Antithrombotic peptide Bannakunin of Simulium bannaense and application of antithrombotic peptide Bannakunin

Abstract

The invention discloses an antithrombotic peptide Bannakunin of Simulium bannaense and application of the antithrombotic peptide Bannakunin, and belongs to the field of biomedicine. The anti-thrombus peptide Bannakunin is cyclic protein of three pairs of intramolecular disulfide bonds, wherein the cyclic protein is coded by a blood sucking insect Simulium bannaense anti-thrombus peptide gene, and the cyclic protein is composed of sixth cysteine and fifteenth cysteine, the cyclic protein is composed of fifteenth cysteine and thirty-ninth cysteine, and the cyclic protein is composed of thirty-first cysteine and fifty-second cysteine. The amino acid sequence of the gene is shown as SEQ ID NO: 1. The antithrombotic peptide provided by the invention can inhibit platelet aggregation induced by ADP or collagen, and has a significant thrombus formation inhibition function in vivo; in addition, the antithrombotic peptide is obtained through prokaryotic expression, large-scale industrial production is easy, and the antithrombotic peptide can be applied to preparation of drugs for inhibiting platelet aggregation and treating thrombotic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a Bannakunin anti-thrombotic peptide from Simulium bannaense and its applications. Background Art

[0002] When hematophagous arthropods suck the blood of mammalian hosts, the host organism responds to vascular injury through a multi-level hemostasis mechanism. This physiological process can be divided into three progressive stages: First, the damaged blood vessel reduces its diameter through nerve reflex and smooth muscle contraction; Second, platelets adhere to and aggregate on the surface of collagen fibers exposed by the vascular endothelium, forming a primary hemostatic plug (i.e., primary hemostasis); Subsequently, the coagulation cascade reaction is activated, and fibrinogen is converted into a cross-linked fibrin network to consolidate the hemostatic structure (secondary hemostasis). Finally, permanent repair of the injury is achieved through fibrotic tissue hyperplasia. To break through the host's hemostatic barrier, such organisms have evolved special salivary components to specifically interfere with the above mechanisms. For example, hirudin and its derivatives secreted by the salivary glands of leeches have become important anticoagulants for preventing postoperative venous thrombosis in clinical practice, and have also shown significant efficacy in the management of complications after cerebral hemorrhage. In-depth research on such bioactive molecules not only reveals the host-parasite interaction mechanism, but also provides important targets for the development of new hemostasis-regulating drugs.

[0003] Platelets play a crucial role in the process of thrombus formation. Antiplatelet aggregation drugs are currently used clinically to prevent and treat thrombotic diseases. However, they have side effects such as gastrointestinal irritation and an increased risk of bleeding. Therefore, there is an urgent need to develop new anti-thrombotic drugs with fewer side effects. Studying the structure and function of salivary proteins of hematophagous arthropods can help to discover and develop some lead bioactive molecules with potential medicinal value. Simuliidae (Diptera: Simuliidae), commonly known as black flies, are hematophagous arthropods that harass humans and livestock, and obtain nutrients by sucking the blood of hosts for egg-laying and reproduction. Simulium bannaense (scientific name: Simulium bannaense ) is a kind of Simuliidae. Its female individuals suck blood and inhabit environments such as forests, grasslands, and wetlands in tropical and subtropical regions, and are mainly distributed in the Xishuangbanna region of Yunnan Province, China. At present, basic understandings have been obtained on its morphological characteristics, living habits, etc., but the identification and functional research of anti-thrombotic components in its salivary glands have not been reported. Summary of the Invention

[0004] The first object of the present invention is to provide a Bannakunin anti-thrombotic peptide from Simulium bannaense (Simulium bannaense) ; the second object is to provide the applications of the Bannakunin anti-thrombotic peptide from Simulium bannaense.

[0005] The first object of the present invention is achieved as follows. The amino acid sequence of the Bannakunin of Simulium (Simulium) bannaense is shown in SEQ ID No. 1.

[0006] The second object of the present invention is achieved as follows. The application of the Bannakunin of Simulium (Simulium) bannaense in the preparation of a product for inhibiting platelet aggregation.

[0007] The Bannakunin of Simulium (Simulium) bannaense of the present invention is a cyclic polypeptide encoded by the antithrombotic peptide gene of the salivary gland of Simulium (Simulium) bannaense, a blood-sucking insect in China. It consists of three pairs of intramolecular disulfide bonds formed by cysteine at the 6th and 56th positions, cysteine at the 15th and 39th positions, and cysteine at the 31st and 52nd positions. It is composed of 80 amino acid residues, and its amino acid sequence is shown in SEQ ID NO: 1. The sequence is: AKDPVCDLPMDEGVCRAMHKRFYYNSVAKTCKKMYYGGCGGNENNFLTKQECLLKCVGKENLIRTRKRKNKTNTQQTPKP.

[0008] The coding gene of the Bannakunin precursor of Simulium (Simulium) bannaense, GenBank accession PV294742, consists of 435 nucleotides, and its nucleotide sequence is shown in SEQ ID NO: 2. Its sequence from the 5' end to the 3' end is: atgagtatcatcccaatcagtgtcatctttttcctttgccttggtcaaattttggcggccaaggatcctgtatgtgatcttccgatggatgaaggtgtgtgcagagcgatgcataagcgtttttactataactctgtggcgaagacatgtaagaagatgtactatggaggatgcggtggaaacgagaacaactttttaaccaaacaggaatgtctcctcaaatgtgtgggaaaggagaatttgattagaaccaggaaacgtaaaaataagacaaacacgcaacaaacgccaaaaccgtgaggaagttgggacattcgagactgagacgacaattgcccgaaagttctccataatattatggccatgagagaatttagaaaactttggaaatatatttaagatatcaaaataatcaagaaaaaaaaaaaaaaaaaa. Among them, the nucleotides at positions 58–297 are the coding gene of the Bannakunin of Simulium (Simulium) bannaense.

[0009] The beneficial effects of the present invention are as follows: A new antithrombotic peptide, Bannakunin, is provided. The antithrombotic peptide of the present invention can inhibit platelet aggregation and has a significant function of inhibiting thrombus formation in vivo; in addition, the antithrombotic peptide is obtained by prokaryotic expression and is easy to be mass-produced industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is the SDS-PAGE electrophoresis diagram of the prokaryotic expression and purification of the antithrombotic peptide Bannakunin; Among them, A is the SDS-PAGE electrophoresis diagram of the prokaryotic expression of Bannakunin: M represents Marker, lane 1 is the protein band of the lysate of Escherichia coli without IPTG induction, lane 2 is the protein band of the soluble lysate of Escherichia coli after adding 0.8 mM IPTG induction, lane 3 is the protein band of the supernatant of Escherichia coli after IPTG induction, and lane 4 is the protein band of the precipitate after the fragmentation of Escherichia coli after IPTG induction; B is the SDS-PAGE electrophoresis diagram of the purified Bannakunin: M represents Marker, lane 1 is 0.5 mg / mL BSA, and lane 2 is the purified Bannakunin; Figure 2 It is the schematic diagram of the inhibitory effect of the antithrombotic peptide Bannakunin on platelet aggregation; Among them, A - the inhibitory effect of different concentrations of Bannakunin (0.25 μM, 0.5 μM, 1 μM) on ADP-induced platelet aggregation; B - the inhibitory effect of different concentrations of Bannakunin (0.25 μM, 0.5 μM, 1 μM) on Collagen-induced platelet aggregation; Figure 3 It is the schematic diagram of the inhibitory effect of the antithrombotic peptide Bannakunin on the formation of mouse tail vein thrombus; Among them, A - the change in the length of the mouse tail thrombus after the antithrombotic peptide Bannakunin (10 mg / kg) acts for 24 hours; B - the statistical result of the length of the mouse tail thrombus after the antithrombotic peptide Bannakunin acts for 24 hours (n = 6). DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be further described below in conjunction with the embodiments and the drawings, but the present invention is not limited in any way. Any transformation or substitution based on the teachings of the present invention belongs to the protection scope of the present invention.

[0012] The amino acid sequence of the Simulium bannaense antithrombotic peptide Bannakunin described in the present invention is shown in SEQ ID No.1.

[0013] The application of the Bannakunin of Simulium (Gomphostilbia) bannaense in the present invention is the application of the Bannakunin of Simulium (Gomphostilbia) bannaense in the preparation of products for inhibiting platelet aggregation.

[0014] The said product is a medicine, food or health food.

[0015] The application of the Bannakunin of Simulium (Gomphostilbia) bannaense in the preparation of drugs for treating thrombotic diseases.

[0016] The following is a further illustration of the present invention with specific embodiments: Example 1

[0017] Discovery of the Bannakunin of Simulium (Gomphostilbia) bannaense Through retrieval, a gene sequence GenBank accession PV294742 was found in the nucleic acid database on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov), USA. Its species source is Simulium (Gomphostilbia) bannaense ( Simulium bannaense ), its nucleotide sequence is shown as SEQ ID NO:2, with a length of 435 bp, and the protein precursor encoded by it is shown as SEQ ID NO:3.

[0018] The gene sequence derived from Simulium (Gomphostilbia) bannaense was analyzed by sequence alignment using the blastX software (http: / / blast.st-va.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) on the NCBI website. The results showed that the encoded product of this gene might be a precursor of Kunitz family protease inhibitor. Further, the precursor protein sequence encoded by this gene was analyzed using the signal peptide prediction software SignalP (https: / / services.healthtech.dtu.dk / service.php?SignalP), and comparative analysis was carried out in combination with the reported sequences of Kunitz family protease inhibitors from other animal sources. It was determined that the cleavage site during the maturation process of this protease inhibitor precursor was between -Ala19- Ala20-, that is, the 58–297th nucleotides of this gene (SEQ ID NO: 2) were the coding gene for the mature peptide of this protease inhibitor. Thus, the mature peptide sequence of this protease inhibitor was obtained, containing 80 amino acid residues AKDPVCDLPMDEGVCRAMHKRFYYNSVAKTCKKMYYGGCGGNENNFLTKQECLLKCVGKENLIRTRKRKNKTNTQQTPKP (amino acid single-letter abbreviation sequence); among them, the sixth cysteine and the fifty-sixth cysteine, the fifteenth cysteine and the thirty-ninth cysteine, and the thirty-first cysteine and the fifty-second cysteine formed three pairs of intramolecular disulfide bonds, and it was named Bannakunin.

[0019] Example 2

[0020] Prokaryotic expression of the antithrombotic peptide Bannakunin from Simulium (Gomphostilbia) bannaense (I) Construction of the recombinant plasmid pET-30a-Bannakunin 1) Preparation of pET-30a plasmid DNA The Escherichia coli vector strain carrying the pET-30a plasmid (Novagen, USA) was inoculated into 5 mL of LB medium containing ampicillin and cultured overnight in a shaker at 37 °C. The plasmid was extracted using a plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd., China).

[0021] 2) Double digestion of pET-30a plasmid DNA At 37 °C, the pET-30a+ plasmid was double-digested with Nde I and Xho I (TaKaRa, Japan), and the double-digestion system was 20 μL. After the digestion products were electrophoresed on a 1% agarose gel, the target bands were cut out, and the double-digested products were recovered using a Gel DNA Recovery Kit (Beijing Bioteke Corporation, China) and stored at -20 °C for later use.

[0022] 3) Preparation of the Bannakunin target gene The nucleotide sequence of Bannakunin was gene-synthesized by Shanghai Sangon Biotech Co., Ltd., which is the 58–297th nucleotide of the gene sequence of the Bannakunin protein precursor (SEQ ID NO: 2), and a 6×His fusion expression tag sequence was added to the 5' end.

[0023] 4) Ligation of the digestion products The recovered double-digested product linear pET-30a+ plasmid and the target gene were reacted under the action of T4 DNA ligase (Takara, Japan), and the ligation was carried out overnight at 16 °C.

[0024] (II) Transformation of the recombinant plasmid The ligation product was transformed into Escherichia coli BL21(DE3) competent cells prepared by the CaCl2-MgCl2 method. An appropriate amount of the transformation product was spread on an LB medium plate containing 100 μg / mL ampicillin and cultured at 37 °C for 16 hours. It was detected by PCR, and the positive clones were sent to a biological company for sequencing and confirmed to contain the Bannakunin mature peptide gene sequence.

[0025] (III) Induced expression of the Bannakunin recombinant protein The recombinant positive clones were picked and inoculated into a liquid LB medium containing 100 μg / mL ampicillin and cultured overnight at 37 °C. The next day, the above bacterial solution was inoculated into 1 L of fresh LB medium at a ratio of 1:100 and cultured with shaking at 37 °C for about 3 hours until the OD 600 reached 0.6, and IPTG was added to a final concentration of 0.4 mmol / L, and the culture was continued to be induced at 28 °C for 3 hours.

[0026] (IV) Isolation and purification of the recombinant expression product Bannakunin 1) Collection of His-Tag fusion protein The induced product was harvested by centrifugation at 10,000 rpm for 10 minutes. The cells were resuspended in 100 mL of 0.1 mol / L PBS at pH 8.0, sonicated for 10 minutes, centrifuged at 8,000 rpm for 10 minutes, and the supernatant was discarded. The collected induced precipitate was resuspended in the above buffer, then sonicated and centrifuged under the same conditions as before. The sonicated precipitate was resuspended in a buffer containing 6 mol / L urea, incubated on ice for 60 minutes, centrifuged at 16,000 rpm for 30 minutes at 4°C, filtered through a 0.45 μm filter membrane, and loaded onto a His Bind Resin (Merck, Germany) affinity chromatography column pre-equilibrated with denaturing buffer to collect the His-Tag fusion protein.

[0027] 2) Renaturation of inclusion bodies The inclusion bodies were renatured by gradient dilution. The dialysis buffers were 20 mmol / L Tris-HCl containing 6 mol / L urea, pH 6.0; 20 mmol / L Tris-HCl containing 4 mol / L urea, pH 6.5; 20 mmol / L Tris-HCl containing 3 mol / L urea, pH 6.8; 20 mmol / L Tris-HCl containing 2 mol / L urea, pH 7.2; 20 mmol / L Tris-HCl containing 1 mol / L urea, pH 7.4; and 20 mmol / L Tris-HCl without urea, pH 7.4. The buffer was changed twice each time during dialysis, and the time interval between buffer changes was 5 hours.

[0028] 3) Isolation and purification of Bannakunin The renatured recombinant protein was cleaved with rTEV protease to remove the His-tag. After dialysis of the cleavage product in 0.1 M PBS buffer at pH 6.0 for 12 hours, it was loaded onto a Superdex 75 10 / 300 GL column (10 × 300 mm, 24 mL volume, GE, USA) of the fast protein liquid chromatography GE ÄKTA purification system, eluted with 0.1 M PBS buffer at pH 6.0, and the collected fractions were identified for the quality of the samples by SDS-PAGE gel electrophoresis. The results were as Figure 1 shown.

[0029] Example 3

[0030] Pharmacological experiments of antithrombotic peptide Bannakunin The obtained recombinant protein Bannakunin of Simulium bannaense was used for the following pharmacological activity detection.

[0031] (I) Inhibitory effect of antithrombotic peptide Bannakunin on ADP- or Collagen-induced platelet aggregation Healthy human platelets were diluted with plasma to 2.5 × 10 8 per mL. 300 μL of platelet-rich plasma was taken, incubated with Bannakunin at 37°C for 5 minutes, then 2 μM ADP or 1 μg / μL Collagen (Sigma, USA) was added to induce aggregation, and the change in the aggregation curve within 5 minutes was detected on a platelet aggregometer. The blank group was platelets without incubation with Bannakunin. The experimental results showed that the inhibition rates of 0.25 μM, 0.5 μM, and 1 μM Bannakunin on ADP-induced platelet aggregation were 49.05%, 63.95%, and 49.05% respectively; the inhibition rates of 0.25 μM, 0.5 μM, and 1 μM Bannakunin on Collagen-induced platelet aggregation were 22.08%, 39.40%, and 57.38% respectively. The antithrombotic peptide Bannakunin of Simulium bannaense can be used in the preparation of drugs for inhibiting platelet aggregation.

[0032] (2) Inhibitory effect of antithrombotic peptide Bannakunin on mouse tail vein thrombosis A carrageenan (Carrageenan, type I, Sigma, USA)-induced mouse tail thrombosis model was used to detect the effect of antithrombotic peptide Bannakunin. 24 male BALB / c mice (weighing 17 - 22 g) were randomly divided into 3 groups (n = 6). The first group was the normal saline negative control group, the second group was the positive control group with 0.2 mg / Kg Apixaban (Bristol Myers Squibb & Pfizer, USA), and the third group was given 10 mg / Kg of the Bannakunin sample. After 30 minutes, the mice were intraperitoneally injected with 1% carrageenan dissolved in normal saline. After 6 hours, the same dose of the sample was reinjected into the tail vein. After 48 hours, the incidence and average length of thrombus formation were determined according to the change in the color of the tail skin. The in vivo pharmacological experiment of Bannakunin showed that the mouse tail vein thrombosis induced by carrageenan could be completely cleared after treatment with 10 mg / Kg Bannakunin for 48 hours. The antithrombotic peptide Bannakunin of Simulium bannaense can be used in the preparation of drugs for treating thrombotic diseases.

[0033] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Bannakunin, a thrombotic peptide of Simulium bannaense, characterized in that The amino acid sequence of the described Simulium bannaense anti-thrombotic peptide Bannakunin is shown in Sequence Listing SEQ ID NO:

1.

2. Use of the Bannakunin of claim 1, characterized in that The application of the described Simulium bannaense anti-thrombotic peptide Bannakunin in the preparation of products for inhibiting platelet aggregation.

3. The application according to claim 2, wherein The described products are drugs, foods or health foods.

4. The application according to claim 2, characterized in that, The application of the described Simulium bannaense anti-thrombotic peptide Bannakunin in the preparation of drugs for treating thrombotic diseases.

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