HAEMADIPSA SYLVESTRIS ANTITHROMBOTIC PEPTIDE SYLVESTIN AND USE THEREOF

Abstract

Provided are a Haemadipsa sylvestris antithrombotic peptide Sylvestin and the use thereof, falling within the technical field of biomedicine. The Haemadipsa sylvestris antithrombotic peptide sylvestin can inhibit FXIIa and kallikrein, has anti-thrombus/infarction effects and alleviates injuries caused by cerebral ischemia, and can also be used in the preparation of inhibitors of FXIIa and kallikrein and drugs for anti-thrombus/infarction and anti-cerebral ischemic injuries.

Claims

1. A polypeptide having anti-thrombus activity, which is derived from Haemadipsa sylvestris antithrombotic peptide Sylvestin, comprising (1) a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1, or (2) a sequence obtained by adding, deleting, or replacing one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1.

2. The polypeptide according to claim 1, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1.

3. The polypeptide according to claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 1, wherein the amino acid other than the amino acid set forth in SEQ ID NO: 1 is identical to the amino acid at the corresponding position of the amino acid sequence encoded by Haemadipsa sylvestris antithrombotic peptide cDNA in nature.

4. The polypeptide according to claim 1, which is produced by genetic engineering or chemical synthesis.

5.-7. (canceled)

8. A method for inhibiting FXIIa and/or kallikrein, comprising exposing the polypeptide according to claim 1 to FXIIa and/or kallikrein.

9. A method of preventing and/or treating a disease caused by blood coagulation, comprising administering the polypeptide according to claim 1 to a subject in need thereof.

10. The method according to claim 9, wherein the disease caused by blood coagulation is a disease caused by intravascular infarction and/or thrombus, for example, a disease selected from the group consisting of arteriosclerosis, atherosclerosis, coronary artery disease, stroke, myocardial infarction, cerebral infarction, cerebral ischemia and acute cerebral ischemia.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0020] FIG. 1 shows the anti-thrombus effect of Haemadipsa sylvestris antithrombotic peptide Sylvestin.

[0021] FIG. 2 shows the anti-acute cerebral ischemic effect of Haemadipsa sylvestris antithrombotic peptide Sylvestin.

DETAILED DESCRIPTION

[0022] The substantial content of the present disclosure will be further illustrated by the following embodiments, but the scope of the present disclosure is not limited thereto.

Example 1 Isolation and Purification of the Haemadipsa sylvestris Antithrombotic Peptide Sylvestin

1.1 Preparation of Haemadipsa sylvestris Sample

[0023] Living Haemadipsa sylvestris was quickly frozen by liquid nitrogen directly. At a low temperature, the haemadipsa was wrapped with cloth and broken into pieces with a hammer. The haemadipsa pieces and an appropriate amount of 50 mM Tris-HCl (pH 8.0) buffer were mixed and homogenized. The mixture was centrifuged at 12000 g at 4 C. for 30 minutes, and the supernatant was a crude sample of the Haemadipsa sylvestris body. All the supernatants were mixed together, divided into aliquots (3 ml/tube) and stored at 80 C.

1.2 Sephadex G-50 Gel Isolation and Reversed Phase High-Pressure Chromatography Isolation

[0024] Step 1. Sephadex G-50 gel chromatography. 2 mL of the haemadipsa homogenate was loaded to a Sephadex G-50 (Amersham Bioscience) gel column (26 mm100 cm) equilibrated with a Tris-HCl (0.02 mol/l, pH 7.8) buffer. Elution was carried out with the same concentration of an equilibration buffer at a flow rate of 0.3 ml/min, 3 ml/tube, and fractions were collected using a CBS-A program-controlled automatic fraction collector (Shanghai Qingpu Huxi Instrument Factory). The O.D. values at 280 nm and 215 nm were measured using an Ultrospec 2100 pro spectrophotometer (Amersham Biosciences). Each peak fraction was collected and stored at 20 C. for use.

[0025] Step 2. C4 reversed phase high-pressure chromatography. The fraction having activity was continued to be separated using a reversed phase high-pressure chromatography (Waters 1525 Binary HPLC Pump) C4 column (Lichrospher 10250 mm); solvent A: 0.1% TFA in ultrapure water solution, solvent B: 0.1% TFA in acetonitrile; linear concentration gradient for elution: 0-10 min, B: 0%; 10-11 min, B: 0-5%; 11-40 min, B: 5-33%; 40-50 min, B: 33-38%; 50-60 min, B: 38-70%; 60-70 min, B: 70-100%; flow rate: 1.5 ml/min; loading sample: 3 mg of crude protein. Peaks were detected using a Waters 2489 Visible/UV detector (215 nm), and each peak was collected as a unit.

[0026] Step 3. C8 reversed phase high-pressure chromatography. C8 column (X Bridge 4.6250 mm) was used. Solvent A: 0.1% TFA in ultrapure water solution, solvent B: 0.1% TFA in acetonitrile; linear concentration gradient for elution: 0-10 min, B: 0%; 10-14 min, B: 0-20%; 14-24 min, B: 20%; 24-55 min, B: 20-35%; 55-60 min, B: 35-100%; flow rate: 0.7 ml/min; loading sample: 1 mg of crude protein. Peaks were detected using a Waters 2489 Visible/UV detector (215 nm), and each peak was collected as a unit. The fraction having FXIIa and kallikrein inhibition activity was detected using the above steps. The purified Haemadipsa sylvestris antithrombotic peptide obtained by the purification was subjected to N-terminal sequencing using Edman degradation (model 491, ABI, USA). The molecular weight of the antithrombotic peptide was determined by Electrospray Ionisation Mass Spectrometry (ESI-MS).

Example 2 Haemadipsa Sylvestris Antithrombotic Peptide cDNA Cloning

2.1 Total RNA Extraction from Haemadipsa sylvestris

[0027] (1) Several haemadipsa were removed from liquid nitrogen quickly, and the front end (including the mouth and throat) of the haemadipsa was cut with scissors; about 100 mg (total weight) sample was put into a mortar, ground thoroughly after liquid nitrogen was added. After the sample became powders, 1 mL of Trizol extraction buffer (Invitrogen) was added and ground together with liquid nitrogen. After the Trizol melted, all the liquid in the mortar was transferred to a 1.5 mL centrifuge tube and allowed to stand at room temperature for 5 min.

[0028] (2) 200 L of chloroform was added to the tube, vigorously vortexed and mixed for 15 s, allowed to stand at room temperature for 5 min; the tube was centrifuged at 12000 g at 4 C. for 10 min; the upper colorless aqueous phase was pipetted to a new 1.5 mL centrifuge tube (pipetted as much upper phase liquid as possible, but avoided the middle or lower phase).

[0029] (3) An equal volume of isopropanol (pre-cooled at 4 C.) was added to the tube, inversed and mixed, allowed to stand at room temperature for 15 min; the tube was centrifuged at 12000 g at 4 C. for 10 min; the small amount of precipitation at the bottom of the tube was RNA, and the supernatant was removed with a pipette.

[0030] (4) 1 mL of 75% ethanol (pre-cooled on ice) was added to the precipitation for washing; the tube was centrifuged at 7500 g at 4 C. for 5 min and the supernatant was discarded. The washing was repeated twice. The tube was placed in a laminar flow cabinet for 5 to 10 min to dry (until no ethanol) to obtain the Haemadipsa sylvestris total RNA. 30 L of 0.1% DEPC water was added to the tube after drying, gently vortexed to dissolve the RNA. 2 L of RNA sample was added to 98 L of DEPC water, and the absorbance was measured at 230 nm, 260 nm, and 280 nm wavelength. 10 L of RNA sample was subjected to 1% agarose gel electrophoresis. The other total RNA was frozen and stored at 80 C.

2.2 cDNA Library Construction

[0031] Haemadipsa sylvestris cDNA library was constructed according to the instruction of Creator SMART cDNA Library Construction Kit (Clontech). The specific operations are as follows:

(1) First-Strand cDNA Synthesis (mRNA Reverse Transcription)

[0032] 2 L of Haemadipsa sylvestris total RNA, 1 L of SMART IV oligonucleotide and 1 L of CDS III/3 Primer were added to a RNase-free PCR tube, 1 L of DEPC water was added to bring total volume up to 5 l, mixed well and centrifuged for 10 s; the tube was heated at 72 C. for 2 min, and then incubated on ice for 2 min; 2 L of 5first strand buffer, 1 L of 20 mM DTT, 1 L of 10 mM dNTP Mix and 1 L of PowerScript reverse transcriptase were added to the PCR tube, mixed well and centrifuged for 10 s. The tube was insulated at 42 C. for 1 h in a PCR machine, and then put on ice to terminate the reaction.

(2) Amplification of the Second-Strand cDNA Using Long-Distance PCR (LD-PCR)

[0033] 1 L of first-strand cDNA, 40 L of deionized H.sub.2O, 5 L of 10buffer, 1 L of 50dNTP Mix, 1 L of 5 PCR primer, 1 L of CDS III/3 PCR primer and 1 L of polymerase were mixed well in a PCR tube. PCR amplification was performed as follows:

[0034] i) 95 C. 1 min

[0035] ii) 20 cycles of: [0036] 95 C. 15 sec, [0037] 65 C. 30 sec, [0038] 68 C. 6 min.

[0039] After the amplification, the synthesized double-strand cDNA were put into PCR tubes (10 L/tube). 5 L sample was subjected to 1% agarose electrophoresis, the other was immediately stored at 80 C.

2.3 Preparation of E. coli DH5 Competent Cells

[0040] (1) A single DH5 colony was picked and inoculated in 1 mL of LB liquid medium without ampicillin, cultured at 37 C. overnight. The next day, the bacterial solution was inoculated again in 1 mL of LB medium at a ratio of 1:100, vortexed at 37 C. for 2 h.

[0041] (2) When the OD 600 of the culture reached 0.35, the bacterial solution was allowed to stand on ice for 10 min to be cooled to 0 C.

[0042] (3) The cells were collected by centrifuging at 5000 rpm at 4 C. for 5 min.

[0043] (4) The medium was discarded, and the tube was inverted for 1 min to let the last trace of medium run out.

[0044] (5) For every 1 mL of initial culture, 600 L of pre-cooled 0.1 M CaCl.sub.2-MgCl.sub.2 solution (80 mM MgCl.sub.2, 20 mM CaCl.sub.2) was added to resuspend cell pellet.

[0045] (6) The cells were collected by centrifuging at 5000 rpm at 4 C. for 5 min.

[0046] (7) The medium was discarded, and the tube was inverted for 1 min to let the last trace of medium run out.

[0047] (8) For every 1 mL of initial culture, 60 L of 0.1 M CaCl.sub.2 pre-cooled by ice was added to resuspend cell pellet, and then the tube was put in a refrigerator at 4 C. for 10 to 18 h.

2.4 cDNA Library Screening

(1) Synthesis of Specific Primers

[0048] Two primers were designed using primer blast and synthesized by Sangon Biotech. The amplification primer was 20 nucleotides in length, and the sequence was 5AAACCTCGGAACCGGTATGT 3; the other amplification primer was a 3 primer, and the sequence was 5CCGAGGTTTGGTGGCTCATT 3.

(2) Cloning Target Sequence from Haemadipsa cDNA Library

[0049] 20 L system: 0.1 L of Taq enzyme, 0.4 L of CDSIII and SMART4 respectively, 0.4 L of dNTP, 2 L of buffer, 1.2 L of Mg.sup.2+, 16 L of PCR water.

PCR Condition

[0050]

TABLE-US-00002 1. Pre-denaturation 95 C., 5 min 2. Denaturation 95 C., 30 s 33 Cycles 3. Annealing 56 C., 30 s 4. Extension 72 C., 30 s 5. Final Extension 72 C., 10 min

[0051] Ligation, Transformation and Detection. 0.2 L of Takara PMD19-T vector and 2.3 L of double-strand DNA were added to the a microcentrifuge tube; 2.5 L of the same amount of ligase buffer mixture (dissolved on ice) was added; the tube was incubated at 16 C. overnight; all the 5 L of the ligation product was added to 60 L DH5 competent cells, put on ice for 30 min; the competent cells were subjected to heat shock for 90 s, gently put on ice for 3 to 5 min to let the cell membrane to be repaired; pre-warmed LB medium was added as soon as possible, and the tube was incubated in a 80 rpm shaker at 37 C. for 45 min; 100 L cell was put onto a LB plate containing 100 g/mL ampicillin, incubated at 37 C. for 16 h; after the colonies appeared, single clones were picked and subjected to PCR (10 L reaction system).

2.5 Sequencing and Sequence Screening

[0052] Twenty single colonies, which have similar fragment size with the target sequence in PCR, were picked and subjected to DNA sequencing. An ABI 3730 sequencer was used for the sequencing. The sequencing primer was M13 (+): 5-CGCCAGGGTTTTCCCAGTCACGAC-3, M13 (): 5-GAGCGGATAACAATTTCACACAGG-3. The obtained sequences were subjected to sequence screening.

2.6 Haemadipsa sylvestris Antithrombotic Peptide Sylvestin DNA Sequence Determination

[0053] Plasmid DNA was extracted and the nucleotide sequence was determined by the dideoxy method, the instrument used was a U.S. Applied Biosystems 373A automatic nucleotide sequencer. The sequencing primer was BcaBESTTM Sequencing Primer RV-M and BcaBESTTM Sequencing Primer M13-47. The sequence of BcaBESTTM Sequencing Primer RV-M was: 5 GAGCGGATAACAATTTCACACAGG 3, and the BcaBESTTM Sequencing Primer M13-47 was: 5 CGCCAGGGTTTTCCCAGTCACGAC 3. The cDNA sequencing result from the 5 end to the 3 end (SEQ ID NO: 2) was:

TABLE-US-00003 GGGAACCCGAAACGGGCATTCGAGCTCGGTACCCGGGGATCCTCTA GAGATTAAACCTCGGAACCGGTATGTGCATGCCCAAAAATGCTATT TTGGGTTTGTGGTAAAGATGGTGAGACTTACACCCATCCTTGCATT GCAAAATGCCATAATGTTGAAGTTGAACATGATGGGAAGTGCAAAT GAAAGGGACCATTCTTCGAAATTGCCTGAAACTTAAAAATATTGAT TTGAATTTAATTAATTCTTATTAATTATAACGTTTCATCATAATAA ATGAATTACGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0054] As shown above, the nucleotide sequence of the cDNA encoding Haemadipsa sylvestris antithrombotic peptide Sylvestin cloned in this example has the following characteristics: the sequence length, 316 bases; sequence type: nucleic acid; number of strand: single strand; topology: linear; sequence type: cDNA; source: Haemadipsa sylvestris.

[0055] Analyzing with reference to Edman degradation N-terminal sequencing and mass spectrometry identification, the coding region of the Haemadipsa sylvestris mature antithrombotic peptide Sylvestin is nucleotides 55-183 of the nucleotide sequence SEQ ID NO: 2. The amino acid sequence (SEQ ID NO: 1) of the Haemadipsa sylvestris antithrombotic peptide Sylvestin is:

TABLE-US-00004 TSEPVCACPKMLFWVCGKDGETYTHPCIAKCHNVEVEHDG KCK.

[0056] The present disclosure also provided a use of the polynucleotide encoding the antithrombotic peptide Sylvestin as an antithrombotic peptide prepared in genetic engineering.

[0057] The present disclosure also provided a use of the antithrombotic peptide Sylvestin in the preparation of FXIIa and kallikrein inhibitors and anti-thrombus, anti-acute cerebral ischemia drugs.

Example 3 Chemical Synthesis of Haemadipsa sylvestris Antithrombotic Peptide Sylvestin

[0058] 3.1 Chemical synthesis of the antithrombotic peptide Sylvestin: referring to Example 2, the amino acid sequence was deduced based on the cDNA sequence and the sequencing result of the protein encoding Haemadipsa sylvestris antithrombotic peptide Sylvesti, and the full sequence was synthesized by an automatic peptide synthesizer, desalted and purified by HPLC reverse phase C18 column chromatography.

[0059] Fast atom bombardment mass spectrometry (FAB-MS) was used to determine the molecular weight, Glycerin:3-nitrobenzyl alcohol:dimethyl sulfoxide (1:1:1, V:V:V, volume ratio) were used as substrates, Cs.sup.+ was used as a bombardment particle, the current was 1 A and the emission voltage was 25 Kv.

[0060] 3.3 The purity of the purified Haemadipsa sylvestris antithrombotic peptide was determined by HPLC, the molecular weight was determined by fast atom bombardment mass spectrometry, the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence was determined by automatic amino acid sequencer.

[0061] Haemadipsa sylvestris antithrombotic peptide Sylvestin is a polypeptide encoded by the Haemadipsa sylvestris antithrombotic peptide gene, which has a molecular weight of 4790.5 Daltons and an isoelectric point of 6.28. The amino acid sequence of the antithrombotic peptide Sylvestin is: TSEPVCACPK MLFWVCGKDG ETYTHPCIAK CHNVEVEHDG KCK (SEQ ID NO:1).

Example 4 Pharmacological Experiment of Haemadipsa sylvestris Antithrombotic Peptide Sylvestin

4.1 Enzyme Kinetics

[0062] In a 96-well plate, 10 L of the sample and 10 L of FXIIa with a final concentration of 0.2 M were mixed in 40 L of buffer (100 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM CaCl.sub.2), allowed to stand at room temperature for 5 min. A mixture of 30 L of buffer and 10 L of chromogenic substrate with a final concentration of 0.04 mM was added to each well, the final volume was 100 L. The kinetics of the coagulation reaction was measured using an Epoch (BioTek) microplate reader and GEN CHS 1.09 software, OD 405 nm, 20 min, with 47 s intervals. The concentration of the sample was 10 M. The result showed that the Sylvestin synthesized in Example 3 had no inhibitory effect on thrombin, plasminogen FXa or the like, while it can inhibit FXIIa and kallikrein. The inhibition constants of Sylvestin on FXIIa and kallikrein were calculated to be 2.9 M and 17.8 nM, respectively.

4.2 APTT and PT Experiments

[0063] The APTT reagent was equilibrated to room temperature and the APTT reagent was mixed by gentle inversion; 50 L of the reagent, 50 L of normal plasma and 5 L of the sample were mixed and incubated in a 37 C. water bath for 3 min; 50 L of pre-warmed CaCl.sub.2 solution was added, mixed immediately; the OD 650 nm was detected with a microplate reader. PT experiment: prothrombin reagent was pre-warmed at 37 C. for 15 min; 50 L of normal plasma and 5 L of the sample were incubated in a 37 C. water bath for 3 min; 100 L of pre-warmed prothrombin reagent was added, mixed immediately; OD 650 nm was detected with a microplate reader. The result showed that the Sylvestin synthesized in Example 3 had no effect in PT experiment, while had a concentration-dependent effect in APTT experiment, indicating that Sylvestin inhibited the endogenous coagulation pathway, consistent with the result of FXIIa inhibitor.

4.3 Carrageenan-Induced Rat Tail Thrombosis Model

[0064] Kunming mice were used as experimental animals, body weight 25 to 30 g (provided by the Experimental Animal Center of Kunming Medical College). After one week of housing, they were randomly divided into groups (n=6), half male and half female. One group was a physiological saline control group, the sample groups were Sylvestin synthesized in Example 2 at a dose of 2 mg/kg and 4 mg/kg, respective, and the positive control was given heparin sodium (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), 500 U/mouse. 30 minutes after tail vein administration, carrageenan (carrageenan, type I, Sigma, dissolved in physiological saline to a concentration of 1%) was injected from the abdomen of the mice at a dose of 60 mg/kg, since the thrombosis rate was >90% in a low temperature environment, the housing temperature was set at 17.5 C. After 12, 24, 36, and 48 h, the average length of thrombus was determined according to the color change of the tail skin. Referring to FIG. 1, the average length of thrombus in each group increased with time. At 12 h, since thrombosis was not obvious, error in the measurement was relatively high, but the symptom of the control group was more obvious than the other group's; in heparin sodium group, the length of thrombus doubled from 12 h to 24 h; the control group was basically unchanged, possibly due to the length of the rat tail; the length of the sample group increased slightly. At each measurement time point, the effects of Sylvestin and heparin on rat tail thrombosis were statistically significant compared with the control group. Data processing was Graphpad Prime 5, data meanSD, t-test (*p<0.05). Overall, the inhabitation rate of Sylvestin against thrombosis decreased over time, but even after 48 h, the Sylvestin-treated group still showed significant inhibitory effect compared with the control group. Compared with the heparin group and the 2 mg/kg group, the inhibition rate of the 4 mg/kg group did not change much. Moreover, the inhibition rate of Sylvestin against thrombus increased as concentration increased (FIG. 1).

4.4 Acute Cerebral Ischemic Intraluminal Thread Model

[0065] Kunming mice (30 to 35 g) were anesthetized with 2% sodium pentobarbital (80 mg/kg). After medium-deep anesthesia, a midline neck incision was made, the skin and subcutaneous tissue of the mice were incised layer by layer, the sternocleidomastoid muscle was separated, the anterior belly of the digastric muscle was incised off, the right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were exposed, an electric coagulator was used to coagulate thyroid artery and pharyngeal artery over the ECA and they were cut off. Ligation of the distal end of the ECA was performed, seton was placed at the proximal end, CCA and ICA were temporarily closed using a clip, the ECA was cut off, an intraluminal thread was inserted into the ICA from the ECA stump, ligation of the ECA stump was performed, the artery clip of the ICA was removed, pushed inward and upward from the ICA. Direction was appropriately adjusted, inserted to the intraluminal thread mark (10 mm from the CCA junction), timing started, the intraluminal thread was removed after 1 h, after no active bleeding was observed, incision was stitched. The temperature was maintained during the whole process using a heating blanket, the temperature was 36 to 37 C. Awakened animals were put back to the cage and were allowed to eat and drink freely. Cerebral ischemia for 24 h, and the neurological deficit score was assessed and recorded according to the Bederson Scale assessment: 0 is no dysfunction; 1 is being unable to extend the right forelimb; 2 is rotating to right; 3 is dumping to right; 4 is no autonomous activity with disturbance of consciousness; 5 is death. Anesthetized after 24 h and head was cut down, brain tissue was removed, placed in a brain mold (on ice), cut into 2 mm thick sections along the optic chiasm, and placed in a 2% TTC phosphate buffer, dyed in a 37 C. incubator for 30 min, fixed with 4% paraformaldehyde overnight and images were acquired. Percentage of ischemic volume=[ischemic volume(volume of the left hemisphere of the brainvolume of the right hemisphere of the brain]/volume of the right hemisphere of the brain100. 10 min before ischemia reperfusion, intravenously administrated once through tail vein, the experimental group was administrated 1, 3, 5 mg/kg of Sylvestin (synthesized according to Example 2), and the control group was administrated physiological saline, the volume was 100 L. Within 10 min after the ischemia reperfusion, intravenously administrated once through tail vein, the dose was the same as above; administrated again after 6 h. 6 in each group. The result showed that Sylvestin can effectively alleviate the injury caused by cerebral ischemia and reperfusion (FIG. 2).