min), removing 30-80% of the water, and then storing the concentrated liquid in a refrigerator at -8℃ for standby.

[0011] (5) Freezing and drying the concentrated liquid of step (4) by using a freeze dryer to obtain powder-state hirudin, and collecting and sealing the hirudin for storage.

[0012] Preferably, the amount of the leaching solution used in step (1) is 2-4 times the weight of the leech powder; and the composition of the leaching solution is 50mM Tris (tris(hydroxymethyl) aminomethane), 10mM EDTA (ethylenediaminetetraacetic acid), 150mM sodium chloride, 3% SDS (sodium dodecyl sulfate), 2% Triton X-100, and 2% deoxycholic acid sodium.

[0013] Preferably, the thrombin and ethanolamine in the step (2) are dissolved in MES buffer solution with a concentration of 50 mM / L and pH 5.5 in advance; the carboxyl magnetic microspheres are activated by an activator EDC / Sulfo-NHS in advance; the thrombin is reacted with the carboxyl magnetic microspheres at room temperature for 2-3 h, and then the ethanolamine is added to continue the reaction at room temperature for 1-1.5 h.

[0014] More preferably, the activity of the thrombin is 2 ku / mg, the mass-volume ratio of the thrombin to the MES buffer solution is 1:(1-5) mg / mL; the particle size of the carboxyl magnetic microspheres is 2.5-3.0 μm; the mass-volume ratio of the thrombin to the ethanolamine is (4-1):1 mg / mL; and the volume ratio of the ethanolamine to the MES buffer solution is 1:(5-10).

[0015] Preferably, the washing in the step (3) is washing the thrombin-affinity magnetic microspheres with a phosphate solution and deionized water respectively; the pH of the phosphate solution is 7.4; and the eluent used for elution is (0.01-0.03) mol / L KCl-(0.05-0.2) mol / L HCl. The eluent in the concentration range can maintain the activity of hirudin and the stability of the affinity medium, and can effectively separate and elute the hirudin. The washing solution and the eluent used in the step are pure salt solutions, which are non-toxic and non-polluting, have high separation efficiency, and can be adapted to a full-automatic nucleic acid purifier to realize automatic production.

[0016] Preferably, the dialysis in the step (4) is using a dialysis bag with a molecular weight cut-off of less than or equal to 5000 Da, and the water in the dialysis bag is changed 2-8 times at intervals of no less than 2 h, and the total dialysis time is no less than 24 h; and the concentration is that the water removal rate reaches 70-80%.

[0017] The application further includes the hirudin obtained by the separation and purification method.

[0018] Preferably, the activity of the hirudin is 17167 ATU / g.

[0019] The application further includes the application of the hirudin in the preparation of health food, medicine or cosmetics.

[0020] The application has the following beneficial effects:

[0021] The present application uses self-made leaching solution to coarsely extract hirudin from leech powder, and then uses the prepared thrombin affinity magnetic microspheres to further separate and purify, and finally natural hirudin with activity of 17000 ATU / g or above is obtained. The self-made leaching solution can destroy the cell wall of leech, so that the hirudin in leech is fully dissolved, and the efficiency of the coarse extraction of hirudin from leech powder is higher. The thrombin affinity magnetic microspheres are prepared by using carboxyl magnetic microspheres, so that the sample enrichment effect can be fully utilized, and compared with agarose microspheres and polymer microspheres, the hirudin with higher activity can be separated and purified, and the impurities in the obtained hirudin are much less than those in the hirudin coarse solution, and the purity of hirudin is obviously improved. The thrombin affinity magnetic microspheres do not require sample concentration and do not use organic solvents, have high loading capacity, have little harm to the environment, and are convenient for manual operation or automatic operation. The method for separating and purifying hirudin has simple operation and low production cost, the obtained hirudin product has high activity and purity, long storage period and stable quality, can provide safe and high-quality raw materials for health food, medicine or cosmetics, and is beneficial to industrial production and application. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of these drawings.

[0023] Figure 1 The principle diagram for separating and purifying hirudin by using thrombin affinity magnetic microspheres according to the present application.

[0024] Figure 2 The HPLC spectrum of hirudin coarse solution in example 1, wherein the peak 2 is the hirudin peak.

[0025] Figure 3 The HPLC spectrum of hirudin separated and purified in example 1, wherein the peak 1 is the hirudin peak. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0027] The experimental methods used in the embodiments of the present application are conventional methods if no special instructions are given.

[0028] The carboxyl magnetic microspheres used in the embodiments of the present application are all purchased from Suzhou Beaver Biomedical Engineering Co., Ltd., and the particle size is 2.5-3.0 μm.

[0029] The materials, reagents and the like used in the embodiments of the present application can be obtained from commercial channels if no special description is made.

[0030] The present application utilizes the principle of separating and purifying hirudin by using thrombin affinity magnetic microspheres, as shown in the following formula: Figure 1 The interaction between thrombin and macromolecular substrate depends on three different regions: the primary binding site of the catalytic reaction site (C), the non-polar binding site adjacent to the catalytic site (AB), and the anion binding site responsible for the specific action of thrombin and fibrinogen (recognition site) (R). The N-terminal of hirudin can block the active site of thrombin, and its hydrophobic domain is complementary to the non-polar binding site (AB) of thrombin; the non-polar binding site (AB) is close to the catalytic center of thrombin. The C-terminal of hirudin has 6 acidic amino acids, which can form many ionic bonds with the positively charged recognition site (R) of thrombin. The two functional domains of N-terminal and C-terminal bind to thrombin in a synergistic manner, forming a cap at the active site of thrombin and preventing the binding of the substrate. This binding does not affect the activity of hirudin, and the thrombin and hirudin can be separated at a certain pH.

[0031] The conditions for HPLC detection in the present application are as follows:

[0032] Column: Biopearl-HC C18 4.6*200mm Detector: UV 280nm

[0033] Mobile phase: A: water B: acetonitrile Column temperature: room temperature

[0034] Injection volume: 10 uL Flow rate: 1 mL / min

[0035] Elution gradient: 10%-30% acetonitrile.

[0036] Example 1

[0037] The hirudin separation and purification method based on thrombin affinity magnetic microspheres provided in the present embodiment has the following specific steps:

[0038] (1) crude extraction of hirudin from leech powder: leech powder (purchased from Nanning Jinhaikang Biomedicine Technology Co., Ltd.) was taken and 2 times the mass of leech powder was added with extraction solution (obtained by mixing 50 mM Tris, 10 mM EDTA, 150 mM sodium chloride, 3% SDS, 2% Triton X-100 and 2% deoxycholic acid sodium), soaked, ultrasonicated, centrifuged, and the supernatant was reserved. The precipitate was repeatedly extracted twice, and all the supernatants were collected and adjusted to a pH of about 4.5. The supernatant was centrifuged again, and the pH was adjusted to about 7.0. The supernatant was reserved.

[0039] (2) preparation of thrombin affinity magnetic microspheres: 5 mL of carboxyl magnetic microspheres (purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.) with a content of 20 mg / mL were taken into a 15 mL centrifuge tube, placed on a magnetic separation rack, and the liquid was removed. The magnetic microspheres were washed with 3 times the volume of deionized water for 3 times. Then 5 mL of MES (1-morpholinoethanesulfonic acid) buffer with a concentration of 50 mM / L and a pH of 5.5 and 0.06 g of Sulfo-NHS (N-hydroxysuccinimide) and 0.1 g of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) were added. The mixture was reciprocally oscillated at room temperature for 1 h, magnetically separated, and the solution was discarded. Thus, activated carboxyl magnetic microspheres were obtained. 2 mg of thrombin (2 ku / mg, purchased from Hunan Woke Biotechnology Co., Ltd.) was dissolved in 2 mL of MES buffer (50 mM / L, pH 5.5), and the activated carboxyl magnetic microspheres were added. The mixture was oscillated at room temperature for 2 h. Then 0.5 mL of ethanolamine (previously dissolved in 5 mL of MES buffer with a concentration of 50 mM / L and a pH of 5.5) was added to the reaction system, and the mixture was continuously oscillated at room temperature for 1 h. The liquid was removed by magnetic separation, and the magnetic microspheres were washed with MES buffer (concentration of 50 mM / L, pH of 5.5) and PBS buffer (concentration of 20 mM / L, pH of 7.4). Finally, the magnetic microspheres were stored in PBS buffer at 4°C. Thus, thrombin affinity magnetic microspheres were obtained.

[0040] (3) separation and purification of thrombin affinity magnetic microspheres: the supernatant was mixed with the thrombin affinity magnetic microspheres, vortexed, and allowed to stand for adsorption. The mixture was magnetically separated, and the supernatant was discarded. The thrombin affinity magnetic microspheres were washed with 10 times the volume of phosphate buffer with a pH of 7.4 and deionized water, respectively. Then the magnetic microspheres were eluted with 0.02 mol / L KCl-0.05 mol / L HCl solution. Finally, the eluate was collected and reserved.

[0041] (4) desalination and concentration: the eluate was dialyzed. The dialysis membrane was a dialysis bag with a molecular weight cut-off of less than or equal to 5000. The dialysis bag was changed twice with an interval of 2 h. The dialyzed eluate was concentrated by rotary evaporation (70°C, 250 r / min). After removing 70% of the water, the concentrated solution was stored at -8°C. The concentrated solution was reserved.

[0042] (5) The concentrated solution is freeze-dried by a freeze dryer to obtain hirudin powder, which is collected and sealed for storage.

[0043] The hirudin crude extract obtained in step (1) and the hirudin obtained after final separation and purification are detected by HPLC, and the detection results are shown in Figure 2 and Figure 3 It can be seen from Figure 2 that four chromatographic peaks including hirudin are detected in the hirudin crude extract, and the peak values of peaks No. 2 (hirudin) and No. 4 are relatively high. It can be seen that the hirudin crude extract contains a large amount of impure proteins in addition to hirudin, and the purity of hirudin is relatively low. Figure 3 There are no other chromatographic peaks in addition to hirudin in the hirudin obtained in the present embodiment. It can be seen that the purity of the hirudin obtained in the present embodiment is very high.

[0044] According to the principle that hirudin specifically binds to thrombin at a ratio of 1:1, the activity of natural hirudin can be detected by Markwardt thrombin direct titration method. The unit of hirudin is international unit, represented by ATU, and the international unit of thrombin activity is NIH, that is, 1 ATU is equal to the amount of hirudin that neutralizes 1 NIH thrombin. The component with the highest anticoagulant activity in natural leech powder is usually considered to be hirudin.

[0045] The natural hirudin powder product obtained in the present embodiment can be detected for its anticoagulant activity by Markwardt thrombin direct titration method. The specific detection method is as follows: 0.01 g of product powder is accurately weighed and placed in a 1.5 mL centrifuge tube, 400 μL of extraction solution is added, and it is shaken to dissolve, and a 0.025 g / mL test solution is prepared. 100 μL of test solution is accurately measured and placed in a 1.5 mL centrifuge tube, 200 μL of 0.5% bovine fibrinogen-containing tris-hydroxymethyl aminomethane hydrochloride buffer (pH 7.4) is added, shaken, and placed in a water bath (37±0.5) ℃, and 1 mL (middle) of 40 NIH (unit) thrombin solution (5 uL / min, dropwise and gently shake) is slowly added until coagulation occurs in the solution, and the volume of thrombin solution consumed is recorded. The activity is calculated according to the following formula:

[0046] U = C1V1 / (C2V2)

[0047] In the formula: U is the activity unit (ATU / g) of 1 g of hirudin;

[0048] C1 is the concentration of thrombin solution (NIH / mL);

[0049] C2 is the mass concentration of the test solution (g / mL);

[0050] V1 is the volume of thrombin solution consumed (μL);

[0051] V2 is the amount of test sample solution added (μL).

[0052] The activity of the hirudin obtained in this example was measured to be 17167 ATU / g, and the yield was measured to be 1.13%, and the activity yield was 16.17%.

[0053] Example 2

[0054] The hirudin separation and purification method based on thrombin affinity magnetic microspheres provided in this example has the following specific steps:

[0055] (1) Coarsely extract hirudin from leech powder: Take leech powder (purchased from Nanning Jinhaikang Pharmaceutical Technology Co., Ltd.) and add 4 times the mass of leech powder with extraction solution (obtained by mixing 50 mM Tris, 10 mM EDTA, 150 mM sodium chloride, 3% SDS, 2% Triton X-100, and 2% deoxycholic acid sodium), soak, ultrasonic, centrifuge, and reserve the supernatant. The precipitate is continuously extracted twice, and all the supernatants are collected and adjusted to a pH of about 4.5. The supernatant is centrifuged again, the pH is adjusted to about 7.0, and it is reserved.

[0056] (2) Thrombin affinity magnetic microsphere preparation: Take 5 mL of carboxyl magnetic microspheres (purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.) with a content of 20 mg / mL and place them in a 15 mL centrifuge tube on a magnetic separation rack. Remove the liquid and wash repeatedly with 3 times the volume of deionized water for 3 times. Then add 5 mL of MES (1-morpholinoethanesulfonic acid) buffer with a concentration of 50 mM / L and a pH of 5.5 and 0.06 g of Sulfo-NHS (N-hydroxysuccinimide) and 0.1 g of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) to the mixture. Oscillate reciprocally at room temperature for 1 h, magnetically separate, and discard the solution. The activated carboxyl magnetic microspheres are obtained. Take 2 mg of thrombin (2 ku / mg, purchased from Hunan Woke Biological Co., Ltd.) and dissolve it in 2 mL of MES buffer (50 mM / L, pH 5.5). Add the activated carboxyl magnetic microspheres to the solution, oscillate at room temperature for 2 h, then add 0.5 mL of ethanolamine (previously dissolved in 5 mL of MES buffer with a concentration of 50 mM / L and a pH of 5.5) to the reaction system, continue to oscillate at room temperature for 1 h, magnetically separate and remove the liquid, and wash with MES buffer (concentration of 50 mM / L, pH of 5.5) and PBS buffer (concentration of 20 mM / L, pH of 7.4). Finally, store the thrombin affinity magnetic microspheres in PBS buffer at 4°C. The obtained thrombin affinity magnetic microspheres are obtained.

[0057] (3) Thrombin affinity magnetic microspheres separation and purification: the supernatant is mixed with thrombin affinity magnetic microspheres, vortexed, adsorbed, magnetically separated, the supernatant is discarded, the thrombin affinity magnetic microspheres are washed with 10 times the volume of phosphate buffer solution with pH of 7.4 and deionized water, respectively, then eluted with 0.01 mol / L KCl-0.1 mol / L HCl solution, and finally the eluate is collected and stored.

[0058] (4) Desalination and concentration: the eluate is dialyzed with a dialysis bag with a molecular weight cut-off of less than or equal to 5000, and the dialysis bag is replaced with water 4 times at intervals of 4 h; the dialyzed eluate is concentrated by rotary evaporation (50°C, 250 r / min) until 80% of the water is removed, and the obtained concentrated solution is stored at -8°C.

[0059] (5) The concentrated solution is freeze-dried to obtain a powder of hirudin, which is collected and stored in a sealed container.

[0060] According to the determination method described in Example 1, the activity of the hirudin obtained in this example is 16218 ATU / g, the yield is 0.97%, and the activity yield is 13.11%.

[0061] Example 3

[0062] The hirudin separation and purification method based on thrombin affinity magnetic microspheres provided in this example has the following specific steps:

[0063] (1) Crude extraction of hirudin from leech powder: leech powder (purchased from Nanning Jinhaikang Pharmaceutical Technology Co., Ltd.) is taken and 3 times the mass of the leech powder is added to the extraction solution (obtained by mixing 50 mM Tris, 10 mM EDTA, 150 mM sodium chloride, 3% SDS, 2% Triton X-100 and 2% deoxycholic acid sodium), soaked, ultrasonicated, centrifuged, and the supernatant is collected and stored. The precipitate is repeatedly extracted twice, and all the supernatants are collected and adjusted to a pH of about 4.5, then centrifuged again to obtain the supernatant, which is adjusted to a pH of about 7.0 and stored.

[0064] (2) Preparation of thrombin affinity magnetic microspheres: 5 mL of carboxyl magnetic microspheres (purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.) with a content of 20 mg / mL were taken into a 15 mL centrifuge tube, placed on a magnetic separation rack, and the liquid was removed. The microspheres were washed repeatedly with 3 times the volume of deionized water for 3 times. Then 5 mL of MES (1-morpholinoethanesulfonic acid) buffer with a concentration of 50 mM / L and a pH of 5.5 and 0.06 g of Sulfo-NHS (N-hydroxysuccinimide) and 0.1 g of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) were added. The mixture was reciprocally shaken at room temperature for 1 h, magnetically separated, and the solution was discarded. Thus, activated carboxyl magnetic microspheres were obtained. 2 mg of thrombin (2 ku / mg, purchased from Hunan Woke Biological Co., Ltd.) was dissolved in 2 mL of MES buffer (50 mM / L, pH 5.5), and the activated carboxyl magnetic microspheres were added. The mixture was shaken at room temperature for 2 h. Then 0.5 mL of ethanolamine (previously dissolved in 5 mL of MES buffer with a concentration of 50 mM / L and a pH of 5.5) was added to the reaction system, and the shaking was continued at room temperature for 1 h. The liquid was removed by magnetic separation, and the microspheres were washed with MES buffer (concentration of 50 mM / L, pH of 5.5) and PBS buffer (concentration of 20 mM / L, pH of 7.4). Finally, the microspheres were stored in PBS buffer at 4°C. Thus, thrombin affinity magnetic microspheres were obtained.

[0065] (3) Separation and purification of thrombin affinity magnetic microspheres: The supernatant was mixed with the thrombin affinity magnetic microspheres, vortexed, allowed to stand for adsorption, magnetically separated, and the supernatant was discarded. The thrombin affinity magnetic microspheres were washed with 10 times the volume of phosphate buffer with a pH of 7.4 and deionized water, respectively. Then the microspheres were eluted with 0.03 mol / L KCl-0.2 mol / L HCl solution. Finally, the eluate was collected for use.

[0066] (4) Desalination and concentration: The eluate was dialyzed using a dialysis bag with a molecular weight cut-off of less than or equal to 5000. The dialysis bag was changed with water 8 times at intervals of 3 h. The dialyzed eluate was concentrated by rotary evaporation (40°C, 150 r / min). After removing 75% of the water, the concentrated solution was stored at -8°C for use.

[0067] (5) The concentrated solution was freeze-dried to obtain powdered hirudin, which was collected and stored in a sealed container.

[0068] The activity of the hirudin obtained in this example was measured by the method described in Reference Example 1 to be 16528 ATU / g, the yield was 0.88%, and the activity yield was 12.12%.

[0069] The hirudin activity and yield obtained by the three different concentrations of eluent in Examples 1, 2 and 3 are slightly different, but the difference is not large. However, the magnetic microspheres contain Fe3O4, and HCl in the eluent can react with Fe3O4, thereby affecting the magnetic microsphere medium, resulting in a shortened service life of the medium. Therefore, the concentration of HCl in the eluent should be as low as possible without affecting the elution, so the eluent (0.02 mol / L KCl-0.05 mol / L HCl solution) described in Example 1 is the most preferred.

[0070] Example 4

[0071] The hirudin separation and purification method based on thrombin affinity magnetic microspheres provided in the present embodiment has the following specific steps:

[0072] (1) Coarsely extract hirudin from leech powder: Take leech powder (purchased from Nanning Jinhaikaoke Kang Medical Technology Co., Ltd.) and add 2 times the mass of leech powder of the leaching solution (obtained by mixing 50 mM Tris, 10 mM EDTA, 150 mM sodium chloride, 3% SDS, 2% Triton X-100 and 2% deoxycholic acid sodium), soak, ultrasonic, centrifuge, and reserve the supernatant. The precipitate is continuously extracted twice, and all the supernatants are collected and adjusted to a pH of about 4.5. The supernatant is centrifuged again, the pH is adjusted to about 7.0, and it is reserved for use.

[0073] (2) Preparation of thrombin affinity magnetic microspheres: Take 5 mL of carboxyl magnetic microspheres (purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.) with a content of 20 mg / mL and place them in a 15 mL centrifuge tube on a magnetic separation rack. Remove the liquid and wash repeatedly with 3 times the volume of deionized water for 3 times. Then add 5 mL of MES (1-morpholinoethanesulfonic acid) buffer solution with a concentration of 50 mM / L and a pH of 5.5 and 0.06 g of Sulfo-NHS (N-hydroxysuccinimide) and 0.1 g of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) to the mixture. Oscillate reciprocally at room temperature for 1 h, magnetically separate, and discard the solution. The activated carboxyl magnetic microspheres are obtained. Take 2 mg of thrombin (2 ku / mg, purchased from Hunan Woke Biological Co., Ltd.) and dissolve it in 4 mL of MES buffer solution (50 mM / L, pH 5.5). Add the activated carboxyl magnetic microspheres to the solution, oscillate at room temperature for 2.5 h, then add 1 mL of ethanolamine (pre-dissolved in 6 mL of MES buffer solution with a concentration of 50 mM / L and a pH of 5.5), continue to oscillate at room temperature for 1.2 h, magnetically separate and remove the liquid, and wash with MES buffer solution (concentration of 50 mM / L, pH of 5.5) and PBS buffer solution (concentration of 20 mM / L, pH of 7.4). Finally, store the obtained thrombin affinity magnetic microspheres in PBS buffer solution at 4°C.

[0074] (3) Thrombin affinity magnetic microspheres separation and purification: the supernatant is mixed with thrombin affinity magnetic microspheres, vortexed, adsorbed, magnetically separated, the supernatant is discarded, the thrombin affinity magnetic microspheres are washed with 10 times the volume of phosphate buffer solution with pH of 7.4 and deionized water, respectively, then eluted with 0.02 mol / L KCl-0.05 mol / L HCl solution, and finally the eluate is collected and used.

[0075] (4) Desalination and concentration: the eluate is dialyzed, the dialysis membrane is a dialysis bag with a molecular weight cut-off of less than or equal to 5000, and the dialysis bag is changed twice with water at an interval of 2 hours; the dialyzed eluate is concentrated by rotary evaporation (70°C, 250 r / min), until 30% of the water is removed, then the obtained concentrated solution is stored at -8°C, and used.

[0076] (5) The concentrated solution is freeze-dried to obtain a powder of hirudin, which is collected and stored in a sealed container.

[0077] According to the determination method described in Example 1, the activity of the hirudin obtained in this example is 17012 ATU / g, the yield is 0.98%, and the activity yield is 15.12%.

[0078] Example 5

[0079] The hirudin separation and purification method based on thrombin affinity magnetic microspheres provided in this example has the following specific steps:

[0080] (1) Crude extraction of hirudin from leech powder: leech powder (purchased from Nanning Jinhaikang Pharmaceutical Technology Co., Ltd.) is taken and 2 times the mass of the leech powder is added to the extraction solution (obtained by mixing 50 mM Tris, 10 mM EDTA, 150 mM sodium chloride, 3% SDS, 2% Triton X-100 and 2% deoxycholic acid sodium), soaked, ultrasonicated, centrifuged, and the supernatant is used, the precipitate is repeatedly extracted twice, all the supernatants are collected and the pH is adjusted to about 4.5, centrifuged again to obtain the supernatant, and the pH is adjusted to about 7.0, and used.

[0081] (2) Preparation of thrombin affinity magnetic microspheres: 5 mL of carboxyl magnetic microspheres (purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.) with a content of 20 mg / mL were taken into a 15 mL centrifuge tube, placed on a magnetic separation rack, and the liquid was removed. The microspheres were washed repeatedly with 3 times the volume of deionized water for 3 times. Then 5 mL of MES (1-morpholinoethanesulfonic acid) buffer with a concentration of 50 mM / L and a pH of 5.5 and 0.06 g of Sulfo-NHS (N-hydroxysuccinimide) and 0.1 g of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) were added. The mixture was reciprocally shaken at room temperature for 1 h, magnetically separated, and the solution was discarded to obtain activated carboxyl magnetic microspheres. 2 mg of thrombin (2 ku / mg, purchased from Hunan Woke Biological Co., Ltd.) was dissolved in 10 mL of MES buffer (50 mM / L, pH 5.5), and the activated carboxyl magnetic microspheres were added. The mixture was shaken at room temperature for 3 h. Then 2 mL of ethanolamine (previously dissolved in 10 mL of MES buffer with a concentration of 50 mM / L and a pH of 5.5) was added to the reaction system, and the shaking was continued at room temperature for 1.5 h. The liquid was removed by magnetic separation, and the microspheres were washed with MES buffer (concentration of 50 mM / L, pH of 5.5) and PBS buffer (concentration of 20 mM / L, pH of 7.4). Finally, the microspheres were stored in PBS buffer at 4°C. The obtained product was thrombin affinity magnetic microspheres.

[0082] (3) Separation and purification of thrombin affinity magnetic microspheres: The supernatant was mixed with the thrombin affinity magnetic microspheres, vortexed, allowed to stand for adsorption, magnetically separated, and the supernatant was discarded. The thrombin affinity magnetic microspheres were washed with 10 times the volume of phosphate buffer with a pH of 7.4 and deionized water, respectively. Then the microspheres were eluted with 0.02 mol / L KCl-0.05 mol / L HCl solution. Finally, the eluate was collected for use.

[0083] (4) Desalination and concentration: The eluate was dialyzed using a dialysis bag with a molecular weight cut-off of less than or equal to 5000. The dialysis bag was changed with water 2 times at an interval of 2 h. The dialyzed eluate was concentrated by rotary evaporation (70°C, 250 r / min). After removing 70% of the water, the concentrated solution was stored at -8°C for use.

[0084] The activity of the leech-derived hirudin obtained in this example was measured to be 16998 ATU / g, the yield was 1.08%, and the activity yield was 16.02% according to the determination method described in Reference Example 1.

[0085] Comparative Example 1

[0086] The separation and purification method of Reference Example 1 was used, but different extraction solutions (normal saline, extraction solution 1, and extraction solution 2) were used to treat the powder of leeches.

[0087] Extraction solution 1 (i.e. the extraction solution used in the present application): 50 mM Tris, 10 mM EDTA, 150 mM NaCl, 3% SDS, 2% Triton X-100, 2% deoxycholate sodium.

[0088] Extraction solution 2 (adjusted according to the RIPA lysis buffer formula): 1.2 M guanidine hydrochloride, 50 mM Tris-HCL, 30 mM EDTA, pH 7.0, 10% Tween-20, 1% Triton X-100.

[0089] Normal saline: 0.9% NaCl aqueous solution.

[0090] Extraction solution 1 (i.e. the extraction solution used in the present application) has a relatively mild effect and has little effect on the properties of the proteins dissolved after the cell wall is destroyed; extraction solution 2 can destroy the cell wall, but the different components contained therein result in different extraction effects on the proteins, and the relatively high salt concentration has a certain effect on the subsequent combination of the proteins and the magnetic balls; normal saline only has a simple dissolving effect and can only dissolve the proteins free in the cells outside, and cannot dissolve the effective components in the cells. According to the determination method described in Reference Example 1, it is determined that the activity of hirudin obtained after the next step experiment of the leech powder treated by extraction solution 1 (i.e. the extraction solution used in the present application) is 17167 ATU / g, and the yield is 1.13%; the activity of hirudin obtained after the next step experiment of the leech powder treated by extraction solution 2 is 2780 ATU / g, and the yield is 1.24%; the activity of hirudin obtained after the next step experiment of the leech powder treated by normal saline is 1320 ATU / g, and the yield is 1.27%. It can be seen that the extraction solution 1 used in the present application has good extraction effect, can destroy the cell wall of the leech and dissolve the hirudin therein, but does not damage the effective components therein, and can provide higher efficiency for the crude extraction of hirudin in the leech powder.

[0091] Comparative Example 2

[0092] The hirudin is extracted from the leech powder by using the affinity microspheres prepared by using different substrates (agarose, polymer microspheres, magnetic microspheres) according to the separation and purification method of Reference Example 1, and the activity and yield of the finally obtained hirudin are determined according to the determination method described in Reference Example 1.

[0093] The activity of the finally obtained hirudin is 7329 ATU / g, and the yield is 0.48% by using the thrombin affinity agarose microspheres prepared by using agarose as the substrate. However, the thrombin affinity agarose microspheres have certain requirements for the loading concentration, and the hirudin is precipitated by using acetone when the hirudin is crudely extracted from the leech powder, and the pressure resistance of the agarose microspheres is low, the flow rate is small, and the efficiency is low during the separation.

[0094] Using the thrombin affinity polymer microspheres prepared by taking polymer microspheres as the matrix, the activity of the hirudin obtained is 6730 ATU / g, and the yield is 0.26%. However, the thrombin affinity polymer microspheres also have certain requirements for the loading concentration, and the hirudin is required to be precipitated by using acetone when the hirudin is coarsely extracted from the limnatis powder, and the loading capacity of the polymer microspheres is low, and the manual operation is inconvenient.

[0095] Using the thrombin affinity magnetic microspheres prepared by taking magnetic microspheres as the matrix, the activity of the hirudin obtained is 17167 ATU / g, and the yield is 1.13%. The hirudin obtained has high purity and few impurities, although the yield is limited, the activity per unit mass is high, and the activity yield is as high as 16.17%, and the high-activity components in the limnatis dry powder can be fully utilized. The thrombin affinity magnetic microspheres prepared by taking magnetic microspheres as the matrix are used for the separation and purification of the hirudin, have the effect of sample enrichment, do not have requirements for the sample concentration, can not use organic solvents, have small harm to the environment, have high loading capacity, and are convenient to operate, and manual and automatic operations are both very convenient.

[0096] The above only describes the preferred embodiments of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for separating and purifying hirudin based on thrombin-affinity magnetic microspheres, characterized in that, Includes the following steps: (1) Weigh the leech powder, soak it in the extract, then sonicate and centrifuge it. Collect the supernatant and adjust the pH to obtain the crude extract of hirudin. (2) Thrombin was mixed with carboxyl magnetic microspheres and reacted. Then ethanolamine was added to continue the reaction. The liquid was separated and removed. After washing, thrombin-affinity magnetic microspheres were obtained. (3) Add the thrombin affinity magnetic microspheres from step (2) to the crude extract of hirudin from step (1), mix well, let stand, then separate and remove the supernatant, wash the precipitate, elute and collect the eluent. (4) Dialyze the eluent from step (3), then concentrate and freeze dry to obtain hirudin; In step (1), the extract is 2-6 times the weight of the leech powder; the extract consists of 50mM Tris, 10mM EDTA, 150mM sodium chloride, 3% SDS, 2% Triton X-100 and 2% sodium deoxycholate.

2. The method for separating and purifying hirudin according to claim 1, characterized in that, The process of collecting the supernatant and adjusting the pH involves: collecting the supernatant and adjusting the pH to 4.5, centrifuging again to collect the supernatant, and adjusting the pH to 7.

0.

3. The method for separating and purifying hirudin according to claim 2, characterized in that: In step (2), thrombin and ethanolamine are both pre-dissolved in MES buffer at a concentration of 50 mM / L and pH 5.

5. The carboxyl magnetic microspheres are pre-activated with activator EDC / Sulfo-NHS. Thrombin reacts with the carboxyl magnetic microspheres at room temperature for 2-3 hours, and then ethanolamine is added to continue the reaction at room temperature for 1-1.5 hours.

4. The method for separating and purifying hirudin according to claim 3, characterized in that: The thrombin activity is 2 kDa / mg, the mass-to-volume ratio of thrombin to MES buffer is 1:(1-5) mg / mL; the particle size of the carboxyl magnetic microspheres is 2.5-3.0 μm; the mass-to-volume ratio of thrombin to ethanolamine is (4-1):1 mg / mL; and the volume ratio of ethanolamine to MES buffer is 1:(5-10).

5. The method for separating and purifying hirudin according to claim 4, characterized in that: The eluent used in step (3) is an aqueous solution containing (0.01-0.03) mol / L KCl and (0.05-0.2) mol / L HCl.

6. The method for separating and purifying hirudin according to claim 5, characterized in that: In step (4), dialysis specifically involves using a dialysis bag with a molecular weight cutoff of less than or equal to 5000 Da, changing the water 2-8 times in between, with an interval of not less than 2 hours between each change; concentration specifically involves achieving a water removal rate of 30-80%.

7. The hirudin obtained by the separation and purification method according to claim 6, characterized in that: The activity of the hirudin is 17167 ATU / g.

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