Simulium bannaense antithrombotic peptide Bannaensin and application thereof

Abstract

The invention discloses an antithrombotic peptide Bannaensin of Simulium bannaense and application thereof, and belongs to the field of biomedicine. The anti-thrombus peptide Bannaensin is one of the twenty-second cysteine, the seventy-second cysteine, the thirty-first cysteine, the fifty-fifth cysteine, the forty-eighth cysteine and the sixty-eighth cysteine coded by a blood sucking insect Simulium bannaense anti-thrombus peptide gene, and the other is one of the twenty-second cysteine, the thirty-first cysteine, the fifty-fifth cysteine, the forty-eighth cysteine and the sixty-eighth cysteine coded by a blood sucking insect Simulium bannaense anti-thrombus peptide gene. The 110th cysteine and the 160th cysteine, the 119th cysteine and the 1343rd cysteine, and the 1336th cysteine and the 1366th cysteine form six pairs of cyclic proteins with intramolecular disulfide bonds, and the amino acid sequence of the cyclic proteins is shown as SEQ ID NO: 1. The antithrombotic peptide of the present invention can inhibit platelet aggregation induced by ADP or collagen, and shows significant antithrombotic activity in vivo; the antithrombotic peptide is obtained through prokaryotic expression, large-scale production can be achieved, 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 technology, and particularly relates to a Banna black fly antithrombotic peptide Bannaensin and its application. Background Art

[0002] When blood-sucking arthropods bite hosts such as cattle, sheep, horses, and humans, they cause damage to their blood vessels, and the host will immediately initiate a physiological hemostasis reaction to stop bleeding. Physiological hemostasis mainly includes three processes: (1) Vasoconstriction: The blood vessels produce neurogenic and myogenic contractions. (2) Formation of platelet hemostatic plugs: Platelets adhere and aggregate on the exposed subendothelial collagen, and finally form platelet hemostatic plugs to block the wound, achieving initial hemostasis, also known as primary hemostasis. (3) Blood coagulation: Coagulation factors are successively activated in a certain order, and finally the soluble fibrinogen is converted into insoluble fibrin, which interweaves into a network to strengthen the hemostatic plug, known as secondary hemostasis. Finally, local fibrous tissue proliferates and grows into the blood clot to achieve permanent hemostasis. Therefore, for blood-sucking arthropods to successfully suck blood from the host, their salivary glands must contain some active molecules that can inhibit these three processes of host physiological hemostasis. The functional study of the active components in the salivary glands of blood-sucking arthropods has positive potential application values. 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 surgical operations and to reduce the symptoms of cerebral edema after cerebral hemorrhage, etc.

[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 irritating the gastrointestinal tract and increasing the risk of bleeding. Therefore, there is an urgent need to develop new antithrombotic drugs with fewer side effects. Studying the structure and function of the salivary proteins of blood-sucking arthropods can explore and develop some lead active molecules with potential medicinal value. Simuliidae (Diptera: Simuliidae), commonly known as black flies, are blood-sucking arthropods that harass humans and livestock and obtain nutrients by sucking the blood of the host for egg-laying and reproduction. The antithrombotic peptide Bannaensin involved in the present invention is derived from the unique blood-sucking insect Banna Simulium bannaense () in Xishuangbanna area of Yunnan Province, China. During the long-term adaptive evolution process, the female adult salivary glands of this species specifically secrete active components with highly efficient antagonistic host hemostasis function, ensuring the continuous fluidity of the host blood during the blood-sucking process by inhibiting the coagulation cascade reaction and platelet aggregation. It is worth noting that there is no literature report on the molecular structure analysis, functional characterization, and mechanism of action of the antithrombotic active components in the salivary glands of Banna black flies. Summary of the Invention

[0004] The first object of the present invention is to provide a Banna black fly(Simulium bannaense) Bannaensin; The second object is to provide the application of the Bannaensin of Simulium (Gomphostilbia) bannaense

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

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

[0007] The Bannaensin of Simulium (Gomphostilbia) bannaense of the present invention is a cyclic polypeptide composed of six pairs of intramolecular disulfide bonds formed by cysteine at the 22nd and 72nd positions, cysteine at the 31st and 55th positions, cysteine at the 48th and 68th positions, cysteine at the 110th and 160th positions, cysteine at the 119th and 143rd positions, and cysteine at the 136th and 156th positions of the anti-thrombotic peptide gene of the salivary gland of Simulium (Gomphostilbia) bannaense, a Chinese blood-sucking insect. It is composed of 192 amino acid residues, and its amino acid sequence is shown in SEQ ID NO:1, which is: DAAAQRSRSSKAKPKAEPTDICMLPLDLGVCKRPTEWRFHYDSEKKQCLMFPWGCTGNANNFLTKKACEEKCVKRNTQASDAAAPKSRSKKAKPKAKPKAKPKAEPTDICMLPLDLGVCKRPTEWRFHYDSEKKKCLMFPWGCTGNANNF LTKKACEDKC MKRNTPKRRF LK The coding gene of the precursor of Bannaensin, a thrombotic peptide from Simulium (Simulium) bannaense, GenBank accession PV240805, consists of 576 nucleotides, and its nucleotide sequence is shown as SEQ ID NO:2. The sequence from the 5'-end to the 3'-end is: ATGTCTATTTCCAAAATCTCAGTTATATTATTGCTTTGTATTGTCTGCTTTTCGACTAGTGATGCTGCCGCACAACGTAGCCGCTCTTCCAAGGCGAAACCTAAGGCTGAACCCACCGACATCTGTATGCTCCCACTAGATCTGGGTGTATGCAAACGACCGACAGAGTGGCGGTTCCATTATGACTCAGAAAAGAAACAGTGTTTAATGTTTCCTTGGGGCTGCACGGGAAATGCCAACAATTTCTTGACTAAGAAAGCATGTGAGGAAAAGTGCGTGAAAAGAAACACCCAAGCGTCGGATGCTGCCGCACCCAAGAGTAGGAGCAAAAAGGCGAAACCAAAGGCTAAACCGAAGGCCAAACCTAAGGCAGAGCCCACAGACATATGTATGCTTCCACTCGATCTAGGGGTTTGCAAACGTCCGACGGAATGGCGCTTTCACTACGACTCTGAGAAGAAAAAGTGTCTGATGTTCCCTTGGGGTTGCACTGGCAATGCGAACAATTTTTTAACCAAAAAGGCTTGTGAAGATAAATGCATGAAGCGAAACACACCCAAACGGAGATTCTTGAAG. Among them, the nucleotides at positions 61–576 are the coding gene of Bannaensin, a thrombotic peptide from Simulium (Simulium) bannaense.

[0008] The beneficial effects of the present invention are as follows: A new thrombotic peptide, Bannaensin, is provided. The thrombotic peptide of the present invention can inhibit platelet aggregation and has a significant function of inhibiting thrombus formation in vivo; in addition, the thrombotic peptide is obtained by prokaryotic expression and is easy to be mass-produced industrially. Description of the Drawings

[0009] Figure 1 SDS-PAGE electrophoresis pattern of the prokaryotic expression and purification of the thrombotic peptide Bannaensin; Among them, A is the SDS-PAGE electrophoresis pattern of prokaryotic expressed Bannaensin: M represents Marker, lane 1 is the protein band of pET-30a induced (empty vector), lane 2 is the protein band of the lysate of Escherichia coli without IPTG induction, lane 3 is the protein band of the soluble lysate of Escherichia coli after induction with 0.8 mM IPTG, lane 4 is the protein band of the supernatant of Escherichia coli after IPTG induction, and lane 5 is the protein band of the precipitate after disruption of Escherichia coli after IPTG induction; B is the SDS-PAGE electrophoresis pattern of purified Bannaensin: M represents Marker, lane 1 is 0.5 mg / mL BSA, and lane 2 is purified Bannaensin; Figure 2 It is a schematic diagram of the inhibitory effect of the antithrombotic peptide Bannaensin on platelet aggregation; Among them, A - the inhibitory effect of different concentrations of Bannaensin (50 nM, 100 nM, 200 nM) on ADP-induced platelet aggregation; B - the inhibitory effect of different concentrations of Bannaensin (50 nM, 100 nM, 200 nM) on Collagen-induced platelet aggregation; Figure 3 It is a schematic diagram of the inhibitory effect of the antithrombotic peptide Bannaensin on the formation of mouse tail vein thrombosis; Among them, A - representative pictures of the changes in the length of mouse tail thrombus after the action of different concentrations of the antithrombotic peptide Bannaensin (2.5 mg / kg, 5 mg / kg) for 12 and 24 hours; B - statistics of the length of mouse tail thrombus after the action of the antithrombotic peptide Bannaensin for 12 and 24 hours (n = 4). Detailed implementation mode

[0010] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0011] The amino acid sequence of the Bannaensis antithrombotic peptide Bannaensin described in the present invention is shown as SEQ ID No.1.

[0012] The application of the Bannaensis antithrombotic peptide Bannaensin described in the present invention is the application of the Bannaensis antithrombotic peptide Bannaensin in the preparation of products for inhibiting platelet aggregation.

[0013] The said products are drugs, foods or health foods.

[0014] Use of the described Bannaensin of Simulium (Gomphostilbia) bannaense in the preparation of a medicament for treating thrombotic diseases.

[0015] The following further illustrates the present invention with specific implementation cases: Example 1

[0016] Discovery of Bannaensin of Simulium (Gomphostilbia) bannaense Through retrieval, a gene sequence GenBank accession PV240805 encoding the precursor of the antithrombotic peptide Bannaensin that is specifically expressed in the salivary glands of Simulium (Gomphostilbia) bannaense was found in the nucleic acid database of the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). Its nucleotide sequence is shown as SEQ ID NO:2, with a length of 576 bp, and the encoded protein precursor is shown as SEQ ID NO:3.

[0017] The gene sequence was analyzed by sequence alignment using the blastX software on the NCBI website (http: / / blast.st-va.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastx&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). The results showed that the coding 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 performed 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 of this protease inhibitor precursor was between -Ser20-Asp21-, that is, the 61–576th 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 172 amino acid residues DAAAQRSRSSKAKPKAEPTDICMLPLDLGVCKRPTEWRFHYDSEKKQCLMFPWGCTGNANNFLTKKACEEKCVKRNTQASDAAAPKSRSKKAKPKAKPKAKPKAEPTDICMLPLDLGVCKRPTEWRFHYDSEKKKCLMFPWGCTGNANNFLTKKACEDKC (amino acid single-letter abbreviation sequence); among them, 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, the 136th cysteine and the 156th cysteine formed six pairs of intramolecular disulfide bonds, and it was named Bannaensin.

[0018] Example 2

[0019] Prokaryotic expression of the antithrombotic peptide Bannaensin from Simulium bannaense (I) Construction of the recombinant plasmid pET-30a-Bannaensin 1) Preparation of pET-30a plasmid DNA Inoculate the Escherichia coli vector strain carrying the pET-30a plasmid (Novagen, USA) into 5 mL of LB medium containing ampicillin and culture overnight in a shaker at 37°C. Extract the plasmid using a plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd., China).

[0020] 2) Double digestion of pET-30a plasmid DNA At 37 °C, 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 1% agarose gel, the target bands were cut out, and the double digestion products were recovered using a Gel DNA Recovery Kit (Beijing Bioteke Corporation, China) and stored at -20 °C for later use.

[0021] 3) Preparation of Bannaensin target gene The nucleotide sequence of Bannaensin was synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., which was the 61–576th nucleotides of the gene sequence of Bannaensin protein precursor (SEQ ID NO: 2), and a 6×His fusion expression tag sequence was added to the 3' end.

[0022] 4) Ligation of digestion products The recovered double digestion 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.

[0023] (II) Transformation of 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 Bannaensin mature peptide gene sequence.

[0024] (III) Induced expression of Bannaensin recombinant protein A recombinant positive clone was 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 liquid 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.

[0025] (IV) Isolation and purification of recombinant expression product Bannaensin 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, and 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 a denaturing buffer to collect the His-Tag fusion protein.

[0026] 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.

[0027] 3) Isolation and purification of Bannaensin 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 by SDS-PAGE gel electrophoresis for the quality of the samples. The results were as Figure 1 shown.

[0028] Example 3

[0029] Pharmacological experiment of antithrombotic peptide Bannaensin The obtained recombinant protein Bannaensin of Simulium bannaense was used for the following pharmacological activity detection.

[0030] (I) Inhibitory effect of antithrombotic peptide Bannaensin 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 and incubated with Bannaensin (50 nM, 100 nM, and 200 nM) 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 incubated without Bannaensin. The experimental results showed that the inhibition rates of 50 nM, 100 nM, and 200 nM Bannaensin on ADP-induced platelet aggregation were 61.43%, 84.52%, and 91.53% respectively; the inhibition rates of 50 nM, 100 nM, and 200 nM Bannaensin on Collagen-induced platelet aggregation were 35.19%, 43.38%, and 81.48% respectively. Bannaensin, an antithrombotic peptide from Simulium bannaense, can be used in the preparation of drugs for inhibiting platelet aggregation.

[0031] (II) Inhibitory effect of antithrombotic peptide Bannaensin on the formation of mouse tail vein thrombosis The carrageenan (Carrageenan, type I, Sigma, USA)-induced mouse tail thrombosis model was used to detect the effect of antithrombotic peptide Bannaensin. 24 male BALB / c mice (weighing 18 - 22 g) were randomly divided into 4 groups (n = 4). The first group was the normal saline negative control group, the second group was the positive control group with 1000 U / kg heparin (GLPBIO, USA), and the third and fourth groups were treated with 2.5 and 5 mg / kg Bannaensin samples respectively. After 1 hour, the mice were intraperitoneally injected with 1% carrageenan dissolved in normal saline. After 12 hours, the incidence and average length of thrombosis were determined according to the change in the color of the tail skin. The in vivo pharmacological experiment of Bannaensin showed that Bannaensin at a concentration of 5 mg / kg could significantly inhibit carrageenan-induced mouse tail vein thrombosis after 24 hours of treatment. Bannaensin, an antithrombotic peptide from Simulium bannaense, can be used in the preparation of drugs for treating thrombotic diseases.

[0032] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Bannaensin, a thrombus-resistant peptide of Simulium bannaense, is characterized in that The amino acid sequence of the described Bannaensin, a thrombostasin from Simulium bannaense, is shown in SEQ ID NO: 1 in the sequence listing.

2. Use of the Bannaensin of claim 1 for anti-thrombosis, characterized in that The application of the described Bannaensin, a thrombostasin from Simulium bannaense, in the preparation of a product for inhibiting platelet aggregation.

3. The application according to claim 2, characterized in that, The product is a drug, food, or health food.

4. The application according to claim 2, characterized in that, The application of the described Bannaensin, a thrombostasin from Simulium bannaense, in the preparation of a drug for treating thrombotic diseases.

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