Earthworm protein with fibrinolytic activity and application thereof

Abstract

The invention relates to the field of protein, in particular to earthworm protein with fibrinolytic activity and application of the earthworm protein. The earthworm protein with fibrinolytic activity has at least one of amino acid sequences as shown in 1)-3): 1) at least one of amino acid sequences as shown in SEQ ID NO: 1 or SEQ ID NO: 2; 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; and 3) an amino acid sequence having at least 90% consistency with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The earthworm protein has the activity of directly hydrolyzing fibrin, and can activate plasminogen, so that the activity of hydrolyzing fibrin is further enhanced, the formation of thrombus can be effectively inhibited, the dissolution of thrombus can be promoted, and the earthworm protein can be applied to preparation of food, medicines or health care products for preventing and / or treating thrombotic diseases.

Description

Technical Field

[0001] The invention relates to the field of proteins, and in particular to earthworm protein with fibrinolytic activity and application thereof. Background Art

[0002] Earthworms, also known as earth dragons, are rich in functional ingredients such as protein, amino acids, trace elements, enzymes, hormones, etc. The protein content of dried earthworms reaches 70%. They are the raw material for preparing medicines, cosmetics, health foods, etc. and have great economic value.

[0003] Earthworm extract is a bioactive substance with multiple physiological effects and functions. It is widely used in the fields of medicine, health products and agriculture, and shows significant therapeutic effects and promotion. In medicine, earthworm extract can enhance human immunity, promote the production and activation of white blood cells, and increase the resistance of immune cells. In addition, earthworm peptides also have antibacterial, antiviral and anti-tumor properties. It can inhibit the growth of bacteria and viruses, reduce the occurrence of infection and disease, and is currently often used in the preparation of cardiovascular and anti-tumor drugs.

[0004] It is reported that the enzyme secreted by the digestive tract of earthworms can directly dissolve fibrin and activate plasminogen. This proteolytic enzyme is generally called lumbrokinase. On the one hand, lumbrokinase can dissolve fibrin by activating plasminogen, and on the other hand, it can inhibit the formation of fibrin by directly degrading fibrinogen. Therefore, lumbrokinase can prevent and dissolve fibrin thrombus. At the same time, due to its small side effects, it has been widely used to prevent and treat thrombotic diseases. However, lumbrokinase is a protein mixture, and it is urgent to isolate a single protein with strong fibrinolytic activity from earthworms and develop it as an ideal anti-thrombotic drug. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a fibrinolytic active protein, which is a single-component protein with strong fibrinolytic activity and high specific activity, and can be used in the preparation of drugs for treating thrombotic diseases.

[0006] The specific scheme of the present invention is as follows:

[0007] The first aspect of the present invention provides an earthworm protein having fibrinolytic activity, wherein the earthworm protein is selected from at least one of the following amino acid sequences:

[0008] 1) at least one of the amino acid sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0009] 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0010] 3) An amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] Among them, the molecular weight of the earthworm protein with a sequence as shown in SEQ ID NO: 1 is 27.5 KDa, and the molecular weight of the earthworm protein with a sequence as shown in SEQ ID NO: 2 is 24.8 KDa.

[0012] The second aspect of the present invention provides a nucleic acid encoding any of the earthworm proteins. In a specific embodiment of the present invention, a nucleotide sequence is designed according to the codon preference of Escherichia coli. Specifically, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 4.

[0013] The third aspect of the present invention provides a biomaterial, wherein the biomaterial is selected from any one of the following I-III:

[0014] I. A vector comprising a nucleic acid encoding at least one of said earthworm proteins;

[0015] II. A transformant comprising the vector described in I;

[0016] III. A recombinant cell capable of producing at least one earthworm protein, and / or containing at least one nucleic acid encoding the earthworm protein.

[0017] The fourth aspect of the present invention provides the use of the earthworm protein or the nucleic acid or the biological material in promoting thrombus dissolution and / or inhibiting thrombus formation.

[0018] The fifth aspect of the present invention provides the use of the earthworm protein or the nucleic acid or the biological material in the preparation of medicines, foods, and health products for treating and / or preventing thrombotic diseases. Preferably, the food, medicine, and health product also include excipients acceptable in the field of medicine, pharmacy, or food. Preferably, the dosage form of the medicine includes at least one of powder, granules, pills, tablets, capsules, pastes, or decoctions.

[0019] The sixth aspect of the present invention provides a method for preparing the earthworm protein by genetic engineering, the method comprising the step of introducing at least one nucleic acid encoding the earthworm protein into a target cell or a target microorganism. The target cell or target microorganism is selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, and mammalian cells.

[0020] The beneficial effects of the present invention include but are not limited to:

[0021] The present invention separates a new single-component protein from earthworms, which has the activity of directly hydrolyzing fibrin and can activate plasminogen, thereby further enhancing its activity of hydrolyzing fibrin. In vitro thrombolytic experiments and animal model experiments have proved that the earthworm protein of the present invention plays an important role in inhibiting thrombosis and promoting thrombolysis, and can be used in the preparation of food, medicine or health products for preventing and / or treating thrombotic diseases. At the same time, it also lays a foundation for large-scale production by recombinant protein expression, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 1 is an electrophoresis diagram of two earthworm protein sequences in the embodiment of the present invention, wherein 1 is an electrophoresis diagram of an earthworm protein with a sequence as shown in SEQ ID NO: 1, and 2 is an electrophoresis diagram of an earthworm protein with a sequence as shown in SEQ ID NO: 2;

[0024] Figure 2 This is a picture of the fibrinolytic ring when the fibrinolytic active protein in the embodiment of the present invention hydrolyzes fibrin for 6 hours, wherein 37-40 in the figure correspond to the ad group in the embodiment respectively;

[0025] Figure 3 This is a schematic diagram of the results of investigating the effects of fibrinolytic active protein on hydrolyzing fibrin and activating plasmin in the examples of the present invention. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0027] Example 1 Separation and purification of earthworm protein

[0028] (1) Wash fresh earthworms, extract the earthworms and pure water at a ratio of 1:20 at 35° C. for 16 hours to obtain a solution containing earthworm extracts, wherein the earthworms are Eisenia fetida;

[0029] (2) adjusting the pH value of the solution containing the earthworm extract obtained in step (1) to 7.5, stirring for 15 minutes, and collecting the supernatant by centrifugation; adding anhydrous ethanol to the supernatant until the ethanol concentration is 70%, and refrigerating in a refrigerator at 4°C for 12 hours; collecting the supernatant by centrifugation, adding anhydrous ethanol to the supernatant until the ethanol concentration is 78%, and refrigerating in a refrigerator at 4°C for 12 hours; collecting the precipitate by centrifugation, adding an appropriate amount of deionized water to dissolve the precipitate, and freeze-drying the solution to obtain the primary extract of earthworm fibrinolytic activity protein;

[0030] (3) The initial extract of the fibrinolytic protein of earthworms was separated and purified by ion exchange method: NGC protein chromatography system was used as the separation and purification device, and the chromatography column was selected from GE's ion exchange chromatography column (HiTrap Q HP, 5 mL). The detector wavelength was set to 280 nm, the flow rate was 4.5 mL / min, and different proportions of 20 mM Tris-HCl (pH 7.5) (mobile phase A liquid) and 20 mM Tris-HCl and 1 M NaCl (pH 7.5) (mobile phase B liquid) were used as elution solvents. Gradient elution was performed with an eluent containing 100-60% A liquid and 0-40% B liquid. The fraction containing 10%-30% B liquid was collected, and deionized water was used as the replacement solvent. Ultrafiltration was used for desalination by ultrafiltration tube (cut-off molecular weight 3 KDa) until no white flocs were produced when 1% AgNO3 was added to the filtrate; finally, the protein solution was concentrated by ultrafiltration to obtain a certain concentration of the active part of the fibrinolytic protein;

[0031] (4) The active part of the fibrinolytic protein was separated and purified by molecular sieve chromatography: the NGC protein chromatography system was used as the separation and purification device, the chromatographic column was a molecular sieve gel chromatography column (Superdex 75 Increase 10 / 300 GL) of GE Company, the detector wavelength was set to 280 nm, 20 mM Tris-HCl and 0.3 M NaCl (pH 7.5) solution was used as the elution solvent; the flow rate was 0.5 mL / min; the main peak corresponding fraction was collected, and deionized water was used as the replacement solvent and ultrafiltration was performed by ultrafiltration tube (cut-off molecular weight 3 KDa) for desalting until no white floccules were produced when 1% AgNO3 was added to the filtrate; finally, the protein solution was concentrated by ultrafiltration to obtain a certain concentration of fibrinolytic protein, and the fibrinolytic protein powder was obtained by freeze drying. The obtained fibrinolytic protein powder had the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO:2 as verified by sequencing. The earthworm protein with the amino acid sequence shown in SEQ ID NO.1 is named QY-1, and the earthworm protein with the amino acid sequence shown in SEQ ID NO.2 is named QY-2. After comparison, the amino acid sequences of QY-1 and QY-2 have 80% homology with lumbrokinase.

[0032] Example 2 Preparation of earthworm protein by genetic engineering

[0033] The plasmid, endonuclease, PCR enzyme, column-type DNA extraction kit and DNA gel recovery kit used in this example are commercial products, and the specific operations are carried out according to the instructions of the kit.

[0034] Conventional operations such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0035] (1) Construction of vector and recombinant engineering bacteria

[0036] The obtained earthworm protein was optimized according to the codon preference of Escherichia coli to obtain the corresponding nucleotide sequence, and the whole gene was synthesized and constructed into the plasmid vector pET-32a(+) (purchased from Hunan Fenghui Biotechnology Co., Ltd.), and then transformed into competent cells BL21(DE3) (purchased from Shanghai Maokang Biotechnology Co., Ltd.), and then the genetically engineered bacteria were obtained by restriction digestion and sequencing. Specifically, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 4.

[0037] (2) Fermentation and cultivation of engineered bacteria

[0038] The culture medium formula used in this example is:

[0039] LB medium formula: peptone 10g / L, yeast extract powder 5g / L, sodium chloride 10g / L, ampicillin 100mg / L.

[0040] Trace element formula: ferrous sulfate heptahydrate 2g / L, manganese sulfate 0.52g / L, zinc sulfate 0.75g / L, cobalt chloride 0.13g / L, copper sulfate 0.1g / L, sodium molybdate 0.13g / L, calcium chloride dihydrate 8.37g / L.

[0041] Basic culture medium formula: glucose 2g / L, yeast powder 12g / L, potassium dihydrogen phosphate 15g / L, magnesium sulfate 0.25g / L, trace elements 10mL / L.

[0042] Feed medium formula: trace elements 100mL / L, glucose 400g / L, yeast powder 100g / L, magnesium sulfate 15.5g / L.

[0043] The engineered bacteria obtained above were inoculated into LB medium at a 1% inoculum and cultured at 37°C and 220 rpm in a shaking incubator until OD 600 ≥1.2, obtain seed solution; transfer the seed solution to a 50L fermenter containing 25L basal medium at a 5% inoculation rate, set the temperature to 37°C, ventilation to 25L / min, pH to 7.0, pressure to 0.050Mpa, speed to 300rpm, and control mode to automatic. During fermentation, adjust the speed and ventilation to control the dissolved oxygen to more than 30%. When OD 600 When it stops growing, start feeding; when OD 600 When OD ≥30, stop feeding, lower the tank temperature to 20℃ within 1 hour, add IPTG to a final concentration of 0.5mmol / L for induction culture, observe the change of dissolved oxygen, continue feeding, and control the feeding rate to maintain the dissolved oxygen content at about 30%. Ferment until OD 600 When the wet weight stops increasing, the material is collected and can be put into the tank.

[0044] (3) Protein separation and purification

[0045] Step (4) in Example 1 was used to separate and purify earthworm protein for subsequent experiments.

[0046] Example 3 Physical and chemical properties test of earthworm protein

[0047] Test 1

[0048] Test the molecular weight of earthworm protein: Use 15% SDS-PAGE denaturing gel electrophoresis Coomassie brilliant blue staining method to analyze the protein of earthworm protein. Figure 1 As shown, it is shown that the molecular weight of the protein shown in SEQ ID NO: 1 is about 27.5 KDa, and the molecular weight of the protein shown in SEQ ID NO: 2 is about 24.8 KDa.

[0049] Test 2

[0050] The fibrin plate method was used to investigate the activation of plasminogen by fibrinolytic protein. The fibrinolytic protein solution was mixed with the plasminogen solution and incubated at 37°C for 1 hour to obtain the sample to be tested. 10 μL of the mixed solution and the fibrinolytic protein solution (the final concentrations of the two were the same) were added to the fibrin plate, and the diameter of the fibrinolytic ring was measured at 3, 6, 12, and 18 hours according to the fibrin plate method.

[0051] The experimental results are shown in Figure 2-Figure 3(Group a is low concentration of lumbrokinase; Group b is high concentration of lumbrokinase; Group c is co-incubation of QY-1 earthworm protein with plasminogen; Group b is co-incubation of QY-2 earthworm protein with plasminogen) and Table 1, the results show that: the fibrinolytic ring area corresponding to the fibrinolytic activity protein increases with time; at the same time, after co-incubation with plasminogen, its fibrinolytic ring area on the fibrin plate increases significantly. After incubation on the fibrin plate for 18 hours, the fibrinolytic ring area of ​​the sample co-incubated with plasminogen increases by 27.4% and 30.8%, respectively. Therefore, QY-1 and QY-2 earthworm proteins can directly and continuously hydrolyze fibrin, and can activate plasminogen, thereby further enhancing its fibrinolytic activity.

[0052] Table 1

[0053] Time (h) concentration 3 6 12 18 <![CDATA[Area of fibrinolytic ring in group a (mm 2 )]]> 0.1mM 45.8 51.6 103.6 163.4 <![CDATA[Area of fibrinolytic ring in group B (mm 2 )]]> 0.2mM 51.2 57.9 124.8 206.4 <![CDATA[Area of fibrinolytic ring in group C (mm 2 )]]> 0.1mM 52.6 58.2 126.3 208.2 <![CDATA[Area of fibrinolytic ring in group D (mm 2 )]]> 0.1mM 54.6 60.9 130.4 213.8

[0054] Test 3

[0055] In vitro thrombus dissolution experiment: In vitro thrombus dissolution experiment: Normal SD rats were anesthetized with chloral hydrate, and blood was collected from the abdominal aorta in a common blood collection tube, which was kept in a 37°C water bath for 4 hours. After the serum was coagulated and discarded, it was rinsed with physiological saline and dried with filter paper to obtain thrombus strips. The thrombus strips were cut into thrombus blocks of similar size and placed in a pre-weighed 1.5mL centrifuge tube to calculate the weight of the thrombus block.

[0056] The following groups were set up for the thrombus dissolution experiment: PBS, lumbrokinase (0.260 mg / mL, 0.520 mg / mL), QY-1 group (0.130 mg / mL, 0.260 mg / mL, 0.520 mg / mL, 1.04 mg / mL), QY-2 group (0.130 mg / mL, 0.260 mg / mL, 0.520 mg / mL, 1.04 mg / mL). 100 μL of each drug solution was added to the thrombus, incubated in a 37°C water bath, and the thrombus was weighed at 0, 0.5, 1, 2, and 4 h to calculate the thrombus dissolution rate, which was thrombus dissolution rate = (initial weight of thrombus - weight of thrombus after drug action) / initial weight of thrombus × 100%.

[0057] Experimental results: The dissolution rates of thrombi by different concentrations of EPF3 are shown in Table 2.

[0058] Table 2

[0059]

[0060] From the results in Table 2, it can be seen that QY-1 and QY-2 have a strong effect on dissolving thrombus in vitro, and are dose-dependent and time-dependent. At 4 hours, the thrombolytic rates of low concentrations of QY-1 and QY-2 were 30.8% and 32.4%, respectively, and the thrombolytic rates of high concentrations of QY-1 and QY-2 were 65.2% and 68.6%, respectively. At the same concentration, the thrombolytic rates of QY-1 and QY-2 are much higher than that of lumbrokinase.

[0061] Test 4

[0062] Animal experiments on earthworm protein inhibiting thrombosis:

[0063] 1) Reagents and instruments

[0064] Carrageenan was provided by Sagma; earthworm polypeptide was chemically synthesized by Gill Biochemical (Shanghai) Co., Ltd.; aspirin standard, brand: TargetMol, batch number T0005-1; Bio-lumbrokinase capsules, yellow capsules, provided by Beijing Bio-Pharmaceutical Co., Ltd., batch number H11021129; lidocaine hydrochloride injection, injection, batch number H37022147.

[0065] 2) Modeling method

[0066] Weigh carrageenan and prepare a 2 mg / mL carrageenan solution (2%) with 0.9% NaCl; weigh the rats, inject carrageenan subcutaneously at a dose of 80 mg / kg, and perform gavage on each group of mice according to the relevant dose 30 minutes after modeling, and then place the rats in a constant temperature environment of (18±2)℃, with free food and water.

[0067] Fifty SD rats were randomly selected for thrombosis modeling using carrageenan and randomly divided into 5 groups, with 10 SD rats in each group. The physiological activities of the rats were observed. The normal saline was used as the blank control group, the aspirin group was used as the positive control group, the earthworm protein QY-1, QY-2 groups and related drugs were dissolved in normal saline, and the gavage dose was operated according to Table 3. The length of the black tail of the thrombus was measured with a vernier caliper at 24, 36, 48, 60, 72, 84, and 96 hours, and the thrombus black tail ratio was calculated. After the injection of carrageenan, rats in each experimental group and the normal saline group had different degrees of stasis and black tails 3 to 14 hours after modeling, and gradually expanded to the root of the tail. The measured data were processed using SPSS 18.0 software. In order to compare the differences between different groups, one-way analysis of variance was used. The results showed that there were significant differences between different groups, and P<0.05 indicated significant differences. After 10 days of feeding, blood was collected from the inferior vena cava of the rats and four coagulation tests and blood routine tests were performed. The test results are shown in Tables 4 and 5.

[0068] Table 3

[0069] Group type Dosage (mg / kg) Normal saline group Normal saline Lumbrokinase Lumbrokinase 61.73 QY-1 Earthworm protein QY-1 61.73 QY-2 Earthworm protein QY-2 61.73 Aspirin group aspirin 7.72

[0070] Table 4

[0071]

[0072] Table 4 shows the black tail rate of rats in different groups. It can be seen from Table 4 that after 48 hours, the proportion of rats with black tails in the normal saline group reached 80%, and the black tail rate in the lumbrokinase and earthworm protein QY-1 and QY-2 groups was significantly lower than that in the normal saline group.

[0073] Table 5

[0074] Group Activated partial thromboplastin time (APTT) Fibrinogen concentration FIB (g / L) Normal saline group 16.8 3.52 Lumbrokinase 20.1 2.47 QY-1 21.4 2.38 QY-2 22.2 2.28

[0075] Table 5 shows the blood coagulation four items and blood routine test data of rats in each group. It can be seen from Table 5 that compared with the normal control group, the prothrombin time (APPT) of the rats in the normal saline group was significantly shortened (P<0.01), and the fibrinogen content (FIB) was significantly increased (P<0.01), and the difference was statistically significant. Compared with the normal saline group, the earthworm protein QY-1 and QY-2 groups can prolong APPT and reduce the fibrinogen content. Among them, the high-dose group APPT was significantly prolonged (P<0.05), and the fibrinogen content was significantly reduced (P<0.05), and the difference was statistically significant. The results show that earthworm protein Y-1 and QY-2 improve the body's fibrinolytic function and have the effect of preventing and inhibiting thrombosis.

[0076] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An earthworm protein having fibrinolytic activity, characterized in that: The earthworm protein is selected from at least one of the following amino acid sequences: 1) at least one of the amino acid sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2; 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; 3) An amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A nucleic acid, characterized in that The nucleic acid encodes any one of the earthworm proteins described in claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO:

4.

4. A biomaterial, characterized in that: The biological material is selected from any one of the following I-III:

1. A vector comprising at least one nucleic acid according to claim 2 or 3; II. A transformant comprising the vector described in I; III. A recombinant cell capable of producing at least one earthworm protein according to claim 1 and / or containing at least one nucleic acid according to claim 2 or 3.

5. Use of the earthworm protein according to claim 1, the nucleic acid according to claim 2 or 3, or the biomaterial according to claim 4 in promoting thrombus dissolution and / or inhibiting thrombus formation.

6. Use of the earthworm protein according to claim 1, the nucleic acid according to claim 2 or 3, or the biomaterial according to claim 4 in the preparation of medicines, foods, and health products for treating and / or preventing thrombotic diseases.

7. The use according to claim 6, characterized in that: The food, medicine and health care product also include excipients acceptable in the field of medicine, pharmacy or food.

8. The use according to claim 6, characterized in that: The dosage form of the medicine includes at least one of powder, granules, pills, tablets, capsules, paste or decoction.

9. A method for preparing the earthworm protein according to claim 1 by genetic engineering, characterized in that: The method comprises the step of introducing the nucleic acid according to claim 2 or 3 into a target cell or a target microorganism.

10. The method according to claim 9, characterized in that The target cell or target microorganism is selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, and mammalian cells.

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