Earthworm polypeptide for treating thrombotic diseases and application thereof

Abstract

The invention relates to the field of biological polypeptides, in particular to an earthworm polypeptide for treating thrombotic diseases and application of the earthworm polypeptide. The amino acid sequence of the earthworm polypeptide disclosed by the invention is selected from at least one of sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6. The polypeptide disclosed by the invention can significantly promote fibrinolysis, has significant effects in inhibiting thrombosis and anticoagulation, and can effectively prevent and treat thrombotic diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological polypeptides, and in particular to an earthworm polypeptide for treating thrombotic diseases and an 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 polypeptide is a biologically active 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 polypeptide can enhance human immunity, promote the production and activation of white blood cells, and increase the resistance of immune cells. In addition, earthworm polypeptide also has hemolytic activity, 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. Among cardiovascular diseases, thrombotic diseases (diseases caused by two pathological processes of thrombosis and thromboembolism, clinically called thrombotic diseases) seriously threaten human life and health. Its incidence rate ranks first among various diseases, and it has been increasing in recent years. It is one of the focuses and hotspots of contemporary medical research.

[0004] However, the current use of earthworm polypeptides to treat cardiovascular diseases is still at the level of earthworm extracts, and often a combination of multiple proteins or polypeptides are used together to treat the disease. There are currently few reports on which specific polypeptides in the earthworm polypeptides play a role, and the mechanism is still unclear. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide an earthworm polypeptide capable of promoting fibrinolysis and a nucleotide sequence encoding the polypeptide, thereby providing a new option for treating thrombotic diseases with fewer side effects and better therapeutic effects.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides an earthworm polypeptide for treating thrombotic diseases, wherein the amino acid sequence of the earthworm polypeptide is selected from at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6.

[0008] The second aspect of the present invention provides a nucleic acid encoding the earthworm polypeptide. Preferably, the nucleotide sequence includes a published nucleotide sequence and / or a nucleotide sequence optimized according to specific conditions.

[0009] The third aspect of the present invention provides a biomaterial, wherein the biomaterial is selected from any one of the following 1) to 2):

[0010] 1) A vector having a nucleic acid encoding one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6;

[0011] 2) A recombinant bacterium or recombinant cell capable of producing one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6, and / or containing a nucleic acid encoding one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6.

[0012] The fourth aspect of the present invention provides the use of the earthworm polypeptide or the nucleic acid or the biomaterial in promoting thrombus dissolution and / or inhibiting thrombus formation. Further, the present invention provides the use of the earthworm polypeptide or the nucleic acid or the biomaterial in the preparation of medicines, foods, and health products for treating and / or preventing thrombotic diseases.

[0013] The fifth aspect of the present invention provides a composition, which comprises the earthworm polypeptide or the nucleic acid or the biological material. Further, the composition also comprises an adjuvant acceptable in the field of medicine, pharmacy or food. Further, the dosage form of the composition comprises at least one of powder, granules, pills, tablets, capsules, pastes or decoctions.

[0014] The sixth aspect of the present invention provides the use of the composition in promoting fibrinolysis and / or provides the use of the composition in preparing medicines, foods, and health products for treating and / or preventing thrombotic diseases.

[0015] The last aspect of the present invention provides a method for preparing the polypeptide, which comprises at least one of a solid phase polypeptide synthesis method, a liquid phase polypeptide synthesis method, and a recombinant genetic engineering synthesis method.

[0016] The beneficial effects of the present invention include but are not limited to:

[0017] The earthworm polypeptide provided by the present invention can promote the dissolution of thrombus and inhibit thrombus formation, can be widely used in the treatment of thrombotic diseases, and has broad prospects in the preparation of drugs for treating and / or preventing thrombotic diseases. Compared with the existing traditional anti-thrombotic drug aspirin, the earthworm polypeptide provided by the present invention has fewer side effects and can treat thrombus from multiple angles, with no significant difference in effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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:

[0019] Figure 1 It is a schematic diagram of the results of the longest length of the black tail of rats at 48 hours in the rat carrageenan thrombosis model in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0021] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0022] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0023] Example 1 Screening and preparation of earthworm polypeptides

[0024] According to domestic and foreign patents and literature reports, a group of earthworm proteins with fibrinolytic activity were screened out. The spatial structural characteristics and specific recognition of lysine binding sites of these earthworm proteins were analyzed using bioinformatics methods, and a series of new polypeptide molecules were designed. Direct synthesis was carried out using chemical synthesis (Gill Biochemical (Shanghai) Co., Ltd.); after the above steps, 8 possible polypeptide sequences were screened out (as shown in Table 1), namely QY-1, QY-2, QY-3, QY-4, QY-5, QY-6, QY-7, and QY-8.

[0025] Table 1

[0026] name Amino acid sequence QY-1 SHSCGASLL (SEQ ID NO: 1) QY-2 PWQLSQQR (SEQ ID NO: 2) QY-3 SGWGRTSASN (SEQ ID NO: 3) QY-4 GSCNGDSGGP (SEQ ID NO: 4) QY-5 LRPGSALSAAH (SEQ ID NO: 5) QY-6 APPADVRIVAG (SEQ ID NO: 6) QY-7 AVASLAESF (SEQ ID NO: 7) QY-8 IIYLLTNIN (SEQ ID NO: 8)

[0027] The earthworm polypeptides QY-1 to QY-8 in Table 1 were tested for in vitro thrombolysis: normal SD rats were anesthetized with chloral hydrate, blood was collected from the abdominal aorta in a common blood collection tube, and 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 1.5 mL centrifuge tube to calculate the weight of the thrombus block.

[0028] The following groups were set up for the thrombus dissolution experiment: PBS, lumbrokinase (0.260 mg / mL), earthworm polypeptide QY-1, QY-2, QY-3, QY-4 group (0.260 mg / mL). 100 μL of each drug solution was added to the thrombus, incubated in a 37°C water bath, and the thrombus weight was weighed at 0, 0.5, 1, 2, and 4 hours to calculate the thrombus dissolution rate, thrombus dissolution rate = (initial weight of thrombus × weight of thrombus after drug action) / initial weight of thrombus × 100%. The measured data were processed using spss26.0 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.

[0029] Experimental results: The dissolution rate of earthworm polypeptide on blood clots is shown in Table 2.

[0030] Table 2

[0031]

[0032] “*” indicates P<0.05, and “**” indicates P<0.01.

[0033] As shown in Table 2, earthworm peptides QY-1, QY-2, QY-5 and QY-6 can significantly promote the dissolution of thrombus, while earthworm peptides QY-3, QY-4, QY-7 and QY-8 have little effect on promoting the dissolution of thrombus. Therefore, QY-1, QY-2, QY-5 and QY-6 were selected for subsequent research.

[0034] Example 2 Test on the efficacy of earthworm polypeptide in promoting fibrinolysis

[0035] Test 1 In vitro thrombolysis experiment

[0036] 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 1.5 mL centrifuge tube to calculate the weight of the thrombus blocks.

[0037] The following groups were set up for the thrombus dissolution experiment: PBS, lumbrokinase (0.260 mg / mL, 0.520 mg / mL), earthworm polypeptide QY-1, QY-2, QY-5, QY-6 groups (0.130 mg / mL, 0.260 mg / mL, 0.520 mg / mL). 100 μL of each drug solution was added to the thrombus, incubated in a 37°C water bath, and the thrombus weight was weighed at 0, 0.5, 1, 2, and 4 hours to calculate the thrombus dissolution rate, thrombus dissolution rate = (initial weight of thrombus × weight of thrombus after drug action) / initial weight of thrombus × 100%. The measured data were processed using spss26.0 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.

[0038] Experimental results: The dissolution rate of earthworm polypeptide on blood clots is shown in Table 3.

[0039] Table 3

[0040]

[0041] “*” indicates P<0.05, and “**” indicates P<0.01.

[0042] From the results in Table 3, it can be seen that earthworm polypeptides QY-1, QY-2, QY-5, and QY-6 have a promoting effect on the dissolution of thrombi in vitro. Test 2 Animal experiment on the inhibition of thrombus formation by earthworm polypeptides

[0043] 1) Reagents and instruments

[0044] 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.

[0045] 2) Modeling method

[0046] 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.

[0047] 70 SD rats were randomly selected for thrombosis modeling using carrageenan and randomly divided into 7 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 peptide QY-1, QY-2, QY-5, QY-6 groups and related drugs were dissolved in normal saline, and the gavage dose was operated according to Table 4. 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 spss26.0 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 5, 6 and Figure 1 .

[0048] Table 4

[0049] Group type Dosage (mg / kg) Normal saline group Normal saline Lumbrokinase Lumbrokinase 61.73 QY-1 Earthworm polypeptide QY-1 61.73 QY-2 Earthworm polypeptide QY-2 61.73 QY-3 Earthworm polypeptide QY-3 61.73 QY-4 Earthworm polypeptide QY-4 61.73 Aspirin group aspirin 7.72

[0050] Table 5

[0051]

[0052] Table 5 shows the black tail rate of rats in different groups. It can be seen from Table 5 that 80% of rats in the saline group had black tails after 48 hours. The black tail rate of rats in the lumbrokinase and earthworm peptide QY-1, QY-2, QY-5, and QY-6 groups was significantly lower than that in the saline group. Figure 1 It can be seen that the length of the longest black tail of rats has been significantly reduced.

[0053] Table 6

[0054] 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 18.4* 2.88* QY-2 19.2* 3.05* QY-5 18.2* 2.72* QY-6 18.9* 2.85*

[0055] “*” indicates P<0.05, and “**” indicates P<0.01.

[0056] Table 6 shows the blood coagulation four items and blood routine test data of rats in each group. It can be seen from Table 6 that compared with the normal saline group, the blood prothrombin time (APPT) of the lumbrokinase and earthworm polypeptide QY-1, QY-2, QY-5, QY-6 groups increased (P<0.05), and the fibrinogen content (FIB) decreased significantly (P<0.05), and the difference was statistically significant. The results show that earthworm polypeptides Y-1, QY-2, QY-5, and QY-6 improve the body's fibrinolytic function and have the effect of preventing and inhibiting thrombosis.

[0057] 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 polypeptide for treating thrombotic diseases, characterized in that: The amino acid sequence of the earthworm polypeptide is selected from at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO:

6.

2. A nucleic acid encoding the earthworm polypeptide of claim 1.

3. A biomaterial, characterized in that: The biological material is selected from any one of the following 1) to 2): 1) A vector comprising one or more nucleic acids according to claim 2; 2) A recombinant cell capable of producing one or more of the earthworm polypeptides as described in claim 1 and / or containing one or more of the nucleic acids as described in claim 2.

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

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

6. A composition, characterized in that The composition comprises the earthworm polypeptide of claim 1 or the nucleic acid of claim 2 or the biological material of claim 3.

7. The composition according to claim 6, characterized in that The composition further comprises excipients acceptable in the fields of medicine, pharmacy or food.

8. The composition according to claim 6, characterized in that The dosage form of the composition includes at least one of powder, granules, pills, tablets, capsules, pastes or decoctions.

9. Use of the composition according to any one of claims 6 to 8 in the preparation of medicines, foods and health products for preventing and / or treating thrombotic diseases.

10. A method for preparing the polypeptide according to claim 1, characterized in that: The method comprises at least one of a solid phase polypeptide synthesis method, a liquid phase polypeptide synthesis method, and a recombinant genetic engineering synthesis method. Earthworm polypeptide for treating thrombotic diseases and its application Technical Field The present invention relates to the field of biological polypeptides, and in particular to an earthworm polypeptide for treating thrombotic diseases and an application thereof. Background Art 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. Earthworm peptide is a bioactive substance with multiple physiological effects and functions. It is widely used in medicine, health products and agriculture, and has shown significant therapeutic and promoting effects. In medicine, earthworm peptide can enhance human immunity, promote the generation and activation of white blood cells, and increase the resistance of immune cells. In addition, earthworm polypeptides also have hemolytic activity, antibacterial, antiviral and antitumor 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. Among cardiovascular diseases, thrombotic diseases (diseases caused by two pathological processes, thrombosis and thromboembolism, clinically known as thrombotic diseases) seriously threaten human life and health. Its incidence rate ranks first among various diseases, and it has been increasing in recent years. It is one of the focuses and hotspots of contemporary medical research. However, the current use of earthworm polypeptides to treat cardiovascular diseases is still at the level of earthworm extracts, and often a combination of multiple proteins or polypeptides are used together to treat the disease. There are currently few reports on which specific polypeptides in the earthworm polypeptides play a role, and the mechanism is still unclear. Summary of the invention In view of the deficiencies of the prior art, the purpose of the present invention is to provide an earthworm polypeptide capable of promoting fibrinolysis and a nucleotide sequence encoding the polypeptide, thereby providing a new option for treating thrombotic diseases with fewer side effects and better therapeutic effects. In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions: In a first aspect, the present invention provides an earthworm polypeptide for treating thrombotic diseases, wherein the amino acid sequence of the earthworm polypeptide is selected from at least one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO:

6. The second aspect of the present invention provides a nucleic acid encoding the earthworm polypeptide. Preferably, the nucleotide sequence includes a published nucleotide sequence and / or a nucleotide sequence optimized according to specific conditions. The third aspect of the present invention provides a biomaterial, wherein the biomaterial is selected from any one of the following 1) to 2): 1) A vector having a nucleic acid encoding one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 6.

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