A Bannaensin, an anti-thrombotic peptide from Simulium bannaense, and its applications

Abstract

The present invention discloses an antithrombotic peptide, Bannaensin, from the Banna moth and its application, belonging to the field of biomedicine. The antithrombotic peptide, Bannaensin, is encoded by the antithrombotic peptide gene of the blood-sucking insect Banna moth. It is a cyclic protein with six pairs of intramolecular disulfide bonds formed by cysteine at positions 22 and 72, cysteine at positions 31 and 55, cysteine at positions 48 and 68, cysteine at positions 110 and 160, cysteine at positions 119 and 143, and cysteine at positions 136 and 156. 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 exhibits significant antithrombotic activity in vivo. The antithrombotic peptide is obtained through prokaryotic expression, can be produced on a large scale, and 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 particularly relates to Bannaensin, an antithrombotic peptide derived from Banna midge, and applications thereof. Background Art

[0002] When blood-sucking arthropods bite hosts such as cattle, sheep, horses, and humans, they damage their blood vessels, and the host will immediately initiate a physiological hemostatic response to stop bleeding. Physiological hemostasis mainly includes three processes: (1) Vasoconstriction: The blood vessels produce neurogenic contraction and myogenic contraction. (2) Formation of platelet hemostatic plug: Platelets adhere and aggregate on the exposed subendothelial collagen, eventually forming a platelet hemostatic plug to block the wound, achieving initial hemostasis, also known as primary hemostasis. (3) Blood coagulation: Coagulation factors are activated in a certain order, eventually converting soluble fibrinogen into insoluble fibrin and interweaving into a network to strengthen the hemostatic plug, known as secondary hemostasis. Finally, local fibrous tissue proliferates and grows into a blood clot, achieving permanent hemostasis. Therefore, in order for blood-sucking arthropods to successfully suck blood from their hosts, their salivary glands must contain some active molecules that can inhibit these three processes of the host's physiological hemostasis. Functional research on active ingredients in the salivary glands of hematuria arthropods has positive potential application value. For example, the anticoagulant protein hirudin and its derivative bivalirudin found in the salivary glands of leeches have been clinically used to prevent deep vein thrombosis after surgery and alleviate the symptoms of brain edema after cerebral hemorrhage.

[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 risk of bleeding. 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 to explore and develop some lead active molecules with potential medicinal value. Blackfly (Diptera: Simulidae), commonly known as black fly, is a blood-sucking arthropod that harasses humans and livestock. It obtains nutrients for laying eggs and reproduction by sucking the blood of the host. The antithrombotic peptide Bannaensin involved in the present invention is derived from the blood-sucking insect Banna ( Simulium bannaense ) Through long-term adaptive evolution, the salivary glands of adult females of this species specifically secrete active ingredients that effectively antagonize the host's hemostatic function. This active ingredient inhibits the coagulation cascade and platelet aggregation, ensuring continuous blood flow during the blood-feeding process. Notably, the molecular structure elucidation, functional characterization, and mechanism of action of the antithrombotic active ingredients in the salivary glands of the Banna rope fly 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 Bannaensin; The second purpose is to provide the application of the antithrombotic peptide Bannaensin from the Banna rope fly.

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

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

[0007] The Bannaensin 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 Bannaensin, which comprises six pairs of intramolecular disulfide bonds formed by cysteine at positions 22 and 72, cysteine at positions 31 and 55, cysteine at positions 48 and 68, cysteine at positions 110 and 160, cysteine at positions 119 and 143, and cysteine at positions 136 and 156. The polypeptide consists of 172 amino acid residues, and its amino acid sequence is shown in SEQ ID NO: 1, which is:

[0008] DAAAQRSRSSKAKPKAEPDICMLPLDLGVCKRPTEWRFHYDSEKKQCLMFPWGCTGNANNFLTKKACEEKCVKRNTQASDAAAPKSRSKKAKPKAKPKAKPKAEPTDICMLPLDLGVCKRPTEWRFHYD SEKKKCLMFPWGCTGNANNF LTKKACEDKC MKRNTPKRRF LK

[0009] The gene encoding the Bannaensin antithrombotic peptide precursor, GenBank accession PV240805, consists of 576 nucleotides, and its nucleotide sequence is shown in SEQ ID NO: 2. The sequence from the 5' end to the 3' end is: wherein the nucleotides at positions 61-576 are the gene encoding the Bannaensin antithrombotic peptide.

[0010] The beneficial effects of the present invention are: providing a new antithrombotic peptide Bannaensin, 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 produce on a large scale industrially. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0012] Among them, A is the SDS-PAGE electrophoresis of prokaryotic expression of Bannaensin: M represents marker, lane 1 is the protein band induced by pET-30a (empty load), lane 2 is the protein band of E. coli lysis product without IPTG induction, lane 3 is the protein band of E. coli soluble lysis product after induction with 0.8 mM IPTG, lane 4 is the protein band of E. coli supernatant after IPTG induction, and lane 5 is the protein band of the precipitate after IPTG-induced E. coli disruption; B is the SDS-PAGE electrophoresis of purified Bannaensin: M represents marker, lane 1 is 0.5 mg / mL BSA, and lane 2 is purified Bannaensin;

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

[0014] Among them, A-Inhibitory effect of different concentrations of Bannaensin (50 nM, 100 nM, 200 nM) on ADP-induced platelet aggregation; B-Inhibitory effect of different concentrations of Bannaensin (50 nM, 100 nM, 200 nM) on collagen-induced platelet aggregation;

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

[0016] Among them, A-representative pictures of the changes in rat tail thrombus length after 12 and 24 hours of treatment with different concentrations of the antithrombotic peptide Bannaensin (2.5 mg / kg, 5 mg / kg); B-statistics of rat tail thrombus length after 12 and 24 hours of treatment with the antithrombotic peptide Bannaensin (n=4). 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 Bannaensin antithrombotic peptide of the present invention is shown in SEQ ID No. 1.

[0019] The application of the Bannaensin antithrombotic peptide of the present invention is the application of the Bannaensin antithrombotic peptide in the preparation of a drug for inhibiting platelet aggregation.

[0020] The invention relates to the application of the Bannaensin antithrombotic peptide 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 Bannaensin, an antithrombotic peptide from the Banna fly

[0024] Through searching the nucleic acid database of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov), we found a gene sequence, GenBank accession PV240805, encoding the antithrombotic peptide Bannaensin precursor, which is specifically expressed in the salivary glands of the Banna fly. The nucleotide sequence is shown in SEQ ID NO: 2, with a length of 576 bp, and the protein precursor it encodes is shown in SEQ ID NO: 3.

[0025] Sequence alignment analysis of the gene 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 proproteinase inhibitor of the Kunitz family. Further analysis of the protein precursor sequence encoded by the gene using the signal peptide prediction software SignalP (https: / / services.healthtech.dtu.dk / service.php?SignalP) and comparison with reported sequences of Kunitz family proteases from other animal sources revealed that the cleavage site during the maturation of the proproteinase inhibitor is between -Ser20 and -Asp21, indicating that nucleotides 61–576 of the gene (SEQ ID NO: 2) encode the mature peptide of the proproteinase inhibitor. The obtained mature peptide sequence of the protease inhibitor contains 172 amino acid residues: DAAAQRSRSSKAKPKAEPTDICMLPLDLGVCKRPTEWRFHYDSEKKQCLMFPWGCTGNANNFLTKKACEEKCVKRNTQASDAAAPKSRSKKAKPKAKPKAKPKAEPTDICMLPLDLGVCKRPTEWRFHYDSEKKKCLMFPWGCTGNANNFLTKKACEDKC MKRNTPKRRF LK (amino acid single-letter abbreviation sequence); among which, the 22nd and 72nd cysteine, the 31st and 55th cysteine, the 48th and 68th cysteine, the 110th and 160th cysteine, the 119th and 143rd cysteine, and the 136th and 156th cysteine form six pairs of intramolecular disulfide bonds, and the protein is named Bannaensin.

[0026] Example 2

[0027] Prokaryotic expression of Bannaensin, an antithrombotic peptide from the banna flies

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

[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 Bannaensin target gene

[0034] The nucleotide sequence of Bannaensin, i.e., nucleotides 61–576 of the gene sequence of the Bannaensin 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 3′ 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 confirmation that they contained the Bannaensin mature peptide gene sequence.

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

[0040] Pick the recombinant positive clones and inoculate them into liquid LB medium containing 100 μg / mL ampicillin, culture at 37°C overnight, and take the above bacterial liquid and inoculate it into 1 L fresh LB medium at a ratio of 1:100 the next day, and culture 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 Bannaensin

[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 Bannaensin

[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 Bannaensin

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

[0051] (I) Inhibitory effect of the antithrombotic peptide Bannaensin 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 bannaensin (50 nM, 100 nM, and 200 nM) 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 incubated without bannaensin were used as blank controls. The experimental results showed that bannaensin at 50 nM, 100 nM, and 200 nM inhibited ADP-induced platelet aggregation by 61.43%, 84.52%, and 91.53%, respectively; and that at 50 nM, 100 nM, and 200 nM, inhibited collagen-induced platelet aggregation by 35.19%, 43.38%, and 81.48%, respectively. Bannaensin, an antithrombotic peptide from the gnatoma serrata, can be used as a drug to inhibit platelet aggregation.

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

[0054] The antithrombotic peptide, bannaensin, was tested in a carrageenan (type I, Sigma, USA)-induced rat tail thrombosis model. Twenty-four BALB / c male mice (weighing 18-22 g) were randomly divided into four groups (n=4). Group 1 served as a negative control group with saline, Group 2 served as a positive control group with 1000 U / kg heparin (GLPBIO, USA), and Groups 3 and 4 were treated with 2.5 and 5 mg / kg bannaensin, respectively. One hour later, the mice were intraperitoneally injected with 1% carrageenan dissolved in saline. Twelve hours later, the incidence and average length of thrombi were assessed by tail skin color changes. In vivo pharmacological studies of bannaensin demonstrated that bannaensin significantly inhibited bannaensin-induced tail vein thrombosis in rats at a concentration of 5 mg / kg for 24 hours. Bannaensin, an antithrombotic peptide from the gnatoma serrata, may be used as a therapeutic agent for 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 within the scope of protection of the present invention.

Claims

1. A Bannaensin antithrombotic peptide, characterized in that: The amino acid sequence of the Banna rope fly antithrombotic peptide Bannaensin is shown in SEQ ID No. 1; wherein, the 22nd cysteine and the 72nd cysteine, the 31st cysteine and the 55th cysteine, the 48th cysteine and the 68th cysteine, the 110th cysteine and the 160th cysteine, the 119th cysteine and the 143rd cysteine, and the 136th cysteine and the 156th cysteine form six pairs of intramolecular disulfide bonds.

2. A use of the Bannaensin antithrombotic peptide according to claim 1, characterized in that: The invention relates to an application of the Bannaensin antithrombotic peptide in the preparation of drugs for inhibiting platelet aggregation.

3. The use of the Bannaensin antithrombotic peptide according to claim 2, characterized in that: The invention relates to the application of the Bannaensin antithrombotic peptide in the preparation of medicines for treating thrombotic diseases.

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