阅读说明：本技术 蛆激酶制备方法及其用途 (Preparation method and application of maggot kinase ) 是由 刘灿 刘秋荻 马彤瑶 马兰青 荣龙 张凯欣 戴隆海 雷梦 孙祎振 郭蓓 孙媛霞 于 2021-10-14 设计创作，主要内容包括：本发明提供蛆激酶的制备方法,所述方法包括蝇蛆的预处理、盐析、透析或超滤、离子交换层析柱层析、洗脱,以获得蛆激酶粗制品。所述方法还包括采用含有丝氨酸蛋白吸附配体的填料进行亲和层析、洗脱,从而获得蛆激酶精制品。本发明获得的蛆激酶具有将纤维蛋白及纤维蛋白原降解的高活力,为溶栓药物的开发和利用提供了坚实的基础。(The invention provides a preparation method of maggot kinase, which comprises pretreatment of maggots, salting out, dialysis or ultrafiltration, ion exchange chromatography column chromatography and elution to obtain a maggot kinase crude product. The method also comprises the steps of carrying out affinity chromatography and elution by adopting a filler containing a serine protein adsorption ligand, thereby obtaining a refined product of the maggot kinase. The maggot kinase obtained by the invention has high activity of degrading fibrin and fibrinogen, and provides a solid foundation for the development and utilization of thrombolytic drugs.)

1. The preparation method of the maggot kinase is characterized by comprising the following steps:

a. cleaning raw material fly maggots, adding a homogenizing agent, and homogenizing;

b. centrifuging the homogenate and collecting a supernatant A;

c. adding ammonium sulfate into the supernatant A until the saturation reaches 25% -35%, standing, separating out protein precipitate, centrifuging, and collecting supernatant B;

d. supplementing ammonium sulfate to 65-75% of saturation degree in the supernatant B, standing, centrifuging, removing supernatant, and collecting precipitate;

e. dissolving the precipitate with distilled water to obtain solution C;

f. dialyzing or ultrafiltering the solution C to remove salt ions in the solution C until the conductivity is less than 200 mus/cm to obtain a solution D;

g. adjusting the pH value of the solution D to 4-8 to obtain a solution E;

h. filtering the solution E with a filter membrane, carrying out chromatography, and adsorbing the target protein on an ion exchange chromatography column;

i. eluting the chromatographic column by using an eluent, and collecting an active elution peak solution F;

j. dialyzing or ultrafiltering the solution F to remove salt ions in the solution F to obtain a solution G;

k. and concentrating the solution G, and freeze-drying to obtain a crude product of the maggot kinase.

2. The preparation method of the maggot kinase is characterized by comprising the following steps:

a. cleaning raw material fly maggots, adding a homogenizing agent, and homogenizing;

b. centrifuging the homogenate and collecting a supernatant A;

c. adding ammonium sulfate into the supernatant A until the saturation reaches 25% -35%, standing, separating out protein precipitate, centrifuging, and collecting supernatant B;

d. supplementing ammonium sulfate to 65-75% of saturation degree in the supernatant B, standing, centrifuging, removing supernatant, and collecting precipitate;

e. dissolving the precipitate with distilled water to obtain solution C;

f. dialyzing or ultrafiltering the solution C to remove salt ions in the solution C until the conductivity is less than 200 mus/cm to obtain a solution D;

g. adjusting the pH value of the solution D to 4-8 to obtain a solution E;

h. filtering the solution E with a filter membrane, carrying out chromatography, and adsorbing the target protein on an ion exchange chromatography column;

i. eluting the chromatographic column by using an eluent, and collecting an active elution peak solution F;

j. dialyzing or ultrafiltering the solution F to remove salt ions in the solution F to obtain a solution G;

l. refining of maggot kinase: balancing the solution G obtained in the step j to pH6.5-8, carrying out affinity adsorption on the maggot kinase in the solution G by using a filler containing a serine protease adsorption ligand, and eluting by using an eluent to obtain an active eluent H;

m, dialyzing or ultrafiltering the solution H to remove salt ions in the solution H to obtain a solution I;

n: and concentrating the solution I, and freeze-drying to obtain a refined product of the maggot kinase.

3. The method for preparing maggot kinase according to claim 1 or 2, wherein the raw material in step a is selected from larvae of muscidae (e.g. chrysomyia megacephala, lucilia sericata), larvae of latrine (summer latrine fly, metalatrine fly); the homogenizing agent is selected from distilled water and PBS buffer.

4. The method for preparing maggot kinase according to any one of claims 1-3, wherein the pH is adjusted using citrate buffer or PBS buffer in step g.

5. The method for preparing maggot kinase according to any one of claims 1-4, wherein the chromatographic column is selected from the group consisting of CM-52, DEAE-32 and DEAE-52 in step h.

6. The method for preparing maggot kinase according to any one of claims 1-5, wherein the eluent is selected from NaCl + PBS and KCl + PBS in step i.

7. The method for preparing maggot kinase according to any one of claims 2-6, wherein the concentration method of the solution G in steps k and n comprises trapping substances with molecular weight of 2000-50000 by using an ultrafiltration device, concentrating, or concentrating by using PEG with molecular weight of 12000.

8. The method for preparing maggot kinase according to any one of claims 2-7, wherein the affinity chromatography packing of step I is grafted with a ligand selected from any one of the following: benzamidine, p-aminobenzamidine hydrochloride, arginine, an arrowhead protease inhibitor, a soybean trypsin inhibitor, and ovomucin.

9. Maggot kinase prepared according to the method of any one of claims 1-8.

10. Use of the maggot kinase prepared according to the method of any one of claims 1-8 in preparing thrombolytic and antithrombotic medicaments.

Technical Field

The invention belongs to the technical field of biochemical pharmacy, and particularly relates to a method for extracting high-activity maggot kinase from maggots.

Background

Normally, the internal hemostatic regulation mechanism and the anticoagulant mechanism are mutually antagonistic, and when the internal hemostatic regulation mechanism is diseased, excessive thrombin is generated to deposit fibrin, thereby initiating the formation of thrombus in vivo. Thrombus, which is a common cardiovascular and cerebrovascular disease, can induce symptoms such as myocardial infarction, ischemic cerebral infarction, coronary arteriosclerotic heart disease and the like, seriously threatens the health of people, and millions of patients die of cerebral infarction, cerebral hemorrhage and myocardial infarction every year.

The antithrombotic drugs commonly used at present are mainly classified into 3 types: firstly, antiplatelet drugs; anticoagulant drugs such as heparin, vitamin K antagonists and the like; and thirdly, thrombolytic drugs. The thrombolytic drug has the advantages of high thrombolysis speed and small side effect, and becomes an important means for treating thrombotic diseases. Thrombolytic enzymes can exert thrombolytic effects by the following pathways: 1. reducing the content of fibrinogen, and inhibiting the original fibrin of fibrin; 2. direct hydrolysis of fibrinogen and fibrin (lysozyme); 3. activating fibrinolysis system, promoting plasminogen conversion to plasmin, promoting vascular endothelial cell to produce tissue plasminogen activator (t-PA), inhibiting Plasminogen Activator Inhibitor (PAI) production, and enhancing endogenous fibrinolysis activity (kinase). The natural resources are natural treasury for obtaining thrombolytic drugs, the sources of thrombolytic enzymes are wide at present, and substances with thrombolytic activity can be separated from microorganisms, plants and animals. Common nattokinase is serine protease produced by bacillus subtilis in the fermentation process of natto, and has the functions of degrading thrombus and softening blood vessels; urokinase separated from healthy human urine can degrade fibrin clot, fibrinogen, blood coagulation factor V, blood coagulation factor VIII and the like in blood circulation; and lumbrukinase widely used in clinic, also has thrombolytic effect, and can be used for treating coronary heart disease, angina pectoris, and cerebral infarction. Studies report that leeches, bats and snake venom can also be used as raw materials for extracting thrombolytic enzyme.

However, the nattokinase produced by using natto has low nattokinase content of wild type bacillus natto, so the production method of the nattokinase obtained by the method is not ideal; the urokinase causes adverse effects on environmental sanitation due to procedures of urine collection, storage and transportation, deodorization and the like, and influences the lives of surrounding residents; lumbrukinase needs to be extracted from a particular species of earthworm.

Therefore, there is still a need to find new extraction sources of thrombolytic enzymes to be able to extract high-activity thrombolytic enzymes in high yield.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the following technical scheme:

in one aspect, the present invention provides a method for preparing maggot kinases, characterized by comprising the steps of:

a. cleaning the raw materials, adding a homogenizing agent, and then homogenizing;

b. centrifuging the homogenate and collecting a supernatant A;

c. adding ammonium sulfate into the supernatant A until the saturation reaches 25% -35%, standing, separating out protein precipitate, centrifuging, and collecting supernatant B;

d. supplementing ammonium sulfate to 65-75% of saturation degree in the supernatant B, standing, centrifuging, removing supernatant, and collecting precipitate;

e. dissolving the precipitate with a solvent to obtain a solution C;

f. dialyzing or ultrafiltering the solution C to remove salt ions in the solution C until the conductivity is lower than 200 mus/cm to obtain a solution D;

g. adjusting the pH value of the solution D to 4-8 to obtain a solution E;

h. filtering the solution E with a filter membrane, carrying out chromatography, and adsorbing the target protein on an ion exchange chromatography column;

i. eluting the chromatographic column by using an eluent, and collecting an active elution peak solution F;

j. dialyzing or ultrafiltering the solution F to remove salt ions in the solution F to obtain a solution G;

k. concentrating the solution G, and freeze-drying to obtain a crude product of the maggot kinase;

l. refining of maggot kinase: balancing the solution G obtained in the step j to pH6.5-8, carrying out affinity adsorption on the maggot kinase in the solution G by using a filler containing a serine protease adsorption ligand, and eluting by using an eluent to obtain an active eluent H;

m, dialyzing or ultrafiltering the solution H to remove salt ions in the solution H to obtain a solution I;

n: and concentrating the solution I, and freeze-drying to obtain a refined product of the maggot kinase.

In some embodiments, the starting material in step a is fly larvae, preferably larvae of the families housefly and callyphoridae, more preferably larvae of housefly and chrysomyia megacephala.

In some embodiments, the homogenizing agent in step a is selected from distilled water and PBS buffer.

In some embodiments, in step e, the solvent is selected from distilled water and PBS buffer.

In some embodiments, in steps f, j, m, the dialysis is performed with a dialysis membrane cut-off of less than 3000 and an ultrafiltration membrane cut-off of less than 3000.

In some embodiments, in step g, the pH is adjusted using citrate buffer or PBS buffer.

In some embodiments, in step h, the size of the filter is 0.20 μm or 0.45 μm.

In some embodiments, in step h, the chromatography column is selected from the group consisting of CM-52, DEAE-32, and DEAE-52.

In some embodiments, in step i, the eluent is selected from NaCl, a glycine salt buffer containing NaCl, and KCl.

In some embodiments, in step i, the concentration of the eluent is 0.5-2M.

In some embodiments, the concentration of solution G in steps k, n comprises trapping of 2000-50000 molecular weight material using an ultrafiltration device, concentration, or concentration using PEG 12000.

In some embodiments, the dialysis membrane or ultrafiltration membrane dialyzed or ultrafiltered in steps f, j, m is the same or different.

In some embodiments, the affinity chromatography packing of step i is grafted with a ligand selected from any one of: benzamidine, p-aminobenzamidine hydrochloride, arginine, an arrowhead protease inhibitor, a soybean trypsin inhibitor, and ovomucin.

In some embodiments, the method for refining maggot kinase includes: refining and purifying a sample by using benzamidine as an affinity chromatography column; equilibrating the solution G to pH7.4, filtering the solution G with a 0.45 μ M filter membrane, subjecting the filtrate to affinity adsorption with benzamidine lipotrope gel 4FF, and collecting the elution peak H, wherein the equilibration solution is 0.1M PBS (pH7.4) containing 0.05M NaCl; eluent is 50mM-pH3 glycine hydrochloric acid buffer solution containing 0.5M NaCl;

in some embodiments, the method for refining maggot kinase includes: purifying with ovomucin affinity chromatographic column, balancing solution G with 0.1M Tris-HCl buffer solution with pH 8, performing affinity adsorption with ovomucin chromatographic column, eluting protein adsorbed on affinity chromatographic column with eluent (0.1M formic acid solution with pH2.5 containing 0.5M KCl), and collecting active elution peak H.

In another aspect, the present invention provides maggot kinases prepared according to the above-described methods.

In another aspect, the present invention provides use of the maggot kinase prepared according to the above method in preparing a thrombolytic drug.

In another aspect, the present invention provides use of the maggot kinase prepared according to the above method in preparing a medicament for preventing or treating diseases associated with thrombus.

In some embodiments, the thrombosis related disorder includes, but is not limited to, coronary atherosclerotic heart disease, thrombophlebitis, stasis of blood flow, mesenteric artery and vein thrombosis, and cerebral thrombosis.

In some embodiments, the thrombosis-related disorder is selected from the group consisting of coronary atherosclerotic heart disease, thrombophlebitis, stasis of blood flow, mesenteric artery and vein thrombosis, and cerebral thrombosis.

In another aspect, the present invention provides a pharmaceutical composition characterized in that it comprises a thrombolytic enzyme prepared according to the above method and a pharmaceutically acceptable carrier.

In some embodiments, the mode of delivery of the pharmaceutical composition includes, but is not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, intraarterial administration), oral administration, topical administration, buccal administration, rectal administration, vaginal administration, subcutaneous administration, intraperitoneal administration, surgical implants, and local injections.

In some embodiments, the pharmaceutical composition is delivered in a manner selected from the group consisting of systemic administration, parenteral administration (including intravascular, intramuscular, intraarterial administration), oral administration, topical administration, buccal administration, rectal administration, vaginal administration, subcutaneous administration, intraperitoneal administration, surgical implantation, and local injection.

In another aspect, the present invention provides a method for treating thrombus, the method comprising administering an effective amount of maggot kinase prepared according to the above-described method to a subject.

In some embodiments, the subject is selected from a vertebrate and an invertebrate.

Definition of

Maggot kinase: the invention extracts protein with thrombolysis ability from fly maggot, named as maggot kinase. According to the evaluation standard of national drug standard WS1- (X-052) -2001Z, the maggot kinase has high activity of degrading fibrin and fibrinogen, and belongs to thrombolytic enzyme.

Advantageous effects

(1) The high-activity maggot kinase is separated from the maggots for the first time, which provides a material basis for the pioneering work and the development of new thrombolytic enzyme.

(2) The maggot kinase is discovered to be serine protease through a system experiment, and a scientific basis is provided for refining and purifying the maggot kinase.

(3) Establishes a technical process for separating high-activity maggot kinase from fly maggots and provides a foundation for the production of thrombolytic enzyme from the insect.

(4) The fly maggots are easy to culture, the produced maggot kinase has high activity and low cost, can provide a foundation for the subsequent preparation of thrombolytic enzyme with high activity and low price, and has great social value.

Drawings

FIG. 1 shows an ion exchange chromatography elution profile. Adsorbing maggot kinase in the extracting solution through a CM52 chromatographic column, and eluting by using NaCl, wherein the abscissa is time, and the ordinate is an ultraviolet absorption peak of the eluent.

FIG. 2 shows an affinity adsorption elution profile. And carrying out affinity purification on the enzyme solution by using a benzamidine affinity chromatographic column, wherein the elution peak is high-purity maggot kinase, and the ordinate is the ultraviolet absorption peak of the product.

Fig. 3 shows enzyme activity evaluation. And (3) enzyme fiber activity determination, wherein the national drug standard WS1- (X-052) -2001Z is approved by reference to the national pharmacopoeia committee of the State food and drug administration. Taking a lumbrukinase standard substance as a positive control, and taking physiological saline as a negative control; the vertical two diameters of the lysis ring are measured by a vernier caliper, and the enzyme activity is measured. Firstly, the thrombolytic effect of the maggot kinase prepared in the example 1; ② the thrombolytic effect of the maggot kinase prepared in the example 2; ③ the thrombolytic effect of the maggot kinase prepared in the example 3; fourthly, the thrombolytic effect of the maggot kinase prepared in the example 4; fifthly, the thrombolytic effect of the maggot kinase prepared in the embodiment 5; sixthly, the thrombolytic effect of the maggot kinase prepared in example 6.

Figure 4 shows a protein SDS electropherogram. The first lane and the fourth lane are protein electrophoresis marker samples, the second lane is the maggot kinase refined by benzamidine in the example 6, and the third lane is the maggot kinase refined by ovomucin in the example 7.

Fig. 5 shows SDS electrophoresis patterns of the degradation of fibrinogen by maggot kinase. Lanes 1-6 are electrophoresis images of the degraded fibrinogen after 15min, 30min, 1h, 1.5h, 2h and 24h of the reaction of maggot kinase with fibrinogen in example 7, respectively.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

Example 1 preparation of maggot kinase

100g of fresh frozen housefly larvae are taken, washed by water, added with 500mL of distilled water for homogenization, centrifuged at 8000r/min to remove bottom sediment, supernatant A is collected, insoluble impurity sediment is removed, and maggot kinase is dissolved in the supernatant.

Ammonium sulfate was added to bring the ammonium sulfate saturation in solution a to 30%. Standing for 4h, centrifuging at 8000r/min after protein precipitation, and collecting supernatant B. Separating out the foreign protein by salting out method, and the maggot kinase is located in the supernatant.

And supplementing ammonium sulfate to the collected supernatant B to 75% saturation, standing, centrifuging at 7000r/min, discarding the supernatant, collecting the precipitate, and dissolving the precipitate with purified water to obtain a precipitate aqueous solution C. Precipitating maggot kinase in the supernatant by using a salting-out method, and dissolving the precipitate (containing maggot kinase in the precipitate) by using water to obtain a maggot kinase-containing aqueous solution C.

And (3) dialyzing or ultrafiltering the aqueous solution C to remove salt ions in the solution C until the conductivity is 50 mu s/cm, so as to obtain a solution D, which is prepared for subsequent ion exchange chromatography.

Solution D was equilibrated in 10mM citric acid buffer at pH 4 to give solution E.

The solution E was filtered through a 0.45 μm filter and chromatographed on a CM52 cation column, and the target protein was adsorbed on CM 52.

Elution was performed with a citrate buffer (pH 4,10mM) containing 0.5M NaCl, and an active elution peak solution F was collected.

The collected solution F was dialyzed (molecular weight cut-off: 2KD) to remove salt ions from the solution F, thereby obtaining a solution G.

The solution G was concentrated with PEG12000 and lyophilized to obtain 0.381G (381mg) of crude enzyme. The determination of the activity of the enzyme in dissolving fiber is carried out according to the national pharmacopoeia committee of the State food and drug administration for examining the national drug standard WS1- (X-052) -2001Z, and the determination of the specific activity of the enzyme is as follows: 15439U/mg.

And (3) fiber dissolving activity determination: taking 39ml of fibrinogen solution (1.5 mg of coagulable protein solution in each 1 ml), placing the fibrinogen solution in a beaker, adding 39ml of 55 ℃ agarose solution and 3.0ml of thrombin solution (1 ml of solution containing 1BP unit) while stirring, immediately mixing, quickly pouring into a plastic culture dish with the diameter of 14cm, placing the mixture horizontally for 1 hour at room temperature, and perforating. Precisely measuring lumbrukinase standard solution and test solution with different concentrations, respectively dropping in the same plate, covering, and reacting in 37 deg.C incubator for 18 hr. And taking out the test sample, measuring two vertical diameters of the lysis ring by using a caliper, calculating a regression equation by using the logarithm of the unit number of the lumbrokinase standard product as a horizontal coordinate and the logarithm of the product of the two vertical diameters as a vertical coordinate, substituting the logarithm of the product of the two vertical diameters of the test sample into the regression equation, and calculating the unit number of the titer of the test sample.

SDS-PAGE electrophoresis method

Protein electrophoresis is carried out on the product, and the formula of the separation gel and the concentrated gel is as follows; electrophoresis conditions: the voltage is adjusted to about 80v to maintain a constant voltage. When the bromophenol blue label moves into the concentrated gel, the voltage is adjusted to about 120v and kept constant.

TABLE 1 separation gel, concentrated gel formulations

Example 2 preparation of maggot kinase

100g of fresh housefly larvae are taken, washed by water, added with 800mL of distilled water, homogenized, centrifuged at 8000r/min to remove bottom sediment, and supernatant A is collected.

Ammonium sulfate was added to achieve 25% saturation of ammonium sulfate in solution a. Standing for 4h, centrifuging at 8000r/min after protein precipitation, and collecting supernatant B.

Supplementing ammonium sulfate to the collected supernatant B to 70% saturation, standing, centrifuging at 8000r/min, removing supernatant, collecting precipitate, and dissolving the precipitate with purified water to obtain precipitate water solution C.

And (3) dialyzing or ultrafiltering the aqueous solution C to remove salt ions in the solution C until the conductivity is 90 mus/cm to obtain a solution D.

The solution D was equilibrated in 10mM citric acid buffer at pH 5 (pH 5 was adjusted to make maggot kinase positively charged in the solution and prepare for the next step of ion exchange chromatography) to obtain a solution E.

The solution E was filtered through a 0.45 μm filter and chromatographed on a CM52 cation column, and the target protein was adsorbed on CM 52.

Elution was performed with a citrate buffer (pH 5,10mM) containing 0.5M NaCl, and an active elution peak solution F was collected.

And (4) dialyzing or ultrafiltering the collected solution F to remove salt ions in the solution F to obtain a solution G.

And concentrating the solution G by using an ultrafiltration device (the molecular weight cutoff is 3000-50000), and freeze-drying to obtain 0.225G (225mg) of the maggot kinase crude enzyme. And (3) determining the activity of the dissolved fiber, namely determining the activity of the dissolved fiber according to the national drug standard WS1- (X-052) -2001Z approved by the national pharmacopoeia committee of the State food and drug administration, and determining the specific activity of enzyme as follows: 28363U/mg (-20 ℃ C.).

Example 3 preparation of maggot kinase

100g of fresh frozen housefly larvae are taken, washed by water, 1600mL of distilled water is added for homogenization, bottom sediment is removed by centrifugation at 8000r/min, and supernatant A is collected.

Ammonium sulfate was added to bring the ammonium sulfate saturation in solution a to 35%. Standing for 4h, centrifuging at 8000r/min after protein precipitation, and collecting supernatant B.

Supplementing ammonium sulfate to the collected supernatant B to 70% saturation, standing, centrifuging at 8000r/min, removing supernatant, collecting precipitate, and dissolving the precipitate with purified water to obtain precipitate water solution C.

And (3) dialyzing or ultrafiltering the aqueous solution C to remove salt ions in the solution C until the conductivity is 70 mu s/cm, thus obtaining a solution D.

Solution D was equilibrated in 10mM PBS buffer at pH6 to give solution E.

The solution E was filtered through a 0.45 μm filter and chromatographed on a CM52 cation column, and the target protein was adsorbed on CM 52.

Elution was performed with PBS buffer (pH 6,10mM) containing 0.5M NaCl, and an active elution peak solution F was collected. And (4) carrying out ultrafiltration on the collected solution F by using a 3KD filter membrane to remove salt ions in the solution F to obtain an interception solution G.

The solution G was concentrated with 12000PEG, and lyophilized to obtain 0.148G (148mg) of crude maggot kinase. And (3) determining the activity of the dissolved fiber, namely determining the activity by referring to the national drug standard WS1- (X-052) -2001Z approved by the national pharmacopoeia committee of the State food and drug administration, and determining the specific activity of the enzyme as follows: 29885U/mg.

Example 4 preparation of maggot kinase

(1) 100g of fresh frozen housefly larvae are taken, washed by water, 1600mL of distilled water is added for homogenization, and the filtrate A is collected after filtration by a filter membrane.

(2) Ammonium sulfate was added to bring the ammonium sulfate saturation in solution a to 30%. Standing for 4h, centrifuging at 8000r/min after protein precipitation, and collecting supernatant B.

(3) Supplementing ammonium sulfate to the collected supernatant B to 70% saturation, standing, centrifuging at 8000r/min, removing supernatant, collecting precipitate, and dissolving the precipitate with purified water to obtain precipitate water solution C.

(4) And (3) dialyzing or ultrafiltering the aqueous solution C to remove salt ions in the solution C until the conductivity is 70 mu s/cm, thus obtaining a solution D.

(5) Solution D was equilibrated in 10mM PBS buffer at pH7 to give solution E.

And filtering the solution E, putting the solution on a CM52 cation chromatographic column, and performing chromatographic separation to adsorb the target protein on CM 52.

(6) Elution was performed with PBS buffer (pH 7,10mM) containing 0.5M NaCl, and an active elution peak solution F was collected. And (3) dialyzing the collected solution F or ultrafiltering with a 3KD filter membrane to remove salt ions in the solution F to obtain a solution G.

(7) The solution G was concentrated and lyophilized to obtain 0.0328G (32.8mg) of crude maggot kinase. And (3) determining the activity of the dissolved fiber, namely determining the activity by referring to the national drug standard WS1- (X-052) -2001Z approved by the national pharmacopoeia committee of the State food and drug administration, and determining the specific activity of the enzyme as follows: 41446U/mg.

Example 5 preparation of maggot kinase

(1) 100g of fresh frozen housefly larvae are taken, washed by water, 1600mL of distilled water is added for homogenization, bottom sediment is removed by centrifugation at 8000r/min, and supernatant A is collected.

(2) Ammonium sulfate was added to bring the ammonium sulfate saturation in solution a to 30%. Standing for 4h, centrifuging at 8000r/min after protein precipitation, and collecting supernatant B.

(3) Supplementing ammonium sulfate to the collected supernatant B to 70% saturation, standing, centrifuging at 8000r/min, removing supernatant, collecting precipitate, and dissolving the precipitate with purified water to obtain precipitate water solution C.

(4) And (3) dialyzing or ultrafiltering the aqueous solution C to remove salt ions in the solution C until the conductivity is 70 mu s/cm, thus obtaining a solution D.

(5) Solution D was equilibrated in 10mM PBS buffer at pH 8 to give solution E.

And filtering the solution E, putting the solution on a CM52 cation chromatographic column, and performing chromatographic separation to adsorb the target protein on CM 52.

(6) Elution was performed with PBS buffer (pH 8,10mM) containing 0.5M NaCl, and an active elution peak solution F was collected.

And (3) dialyzing or ultrafiltering the collected solution F (the molecular weight cut-off of the filter membrane is 2000 daltons), and removing salt ions in the solution F until the conductivity is 50 mus/cm to obtain a solution G.

(7) The eluate G was concentrated and lyophilized to obtain 0.0283G (28.3mg) of maggot kinase. The determination of the activity of the enzyme in dissolving fiber is carried out according to the national pharmacopoeia committee of the State food and drug administration for examining the national drug standard WS1- (X-052) -2001Z, and the determination of the specific activity of the enzyme is as follows: 71649U/mg.

Examples 1-5 show that the pH of solution D is an important parameter affecting the activity of the product, and that the closer the pH of solution D is adjusted to 8, the higher the specific activity of the enzyme obtained.

Example 6 preparation of maggot kinase

(1) 100g of fresh frozen housefly larvae are taken, washed by water, 1800mL of distilled water is added for homogenization, bottom sediment is removed by centrifugation at 8000r/min, and supernatant A is collected.

(2) Ammonium sulfate was added to bring the ammonium sulfate saturation in solution a to 35%. Standing for 4h, centrifuging at 8000r/min after protein precipitation, and collecting supernatant B.

(3) Supplementing ammonium sulfate to the collected supernatant B to 70% saturation, standing, centrifuging at 8000r/min, discarding the supernatant, collecting precipitate, and dissolving the precipitate with purified water to obtain precipitate water solution C.

(4) And (3) dialyzing or ultrafiltering the aqueous solution C to remove salt ions in the solution C until the conductivity is 100 mu s/cm, thus obtaining a solution D.

(5) Solution D was equilibrated in 10mM PBS buffer at pH 8 to give solution E.

And filtering the solution E, loading the solution E on a CM52 cation chromatographic column, performing chromatographic separation, and adsorbing the maggot kinase on CM 52.

(6) Elution was performed with PBS buffer (pH 8,10mM) containing 0.5M NaCl, and an active elution peak solution F was collected. And (3) dialyzing or ultrafiltering (with the molecular weight cutoff of 2000 daltons) the collected solution F to remove salt ions in the solution F to obtain a solution G.

(7) Solution G is equilibrated in 10mM PBS at pH7.5 for affinity chromatography, preferably on benzamidine sepharose containing a filler for adsorption of serine protease ligands, and eluted with 0.05M glycine hydrochloride buffer pH3 containing 0.5M NaCl and the active elution peak H is collected.

(8) And dialyzing the collected solution H (the molecular weight cut-off is 2KD), removing salt ions in the solution H until the conductivity is less than 10 mu s/cm to obtain a solution I, concentrating the solution I by using an ultrafiltration device (the molecular weight cut-off is 2000-50000), and freeze-drying to obtain 0.0031g (3.1mg) of the high-activity maggot kinase.

The fiber dissolving activity of the maggot kinase is determined by examining the national drug standard WS1- (X-052) -2001Z according to the national pharmacopoeia committee of the State food and drug administration, and the activity is determined as follows: 402974U/mg.

Example 7 purification of maggot kinase

(1) 100g of fresh frozen housefly larvae are taken, washed by water, 1800mL of distilled water is added for homogenization, bottom sediment is removed by centrifugation at 8000r/min, and supernatant A is collected.

(2) Ammonium sulfate was added to bring the ammonium sulfate saturation in solution a to 35%. Standing for 4h, centrifuging at 8000r/min after protein precipitation, and collecting supernatant B.

(3) And supplementing ammonium sulfate to the collected supernatant B to 70% saturation, standing, centrifuging at 7500r/min, removing the supernatant, collecting precipitate, and dissolving the precipitate with purified water to obtain a precipitate aqueous solution C.

(4) And (3) dialyzing or ultrafiltering the aqueous solution C to remove salt ions in the solution C until the conductivity is 100 mu s/cm, thus obtaining a solution D.

(5) Solution D was equilibrated in 10mM PBS buffer at pH 8 to give solution E.

And filtering the solution E, loading the solution E on a CM52 cation chromatographic column, performing chromatographic separation, and adsorbing the maggot kinase on CM 52.

(6) Elution was performed with PBS buffer (pH 8,10mM) containing 0.5M NaCl, and an active elution peak solution F was collected. And (3) dialyzing or ultrafiltering (with the molecular weight cutoff of 2000 daltons) the collected solution F to remove salt ions in the solution F to obtain a solution G.

(7) And (3) refining the enzyme: solution G was equilibrated with 0.1M Tris-HCl buffer pH 8 (containing 0.5M KCl) and affinity-chromatographed on an ovomucin column (note: ovomucin grafted on Sepharose 4B); the eluent was 0.1M formic acid solution pH2.5 containing 0.5M KCl. And collecting an activity elution peak H.

(8) Dialyzing the collected solution H (the molecular weight cutoff is 2KD), removing salt ions in the solution H until the conductivity is less than 10 mus/cm to obtain a solution I, concentrating the solution I by PEG12000, freeze-drying to obtain 0.0035g (3.5mg) of high-activity maggot kinase, determining the fibrinolysis activity of the maggot kinase, referring to the national pharmacopoeia committee of the State food and drug administration for examining the national drug standard WS1- (X-052) -2001Z, and determining the activity, wherein the fibrinolysis activity is as follows: 391085U/mg.

(9) The maggot kinases purified in examples 6 and 7 were analyzed by SDS electrophoresis. The electrophoresis bands of the maggot kinase affinity purified by the benzamidine and the ovomucin are consistent, and the benzamidine and the ovomucin are serine protease specific adsorption ligands, which indicates that the product is serine protease.

(10) Fibrinolytic assay: taking 6 centrifuge tubes with the volume of 1.5mL, respectively adding 40 muL of 1.5mg/mL bovine blood fibrinogen (fibrinogen comes from China food and drug testing research institute) and 2 muL of 0.088mg/mL maggot kinase, placing the mixture in a water bath kettle at the temperature of 37 ℃ for heat preservation reaction, wherein the rotating speed of the water bath kettle is about 90 r/min. Measuring bovine blood fibrinogen component after 15min, 30min, 1h, 1.5h, 2h and 24h respectively, and performing SDS electrophoresis analysis. As shown in fig. 5, fibrinogen was gradually degraded over time.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.