Bannakunin, an antithrombotic peptide from Banna gnat and its application

Abstract

The present invention discloses an antithrombotic peptide, Bannakunin, from the Banna moth and its application, belonging to the field of biomedicine. The antithrombotic peptide, Bannakunin, is encoded by the antithrombotic peptide gene of the blood-sucking insect Banna moth. It is a cyclic protein with three pairs of intramolecular disulfide bonds formed by cysteine ​​at positions 6 and 56, cysteine ​​at positions 15 and 39, and cysteine ​​at positions 31 and 52. Its amino acid sequence is shown in SEQ ID NO: 1. The antithrombotic peptide of the present invention can inhibit platelet aggregation induced by ADP or collagen and has significant thrombosis inhibition function in vivo. In addition, the antithrombotic peptide is obtained through prokaryotic expression and is easily produced on a large scale industrially. It can be used to prepare drugs for inhibiting platelet aggregation and treating thrombotic diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an antithrombotic peptide Bannakunin from Banna gnat and an application thereof. Background Art

[0002] When blood-sucking arthropods suck blood from their mammalian hosts, the host responds to vascular damage through a multi-stage hemostatic mechanism. This physiological process can be divided into three progressive stages: First, the damaged vessel narrows in diameter through neural reflexes and smooth muscle contraction; second, platelets adhere and aggregate on the exposed collagen fibers of the vascular endothelium, forming a primary hemostatic plug (primary hemostasis); then, the coagulation cascade is activated, converting fibrinogen into a cross-linked fibrin network, consolidating the hemostatic structure (secondary hemostasis). Finally, fibrosis promotes permanent repair of the injury. To overcome the host's hemostatic barrier, these organisms have evolved specialized salivary components to specifically interfere with these mechanisms. For example, hirudin and its derivatives, secreted by the leech salivary glands, effectively inhibit thrombin and have become clinically important anticoagulants for preventing postoperative venous thrombosis. They also demonstrate significant efficacy in managing complications following intracerebral hemorrhage. In-depth research on these bioactive molecules not only sheds light on the mechanisms of host-parasite interactions but also provides important targets for the development of novel drugs that regulate hemostasis.

[0003] Platelets play a vital role in the process of thrombosis. Antiplatelet aggregation drugs are currently used clinically to prevent and treat thrombotic diseases. However, they have side effects such as gastrointestinal irritation and increased bleeding risk. Therefore, it is urgent to develop new antithrombotic drugs with fewer side effects. Studying the structure and function of salivary proteins of blood-sucking arthropods can help us discover and develop some lead active molecules with potential medicinal value. Blackflies (Diptera: Simuliidae), commonly known as black flies, are blood-sucking arthropods that harass humans and livestock. They obtain nutrients for laying eggs and reproduction by sucking the blood of their hosts. Simulium bannaense The female gnat ( ) is a species of midge. It inhabits forests, grasslands, and wetlands in tropical and subtropical regions, primarily in Xishuangbanna, Yunnan Province, China. While their morphological characteristics and habits are generally understood, the identification and functional investigation of the antithrombotic components in their salivary glands have yet to be reported. Summary of the Invention

[0004] The first object of the present invention is to provide a (Simulium bannaense) Antithrombotic peptide Bannakunin; The second purpose is to provide the application of the antithrombotic peptide Bannakunin from the Banna rope fly.

[0005] The first object of the present invention is achieved in that the amino acid sequence of the Banna gnat antithrombotic peptide Bannakunin is shown in SEQ ID No.1.

[0006] The second object of the present invention is achieved by using the antithrombotic peptide Bannakunin from Banna midge in the preparation of products for inhibiting platelet aggregation.

[0007] The Bannakunin antithrombotic peptide of the present invention is a cyclic polypeptide encoded by the antithrombotic peptide gene of the salivary gland of the Chinese blood-sucking insect Bannakunin, which has three pairs of intramolecular disulfide bonds formed by cysteine ​​at positions 6 and 56, cysteine ​​at positions 15 and 39, and cysteine ​​at positions 31 and 52. The polypeptide is composed of 80 amino acid residues, and its amino acid sequence is shown in SEQ ID NO: 1. The sequence is:

[0008] AKDPPVCDLPMDEGVCRAMHKRFYYNSVAKTCKKMYYGGCGGNENNFLTKQECLLKCVGKENLIRTRKRKNKTNTQQTPKP.

[0009] The gene encoding the Banna thrombus-resistant peptide precursor (GenBank accession PV294742) consists of 435 nucleotides, and its nucleotide sequence is shown in SEQ ID NO: 2. The sequence from the 5' end to the 3' end is: atgagtatcatcccaatcagtgtcatctttttcctttgccttggtcaaattttggcggccaaggatcctgtatgtgatcttccgatggatgaaggtgtgtgcagagcgatgcataagcgtttttactataactctgtggcgaagacatgtaagaagatgtactatggaggatgcggtggaaacgagaacaactttttaaccaaacaggaatgtctcctcaaatgtgt gggaaaggagaatttgattagaaccaggaaacgtaaaaataagacaaacacgcaacaaacgccaaaaccgtgaggaagttgggacattcgagactgagacgacaattgcccgaaagttctccataatattatggccatgagagaatttagaaaactttggaaatatatttaagatatcaaaataatcaagaaaaaaaaaaaaaaaa, among which, nucleotides 58–297 are the coding gene of Bannakunin, an antithrombotic peptide from Banna rope fly.

[0010] The beneficial effects of the present invention are: providing a new antithrombotic peptide Bannakunin, which can inhibit platelet aggregation and has a significant function of inhibiting thrombosis in vivo; in addition, the antithrombotic peptide is obtained through prokaryotic expression and is easy to be produced on a large scale industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the SDS-PAGE electrophoresis diagram of the antithrombotic peptide Bannakunin after prokaryotic expression and purification;

[0012] Among them, A is the SDS-PAGE electrophoresis of prokaryotic expression of Bannakunin: M represents marker, lane 1 is the protein band of E. coli lysis product without IPTG induction, lane 2 is the protein band of E. coli soluble lysis product after induction with 0.8 mM IPTG, lane 3 is the protein band of E. coli supernatant after IPTG induction, and lane 4 is the protein band of the precipitate after IPTG-induced E. coli disruption; B is the SDS-PAGE electrophoresis of purified Bannakunin: M represents marker, lane 1 is 0.5 mg / mL BSA, and lane 2 is purified Bannakunin;

[0013] Figure 2 Schematic diagram of the inhibitory effect of the antithrombotic peptide Bannakunin on platelet aggregation;

[0014] A-Inhibitory effect of different concentrations of Bannakunin (0.25 μM, 0.5 μM, 1 μM) on ADP-induced platelet aggregation; B-Inhibitory effect of different concentrations of Bannakunin (0.25 μM, 0.5 μM, 1 μM) on collagen-induced platelet aggregation;

[0015] Figure 3 Schematic diagram of the inhibitory effect of the antithrombotic peptide Bannakunin on rat tail vein thrombosis;

[0016] Among them, the changes in rat tail thrombus length after 24 hours of treatment with A-antithrombotic peptide Bannakunin (10 mg / kg); the statistics of rat tail thrombus length after 24 hours of treatment with B-antithrombotic peptide Bannakunin (n=6). DETAILED DESCRIPTION

[0017] The present invention is further described below with reference to the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0018] The amino acid sequence of the Banna gnat antithrombotic peptide Bannakunin of the present invention is shown in SEQ ID No. 1.

[0019] The application of the Banna gnat antithrombotic peptide Bannakunin of the present invention is the application of the Banna gnat antithrombotic peptide Bannakunin in the preparation of a product for inhibiting platelet aggregation.

[0020] The invention relates to an application of the Banna gnat antithrombotic peptide Bannakunin in the preparation of medicines for treating thrombotic diseases.

[0021] The present invention will be further described below with reference to specific implementation cases:

[0022] Example 1

[0023] Discovery of Bannakunin, an antithrombotic peptide from the Banna fly

[0024] Through searching, a gene sequence GenBank accession PV294742 was found in the nucleic acid database of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov), and its species origin was Banna rope fly ( Simulium bannaense ), the nucleotide sequence of which is shown in SEQ ID NO: 2, the length is 435 bp, and the protein precursor it encodes is shown in SEQ ID NO: 3.

[0025] Sequence alignment analysis of the gene sequence from the banna fly using blastX software from the NCBI website (http: / / blast.st-va.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) indicated that the gene encodes a protease inhibitor of the Kunitz family. Further analysis of the protein precursor sequence encoded by this gene using the signal peptide prediction software SignalP (https: / / services.healthtech.dtu.dk / service.php?SignalP) and comparison with reported sequences of Kunitz family protease inhibitors from other animals revealed that the cleavage site during the maturation of the protease inhibitor is between -Ala19 and Ala20, indicating that nucleotides 58–297 of this gene (SEQ ID NO: 2) encode the mature protease inhibitor peptide. The obtained mature peptide sequence of the protease inhibitor contains 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 ​​form three pairs of intramolecular disulfide bonds, and it is named Bannakunin.

[0026] Example 2

[0027] Prokaryotic expression of Bannakunin, an antithrombotic peptide from the banna fly

[0028] (I) Construction of recombinant plasmid pET-30a-Bannakunin

[0029] 1) Preparation of pET-30a plasmid DNA

[0030] The Escherichia coli vector strain (Novagen, USA) inoculated with the pET-30a plasmid was cultured in 5 mL of LB medium containing ampicillin in a shaker at 37 °C overnight, and the plasmid was extracted using a plasmid extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd., China).

[0031] 2) Double enzyme digestion of pET-30a plasmid DNA

[0032] The pET-30a+ plasmid was double-digested with Nde I and Xho I (TaKaRa, Japan) at 37°C in a 20 μL digestion system. The digestion product was subjected to 1% agarose gel electrophoresis, and the target band was excised. The double-digested product was recovered using a gel DNA recovery kit (Beijing Biotech Biotechnology Co., Ltd., China) and stored at −20°C until further use.

[0033] 3) Preparation of Bannakunin target gene

[0034] The nucleotide sequence of Bannakunin, i.e., nucleotides 58–297 of the gene sequence of the Bannakunin protein precursor (SEQ ID NO: 2), was synthesized by Shanghai Sangon Biotechnology Service Co., Ltd., and a 6×His fusion expression tag sequence was added to the 5′ end.

[0035] 4) Ligation of enzyme digestion products

[0036] The recovered double-enzyme digestion product, linearized pET-30a+ plasmid, and the target gene were reacted with T4 DNA ligase (Takara, Japan) and ligated at 16°C overnight.

[0037] (II) Transformation of recombinant plasmid

[0038] 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 LB medium plates containing 100 μg / mL ampicillin and cultured at 37°C for 16 hours. PCR detection was performed, and positive clones were sent to a biological company for sequencing and confirmed to contain the Bannakunin mature peptide gene sequence.

[0039] (III) Inducible expression of Bannakunin recombinant protein

[0040] The recombinant positive clones were picked and inoculated into liquid LB medium containing 100 μg / mL ampicillin, and cultured at 37°C overnight. The next day, the above bacterial liquid was inoculated into 1 L of fresh LB medium at a ratio of 1:100, and cultured at 37°C with shaking for about 3 hours to make the OD 600 When the pH value reached 0.6, IPTG was added to a final concentration of 0.4 mmol / L, and the induction culture was continued at 28°C for 3 h.

[0041] (IV) Isolation and purification of the recombinant expression product Bannakunin

[0042] 1) His-Tag fusion protein collection

[0043] 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 (pH 8.0), sonicated for 10 minutes, and centrifuged at 8,000 rpm for 10 minutes. The supernatant was discarded, and the collected induced precipitate was resuspended in the above buffer and sonicated and centrifuged using the same conditions as before. The sonicated precipitate was resuspended in buffer containing 6 mol / L urea, incubated on ice for 60 minutes, and then centrifuged at 16,000 rpm for 30 minutes at 4°C. The precipitate was filtered through a 0.45 μm membrane and loaded onto a His-Bind Resin (Merck, Germany) affinity chromatography column equilibrated with denaturing buffer to collect the His-Tag fusion protein.

[0044] 2) Renaturation of inclusion bodies

[0045] The inclusion bodies were renatured by a gradient dilution method. The dialysis buffer was as follows: 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 during each dialysis, with an interval of 5 hours between each change.

[0046] 3) Isolation and purification of Bannakunin

[0047] The His tag of the refolded recombinant protein was cleaved by rTEV protease. The cleavage product was dialyzed against 0.1 M PBS, pH 6.0, for 12 hours and then loaded onto a Superdex 75 10 / 300 GL column (10 × 300 mm, 24 mL volume, GE, USA) in a GE ÄKTA purification system for fast liquid chromatography. The column was eluted with 0.1 M PBS, pH 6.0, and the quality of the collected fractions was verified by SDS-PAGE gel electrophoresis. The results are shown in Figure 2. Figure 1 shown.

[0048] Example 3

[0049] Pharmacological Experiments of Antithrombotic Peptide Bannakunin

[0050] The obtained recombinant protein Bannakunin from Banna fly was subjected to the following pharmacological activity test.

[0051] (I) Inhibitory effect of the antithrombotic peptide Bannakunin on ADP- or collagen-induced platelet aggregation

[0052] Healthy human platelets were diluted with plasma to 2.5 × 10 8 Platelets (300 μL) of plasma-rich platelets were incubated with Bannakunin at 37°C for 5 minutes. Aggregation was then induced by the addition of 2 μM ADP or 1 μg / μL Collagen (Sigma, USA). Aggregation curves were measured within 5 minutes using a platelet aggregometer. Platelets were incubated without Bannakunin in the blank control group. The experimental results showed that Bannakunin at 0.25 μM, 0.5 μM, and 1 μM inhibited ADP-induced platelet aggregation by 49.05%, 63.95%, and 49.05%, respectively; while Bannakunin at 0.25 μM, 0.5 μM, and 1 μM inhibited Collagen-induced platelet aggregation by 22.08%, 39.40%, and 57.38%, respectively. Bannakunin, an antithrombotic peptide from the gnatoma bannaensis, can be used as a drug to inhibit platelet aggregation.

[0053] (II) Inhibitory effect of antithrombotic peptide Bannakunin on rat tail vein thrombosis

[0054] The antithrombotic peptide bannakunin was tested in a carrageenan (type I, Sigma, USA)-induced rat tail thrombosis model. Twenty-four BALB / c male mice (weighing 17-22 g) were randomly divided into three groups (n=6): Group 1 received a saline negative control, Group 2 received 0.2 mg / kg Apixaban (Bristol Myers Squibb & Pfizer, USA) as a positive control, and Group 3 received a 10 mg / kg dose of bannakunin. Thirty minutes after treatment, mice were intraperitoneally injected with 1% carrageenan dissolved in saline. Six hours later, the same dose was injected again into the tail vein. Forty-eight hours later, the incidence and average length of thrombi were assessed by tail skin color changes. In vivo pharmacological studies of bannakunin demonstrated that a 10 mg / kg dose of bannakunin completely eliminated carrageenan-induced tail vein thrombosis in rats after 48 hours of treatment. Bannakunin, an antithrombotic peptide from the banna midge, can be used as a drug for the treatment of thrombotic diseases.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Banna gnat antithrombotic peptide Bannakunin, characterized in that: The amino acid sequence of the Banna fly antithrombotic peptide Bannakunin is shown in SEQ ID No. 1; wherein, 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 ​​form three pairs of intramolecular disulfide bonds.

2. A use of the Banna gnat antithrombotic peptide Bannakunin according to claim 1, characterized in that: The invention relates to an application of the Banna gnat antithrombotic peptide Bannakunin in the preparation of antithrombotic drugs.

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