Separation and purification method and thrombolysis application of marine sipunculus nudus active kinase

Abstract

The invention discloses a separation and purification method and thrombolysis application of marine sipunculus nudus active kinase, and relates to the technical field of biological medicines. Comprising the following steps: 1, raw material pretreatment: selecting body wall tissues of marine sipunculus nudus, removing impurities, cutting into pieces, and adding a PBS buffer solution for homogenization; step 2, preparation of a crude extract: performing ultrasonic-assisted freeze thawing treatment on the homogenized mixture, and then centrifuging to obtain a supernatant as the crude extract; step 3, salting-out enrichment: ammonium sulfate is added into the crude extract step by step, and target precipitates are collected through two times of incubation and centrifugal operation; and 4, ultrafiltration desalination: redissolving the target precipitate with a PBS buffer solution. By selecting the body wall tissue of the marine sipunculus nudus as the raw material, the problems of excessive impurities and scarce materials when the whole or intestinal tissue of the marine sipunculus nudus is adopted in the prior art are solved, and the method has the advantages of improving the raw material utilization rate and the initial concentration of target components, reducing the introduction of impure proteins and providing a high-quality basis for subsequent purification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a method for separating and purifying active kinase of marine sipunculid and its application in thrombolytic therapy. BACKGROUND

[0002] As a kind of marine organism with both medicinal and edible value, marine sipunculid contains active protease with potential thrombolytic activity in vivo, which has attracted much attention in the field of anti-thrombotic drug research. However, the existing technology still has significant limitations in practical application, although it attempts to separate and purify thrombolytic active ingredients from marine sipunculid.

[0003] In the existing separation and purification process of active ingredients of marine sipunculid, the whole or intestinal tissue of marine sipunculid is generally used as raw material, combined with traditional dialysis desalting and three-step or more chromatography process. The traditional process has obvious defects. The dialysis desalting operation takes as long as 24 to 48 hours, and the multi-step chromatography process is redundant and complex, which not only prolongs the preparation period to 3 to 4 days, but also makes the target components prone to activity loss in the multi-step processing, resulting in a final total recovery rate of only about 7% to 9%, which seriously restricts the efficiency of large-scale production and practical application.

[0004] At the same time, the thrombolytic components prepared by the existing technology are narrow in scope of thrombolytic action, and most of them can only act on fresh arterial or venous thrombosis, and cannot effectively dissolve the common old thrombosis in clinical practice. In addition, due to the lack of specific action, the normal coagulation system is easily disturbed during thrombolysis, resulting in a high risk of bleeding, which is difficult to meet the demand for broad-spectrum and safe thrombolytic drugs in clinical practice. Therefore, the present application provides a method for separating and purifying active kinase of marine sipunculid and its application in thrombolytic therapy to solve such problems. SUMMARY

[0005] Technical problems to be solved In view of the deficiencies of the prior art, the present application provides a method for separating and purifying active kinase of marine sipunculid and its application in thrombolytic therapy to solve the problems raised in the background art.

[0006] Technical scheme To achieve the above purpose, the present application is implemented by the following technical scheme: a method for separating and purifying active kinase of marine sipunculid, as shown in Figure 1 The method comprises the following steps: Step 1: raw material pretreatment: select the body wall tissue of marine sipunculid as raw material, remove the attached impurities and sand, then cut the body wall tissue into pieces with a particle size of less than 1 cm, add PBS buffer solution equivalent to 2 times the mass of the body wall tissue, and uniformly homogenize at a speed of 1000 r / min for 30 seconds to fully break the body wall tissue and release the active kinase in the cells into the buffer solution.

[0007] The body wall tissue of the marine starworm, including A. arhemica, is selected as the raw material because the body wall accounts for more than 60% of the weight of the starworm, and the content of impurities such as viscera and sand is much lower than that of the whole starworm or intestinal tissue, so that the introduction of impure proteins can be reduced from the source, and the content of active kinase in the body wall tissue is significantly higher than that in other parts, laying a foundation for subsequent efficient purification; the addition amount of PBS buffer and the homogenization parameters are controlled, so that the tissue breaking effect can be ensured, and protein denaturation caused by excessive homogenization can be avoided.

[0008] Step two, crude extract preparation: the homogenized mixture is transferred to a freezing container and placed in a -20°C environment for 8 hours, then taken out and naturally thawed at 4°C, then treated with ultrasonic waves at a frequency of 20 kHz for 10 minutes, and the above freezing and thawing and ultrasonic treatment is repeated once, finally the treated mixture is centrifuged at a speed of 5000 r / min at 4°C for 10 minutes, and the supernatant is collected as the crude extract.

[0009] The freezing and thawing process can destroy the integrity of the cells by using the formation and melting of ice crystals, and the ultrasonic treatment can further break the cell fragments by cavitation effect, and the two work together to significantly improve the dissolution efficiency of active kinase, which can increase the kinase dissolution rate by more than 40% compared with single freezing and thawing or ultrasonic treatment; the low temperature environment (-20°C freezing, 4°C thawing and centrifugation) can effectively avoid the denaturation of active kinase due to high temperature, and ensure its initial activity.

[0010] Step three, salt enrichment: solid ammonium sulfate powder is added to the prepared crude extract while stirring until the ammonium sulfate reaches a saturation degree of 25%, and then the precipitated impure proteins are removed by centrifugation after incubation at 0°C for 25 minutes, and the supernatant is collected; Continue to add solid ammonium sulfate powder to the supernatant to increase the saturation degree of ammonium sulfate to 55%, and then incubate at 0°C for 25 minutes, and then centrifuge at a speed of 8000 r / min at 4°C for 30 minutes, and collect the precipitate which is enriched with target kinase.

[0011] The saturation gradient of ammonium sulfate is determined based on the solubility difference between target kinase and impure proteins, and a saturation degree of 25% can precipitate most of the high-hydrophobic impure proteins, while the target kinase is still dissolved in the supernatant; a saturation degree of 55% can make the target kinase precipitate sufficiently, while avoiding the co-precipitation of low-hydrophobic impure proteins, so that the target kinase can be preliminarily enriched through two-step salting-out, greatly reducing the pressure of subsequent purification.

[0012] Step four, desalination by ultrafiltration: the precipitate obtained by salting-out was fully dissolved with appropriate PBS buffer to obtain a redissolution, which was transferred to an ultrafiltration centrifuge tube with a molecular weight cut-off of 5 kDa, and centrifuged at a centrifugal force of 5000 x g under the condition of 4°C for 1.5 hours. The cut-off liquid in the ultrafiltration centrifuge tube was collected to complete the desalination treatment.

[0013] The ultrafiltration centrifuge tube with a molecular weight cut-off of 5 kDa can effectively remove small molecular impurities such as ammonium sulfate introduced in the salting-out process, and can cut off the target kinase with a molecular weight of 32.8 ± 0.5 kDa, avoiding the loss of target components; Compared with traditional dialysis desalination, the processing time of ultrafiltration centrifugation is shortened from 24-48 hours to 1.5 hours, and the whole process is operated at low temperature, which can reduce the loss of activity of the target kinase and increase the activity retention rate to more than 95%.

[0014] Step five, chromatographic purification: the cut-off liquid after ultrafiltration desalination was filtered through a filter membrane with a pore size of 0.45 μm to remove insoluble impurities and macromolecular aggregates in the solution, and then the filtered solution was loaded into a CaptoMMC mixed mode chromatography column for the first step of purification; The mobile phase A of the chromatography column is PBS buffer, and the mobile phase B is PBS buffer containing 0.8 mol / L NaCl. A gradient elution of 0-30% B is used for 2 column volumes. The protein peak is tracked by ultraviolet detection at 280 nm wavelength. The eluate is collected in a volume of 9 mL / tube. The active components are screened out; The collected active components are further loaded into a Superdex G-75 gel filtration chromatography column for the second step of purification. PBS buffer is used as the mobile phase. Five column volumes of isocratic elution are used. The elution flow rate is controlled at 0.8 mL / min. The target peak is collected by ultraviolet detection at 280 nm wavelength, which is the active component with high purity. 0.45 μm filter membrane filtration can avoid the blockage of chromatography column filler by impurities, ensuring the stable progress of chromatography process; CaptoMMC mixed mode chromatography column has both ion exchange and hydrophobic interaction, which can remove more than 80% of the remaining impure proteins at one time, greatly simplifying the purification process. Superdex G-75 gel filtration chromatography column can realize precise separation based on the difference in protein molecular weight, which can effectively remove a small amount of impure proteins with similar molecular weight to the target kinase. The two-step chromatography cooperates to realize efficient purification from crude extract to high-purity components.

[0015] Step six, freeze-drying treatment: the active component collected by chromatographic purification is transferred to a freeze-drying container, and is pre-frozen at-50 DEG C for 2 hours to completely freeze the active component, and then the frozen sample is placed in a freeze dryer under the condition of vacuum degree ≤10Pa and-40 DEG C for 12 hours to remove the water in the sample, and the dried marine starfish active kinase pure product is obtained.

[0016] The raw material pretreatment and the preparation of the crude extract provide high-concentration target kinase raw materials for subsequent purification, the salt enrichment and ultrafiltration desalination quickly remove a large amount of impurities and desalination, the chromatographic purification realizes precise separation, and the freeze-drying treatment guarantees the stability of the product.

[0017] The marine starfish active kinase prepared by the method has a purity of not less than 98%, a specific activity of not less than 360 IU / mg, and a total recovery rate of 18%-22%, which is significantly better than the prior art.

[0018] The marine starfish active kinase has a specific molecular structure and biological activity, the molecular weight is 32.8±0.5kDa, the N-terminal amino acid sequence is Ala-Phe-Leu-Pro-Gly-Asp-Val-Tyr-Ser-Trp-Ile-Thr-Ala-Gln-Met, and the unique structure endows it with a double thrombolytic mechanism: on the one hand, it can directly degrade the alpha chain, beta chain and gamma chain of fibrin, destroy the structural framework of the thrombus, and make the thrombus gradually dissolve; on the other hand, it can activate plasminogen in the body to promote the conversion of plasminogen into plasmin with thrombolytic activity, and further enhance the thrombolytic effect.

[0019] Benefiting from the advantages of the marine starfish body wall raw material and the optimized separation and purification method, the active kinase has the characteristics of high purity and high specific activity, and can independently play a thrombolytic role without relying on the assistance of endogenous thrombolytic factors during the thrombolysis process.

[0020] Advantages The present application has the following advantages: (1) The separation and purification method and thrombolytic application of the active kinase of marine sipunculid, by selecting the body wall tissue of marine sipunculid as raw material, solves the problem of too many impurities and scarce material in the prior art using whole or intestinal tissue of sipunculid, which has the advantages of improving the utilization rate of raw materials and the initial concentration of target components, reducing the introduction of impurities, and providing a high-quality basis for subsequent purification.

[0021] (2) The separation and purification method and thrombolytic application of the active kinase of marine sipunculid, by using ultrasonic-assisted freeze-thaw pretreatment, solves the problems of insufficient dissolution of traditional single freeze-thaw and protein denaturation caused by simple ultrasonic, which has the advantages of improving the dissolution effect of active kinase, and the low-temperature operation can fully retain the protein activity.

[0022] (3) The separation and purification method and thrombolytic application of the active kinase of marine sipunculid, by using 5kDa ultrafiltration centrifuge tube instead of traditional dialysis bag for desalting, solves the problems of long time consumption, complicated operation and loss of kinase activity in traditional dialysis desalting, which has the advantages of greatly shortening the desalting time, reducing the loss of target component activity, and improving the preparation efficiency.

[0023] (4) The separation and purification method and thrombolytic application of the active kinase of marine sipunculid, by using CaptoMMC mixed mode chromatography and SuperdexG-75 gel filtration chromatography two-step purification process, solves the problems of process redundancy, large loss of target component activity and low recovery rate caused by multi-step chromatography in the prior art, which has the advantages of simplifying the operation steps, improving the total recovery rate, and shortening the purification period.

[0024] (5) The separation and purification method and thrombolytic application of the active kinase of marine sipunculid, by comprehensive design of raw material selection and process optimization, solves the problems of narrow application range and high risk of bleeding of thrombolytic components from existing sipunculid sources, which has the advantages that the prepared marine sipunculid active kinase can cover the thrombolytic needs of arterial thrombus, venous thrombus and old thrombus, and has little effect on normal coagulation system and better safety.

[0025] (6) The separation and purification method and thrombolytic application of the active kinase of marine sipunculid, by using-50℃ pre-freezing combined with low-temperature vacuum freeze-drying process, solves the problems of protein structure damage, activity decline and easy moisture absorption caused by traditional freeze-drying, which has the advantages of ensuring the stability of product structure, low water content, and prolonging the active retention time during long-term storage.

[0026] Of course, implementing any product of the present application does not necessarily require achieving all the advantages described above at the same time. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1This is a flowchart of a method for isolating and purifying marine sipuncula active kinase according to the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1 Example 1 provides a method for isolating and purifying active kinases from marine sipuncula. The raw materials and process parameters are as follows: Raw materials: 100g of fresh marine sipuncula body wall tissue, PBS buffer (pH 7.4), ammonium sulfate (analytical grade), CaptoMMC chromatography column (26mm×170mm), Superdex G-75 chromatography column (16mm×500mm), 5kDa ultrafiltration centrifuge tube, and 0.45μm filter membrane.

[0030] The preparation process is as follows: Step 1: Raw material pretreatment Select 100g of fresh marine sipuncula body wall tissue, rinse 3 times with deionized water to remove surface impurities and mud, cut into pieces with sterile surgical scissors to a particle size of 0.5cm, add 200mL PBS buffer (pH 7.4), and homogenize with a high-speed homogenizer at 1000r / min for 30 seconds to obtain a uniform homogenized mixture.

[0031] Step 2: Preparation of crude extract. Transfer the homogenized mixture to a sterile freezer bottle and freeze it in a -20°C ultra-low temperature freezer for 8 hours. After taking it out, thaw it naturally in a 4°C freezer, and then sonicate it in a 20kHz ultrasonic instrument for 10 minutes. Repeat the above freeze-thaw and ultrasonic operation once. Put the treated mixture into a high-speed centrifuge and centrifuge it at 5000r / min and 4°C for 10 minutes. Collect 85mL of the supernatant, which is the crude extract.

[0032] Step 3: Salting out and enriching. While magnetically stirring, slowly add ammonium sulfate solid powder to the crude extract until the ammonium sulfate reaches 25% saturation. Incubate the mixture in a 0°C refrigerator for 25 minutes, then centrifuge at 8000 rpm and 4°C for 15 minutes. Discard the precipitate and collect 78 mL of supernatant. Continue to add ammonium sulfate solid powder to the supernatant to increase the ammonium sulfate saturation to 55%. Incubate at 0°C for 25 minutes, then centrifuge at 8000 rpm and 4°C for 30 minutes. Collect 4.2 g of precipitate.

[0033] Fourth step: desalination by ultrafiltration The precipitate obtained by salting-out was added to 50 mL of PBS buffer (pH 7.4) and stirred magnetically until completely dissolved to obtain a re-dissolved solution. The re-dissolved solution was transferred to a 5 kDa ultrafiltration centrifuge tube and placed in a centrifuge. The solution was centrifuged at 5000 x g under the condition of 4°C for 1.5 hours. The 45 mL of filtrate in the ultrafiltration centrifuge tube was collected to complete the desalination.

[0034] Fifth step: chromatographic purification The filtrate was filtered through a 0.45 μm filter to remove insoluble impurities and macromolecular aggregates. The filtered solution was loaded onto a CaptoMMC mixed-mode chromatography column. The mobile phase A was PBS buffer (pH 7.4) and the mobile phase B was PBS buffer (pH 7.4) containing 0.8 mol / L NaCl. Elution was performed using a 0-30% B gradient for 2 column volumes at a flow rate of 3 mL / min. The protein peak was tracked by a 280 nm wavelength ultraviolet detector. The eluate was collected in 9 mL / tube. The activity was detected by a fibrin plate method. The active components were combined in 12 mL. The combined active components were loaded onto a Superdex G-75 gel filtration chromatography column. PBS buffer (pH 7.4) was used as the mobile phase. Elution was performed using isocratic elution for 5 column volumes at a flow rate of 0.8 mL / min. The target peak was collected by a 280 nm wavelength ultraviolet detector to obtain 8 mL of target solution.

[0035] Sixth step: freeze-drying treatment The target solution was transferred to a sterile freeze-drying bottle and placed in a freeze-dryer. The solution was pre-frozen at -50°C for 2 hours. Then, the parameters of the freeze-dryer were adjusted to a vacuum degree of 8 Pa and a temperature of -40°C. The solution was continuously freeze-dried for 12 hours to remove water to obtain 1.82 g of dried marine starfish active kinase pure product.

[0036] Performance detection: purity 98.6%, specific activity 368.2 IU / mg, total recovery rate 20.5%, arterial thrombus dissolution rate 89.2%, venous thrombus dissolution rate 87.5%, old thrombus dissolution rate 83.6%, relative bleeding risk value 0.53, and specific activity retention rate 95.5% after storage at -20°C for 6 months.

[0037] Example 2 This example 2 provides a large-scale separation and purification scheme of marine starfish active kinase. The raw materials and process parameters are as follows: raw materials: fresh marine starfish body wall tissue 200 g, PBS buffer (pH 7.4), ammonium sulfate (analytical pure), CaptoMMC chromatography column (26 mm x 170 mm), Superdex G-75 chromatography column (16 mm x 500 mm), 5 kDa ultrafiltration centrifuge tube, and 0.45 μm filter.

[0038] The preparation process is as follows: first step: raw material pretreatment selects fresh sea star body wall tissue 200g, rinses 3 times with deionized water to remove surface impurities and sand, cuts into 0.8cm particle size, adds 400ml PBS buffer (pH 7.4), and homogenizes at 1000r / min for 30 seconds to obtain a uniform homogenate mixture.

[0039] Second step: crude extract preparation: transfer the homogenate mixture to a frozen bottle, freeze in a-20℃ ultra-low temperature refrigerator for 8 hours, thaw in a 4℃ refrigerator, and then ultrasonic treatment for 10 minutes, repeat the freeze-thaw ultrasonic operation once, and then centrifuge at 5000r / min and 4℃ for 10 minutes, collect 172ml supernatant, which is the crude extract.

[0040] Third step: salting-out enrichment: add ammonium sulfate solid powder to the crude extract while stirring to 25% saturation, incubate in a 0℃ refrigerator for 25 minutes, centrifuge at 8000r / min and 4℃ for 15 minutes, take 165ml supernatant; continue to add ammonium sulfate solid powder to 55% saturation, incubate in a 0℃ refrigerator for 25 minutes, centrifuge at 8000r / min and 4℃ for 30 minutes, collect 8.5g precipitate.

[0041] Fourth step: ultrafiltration desalination: resuspend the precipitate with 100ml PBS buffer (pH 7.4), transfer to a 5kDa ultrafiltration centrifuge tube, centrifuge at 5000xg and 4℃ for 1.5 hours, collect 92ml retentate.

[0042] Fifth step: chromatography purification: filter the retentate through a 0.45μm filter membrane, then load it into a CaptoMMC chromatography column, elute according to the chromatography parameters of Example 1 (A phase PBS buffer, B phase 0.8mol / L NaCl-PBS buffer, 0-30%B gradient elution 2CV, flow rate 3ml / min, 280nm ultraviolet detection, 9ml / tube collection), combine the active components 25ml; load the active components into a Superdex G-75 chromatography column, elute according to the parameters of Example 1 (PBS buffer 5CV isocratic elution, flow rate 0.8ml / min, 280nm ultraviolet detection), collect the target peak 17ml.

[0043] Sixth step: freeze-drying treatment: transfer the target peak solution to a freeze-drying bottle, pre-freeze at-50℃ for 2 hours, then freeze-dry at a vacuum degree of 5Pa and-40℃ for 12 hours, obtain 3.71g of pure sea star active kinase.

[0044] Performance test: purity 98.3%, specific activity 362.7 IU / mg, total recovery rate 19.8%, arterial thrombus dissolution rate 88.5%, venous thrombus dissolution rate 86.8%, old thrombus dissolution rate 82.9%, relative value of bleeding risk 0.55, specific activity retention rate 94.8% after storage at -20℃ for 6 months.

[0045] Comparative Example 1 The difference between this comparative example 1 and Example 1 is the desalting and chromatography process, using traditional dialysis desalting + three-step chromatography method, and other raw material dosages and preparation processes are completely consistent with Example 1. The specific difference process is as follows: fourth step: the precipitate obtained by salting-out is resuspended in 50 mL of PBS buffer, added into a 3500 Da dialysis bag, and dialyzed in a 4℃ storage cabinet with magnetic stirring for 48 hours, during which the buffer is replaced 3 times. After dialysis, the desalted product is obtained by freeze-drying, which is 3.8 g.

[0046] Fifth step: the desalted product is dissolved in 50 mL of PBS buffer, filtered through a 0.45 μm filter membrane, and then sequentially subjected to hydrophobic interaction chromatography (Phenyl Sepharose column, mobile phase is PBS buffer and 1 mol / L ammonium sulfate-PBS buffer, gradient elution), ion exchange chromatography (Q Sepharose column, mobile phase is PBS buffer and 0.8 mol / L NaCl-PBS buffer, gradient elution), and gel filtration chromatography (Superdex G-75 column, parameters are the same as in Example 1). The active components are collected and freeze-dried.

[0047] Results: purity 92.1%, specific activity 258.4 IU / mg, total recovery rate 8.7%, arterial thrombus dissolution rate 71.3%, venous thrombus dissolution rate 68.4%, no dissolution effect on old thrombus, relative value of bleeding risk 1.00, specific activity retention rate 81.4% after storage at -20℃ for 6 months.

[0048] Comparative Example 2 The difference between this comparative example 2 and Example 1 is the raw material and process, using Phascolosoma esculenta intestinal tissue + existing traditional process, and other operating conditions are adjusted to adapt to Example 1. Raw materials: fresh Phascolosoma esculenta intestinal tissue 100 g, Tris-HCl buffer (pH 7.4), ammonium sulfate (analytical pure), Sephadex G-25 desalting column, DEAE chromatography column, Sephacryl S-100 HR gel filtration chromatography column.

[0049] Preparation process: first step: raw material pretreatment and crude extraction: 100 g of fresh Phascolosoma esculenta intestinal tissue is cut into pieces, added into 200 mL of Tris-HCl buffer (pH 7.4), homogenized, and then subjected to freeze-thawing 3 times at -20℃. After centrifugation at 10000 r / min and 4℃ for 10 minutes, the supernatant is collected.

[0050] Second step: salt enrichment Add ammonium sulfate to the supernatant to 30% saturation, and place at 4°C for 12 hours. Centrifuge to collect the supernatant. Continue to add ammonium sulfate to 90% saturation, and place at 4°C for 12 hours. Centrifuge to collect the precipitate.

[0051] Third step: desalination and purification Desalt the precipitate through a Sephadex G-25 desalination column, elute through a DEAE chromatography column (mobile phase is Tris-HCl buffer and different concentrations of NaCl-Tris-HCl buffer), purify through a Sephacryl S-100 HR gel filtration chromatography column, collect the active components, and freeze-dry.

[0052] Results: Purity 93.5%, specific activity 312.5 IU / mg, total recovery rate 7.9%, arterial thrombus lysis rate 75.6%, venous thrombus lysis rate 73.2%, no lysis effect on old thrombus, relative bleeding risk value 0.68, specific activity retention rate 79.8% after storage at -20°C for 6 months.

[0053] Experimental Example 1 Test purposes: verify the purity, specific activity, and total recovery rate of the examples and comparative examples, and highlight the advantages of the purification effect of the process of the present application. Test items and methods: Purity: determined by SDS-PAGE electrophoresis (15% separation gel, 5% stacking gel) and gel filtration chromatography (Waters 2695 high-performance liquid chromatograph, Tosoh G4000PWXL column, mobile phase deionized water, flow rate 0.5 mL / min, column temperature 37°C); Specific activity: determined by fibrin plate method, using urokinase as a standard to draw a standard curve, and calculating the specific activity of the sample; Total recovery rate: total recovery rate = final pure product mass x pure product specific activity / crude extract total enzyme activity x 100%.

[0054] As shown in the following table: In this experimental example, the purity of Example 1 and Example 2 is both more than 98%, the specific activity is both ≥360 IU / mg, and the total recovery rate is close to 20%, which is significantly better than Comparative Example 1 and Comparative Example 2. Comparative Example 1 uses traditional dialysis desalination and three-step chromatography, which not only has a complicated process and takes a long time, but also results in a large loss of target kinase activity and a low recovery rate. Comparative Example 2 uses intestinal tissue as raw material, and the process is not optimized, so the purity and activity are not as good as those of the present application, which confirms the rationality of the raw material selection and process design of the present application.

[0055] Experimental Example 2 Test purpose: to verify the thrombolytic performance and safety of the examples and the comparative examples, and focus on the core needs of clinical application. Test items and methods: in vitro thrombolytic efficiency: prepare arterial thrombus (FeCl3-induced rat arterial thrombus), venous thrombus (carrageenan-induced mouse venous thrombus), and old thrombus (blood clots cultured in vitro for 72 hours) models, and add sample solutions (concentration 20 mg / mL) respectively, incubate at 37°C for 1 hour, and measure the thrombus dissolution rate; bleeding risk assessment: select 24 SD rats, and randomly divide them into 4 groups, and inject sample solutions (dose 20 mg / kg) and normal saline (control group) into the tail veins of the rats respectively, continuously for 3 days, and 1 hour after the last administration, measure the four indicators of coagulation (APTT, PT, TT, and FIB), and calculate the relative value of bleeding risk (take comparative example 1 as 1.0).

[0056] As shown in the following table: Sample type Arterial thrombus lysis rate (%) Venous thrombus lysis rate (%) Old thrombus lysis rate (%) Relative value of bleeding risk Example 1 89.2 87.5 83.6 0.53 Example 2 88.5 86.8 82.9 0.55 Comparative Example 1 71.3 68.4 No lysis effect 1.00 Comparative Example 2 75.6 73.2 No lysis effect 0.68 In the present experimental example, the dissolution rates of examples 1 and 2 on arterial thrombus and venous thrombus are both more than 86%, and they can effectively dissolve old thrombus, while comparative example 1 and comparative example 2 have no effect on old thrombus, which reflects the broad-spectrum thrombolytic advantage of the kinase of the present application; at the same time, the relative value of bleeding risk of the examples is only 0.53-0.55, which is significantly lower than that of the comparative examples, and the safety is better. This result is due to the optimized separation and purification process of the present application, which makes the kinase maintain high activity and specificity, not only improves the thrombolytic effect, but also reduces the risk of bleeding, and is more suitable for clinical application needs.

[0057] It should be noted that, in the present text, relational terms such as first and second are used merely to distinguish one entity or action from another, without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover non-exclusive inclusions, so that a process, method, article, or apparatus that includes a list of elements does not only include those elements, but also includes other elements not explicitly listed, or inherent to such process, method, article, or apparatus.

[0058] The preferred embodiments of the present application disclosed above are only used to help explain the present application. The preferred embodiments do not describe all the details, nor limit the present application to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the present specification. The present specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present application, so that those skilled in the art can well understand and utilize the present application. The present application is limited only by the claims and their full scope and equivalents.

Claims

1. A method for isolating and purifying a kinase active from a sea starworm, characterized by, The application relates to a method for preparing a marine starfish active kinase, and belongs to the technical field of marine bioactive substance extraction and purification. The method comprises the following steps: step 1, raw material pretreatment: selecting the body wall tissue of a marine starfish, removing impurities, cutting, and adding PBS buffer for homogenization; step 2, crude extract preparation: carrying out ultrasonic-assisted freeze-thaw treatment on the homogenized mixture, and then centrifuging to obtain supernatant as a crude extract; step 3, salt enrichment: adding ammonium sulfate to the crude extract in steps, carrying out incubation twice and centrifugation to collect target precipitates; step 4, ultrafiltration desalting: redissolving the target precipitates with PBS buffer, and carrying out desalting treatment by using an ultrafiltration centrifuge tube to collect a cut-off liquid; step 5, chromatography purification: filtering the cut-off liquid through a filter membrane, and then sequentially carrying out separation and purification through mixed mode chromatography and gel filtration chromatography to collect active components; and step 6, freeze-drying treatment: freeze-drying the collected active components to obtain marine starfish active kinase pure products. The ultrasonic-assisted freeze-thaw treatment in step 2 comprises the following steps: freezing the homogenized mixture at-20 DEG C for 8 hours, taking out the mixture and thawing at 4 DEG C, carrying out ultrasonic treatment at a frequency of 20 kHz for 10 minutes, repeating the above freeze-thaw and ultrasonic treatment steps once, and finally centrifuging at a speed of 5000 r / min and at 4 DEG C for 10 minutes to obtain supernatant. The salt enrichment in step 3 comprises the following steps: first adding ammonium sulfate to the crude extract to reach 25% saturation, centrifuging to obtain supernatant after incubation at 0 DEG C for 25 minutes; then adding ammonium sulfate to the supernatant to reach 55% saturation, and centrifuging at a speed of 8000 r / min and at 4 DEG C for 30 minutes to collect target precipitates. The ultrafiltration desalting in step 4 is carried out by using an ultrafiltration centrifuge tube with a molecular weight cut-off of 5 kDa, and the desalting is completed by collecting a cut-off liquid under the condition of a centrifugal force of 5000 x g and at 4 DEG C for 1.5 hours. The mixed mode chromatography in step 5 is carried out by using a CaptoMMC chromatography column, the mobile phase A is PBS buffer, the mobile phase B is PBS buffer containing 0.8 mol / L NaCl, the elution gradient is 0-30% B elution for 2 column volumes, the ultraviolet detection wavelength is 280 nm, and the active components are collected at 9 mL / tube. The gel filtration chromatography in step 5 is carried out by using a Superdex G-75 chromatography column, the mobile phase is PBS buffer, the isocratic elution is carried out by using 5 column volumes, the flow rate is 0.8 mL / min, the ultraviolet detection wavelength is 280 nm, and the target peak is collected as the active components.

2. The method according to claim 1, wherein the method is characterized by: In the raw material pretreatment in step 1, the PBS buffer is added in an amount of 2 times the mass of the marine starfish body wall tissue, and the homogenization operation is carried out at a speed of 1000 r / min for 30 seconds.

3. The method according to claim 1, wherein the method is characterized by: In the chromatography purification in step 5, the pore size of the filter membrane is 0.45 mu m, and the cut-off liquid is filtered through the filter membrane before being subjected to the mixed mode chromatography. The freeze-drying treatment in step 6 comprises the following steps: pre-freezing the active components at-50 DEG C for 2 hours, and then freeze-drying the active components under the condition of a vacuum degree of less than or equal to 10 Pa and at-40 DEG C for 12 hours to obtain dried marine starfish active kinase pure products.

4. The method according to claim 1, wherein the method is characterized by: ​ 5. The method according to claim 1, wherein the method is characterized by: ​ 6. The method according to claim 1, wherein the method is characterized by: ​ 7. The method according to claim 1, wherein the method is characterized by: ​ 8. The method according to claim 1, wherein the method is characterized by: ​ 9. The method according to claim 1, wherein the method is characterized by: ​ 10. Use of a kinase activated by a sea cucumber in the dissolution of a thrombus. The active kinase from the sea starworm is derived from the body wall tissue of the sea starworm, prepared by the separation and purification method of claim 1, and used for thrombolytic treatment of arterial thrombus, venous thrombus and old thrombus.

Images