阅读说明：本技术 从蝇蛆中分离蛆激酶的方法及应用 (Method for separating maggot kinase from fly maggots and application thereof ) 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 杨明峰 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,所述方法包括将蝇蛆进行匀浆、利用调节杂蛋白等电点沉淀法除去杂蛋白,通过离子交换层析与亲和层析纯化,获得蛆激酶,溶纤实验表明制备的蛆激酶具有高活性溶纤效果。本发明的工艺简单,获得的酶比活高,为抗血栓药物研发提供了基础。(The invention provides a preparation method of maggot kinase, which comprises homogenizing maggots, removing impure protein by using an isoelectric point precipitation method for regulating impure protein, and purifying by ion exchange chromatography and affinity chromatography to obtain maggot kinase, wherein a fiber dissolving experiment shows that the prepared maggot kinase has high-activity fiber dissolving effect. The method has simple process, and the obtained enzyme has high specific activity, thereby providing a foundation for the research and development of antithrombotic drugs.)

1. A method for preparing maggot kinase, characterized by comprising the step of adjusting pH of maggot kinase extract solution to 4.5-6.0 to remove foreign proteins.

2. The method as claimed in claim 1, further comprising the step of adjusting the pH of the maggot kinase extracting solution to 4.5-6.0 and standing at 4-8 ℃ to remove foreign proteins.

3. The method as claimed in claim 1, further comprising the step of refining the maggot kinase using an affinity filler.

4. The method according to claim 2, characterized in that it further comprises a step of subjecting the maggot kinase preparation to affinity chromatography, preferably cation exchange resin CM 52.

5. The method as claimed in claim 1, further comprising a step of desalting to a conductivity of <100 μ s/cm before obtaining the crude maggot kinase product.

6. The method as set forth in claim 1, further comprising the step of irradiating or filter sterilizing the obtained maggot kinase.

7. Method according to any one of claims 1-6, characterized in that it comprises the following steps:

(1) cleaning raw material fly maggots with clear water, and homogenizing according to the mass ratio of the raw material to water or buffer solution of 1: 3-15;

(2) centrifuging or filtering the homogenate at a high speed to remove impurities and insoluble substances to obtain a maggot kinase extract solution;

(3) adjusting pH of the extractive solution, preferably pH 4.5-6.0, standing at low temperature (4-8 deg.C) until protein precipitate is separated out, centrifuging to remove precipitate, and collecting supernatant;

(4) equilibrating the supernatant to pH 5-8 with a buffer (e.g., PBS buffer), preferably 10-50mM, and filtering with a 0.45 μm filter to obtain an equilibrated clear solution;

(5) adsorbing the zymoprotein molecules in the clear solution obtained in the step (4) by using ion exchange resin, eluting and collecting active peak solution, preferably cation exchange resin CM 52;

(6) desalting the solution by ultrafiltration or dialysis to obtain a protein solution with the molecular weight cutoff of 10000-50000 dalton, and concentrating and desalting until the conductivity is less than 100 mus/cm to obtain a protein concentrated solution;

(7) freezing or vacuum drying the concentrated solution to obtain crude enzyme;

(8) and (3) refining the enzyme: dialyzing and desalting the active peak solution obtained in the step (5), performing affinity adsorption, and collecting an elution peak, wherein the used affinity filler is a filler of which a ligand can adsorb serine protein;

(9) the eluted protein is purified and dried (preferably freeze-dried) to obtain the refined maggot kinase.

8. The process according to claim 7, wherein the affinity filler of step (8) is grafted with a ligand capable of adsorbing serine proteases selected from any one of the group consisting of benzamidine, p-aminobenzamidine hydrochloride, arginine, arrowhead protease inhibitor, soybean trypsin inhibitor and ovomucin.

9. The method according to claim 7, wherein the raw material in step (1) is fly maggots selected from the group consisting of Musca, Drosophila, Musca, and Calibromyidae.

10. Use of maggot kinase obtained by the method according to any one of claims 1-9 as raw material and adjuvant ingredient of antithrombotic medicine.

Technical Field

The invention relates to the field of medicines, in particular to a method for separating maggot kinase from fly maggots and application thereof.

Background

The formation of a thrombus is caused by local blood coagulation, and the main components of the thrombus are insoluble fibrin, platelets, white blood cells and red blood cells. Thrombosis is a pathological process, associated with blood vessels, platelets and thrombin, as well as blood flow velocity. Millions of people die every year in the world from diseases related to thrombus, such as cerebral infarction, cerebral hemorrhage, myocardial infarction and the like, so that the development of medicaments with thrombolytic activity is urgently needed.

The thrombolytic drug can be obtained by artificial chemical synthesis and natural extraction. The nature is a natural treasure house for obtaining thrombolytic drugs, and the natural thrombolytic enzyme has wide sources, small to terrestrial microorganisms, and large to marine organisms, animals and plants and has discovery of thrombolytic active ingredients. The different sources of natural thrombolytics have different thrombus degradation mechanisms, and from the current research reports, most of the thrombolytics can efficiently degrade thrombus and inhibit thrombus formation by directly acting on fibrin and activating an in vivo fibrinolytic system. In 1980, foreign and other people need to find nattokinase with high thrombolysis activity from fermented natto, and subsequent researches show that the nattokinase not only can dissolve thrombus, but also can inhibit platelet coagulation, and has the function of preventing thrombus formation. In 1983, the constant professor of Megasaki university, Japan, extracts thrombolytic protease lumbrokinase from earthworms, and separates the lumbrokinase with thrombolytic activity from the earthworms for the first time. In addition, enzymes with thrombolysis and anticoagulation effects are also found in urechis unicinctus, snake venom, yellow mealworm and human urine in marine organisms.

Disclosure of Invention

The fly maggots are easy to raise and low in raw material cost, the fly maggots are usually sold by taking feed as a commodity, and a research method and a production process of maggot kinase are not available at present. The invention provides a method for separating maggot kinase from fly maggots, the enzyme shows high fibrinolysis and anti-thrombus activity, provides a foundation for the development of high added value of fly maggots, provides thinking and technology for the development of new thrombolytic drugs, and has great social value and economic value.

Specifically, the invention provides the following technical scheme:

in one aspect, the present invention provides a method for preparing maggot kinase, characterized by comprising the step of adjusting pH of maggot kinase extract solution to 4.5-6.0 to remove foreign proteins.

In some embodiments, the method further comprises the step of adjusting the pH of the maggot kinase extraction solution to 4.5-6.0, and standing at 4-8 ℃ to remove foreign proteins.

In some embodiments, the method further comprises a step of refining the maggot kinase with an affinity filler.

In some embodiments, the method further comprises the step of performing affinity chromatography on the maggot kinase crude product, wherein the affinity chromatography is preferably cation exchange resin CM 52.

In some embodiments, the method further comprises a step of desalting to a conductivity of <100 μ s/cm before obtaining the crude maggot kinase preparation.

In some embodiments, the method further comprises a step of irradiating or filter sterilization of the obtained maggot kinase.

In some embodiments, the method comprises the steps of:

(1) cleaning the fly maggots with clear water, and homogenizing according to the mass ratio of the raw materials to water or buffer solution of 1: 3-15;

(2) centrifuging or filtering the homogenate at a high speed to remove impurities and insoluble substances to obtain a maggot kinase extract solution;

(3) adjusting pH of the extractive solution, preferably pH 4.5-6.0, standing at low temperature (4-8 deg.C) until protein precipitate is separated out, centrifuging to remove precipitate, and collecting supernatant;

(4) equilibrating the supernatant to pH 5-8 with a buffer (e.g., PBS buffer), preferably 10-50mM, and filtering with a 0.45 μm filter to obtain an equilibrated clear solution;

(5) adsorbing maggot kinase protein molecules in the clear solution obtained in the step (4) by using ion exchange resin, eluting and collecting active peak solution, wherein the cation exchange resin CM52 is preferred;

(6) desalting the solution by ultrafiltration or dialysis to obtain a protein solution with the molecular weight cutoff of 10000-50000 dalton, and concentrating and desalting until the conductivity is less than 100 mus/cm to obtain a maggot kinase concentrated solution;

(7) freezing or vacuum drying the concentrated solution to obtain crude enzyme;

(8) and (3) refining the enzyme: desalting the active peak solution obtained in the step (5), performing affinity adsorption, and collecting an elution peak, wherein the used affinity filler is a filler for adsorbing serine protein;

(9) the eluted protein is purified and dried (preferably freeze-dried) to obtain the maggot kinase.

In some embodiments, the affinity filler of step (8) is grafted with a ligand capable of adsorbing a serine protease selected from any one of the following, preferably selected from benzamidine, p-aminobenzamidine hydrochloride, arginine, an arrowhead protease inhibitor, a soybean trypsin inhibitor, and ovomucin.

In some embodiments, the raw material in step (1) is a larva selected from the group consisting of housefly, city fly, chrysomyia megacephala, red head blowfly, and lucilia sericata.

In some embodiments, the starting material in step (1) may also be larvas of the family sarcophagidae, such as larvae of the genus elaiopsis, such as, for example, acroleidae.

In another aspect, the present invention provides the use of the maggot kinase obtained according to the above-mentioned method as a raw material and an adjuvant component of an antithrombotic drug.

In another aspect, the present invention provides a method for treating thrombotic diseases, which comprises administering the maggot kinase obtained according to the above method to a subject in need thereof.

Advantageous effects

(1) The isoelectric points of the impure proteins in the fly maggots are mainly distributed at the pH value of 4.5-6, and when the extracting solution is pretreated, the impure proteins are precipitated by using a method for adjusting the pH value, the maggot kinase is reserved, the process is simple, and the reagent cost is low.

(2) The high-activity thrombolytic enzyme is obtained by utilizing the fly maggots for the first time, and a foundation is provided for the high-value development of the fly maggots.

(3) The maggot kinase has cheap and easily obtained raw materials and high activity, provides a foundation for developing low-price and high-activity antithrombotic medicaments, and has important social value.

Drawings

Figure 1 shows a schematic of pH 5.5 adjustment in pretreatment to precipitate the hetero-protein;

FIG. 2 shows a schematic of chromatographic elution with CM52 packing, showing multiple protein peaks;

FIG. 3 shows a schematic diagram of affinity adsorption elution using ovomucin, and it can be seen that a single elution peak is obtained by affinity chromatography, and maggot kinases of high purity are prepared;

FIG. 4 shows a schematic diagram of elution after affinity adsorption using benzamidine, and it can be seen that a single elution peak is obtained by affinity chromatography, and high-purity maggot kinase is prepared;

FIG. 5 shows the SDS protein electrophoresis of example 5 in which the raw material is purified into maggot kinase, the first lane from left to right is electrophoretic marker, the second lane is raw material homogenate, the third lane is supernatant after acid precipitation, the fourth lane is sample after CM52 chromatography, the fifth lane is benzamidine affinity purification sample, and the sixth lane is marker;

FIG. 6 shows a plate diagram of activity assay by fiber plate method according to national drug Standard WS1- (X-052) -2001Z, where fibrinolysin 1, 2 are lumbrokinase standards (purchased from China food and drug testing institute) and fibrinolysin 3 is a maggot kinase sample obtained after affinity chromatography of benzamidine of the present invention.

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

In the pretreatment process, the method does not remove the foreign proteins by an ammonium sulfate precipitation method and retains the maggot kinase, but during pretreatment, "the pH value of a supernatant solution is adjusted, preferably the pH value is 4.5-6.0, the supernatant solution is kept at a low temperature of 4-8 ℃ for centrifugal precipitation after protein precipitation is carried out, and the foreign proteins are removed by adjusting the pH value, so that the process is simpler and the cost is low.

Specifically, example 1 includes the following steps:

(1) 100g of housefly larvae are washed with water or sodium chloride and homogenized by adding 500mL of water.

(2) Centrifuging the homogenate at 8000r/min, removing impurities and insoluble substances, collecting supernatant, and filtering with 500 mesh filter membrane to obtain maggot kinase extract solution A.

(3) Collecting extractive solution A, adjusting pH to 5.5 with dilute hydrochloric acid, standing at 4 deg.C for 2 hr to obtain flocculent protein and precipitate, centrifuging at 8000r/min, discarding precipitate (impurity protein), collecting supernatant, and filtering with 0.45 μm filter membrane to obtain clear solution B.

(4) Solution B was equilibrated to pH 8 with 0.01M PBS and pH 8 buffer, and filtered through a 0.45 μ M filter to obtain solution C.

(5) The maggot kinase molecules in the solution C were adsorbed using a cation exchange resin CM-52, and the active peak solution D was collected after elution using a PBS (0.01M PBS, pH 8, and in some embodiments, KCl) buffer containing 0.5M NaCl.

(6) And (3) performing ultrafiltration on the solution D to obtain a molecular cut-off solution with the cut-off molecular weight of 10000-50000 dalton, measuring the conductivity of the cut-off solution by using a conductivity meter, and concentrating and desalting until the conductivity is less than 100 mu s/cm to obtain a protein concentrated solution E.

(7) After the concentrate E was frozen or dried under vacuum, 0.0071g (7.1mg) of the crude enzyme was obtained, which had a specific activity of 68872U/mg.

(8) Or (3) dialyzing the solution D obtained in the step (5), placing the solution D in PBS to be balanced to pH7.4, filtering the solution by using a 0.45 mu m filter membrane, carrying out affinity adsorption on the filtrate by using benzamidine sepharose 4FF, and collecting the solution of an elution peak F.

Note: benzamidine Sepharose 4FF Filler (Solebao Co.), equilibrium liquid 0.1M pH7.4PBS, contain 0.05M NaCl; eluent 50mM glycine hydrochloride buffer solution with pH3 containing 0.5 MNaCl;

(9) and (3) dialyzing and desalting the F solution, and freeze-drying to obtain 0.0005g (0.5mg) of high-activity maggot kinase molecules. The activity of the product is determined according to the national drug standard WS1- (X-052) -2001Z approved by the national Committee of pharmacopoeia of the State food and drug administration. The specific activity is as follows: 451512U/mg.

Determination of fibrinolytic Activity of maggot kinase

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. )

(9) Sterilization is performed by irradiation or filtration.

Note: when ion exchange chromatography and affinity chromatography are carried out, a JP-3000 type protein nucleic acid detection instrument is used for real-time determination of the light absorption value of the eluted protein

Example 2 preparation of maggot kinase-unrefined

(1) 100g of the C.martensii larvae were washed with water or sodium chloride and homogenized by adding 1000mL of water.

(2) Centrifuging the homogenate at 7000r/min to remove impurities and insoluble substances, collecting supernatant, and filtering with 500 mesh filter membrane to obtain filtrate A.

(3) Collecting solution A, adjusting pH of solution A to 6 with dilute hydrochloric acid, standing at 4 deg.C for 2 hr to obtain flocculent protein and precipitate, centrifuging at 8000r/min, discarding precipitate, and collecting supernatant B.

(4) Solution B was equilibrated to pH 8 with 0.01M PBS and pH 8 buffer, and filtered through a 0.45 μ M filter to obtain solution C.

(5) The maggot kinase molecules in the solution C were adsorbed by using a cation exchange resin CM-52, and eluted with a PBS buffer (0.01M PBS, pH 8) containing 0.5M NaCl, and then an active peak solution D was collected.

(6) And (3) performing ultrafiltration on the solution D to obtain a protein solution with the molecular weight cutoff of 10000-50000 dalton molecules, and concentrating and desalting the solution until the conductivity is less than 100 mu s/cm to obtain a protein concentrated solution E.

(7) After freeze-drying or vacuum-drying the concentrate E, 0.0081g (8.1mg) of crude enzyme was obtained, with a specific activity of: 71181U/mg.

EXAMPLE 3 preparation of maggot kinase-benzamidine agarose gel refining

(1) 100g of housefly larvae are washed with water or sodium chloride and homogenized by adding 1000mL of water.

(2) Centrifuging the homogenate at 7000r/min to remove impurities and insoluble substances, collecting supernatant, and filtering with 500 mesh filter membrane to obtain filtrate A.

(3) Adjusting pH of filtrate A to 6 with dilute hydrochloric acid, standing at 4 deg.C for 2 hr to obtain flocculent protein and precipitate, centrifuging at 8000r/min, discarding the precipitate, and collecting supernatant B.

(4) Solution B was equilibrated to pH 8 with 0.01M PBS and pH 8 buffer, and filtered through a 0.45 μ M filter to obtain solution C.

(4) Adsorbing maggot kinase molecules in the solution C by using cation exchange resin CM-52, eluting by using an aqueous solution containing 0.5M NaCl, and collecting an active peak solution D.

(5) The solution D was dialyzed, then equilibrated to pH7.4 with PBS buffer, filtered through a 0.45 μm filter, and the filtrate was subjected to affinity adsorption with benzamidine sepharose 4FF, and the peak E elution solution was collected.

Note: benzamidine Sepharose 4FF Filler (Solebao Co.), equilibrium liquid 0.1M pH7.4PBS, contain 0.05M NaCl; eluent is 50mM glycine hydrochloric acid buffer solution with pH3 and containing 0.5M NaCl;

(8) the E solution is dialyzed and desalted, and 0.0006g (6mg) of high-activity maggot kinase is obtained after freeze drying, preferably freeze drying. The specific activity is as follows: 453753U/mg. The products were analyzed for molecular weight by SDS-PAGE electrophoresis. The activity of the product is determined according to the national drug standard WS1- (X-052) -2001Z approved by the national Committee of pharmacopoeia of the State food and drug administration.

Note: SDS-PAGE conditions were: 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 4 preparation of maggot kinase-pretreatment pH of solution a was adjusted to 4.5, and ovomucin was subjected to affinity chromatography and refined

(1) 100g of housefly larvae were washed with water or sodium chloride and homogenized by adding 1500mL of water.

(2) Centrifuging the homogenate at 8000r/min, or filtering to remove impurities and insoluble substances to obtain extractive solution A.

(3) Adjusting pH of solution A to 4.5 with dilute hydrochloric acid, standing at 4 deg.C for 2 hr to obtain flocculent protein and precipitate, centrifuging at 8000r/min, discarding the precipitate, and collecting supernatant B.

(4) The pH of the supernatant B was adjusted to 8 with a PBS buffer solution having an ionic strength of 10mM and pH 8, and the mixture was filtered through a 0.45 μm filter to obtain a solution C.

(5) And adsorbing the zymoprotein molecules in the solution C by using cation exchange resin CM-52, eluting and collecting an active peak solution D.

(6) And (3) dialyzing the solution D obtained in the step (5), balancing with a Tris-HCl buffer solution with the pH value of 8, performing affinity adsorption by using an ovomucin chromatographic column, eluting the protein adsorbed on the affinity chromatographic column by using an eluent, and collecting an elution peak solution E with activity.

(7) And E solution is dialyzed and desalted, and after freeze drying, 0.0006g (0.6mg) of high-activity maggot kinase is obtained, and the specific activity is 466571U/mg: and (3) product activity determination: the measurement of the fibrinolytic activity was carried out according to the national drug Standard WS1- (X-052) -2001Z approved by the State Committee on the pharmacopoeia of the State food and drug administration.

Solution D was equilibrated with 0.1M Tris-HCl buffer pH 8, containing 0.5M KCl; the eluent was 0.1M formic acid solution pH2.5 containing 0.5M KCl. The preparation method of the affinity filler ovomucin chromatographic column comprises the following steps:

1 affinity chromatography column (preparation of ovomucin chromatography column)

1.1. Activation of Sepharose 4B

8G of Sepharose 4B, an agarose gel chromatography medium, was weighed, placed in a G-3 glass fritted funnel, and then subjected to suction washing (a small amount of times) with 100ml of 1.0mol/L NaCl solution, 100ml of distilled water, and transferred to a 100ml Erlenmeyer flask for later use after being drained.

1.2 activator formulation

7ml of distilled water, 8ml of 1, 4-dioxane, 6.5ml of 2mol/L NaOH and 1.5ml of epichlorohydrin are sequentially added into 8g of agarose gel chromatography medium.

1.3 activation

And (3) putting the prepared triangular flask of the chromatography medium into a constant-temperature water bath at 45 ℃, and shaking for activation for 2 hours at 160 r/min. Activation was stopped, transferred to a G-3 glass fritted funnel, the activator removed, washed with 100ml of distilled water (several times in small amounts), and 100ml transferred to a Erlenmeyer flask in preparation for coupling.

1.4 coupling

Weighing about 100mg chicken ovomucin (purchased from Sigma Co.) and adding 10ml 0.1mol/L Na with pH of 9.5₂CO₃The buffer was fully dissolved. Then transferring the dissolved chicken ovomucin solution into a 100ml triangular flask to be uniformly mixed with the activated agarose gel medium. The coupling is stopped after shaking for about 22 hours in a constant temperature water bath at 40 ℃ and 140 r/min.

1.5 washing

A500 ml clean filtration flask was taken and the coupled Sepharose chromatography medium was transferred to a G-3 glass fritted funnel and filtered with suction.

The column was then washed with 100mL of 1.0mol/L NaCl solution, 100mL of distilled water, and then 20mL of the affinity chromatography eluent and 50mL of distilled water, respectively. Transferring the affinity medium into a small beaker of 50ml, adding 20ml of affinity chromatography equilibrium solution, soaking for 20 minutes, and degassing for later use.

Examples 1, 3 and 4 all show that the filler containing serine ligand (benzamidine, ovomucin) can adsorb maggot kinase to purify the maggot kinase, which indicates that the maggot kinase is serine protease.

Comparative example

This example is a comparative example, in which the pH of the extract A was not adjusted in the pretreatment, and if the foreign protein in the A solution could not be precipitated, a clear solution could not be obtained, which resulted in difficulty in the subsequent separation process, as shown below.

(1) 100g of raw housefly larvae are washed with water or sodium chloride, and 500mL of water is added for homogenization.

(2) Centrifuging the homogenate at 7000r/min, removing impurities and insoluble substances, collecting supernatant, and filtering with 500-mesh filter membrane to obtain maggot kinase extract solution A, wherein the solution A is turbid liquid.

(3) The cation exchange resin CM-52 is used for adsorbing maggot kinase molecules in the solution A, and the solution A is turbid, liquid containing a large amount of impurities, thick and high in pigment content, so that the CM-52 cannot effectively adsorb maggot kinase, and the subsequent extraction process cannot be carried out. Therefore, the key step of the subsequent process for extracting the maggot kinase from the housefly larvae is to adjust the pH value of the extracting solution A to 4.5-6 to precipitate the foreign proteins so as to obtain a clear solution.

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.