A Banna rope midge antithrombotic peptide Sibakazin and its application

Abstract

The present invention discloses an antithrombotic peptide, Sibakazin, from the Banna moth fly and its application, belonging to the field of biomedicine. The antithrombotic peptide, Sibakazin, is encoded by the antithrombotic peptide gene of the blood-sucking insect Banna moth fly. It is a cyclic protein with three pairs of intramolecular disulfide bonds formed by cysteine ​​at positions 26 and 51, cysteine ​​at positions 28 and 47, and cysteine ​​at positions 36 and 71. 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, making it easy to produce on a large scale industrially, 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 in particular relates to a Banna gnat antithrombotic peptide Sibakazin and application thereof. Background Art

[0002] When blood-sucking arthropods bite hosts such as cattle, sheep, horses, and humans, they damage their blood vessels. Upon bite, the host rapidly initiates a physiological hemostatic response to prevent bleeding. Physiological hemostasis involves three processes: first, vasoconstriction, which includes both neurogenic and myogenic contractions; second, platelets adhere and aggregate to exposed subendothelial collagen, forming a platelet hemostatic plug that initially plugs the wound (this is known as primary hemostasis); third, blood coagulation, in which coagulation factors are sequentially activated, converting soluble fibrinogen into insoluble fibrin, which forms a network that reinforces the hemostatic plug (this is known as secondary hemostasis). Finally, local fibrous tissue proliferates and incorporates into the blood clot, achieving permanent hemostasis. Therefore, for blood-sucking arthropods to successfully feed on blood, their salivary glands must contain active molecules that inhibit these three host hemostatic processes. Studying the functions of active components in the salivary glands of blood-sucking arthropods has potential applications. For example, hirudin, an anticoagulant protein found in the salivary glands of leeches, and its derivative bivalirudin have been used clinically to prevent deep vein thrombosis after surgery and alleviate symptoms of cerebral edema after cerebral hemorrhage.

[0003] Platelets play a key role in thrombosis. Although the antiplatelet aggregation drugs currently used in clinical practice can prevent and treat thrombotic diseases, they have side effects such as irritation of the gastrointestinal tract and increased risk of bleeding. Therefore, it is necessary to develop new antithrombotic drugs with fewer side effects. Studying the structure and function of salivary proteins of blood-sucking arthropods will help to discover and develop lead active molecules with potential medicinal value. Blackfly (Diptera: Simulidae), commonly known as black fly, is a blood-sucking arthropod that obtains nutrients by sucking the host's blood for egg laying and reproduction. Simulium bannaense The schizontidae (Schizontidae) is a blood-sucking Diptera insect endemic to the tropical rainforests of Xishuangbanna, Yunnan, my country, belonging to the subgenus Simulidae of the family Simulidae. The active ingredients secreted by its salivary glands may contain specific antithrombotic substances, but the identification and functional research of these antithrombotic components are limited. Summary of the Invention

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

[0005] The first object of the present invention is achieved as follows: the amino acid sequence of the Banna rope fly antithrombotic peptide Sibakazin is shown in SEQ ID No. 1; wherein, the 26th cysteine ​​and the 51st cysteine, the 28th cysteine ​​and the 47th cysteine, and the 36th cysteine ​​and the 71st cysteine ​​form three pairs of intramolecular disulfide bonds.

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

[0007] The Banna fly antithrombotic peptide Sibakazin of the present invention is encoded by the salivary gland antithrombotic peptide gene of the Chinese blood-sucking insect Banna fly. It is a cyclic protein with three pairs of intramolecular disulfide bonds formed by cysteine ​​at position 26 and cysteine ​​at position 51, cysteine ​​at position 28 and cysteine ​​at position 47, and cysteine ​​at position 36 and cysteine ​​at position 71. It is composed of 75 amino acid residues, and its amino acid sequence is shown in SEQ ID NO: 1. The sequence is:

[0008] DNMDMEWNEPDEELDDQSELADVAMCPCPMIYLPVCGSDNVTYSNKCTLKCEYRTKRGKAINLRMVKEGPCNEEL

[0009] The gene encoding the Banna thrombus-resistant peptide precursor (GenBank accession PV296096) consists of 410 nucleotides, and its nucleotide sequence is shown in SEQ ID NO: 2. The sequence from the 5' end to the 3' end is: atgatgaataaaacaattgggctgttgatggtttgctggtgtatcgttgcggtgggcatggttagtgtgcgagccgacaacatggacatggaatggaatgagcccgacgaggagctagacgaccaatccgaactggctgatgtggcaatgtgtccatgtccgatgatctatttgccagtttgtggtagcgacaatgtgacgtattcaaacaaat gtacgctcaagtgtgagtaccgaacgaagcggggcaaagcgatcaacttgcgaatggtgaaagaaggaccgtgcaacgaggaattgtaattacaaacgatgttatgagttaattaatcggttgtaattttctcattcatcatttgaattgaattataaatgcgtttgccacaatttataaaaaaaaaaaaaaaaaa, among which nucleotides 76–300 are the coding gene for the antithrombotic peptide Sibakazin from Banna rope fly.

[0010] The beneficial effects of the present invention are: providing a new antithrombotic peptide Sibakazin, 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 Sibakazin after prokaryotic expression and purification;

[0012] Among them, A is the SDS-PAGE electrophoresis of prokaryotic expression of Sibakazin: 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 0.2 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 Sibakazin: M represents Marker, lane 1 is 0.5 mg / mL BSA, and lane 2 is the purified Sibakazin;

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

[0014] Among them, A shows the inhibitory effect of different concentrations of Sibakazin (100 nM, 500 nM, 1000 nM) on ADP-induced platelet aggregation; B shows the inhibitory effect of different concentrations of Sibakazin (100 nM, 500 nM, 1000 nM) on collagen-induced platelet aggregation;

[0015] Figure 3 Schematic diagram of the inhibitory effect of antithrombotic peptide Sibakazin on rat carotid artery thrombosis;

[0016] Among them, A shows the inhibitory effect of different concentrations of the antithrombotic peptide Sibakazin (0.5 mg / kg, 1 mg / kg, 2 mg / kg) on ​​rat carotid artery thrombosis; B shows the blood flow in the rat carotid artery after the action of the antithrombotic peptide Sibakazin (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 rope fly antithrombotic peptide Sibakazin of the present invention is shown in SEQ ID No.1; wherein, the 26th cysteine ​​and the 51st cysteine, the 28th cysteine ​​and the 47th cysteine, and the 36th cysteine ​​and the 71st cysteine ​​form three pairs of intramolecular disulfide bonds.

[0019] The application of the Banna rope midge antithrombotic peptide Sibakazin of the present invention is the application of the Banna rope midge antithrombotic peptide Sibakazin in the preparation of drugs for inhibiting platelet aggregation.

[0020] The invention relates to the application of the Banna gnat antithrombotic peptide Sibakazin 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 the antithrombotic peptide Sibakazin from the Banna gnat

[0024] A Kazal-type serine protease inhibitor gene sequence from the banna fly (GenBank accession PV296096) was uploaded to the nucleic acid database of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov) website of the United States. It was named Sibakazin. Its nucleotide sequence is shown in SEQ ID NO:2, with a length of 410 bp. 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 proprotein of the Kazal 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 Kazal family protease inhibitors from other animal sources revealed that the cleavage site during the maturation of the proproteinase inhibitor is between -Ala25 and Asp26, indicating that nucleotides 76-300 of the gene (SEQ ID NO: 2) encode the mature peptide of the proproteinase inhibitor. The mature peptide sequence of the protease inhibitor thus obtained contains 75 amino acid residues DNMDMEWNEPDEELDDQSELADVAMCPCPMIYLPVCGSDNVTYSNKCTLKCEYRTKRGKAINLRMVKEGPCNEEL (amino acid single-letter abbreviation sequence); among them, the 26th cysteine ​​and the 51st cysteine, the 28th cysteine ​​and the 47th cysteine, and the 36th cysteine ​​and the 71st cysteine ​​form three pairs of intramolecular disulfide bonds, and it is named Sibakazin.

[0026] Example 2

[0027] Prokaryotic Expression of Sibakazin, an Antithrombotic Peptide from Banna Ropefly

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

[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 Sibakazin target gene

[0034] The nucleotide sequence of Sibakazin, namely the 76th to 300th nucleotides of the gene sequence of Sibakazin 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 using the CaCl2-MgCl2 method. An appropriate amount of the transformation product was spread onto 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 sibakazin mature peptide gene sequence.

[0039] (III) Inducible expression of Sibakazin 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 0.2 mmol / L, and the culture was continued at 28°C for 3 h.

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

[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 filter 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 Sibakazin

[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 Sibakazin

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

[0051] (I) Inhibitory effect of the antithrombotic peptide Sibakazin 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 sibakazin at 37°C for 5 minutes. Aggregation was then induced by the addition of 5 μM ADP or 1 μg / μL Collagen (Sigma, USA). Aggregation curves were measured within 5 minutes using a platelet aggregometer. Platelets incubated without sibakazin were used as blank controls. The experimental results showed that 100 nM, 500 nM, and 1000 nM sibakazin inhibited ADP-induced platelet aggregation by 21.38%, 40.62%, and 65.42%, respectively; and 100 nM, 500 nM, and 1000 nM sibakazin inhibited collagen-induced platelet aggregation by 21.09%, 39.32%, and 72.00%, respectively. Sibakazin, an antithrombotic peptide from the banna gnat, can be used as a drug to inhibit platelet aggregation.

[0053] (II) Inhibitory effect of antithrombotic peptide Sibakazin on carotid artery thrombosis in mice

[0054] The antithrombotic peptide sibakazin was tested in a ferric chloride-induced carotid artery thrombosis model in mice. Thirty C57BL / 6J male mice (weighing 18-22 g) were randomly divided into five groups (n=6). Group 1 served as a negative control group with normal saline, Group 2 served as a positive control group with 1000 U / kg heparin (GlpBio, USA), and Groups 3, 4, and 5 were treated with 0.5, 1, and 2 mg / kg of sibakazin, respectively. After treatment for one hour, the mice were anesthetized by intraperitoneal injection of 3.0% sodium pentobarbital. The left carotid artery of the mice was bluntly dissected under a stereomicroscope using a laser speckle analyzer. Using a low-absorbency paper pad as a spacer, a filter paper soaked in ferric chloride (10% w / v) was placed on the carotid artery to induce carotid thrombosis. The laser speckle analyzer (RFLSI Pro, RWD Life Science) was activated in flow mode to observe carotid blood flow. In vivo pharmacological experiments of Sibakazin showed that Sibakazin at a concentration of 2 mg / kg could completely inhibit ferric chloride-induced carotid artery thrombosis in rats. The antithrombotic peptide Sibakazin from the banna gnat could 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 within the scope of protection of the present invention.

Claims

1. A Banna rope fly antithrombotic peptide Sibakazin, characterized in that: The amino acid sequence of the Banna rope fly antithrombotic peptide Sibakazin is shown in SEQ ID No. 1; wherein, the 26th cysteine ​​and the 51st cysteine, the 28th cysteine ​​and the 47th cysteine, and the 36th cysteine ​​and the 71st cysteine ​​form three pairs of intramolecular disulfide bonds.

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

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

Images