CN106632632B

CN106632632B - Armadillidium fibrinolytic active protein and application thereof

Info

Publication number: CN106632632B
Application number: CN201610920569.9A
Authority: CN
Inventors: 李博; 吴勉华; 郭立玮; 朱华旭; 王正俊
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2016-10-21
Filing date: 2016-10-21
Publication date: 2020-06-02
Anticipated expiration: 2036-10-21
Also published as: CN106632632A

Abstract

The invention provides a pillbug fibrinolytic active protein and application thereof, relating to the field of pharmacy. A fibrinolytic active protein of Armadillidium vulgare is prepared by sequentially subjecting pulverized Armadillidium vulgare to enzymolysis with pepsin and trypsin, and separating to obtain product with molecular weight of 8k-10k Da and thrombolytic and anticoagulant activities. The pillworm fibrinolytic active protein has thrombolytic and anticoagulant activities, has strong in-vivo antithrombotic activity after being orally taken by rats verified by experiments, has relatively mild thrombolytic effect, and can be applied to preparing oral medicaments for treating thrombotic diseases.

Description

Armadillidium fibrinolytic active protein and application thereof

Technical Field

The invention relates to the field of pharmacy, and in particular relates to a pillbug fibrinolytic active protein and application thereof.

Background

Thrombosis is the root cause of three fatal cardiovascular diseases (heart disease, stroke and venous thromboembolism), has high morbidity and is increasing in the global scope. The international society for thrombosis and hemostasis provides data showing that 10 to 30 million people die of venous thrombosis each year in the united states; in europe, 50 million people die of venous thrombosis each year, exceeding the sum of deaths from aids, breast cancer, prostate cancer and highway traffic accidents; in China, the number of deaths due to thrombotic diseases and their complications is also large. Currently, although clinically applied antithrombotic drugs can greatly reduce mortality and disability rate, these thrombolytic drugs still have the disadvantages such as large bleeding side effects, difficult dose control, and possible occurrence of reocclusion and reocclusion. In addition, in the clinical treatment of thrombotic diseases, thrombolytic drugs are generally used to dissolve a formed thrombus, and then anticoagulant drugs are used to prevent the thrombus from being formed again. However, there are few oral drugs on the market that have both thrombolytic and anticoagulant effects, and therefore, it is imperative to find a thrombolytic anticoagulant drug that has low side effects and avoids reocclusion and reocclusion.

Armadillidium is the dried polypide of the gynaecological animals of the isopoda rat of Crustacea, such as Armadillidium Porcellio scaber Latreille, also known as Tide bug, Dilouse, watermelon insect, pea insect, etc., and has the efficacy of breaking blood and promoting diuresis, detoxifying and relieving pain. Armadillidium is a traditional Chinese medicine for treating moderate and severe cancer pain, flat wart, hemorrhoid, pertussis and the like.

Disclosure of Invention

The invention aims to provide a pillbug fibrinolytic active protein which not only has thrombolytic and anticoagulant activities, but also is suitable for oral administration.

The invention also aims to provide the application of the pillbug fibrinolytic active protein in preparing the oral medicine for treating thrombotic diseases.

The purpose of the invention is realized by adopting the following technical scheme.

A fibrinolytic active protein of Armadillidium vulgare is prepared by sequentially subjecting pulverized Armadillidium vulgare to enzymolysis with pepsin and trypsin, and separating to obtain product with molecular weight of 8k-10k Da and thrombolytic and anticoagulant activities.

After enzymolysis of the pillbug crushed material, ion exchange chromatography, gel filtration chromatography and ultrafiltration concentration are sequentially adopted, and fibrinolytic active protein is obtained through separation.

In the ion exchange chromatography, a HiTrap Capto Q ion exchange chromatography column is adopted for separation, 1.5-3 column volumes of eluent containing 0.1-0.3mol/L NaCl is adopted for elution, and eluent is collected.

The chromatographic columns adopted in the gel filtration chromatography are Sephacryl S-200HR gel chromatographic column and Superdex75pg gel chromatographic column.

In the process of separating the Sephacryl S-200HR gel chromatographic column, an eluent is Tris-HCl buffer solution with the pH value of 7-8 and the concentration of 10-30mmol/L, the flow rate of the eluent is 0.5-1mL/min, and the eluent with fibrinolytic activity is collected.

In the separation process of the Superdex75pg gel chromatographic column, an eluent is Tris-HCl buffer solution with the pH value of 7-8 and the concentration of 10-30mmol/L, the flow rate of the eluent is 0.5-1mL/min, and the eluent with fibrinolytic activity is collected.

Concentrating by ultrafiltration to obtain components with molecular weight of more than 5kDa and less than 10 kDa.

When in enzymolysis, the mass ratio of the pepsin, the trypsin and the pillbug crushed material is 0.5-1.5: 1.

The enzymolysis temperature is 37.5-38.5, and the enzymolysis time is 3.5-4.5 hours.

The pillbug fibrinolytic active protein is applied to preparing oral drugs for treating thrombotic diseases.

The pillworm fibrinolytic active protein provided by the invention not only has thrombolytic and anticoagulant activities, but also is suitable for oral administration. The fibrinolytic active protein of the pillbug is obtained by sequentially carrying out enzymolysis on crushed pillbug by adopting pepsin and trypsin and then separating, thus being suitable for oral administration. The activity of the pillbug fibrinolytic active protein in thrombolysis and anticoagulation is obviously stronger than that of PSLTro01, and the experiment proves that the pillbug fibrinolytic active protein has stronger in-vivo antithrombotic activity after being orally taken by rats. The wood louse fibrinolytic active protein of the invention does not directly dissolve fibrin, but converts plasminogen into plasmin by activating plasminogen so as to dissolve fibrin, therefore, the thrombolytic effect of the wood louse fibrinolytic active protein is relatively mild, thus no massive bleeding tendency occurs and the risk of tissue hemorrhage is reduced. In addition, the pillworm fibrinolytic active protein provided by the invention has the advantages of effective preparation method, simple and convenient process, easy operation and suitability for industrial production. The wood louse fibrinolytic active protein can also be applied to preparing oral drugs for treating thrombotic diseases.

Drawings

FIG. 1 shows the results of Sephacryl S-200HR gel chromatography, ▉ indicates the value of A280 nm. diamond-solid indicates fibrinolytic activity, and the abscissa indicates the number of collected tubes.

FIG. 2 shows the results of Superdex75pg gel chromatography, ▉ denotes the A280nm value,. diamond-solid denotes fibrinolytic activity, and the abscissa denotes the number of collected tubes.

FIG. 3 is an electrophoretogram of fractions obtained at each step in the separation and purification process of PSLTro02, wherein each lane is: 1. zymolyte of pulverized Armadillidium vulgare; 2. passing through HiTrap Capto Q ion exchange chromatography column to obtain component F-A; 3. passing through Sephacryl S-200HR gel chromatography column to obtain component F-C; 4. passing through Superdex75pg gel chromatography column to obtain component F-E; 5. purifying by an ultrafiltration centrifugal tube to obtain a component F-F; m. protein Marker.

FIG. 4 is a chromatogram for HPLC detection of PSLTro 02.

FIG. 5MALDI-TOF MS mass spectrometry spectrum of molecular weight determination

FIG. 6 shows a primary mass spectrum of PSLTro 02.

Fig. 7 shows a secondary mass spectrum of PSLTro 02.

FIG. 8 shows the effect of temperature on the fibrinolytic activity of PSLTro 02.

Fig. 9 shows the temperature stability of PSLTro02, with different colored bars representing different treatment times.

FIG. 10 Effect of Metal ions on the fibrinolytic Activity of PSLTro02

FIG. 11 comparison of fibrinolytic activity of substances, a: urokinase; b: (ii) trypsin; c: lumbrukinase; d: pepsin; e: fibrinolytic crude protein of Armadillidium vulgare; f: PSLTro 01; g is PSLTro 02; h, normal saline.

Detailed Description

The invention may be carried out as follows, without intending to limit the scope of the invention.

Example 1 Armadillidium crushed Material and enzymatic hydrolysate thereof

1. Preparation of wet superfine crushed Armadillidium

Weighing 100.0g of dried pillbug, adding distilled water 8 times of pillbug mass, soaking in water bath at 25 deg.C for 1h, adding into ultramicro pulverizer, and wet-micronizing at 25 deg.C for 10 min. Centrifuging the slurry obtained after grinding at 4 ℃ at 10000r/min for 10min, taking supernatant, and freeze-drying to obtain 9.6g of the pillbug wet-process ultrafine powder.

1. Preparation of zymolyte of comminuted pillbug

Weighing 500mg of pillbug wet-process ultrafine crushed material, suspending in 50mL of artificial gastric juice (the preparation of the artificial gastric juice is shown in 2010 version Chinese pharmacopoeia, the concentration of pepsin is 10mg/mL), ensuring that the quality of the pepsin (batch number 313A0313, Solarbio company) in the artificial gastric juice is equal to that of the pillbug wet-process ultrafine crushed material, putting the pillbug wet-process ultrafine crushed material into a constant-temperature 38 ℃ shaking table for enzymolysis for 4h, and centrifuging at 12000r/min for 10min, wherein the supernatant is the artificial gastric juice enzymatic hydrolysate. Freeze-drying into lyophilized powder, suspending in 50mL artificial intestinal juice again to ensure that the quality of trypsin (batch No. 112B1220, Solarbio company) in the artificial intestinal juice (see preparation of artificial intestinal juice in 2010 version Chinese pharmacopoeia, and the concentration of the trypsin is 10mg/mL) is equal to that of the wet superfine crushed material of the pillbug, performing enzymolysis for 4h in a constant-temperature 38 ℃ shaking table, centrifuging for 10min at 12000r/min to obtain supernatant fluid which is the enzymatic hydrolysate of the artificial intestinal juice, and freeze-drying to obtain the enzymatic hydrolysate of the crushed material of the pillbug.

2. Enzyme activity of pepsin and trypsin in zymolyte

The zymolyte is inactivated with pepsin and trypsin before column chromatography according to the following steps: the zymolyte freeze-dried powder is firstly added into a pH7.0 buffer solution (such as 10-30mmol/L, pH7.0 phosphate buffer solution or Tris-HCl buffer solution) to inactivate pepsin, and then added into a solution (10-30 mmol/L, pH7.2 phosphate buffer solution or Tris-HCl buffer solution) containing 25mg/mL TLCK (p-toluenesulfonyl-L-lysinochloridon) and 1mmol/L EDTA (ethylene diamine tetraacetic acid) to inactivate trypsin.

Example 2 preparation of Armadillidium extract having thrombolytic and anticoagulant activities

(1) HiTrap Capto Q ion exchange chromatography

A HiTrap Capto Q ion exchange chromatography column (100mL, purchased from GE Healthcare, USA) was equilibrated with 5 column volumes of equilibration buffer I (Tris-HCl buffer, pH8.0, 20 mmol/L) at a flow rate of 1.0 mL/min. Preparing lyophilized powder of Armadillidium comminuted hydrolysate (inactivated pepsin and trypsin) with 20mmol/L Tris-HCl buffer solution (pH8.0) to obtain 10mg/mL solution, loading to balanced HiTrap Capto Q ion exchange chromatography column, eluting unadsorbed protein with balance buffer solution I, eluting to baseline, eluting with elution buffer solution I (pH8.0 containing 1mol/LNaCl and 20mmol/LTris-HCl buffer solution) and balance buffer solution I, eluting for 2 column volumes, wherein the volume ratio of elution buffer solution I to balance buffer solution I is 1:4, and collecting each 2mL tube. The elution flow rate was 1.0 mL/min. And (3) measuring the absorbance of the protein solution of each tube at 280nm, detecting the fibrinolytic activity, mixing the tubes, and freeze-drying to obtain the component F-A, wherein each tube has the fibrinolytic activity (the detection method is the same as the invention patent application with the application number of 201510043911.7).

(2) Sephacryl S-200HR gel chromatography

The Sephacryl S-200HR gel chromatography column was equilibrated with 5 column volumes of equilibration buffer II (pH7.4, 20mmol/LTris-HCl buffer) at a flow rate of 0.5 mL/min. The fraction F-A was prepared as a 5mg/mL solution with the equilibration buffer II, and subjected to gel separation by loading on the top of the column by the system pump at a flow rate of 0.5 mL/min. Elution was performed with equilibration buffer II at a flow rate of 0.5mL/min, and collection was performed in 3mL tubes. And (3) measuring the absorbance of each tube of protein liquid at 280nm, detecting the fibrinolytic activity, collecting and mixing eluates with the fibrinolytic activity, and freeze-drying to obtain the component F-C. See fig. 1.

(3) Superdex75pg gel chromatography

The Superdex75pg gel chromatography column was equilibrated with 5 column volumes of equilibration buffer II (pH7.4, 20mmol/L Tris-HCl buffer) at a flow rate of 0.5 mL/min; the fraction F-C was prepared into a 5mg/mL solution with the equilibration buffer II, and applied to the top of the column at a flow rate of 0.5mL/min by a system pump for gel separation. Elution was performed with equilibration buffer II at the same flow rate, and collected in 2mL tubes. The absorbance of each tube of protein solution at 280nm was measured and the fibrinolytic activity was measured. The eluents having fibrinolytic activity were mixed to obtain components F-E, see FIG. 2. Passing the fraction F-E through an ultrafiltration centrifuge tube with a molecular weight cut-off of 10kDa (

ultra-15), taking the permeate, passing through an ultrafiltration centrifugal tube (with molecular weight cut-off of 5 kDa)

ultra-15), and collecting the trapped fluid to obtain the component F-F. The component F-F was named as the wood fibrinolytic active protein PSLTro 02. An electrophoretogram of the fibrinolysin PSLTro02 of Armadillia volvuli, as shown in FIG. 3. As can be seen from FIG. 3, the fibrinolytically active protein PSLTro02, has a molecular weight in the range of 8.0-10.0 kDa.

(4) Separation and purification results and identification of fibrinolytic active protein PSLTro02

separation and purification of the product

0.86mg of fibrinolytic active protein PSLTro02 is obtained by separating and purifying zymolyte of 3000mg of the comminuted pillbug, the relative titer is 1509.8U/mg, the recovery rate is 9.6 percent, and the purification multiple is 334.8. The detailed results are shown in Table 1.

TABLE 1 results of purification of Armadillidium fibrinolytic active protein PSLTro02

② HPLC purity detection

HPLC detection as shown in FIG. 4 gave a symmetrical peak, and the integration of the peak area indicated that the purity of the isolated and purified fibrinolytic protein PSLTro02 reached 93.86%.

③ MALDI-TOF MS mass spectrometry molecular weight determination

The PSLTro02 protein had a relative molecular weight of 8480.55Da as determined by MALDI-TOF MS mass spectrometry, as shown in FIG. 5, which is consistent with SDS-PAGE results.

MALDI-TOF MS mass spectrometry protein identification

The PSLTro02 is sent to the detection center of the life academy of sciences of Shanghai province, and is identified by MALDI-TOF MS mass spectrometry, and the primary mass spectrum and the secondary mass spectrum are shown in figures 6 and 7. Mass spectrum data of fibrinolytic protein of the same species are downloaded in a National Center for Biotechnology Information (NCBI) database, and Mascot software is used for analyzing the matching condition of the mass spectrum number of PSLTro02 and the downloaded mass spectrum data, so that the matching degree of PSLTro02 and the protein obtained from Porcellioscaber and Armadillidium vulgare of the same species is lower than 20%, and therefore, the PSLTro02 of the fibrinolytic protein is different from the protein between the same species included in the database at present, namely PSLTro02 is a novel fibrinolytic protein obtained by first enzymolysis of the fibrinolytic protein of pillbug at present.

Example 2 enzymatic Properties of fibrinolytically active protein PSLTro02

1. Effect of pH on fibrinolytic Activity

The fibrinolytic activity of the pillbug fibrinolytic protein PSLTro02 is obviously different under different pH environments. Under strong acid and strong alkali environment, the fibrinolytic activity is sharply reduced, the activity is stronger between pH6 and pH8, the fibrinolytic activity is strongest at pH7, and the fibrinolytic activity in alkaline environment is stronger than that in acidic environment.

Through experiments, PSLTro02 was found to be more stable in alkaline and neutral environments than in acidic environments. Under strong acid conditions, the fibrinolytic activity gradually decreases with time. The pH of the blood in humans is generally maintained at 7.34-7.45, and the body fluid environment is also weakly alkaline, which coincides with the optimum reaction pH of PSLTro 02. This result also confirms that the optimal conditions for pepsin inactivation have no effect on its activity.

2. Effect of temperature on its fibrinolytic Activity

The effect of temperature on the fibrinolytic activity of PSLTro02 is shown in fig. 8, where it can be seen that the effect of temperature is large, especially at temperatures above 50 ℃ where the activity drops sharply, above 60 ℃ where the activity drops almost to 0. The activity of the product is reduced to 0 at a temperature below 10 ℃. However, at 40 ℃ the activity is maximal.

The effect of PSLTro02 on its fibrinolytic activity when treated at different temperatures for different times, i.e. the stability of PSLTro02, was examined. As can be seen from fig. 9, the activity was most stable at a temperature of 20 c, and gradually decreased with the passage of treatment time after a temperature of more than 20 c, and sharply decreased with the passage of treatment time after a temperature of more than 50 c. Therefore, the optimum temperature for storing PSLTro02 is preferably 20 ℃.

3. Effect of Metal ions on their fibrinolytic Activity

PSLTro02 was added to different metal ions (0.1mmol/L) and the effect of the metal ions on the fibrinolytic activity of PSLTro02 was examined as shown in FIG. 10, where it can be seen that Na⁺，K⁺，Mg²⁺Has no influence on the fibrinolytic activity of PSLTro02, and is Cu²⁺，Mn²⁺，Fe²⁺，Ca²⁺，Zn²⁺，Al³⁺，Fe³⁺All inhibit their fibrinolytic activity to different extents, especially Fe²⁺，Fe³⁺Obviously inhibits the activity of the enzyme, and the activity of the residual enzyme is only less than 12.5 percent of that of a blank control after the action of PSLTro 02.

4. Effect of enzyme inhibitors and surfactants on their fibrinolytic Activity

Pepstaiin A (pepsin inhibitor A), KTI (soybean trypsin inhibitor), SBTI (soybean protease inhibitor), Aprotinin (Aprotinin), β -mercaptoethanol (β -mercaptoethanol) had a greater effect on the fibrinolytic activity of PSLTro02, but PMSF (phenylmethylsulfonyl fluoride, 0.1-100mmol/L), TLCK (p-toluenesulfonyl-L-lysinoyl chloride methanone, 0.1-50mg/mL), TPCK (p-toluenesulfonyl-L-phenylalanine acyl chloride methanone, 0.1-50mg/mL), EDTA (ethylenediaminetetraacetic acid, 0.5-50mmol/L), EGTA (ethylene glycol-bis- (2-aminoethylether) tetraacetic acid, 0.5-50mmol/L) and SDS concentrations had substantially no effect on the fibrinolytic activity of PSLTro 02.

Determination of specificity of 5 Armadillidium fibrinolytic active protein PSLTro02 for chromogenic substrate

To each well of a 96-well plate, 175. mu.L of a PSLTro01 solution (containing 1. mu.g of PSLTro02 in a solvent of pH7.2 and 20 mmol. multidot.L) was added^-1Tris-HCl buffer solution) and then 25. mu.L of each of the solutions was added thereto at a concentration of 0.5 mmol. multidot.mL^-1The thrombin-specific chromogenic substrate S-2238(H-D-Phe-Pip-Arg-pNA), the plasmin-specific chromogenic substrate S-2251(H-D-Val-Leu-Lys-pNA) or the urokinase-specific chromogenic substrate S-2444(pyro-Glu-Gly-Arg-pNA) solution (solvent pH7.2, 20 mmol. L^-1Tris-HCl buffer (chromogenic substrates all available from HYPHEN BioMed, France). Putting the 96-well Plate into an EnSpire Multimode Plate Reader enzyme-linked immunosorbent assay temperature control test area, wherein the temperature of the test area is always kept at 37 DEG CAfter 5min of incubation, absorbance of released pNA (p-nitroaniline) was measured by continuous scanning at 5mim under an ultraviolet wavelength of 405 nm. The released amount of pNA was calculated from the change in absorbance of the solution in each well. PSLTro02 activity was defined as: the protein amount of 1 mu mol pNA released by hydrolyzing the chromogenic substrate within 1min per mg of pillworm plasmin at 37 ℃ is 1U.

The results of the hydrolysis of the fibrinolytic protein PSLTro02 of the pillbug are shown in Table 2, and the results show that PSLTro02 has the highest hydrolytic activity on the plasmin optimum substrate H-D-Val-Leu-Lys-pNA, and has the same hydrolytic activity on the thrombin optimum substrate H-D-Phe-Pip-Arg-pNA and the urokinase optimum substrate pyro-Glu-Gly-Arg-pNA but lower hydrolytic activity than the plasmin optimum substrate, so that the specific hydrolysate of PSLTro02 is presumed to be S-2251.

TABLE 2 amidolytic Activity of PSLTro02 on chromogenic substrates: (

n＝5)

EXAMPLE 3 thrombolytic anticoagulant Activity of fibrinolytically active protein PSLTro02

1. Antithrombotic activity in vivo

40 SD rats, half male and half female, were randomly divided into 5 groups: normal saline group (blank control group), urokinase group and lumbrokinase group (positive control group), fibrinolytic protein PSLTro02 high dose group and low dose group, wherein each group is continuously gavaged for 5 days, 1 time per day, and the dosage is shown in table 3. Anaesthetizing the above 5 groups of SD rats with 2% pentobarbital sodium solution (40 mg/kg), making arteriovenous loop thrombosis model, fixing the rats on an operation plate in supine position, separating out the right common carotid artery and the left external jugular vein, placing a weighed operation line with the length of about 8cm in a polyethylene tube filled with heparin sodium physiological saline (50U/mL), communicating the right common carotid artery and the left external jugular vein with the polyethylene tube, opening the arteriovenous loop for 15min after 30min of medication, and interrupting the blood flow to form thrombosis. Taking out the thrombus after 15min, rolling back and forth on filter paper to remove excessive floating blood, placing on a weighed glass slide, weighing, subtracting the weight of the glass slide and the surgical line to obtain the wet weight of the thrombus, and calculating the thrombus inhibition rate. After 80min in a 75 ℃ drying oven, the thrombus was measured by weighing the dry weight after cooling.

The thrombus inhibition ratio (%) - (blank control thrombus weight-experimental group thrombus weight)/blank control thrombus weight × 100%. The statistical data method comprises the following steps: using SPSS software to process and count the results

(

Mean, s standard deviation), and significance of differences between groups was judged by T-test.

TABLE 3 Effect of PSLTro02 on thrombotic weight of rat arteriovenous circuits (S) ((S))

n＝5)

Note: in table 3, compared to the blank control group,^*P<0.05，^**P<0.01。

as can be seen from Table 3, the inhibition rate of the PSLTro02 high-dose group is obviously higher than that of the positive control urokinase group, and the PSLTro02 high-dose group has very good in-vivo antithrombotic activity.

2. In vitro anticoagulant activity

Selecting male Japanese big ear rabbit, performing local anesthesia with procaine hydrochloride, inserting polyethylene tube into carotid artery, and taking blood according to the volume ratio of 1: 9 adding 3.8% sodium citrate solution, mixing, centrifuging at 3000r/min for 10min, and collecting supernatant. The Thrombin Time (TT), Prothrombin Time (PT) and Activated Partial Thrombin Time (APTT) were measured using in vitro diagnostic kits purchased from Beijing Zhongji Shidi scientific instruments Co., Ltd, TT (lot number STY50301-35), APTT (lot number STY50201-36) and PT (lot number STY50101-58), respectively.

In the detection, the fibrinolytic protein PSLTro02 solution is divided into three dose groups of small, medium and large, and the final concentration of PSLTro02 is 5 mug. mL^-1，10μg·mL^-1，20μg·mL^-1(ii) a Deionized water was added to the blank control group, and heparin sodium (final concentration of 1U. mL) was added to the positive control group^-1). Pillbug plasmin PSLTro01 was prepared according to the method of the invention patent application No. 201510043911.7.

As can be seen from Table 4, PSLTro02 can delay TT, PT and APTT significantly, but the delay effect is different. The delay action time on TT is basically equivalent to that of heparin sodium when the concentration of PSLTro02 reaches 10ug/mL, and the delay action time on PT is equivalent to that of heparin sodium when the concentration of PSLTro02 reaches 20ug/mL, particularly the delay action time on APTT is most obvious along with the increasing of the concentration. The experimental result shows that PSLTro02 acts on three ways of TT, PT and APTT in the blood coagulation process simultaneously, mainly inhibits an intrinsic blood coagulation way and a common blood coagulation way, delays the blood plasma coagulation time, and has stronger anticoagulation effect along with the increase of the concentration of PSLTro 02. Meanwhile, compared with the anticoagulant activity of the pillbug plasmin PSLTro01 in the same environment, the difference of the in vitro anticoagulant activity is obvious.

Table 4 shows the effects of PSLTro02 and PSLTro01 on TT, PT, APTT ((R))

n＝5)

Note: compared to the blank control:^*P<0.05，^**P<0.01。

3. in vitro antithrombotic Activity Studies

100mg of agarose was weighed, 10mL of PBS buffer (pH7.8) was added thereto, the mixture was boiled to be sufficiently dissolved, and then the mixture was placed in a 50 ℃ water bath to be kept at a constant temperature to obtain an agarose solution. Weighing 45mg of fibrinogen, adding 15ml of PBS, and placing in a water bath kettle at 50 ℃ to fully dissolve the fibrinogen to obtain a fibrinogen solution. Thrombin was dispensed in a bottle of 2000U, and dissolved in 4mL of physiological saline (500U/mL) in 50 uL. 20uL of thrombin is taken and added with 3mL of normal saline to be mixed evenly to obtain thrombin solution. And pouring the fibrinogen solution, the thrombin solution and the agarose solution into a culture dish in sequence, quickly and uniformly mixing, standing for 1h at room temperature or standing for 30mn at 4 ℃ in a refrigerator until complete coagulation is achieved, and thus obtaining the fibrin plate. Punching a puncher, respectively sampling 80uL of urokinase, lumbrokinase (10mg/ml), zymolyte (inactivated pepsin and trypsin) (10mg/ml) of a wood louse crushed material, wood louse plasmin PSLTro01(10mg/ml), fibrinolytic active protein PSLTro02(10mg/ml), pepsin (10mg/ml), trypsin (10mg/ml) and normal saline into a plate hole, placing the plate in an oven at 37 ℃, observing whether a soluble ring exists after 18h, and comparing the sizes. Wherein the added mass of the pillbug plasmin PSLTro01 and the fibrinolytic protein PSLTro02 are equal.

As can be seen from fig. 11, fibrinolytic activity is compared: urokinase > trypsin > lumbrokinase > PSLTro02> PSLTro01> pepsin > pillbug fibrinolytic crude protein, the difference of fibrinolytic activity of PSLTro02 and PSLTro01 is obvious, and the lysoloop area PSLTro02 is obviously larger than that of PSLTro 01.

Thrombolytic mechanism of 4-pillworm fibrinolytic active protein PSLTro02

Analytical pure fibrinogen usually contains a small amount of plasminogen, which results in the formulation of fibrin plates containing traces of plasminogen that easily confound the fibrinolysis mechanism of the sample, and therefore the formulation of unheated plates is called positive plates. Heating the positive plate in a constant temperature and humidity incubator at 85 deg.C for 35min to completely inactivate plasminogen, and making into fibrin plate without plasminogen, i.e. negative plate. A plurality of small holes with the diameter of about 2mm are punched on the positive plate and the negative plate, and 10 mu L of the pillbug fibrinolytic active protein PSLTro02 solution (20ug/ml), ultrapure water and urokinase solution (the concentration is 10U/ml) are respectively added. Placing into a constant temperature incubator at 37 ℃, and observing the change of the lysis ring of the fiber plate after 18 h. As a result: urokinase and PSLTro02 were visibly noted as having a lysoloop on the positive plate, while on the negative plate, neither appeared, thus suggesting that PSLTro02 thrombolytic means is activating plasminogen to plasmin, further lysing fibrin, rather than directly lysing fibrin.

Cleavage of fibrinogen by the fibrinolysin PSLTro02 of Armadillidium vulgare

200 μ L of 5mg/mL^-1after fully mixing a fibrinogen solution and 100 mu L of PSLTro02 fibrinolytic active protein solution with the concentration of 0.2mg/mL, placing the mixture in a constant-temperature water bath kettle at 37 ℃ for water bath for 5min, 15min, 30min, 1h, 4h, 8h, 16h and 24h, taking out 20 mu L of sample liquid, adding 4 mu L of sample buffer solution, carrying out SDS-PAGE vertical plate electrophoresis (separation gel T is 12%), using fibrinogen as a blank control during electrophoresis, observing gel imaging after the electrophoresis is finished, and analyzing the degradation conditions of fibrinogen alpha, β and gamma chains.

Claims

1. A wood louse fibrinolytic active protein is characterized in that wood louse crushed material is separated after enzymolysis by pepsin and trypsinase in sequence, the molecular weight is 8k-10k Da, and the wood louse fibrinolytic active protein has thrombolytic and anticoagulant activities;

after enzymolysis of the pillbug crushed material, sequentially adopting ion exchange chromatography, gel filtration chromatography and ultrafiltration concentration to separate and obtain fibrinolytic active protein, wherein HiTrap Capto Q ion exchange chromatography column is adopted in the ion exchange chromatography to separate, 1.5-3 column volumes of eluent containing 0.1-0.3mol/L NaCl is adopted to elute and collect eluent, and the chromatographic columns adopted in the gel filtration chromatography are Sephacryl S-200HR gel chromatography column and Superdex75pg gel chromatography column.

2. The pillbug fibrinolytic activity protein as claimed in claim 1, wherein in the process of separating on the Sephacryl S-200HR gel chromatography column, the eluent is Tris-HCl buffer solution with pH value of 7-8 and 10-30mmol/L, the flow rate of the eluent is 0.5-1mL/min, and the eluent with fibrinolytic activity is collected.

3. The pillbug fibrinolytic activity protein as claimed in claim 2, wherein in the Superdex75pg gel chromatography column separation process, the eluent is Tris-HCl buffer solution with pH of 7-8 and 10-30mmol/L, the eluent flow rate is 0.5-1mL/min, and the eluent with fibrinolytic activity is collected.

4. The fibrinolytic active protein of Armadillidium vulgare according to claim 3, wherein the fraction having a molecular weight of more than 5kDa and less than 10kDa is obtained by concentration by ultrafiltration.

5. The pillbug fibrinolytic active protein according to claim 4, wherein the mass ratio of the pepsin, the trypsin and the pillbug crushed material is 0.5-1.5:1 during enzymolysis.

6. The fibrinolytic active protein of Armadillidium vulgare according to claim 5, wherein the enzymolysis temperature is 37.5-38.5, and the enzymolysis time is 3.5-4.5 hours.

7. Use of the Armadillidium fibrinolytic active protein according to claim 1 for the preparation of an oral medicament for the treatment of thrombotic disorders.