Recombinant hirudin with tyrosine sulfation, preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of genetic engineering. Provided are a recombinant hirudin with tyrosine sulfation, a preparation method therefor and the use thereof. In order to improve the safety and synthesis yield of recombinant hirudin, and reduce the production cost, a recombinant hirudin with tyrosine sulfation is prepared by means of an algae expression vector.

Description

A recombinant hirudin with tyrosine sulfate, its preparation method and its uses Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant hirudin with tyrosine sulfate, its preparation method, and its uses. Background Technology

[0002] Hirudin, a polypeptide extracted from the salivary glands of leeches, can specifically bind to thrombin to prevent thrombus formation and has been developed as an effective anticoagulant. The FDA has approved recombinant hirudin and its derivatives, such as bivalirudin, lepirudin, and desirudin, primarily for the prevention of complications in thromboembolic diseases, prevention of arterial thrombosis after surgery, and prevention of thrombus formation after thrombolysis or angiogenesis. However, as polypeptide biological drugs, conventional recombinant hirudin and its derivatives have a short half-life in vivo after administration via gastrointestinal injection. Frequent injections to achieve an anticoagulant effect in clinical patients pose a risk of infection; bleeding is a common side effect of anticoagulants in clinical use. Therefore, the development of a long-acting formulation is extremely important.

[0003] Hirudin is the most potent known natural thrombin inhibitor and is widely used clinically for the treatment of cardiovascular diseases. However, its application in the medical field is severely limited by the low yield and high extraction cost of natural hirudin. Currently available recombinant hirudin has only one-tenth the efficacy of natural hirudin. The main reason for this difference is that natural hirudin is a short peptide composed of 63-65 amino acids, with a functional domain consisting of three disulfide bonds at its N-terminus. The tyrosine residue at position 63 of the C-terminus is modified by tyrosyl protein sulfotransferase (TPST) in the Golgi apparatus of the cell, forming a sulfonated tyrosine residue. This tyrosine sulfation modification is crucial for hirudin activity. Without this modification, most bioengineered hosts cannot complete the post-translational modification reaction of hirudin, resulting in a significant reduction in the activity of recombinant hirudin lacking sulfonation compared to natural hirudin. Furthermore, since most commercially available recombinant hirudin products are derived from engineered E. coli or yeast, they lack post-translational modification capabilities. Microalgae, as a novel type of chassis organism, possess this function, undergoing sulfation modification on 87Tyr, resulting in higher activity than recombinant hirudin products derived from other yeast, E. coli, and other sources.

[0004] Studies have shown that TPST exists only in higher plants, animals, and microalgae. Traditional plant and microbial production systems each have their limitations: plant systems, while possessing post-translational modification capabilities, are greatly affected by the growing environment, have poor gene expression stability, and have long growth cycles and occupy arable land; microbial systems suffer from problems such as endotoxins and inclusion bodies, and most microorganisms lack post-translational modification capabilities, resulting in limited protein synthesis. In contrast, microalgae have advantages such as rapid growth, clear genetic background, ease of transformation and expression of exogenous genes, and contain TPST enzymes, making them suitable for the expression and production of recombinant hirudin. Currently, in the expression and production of recombinant hirudin, there is no known technical route that uses algae as a host, relies on genetic engineering and metabolic engineering, and induces the secretion of heterologous target proteins by introducing exogenous genes. Recombinant hirudin generated based on this technical route has the characteristic of natural hirudin with sulfation of the tyrosine residue at position 63 of the C-terminus, and has great application potential and economic value. Summary of the Invention

[0005] The purpose of this invention is to provide a tyrosine-sulfated recombinant hirudin, its preparation method, and its uses, so as to solve one or more technical problems existing in the prior art, and at least provide a beneficial option or create conditions.

[0006] The first aspect of this invention provides a recombinant hirudin with tyrosine sulfate.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned tyrosine-sulfated recombinant hirudin.

[0008] A third aspect of the present invention provides the use of the above-described tyrosine-sulfated recombinant hirudin in the preparation of a medicament for the prevention or treatment of thrombin-related physiological abnormalities.

[0009] The fourth aspect of the present invention provides an administration route for the above-mentioned tyrosine-sulfated recombinant hirudin.

[0010] The nucleotide sequence of the tyrosine-sulfated recombinant hirudin described in the first aspect of the present invention is SEQ ID No. 1, and the amino acid sequence is SEQ ID No. 4.

[0011] The method for preparing recombinant hirudin with tyrosine sulfate according to the second aspect of the present invention is characterized in that the recombinant hirudin is expressed through an algal expression vector.

[0012] In some embodiments of the second aspect of the present invention, the algae is Chlamydomonas reinhardtii.

[0013] In some embodiments of the second aspect of the present invention, the following preparation steps are included.

[0014] a) Preparation of the insert: The endogenous promoter beta2-tubulin and RBCS2 intron sequence form the key promoter element, which is linked to the Hygromycin resistance gene. A 2A peptide self-cleaving sequence is added between the recombinant hirudin gene and the Hygromycin resistance gene, and the end is attached with an HA tag.

[0015] b) Plasmid preparation: The insert fragment was ligated to the Phyg3-FMDV2A plasmid vector to construct the Phyg3-FMDV2A-hirudin-HA plasmid vector. The restriction enzyme used for linearization was KpnⅠ-HF.

[0016] c) Plasmid introduction into Chlamydomonas cells: Plasmid DNA was introduced into Chlamydomonas reinhardtii cells by electroporation to obtain Chlamydomonas reinhardtii-derived recombinant hirudin-expressing algal strains.

[0017] d) Screening of algal strains: The obtained Chlamydomonas reinhardtii-derived recombinant hirudin-containing algal strains were screened for resistance using the antibiotic Hygromycin, thereby isolating Chlamydomonas reinhardtii cells that successfully introduced and expressed recombinant hirudin.

[0018] e) Obtain a high-yielding recombinant hirudin-derived Chlamydomonas rhineland strain through fermentation culture.

[0019] In some embodiments of the second aspect of the present invention, the nucleotide sequence of the Hygromycin resistance gene is SEQ ID No. 2; the self-cleaving sequence of the 2A peptide is SEQ ID No. 3; the endogenous promoter beta2-tubulin sequence is SEQ ID No. 8; and the RBCS2 intron sequence is SEQ ID No. 9.

[0020] In some embodiments of the second aspect of the invention, when introducing plasmid DNA into Chlamydomonas reinhardtii cells by electroporation, the concentration of Chlamydomonas reinhardtii cells used is 5 x 10⁻⁶. 7 ~3x10 8 per ml.

[0021] In some embodiments of the second aspect of the present invention, when introducing plasmid DNA into Chlamydomonas reinhardtii cells by electroporation, the solvent used is TAP + 55-95 mM sorbitol.

[0022] In some embodiments of the second aspect of the present invention, when introducing plasmid DNA into Chlamydomonas reinhardtii cells by electroporation, the electroporation device parameters are voltage 750V-1200V, resistance 1200-1800Ω, and capacitance 50uF.

[0023] In some embodiments of the second aspect of the present invention, after plasmid DNA is introduced into Chlamydomonas reinhardtii cells by electroporation, the Chlamydomonas reinhardtii cells are recovered by slow, low-light shaking on a shaker at a temperature of 20-27°C and a rotation speed of 50-150 rpm for 6-8 hours.

[0024] In some embodiments of the second aspect of the present invention, the concentration of the Hygromycin antibiotic is 10-20 mg / L.

[0025] In some embodiments of the second aspect of the present invention, the screening and culture conditions for the algal strain are a temperature of 20-27°C, a photoperiod of light:dark = 16h:8h, and a light intensity of 3000-6000 lux.

[0026] The third aspect of this invention discloses the use of tyrosine-sulfated recombinant hirudin in the preparation of medicaments for the prevention or treatment of physiological abnormalities related to thrombin, including but not limited to venous thrombosis, pulmonary embolism, thrombocytopenia, and myocardial infarction.

[0027] The fourth aspect of this invention discloses an oral administration route for recombinant hirudin with tyrosine sulfate.

[0028] In some embodiments of the fourth aspect of the present invention, tyrosine-sulfated recombinant hirudin can be administered via two or more routes, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal administration. Beneficial effects:

[0029] Through in vivo testing of recombinant hirudin protein from Chlamydomonas reinhardtii, we obtained the following beneficial results: the recombinant hirudin expression system using Chlamydomonas reinhardtii as a chassis cell exhibits significant advantages in terms of production cost, biosafety, production efficiency, ease of genetic manipulation, product activity, and in vivo testing, opening up new avenues for the research and development and production of recombinant protein drugs. Attached Figure Description

[0030] 1. Figure 1. Western blot analysis of protein expression in the recombinant hirudin-derived Chlamydomonas reinhardtii strain.

[0031] 2. Figure 2 shows the identification of recombinant hirudin from Chlamydomonas reinhardtii by Western blot analysis.

[0032] 3. Figure 3 compares the enzyme activities of other similar recombinant hirudin expression products.

[0033] 4. Figure 4 shows the anticoagulant effects of oral administration of 5L (A) and 2T (B) fermented algal powder in the mouse model.

[0034] 5. Figure 5. Mouse model testing of the effect of recombinant hirudin from Chlamydomonas reinhardtii—thrombus weight; A. Inferior vena cava of mice, control group received oral administration of wild-type Chlamydomonas reinhardtii (a), experimental group received oral administration of recombinant hirudin from Chlamydomonas reinhardtii (b); B. Statistical data on the weight of ligated thrombi in the inferior vena cava of mice (same as in Figure A for control and experimental groups).

[0035] 6. Figure 6 compares the adverse drug reactions of Chlamydomonas reinhardtii recombinant hirudin and its derivatives—platelet count.

[0036] 7. Figure 7 compares the specific implementation methods for adverse drug reactions of Chlamydomonas reinhardtii recombinant hirudin and its derivatives, specifically regarding bleeding.

[0037] The terms used in this invention, unless otherwise stated, generally have the meanings commonly understood by those skilled in the art.

[0038] In the following embodiments, various processes and methods not described in detail are conventional methods known in the art.

[0039] The reagents used in the following examples were obtained through common commercial channels. Experimental procedures and conditions not specified are in accordance with conventional procedures and conditions in the art.

[0040] Example 1

[0041] pHyg3-FMDV2A-CrHirudin-HA was used to construct and amplify the target Hirudin fragment.

[0042] 1. Using the artificially synthesized CrHirudin fragment plasmid SEQ ID No. 1 as a template, PCR amplification was performed using high-fidelity enzyme (product number: P505) from Nanjing Novizan Biotechnology Co., Ltd. The amplification primer sequences Primer-S are SEQ ID No. 5 and Primer-AS are SEQ ID No. 6, with a fragment length of 267 bp.

[0043] 2. Add the following ingredients to the PCR amplification system in the following order:

[0044] 3. The PCR program should be set as follows:

[0045] 4. After 1% agarose gel electrophoresis, cut off the gel block of the target size and recover the gel using the gel recovery kit (B518131-0100) from Sangon Biotech (Shanghai) Co., Ltd.

[0046] Homologous recombination to obtain expression vectors

[0047] As shown in Figure 2, using the vector pHyg3 as a template, the fragment was inserted into the vector using homologous recombination. It was positioned after the sequence fragment of the self-cleaved polypeptide P2A and before the tag HA, thus constructing the final target gene expression plasmid SEQ ID No. 7, which was then confirmed by first-generation sequencing.

[0048] 1. Linearize the vector using restriction endonucleases from Takara, adding the following reagents in sequence:

[0049] 2. After 2% agarose gel electrophoresis, a 4.3kb gel block was cut off and the gel was recovered using the gel recovery kit (B518131-0100) from Sangon Biotech (Shanghai) Co., Ltd.

[0050] 3. Homologous recombination was performed using the Nanjing Novizan Biotechnology Co., Ltd. reagent kit (C112-02). Reagents were added in the following order:

[0051] 4. Maintain a constant temperature of 37℃ for 30 minutes.

[0052] 5. Add 10 μL of the recombinant product to E. coli competent cells DH5α, gently tap the tube wall, and let stand on ice for 30 min.

[0053] 6. Incubate in a 42.0℃ water bath for 45 seconds, then incubate on ice for 2 minutes. After that, add SOC, mix well, and incubate at 37℃ and 200 rpm for 1 hour.

[0054] 7. After 1 hour, spread the liquid from the centrifuge tube onto a solid culture plate containing 100 ng / µL Amp+, dry it, and then incubate it upside down in a 37°C incubator for 8 hours.

[0055] 8. Select positive single-clone colonies for liquid culture, shake to mix, and then perform first-generation sequencing for confirmation.

[0056] Preparation of expression vector DNA

[0057] 1. Inoculate an appropriate amount of bacterial culture into a medium containing 100 ng / mL Amp+ and incubate at 37°C and 200 rpm for 12 h.

[0058] 2. Centrifuge at 8000xg for 2 minutes to collect bacteria.

[0059] 3. Using the plasmid extraction kit (B515109-0100) from Sangon Biotech (Shanghai) Co., Ltd., add 250 μL of Buffer P1 to suspend the bacterial culture, then add 250 μL of Buffer P2, let stand at room temperature for 4 min, and finally add 300 μL of Buffer P3 while inverting the container 5-10 times. Centrifuge at 12000 x g for 5-10 min, and pour the supernatant into the adsorption column.

[0060] 4. Add 500uL of wash buffer, centrifuge at 9000xg for 30s, and repeat once.

[0061] 5. Centrifuge the empty adsorption column at 9000xg for 1 min.

[0062] 6. Place the adsorption column into a 1.5 mL centrifuge tube, add 50-100 μL of ddH2O, let stand for 1 min, and centrifuge for 1 min.

[0063] 7. Linearization was performed using NEB KpnI-HF endonuclease (R3142L), with reagents added in the following order:

[0064] 8. Further, the linearized plasmid was subjected to 1% agarose gel electrophoresis, and the target fragment was purified by gel extraction (Biotechnical Gel Extraction Kit, catalog number: B110092; Biotechnical Purification Kit, catalog number: B110093). The purified product was stored at -20°C.

[0065] Algal strain transformation

[0066] 1. Wild-type Chlamydomonas reinhardtii 21gr were continuously cultured in an aeration bottle containing TAP medium until the logarithmic growth phase, then transferred to a 200mL Erlenmeyer flask and placed on a shaker at 200rpm under continuous illumination for 8-16 hours until the cell concentration reached 4x10⁻⁶. 6 cells / ml.

[0067] 2. Clean the electrode cup three times with anhydrous ethanol in a clean bench, then wash it three times with TAP + 60mM sorbitol. After that, place the electrode cup in a refrigerator at -20°C for 30 minutes to pre-cool it for later use.

[0068] 3. Collect cells from step 1 by centrifugation at 2500 rpm for 3 min, and resuspend the cells in TAP medium to a density of 2 x 10⁻⁶. 8 cells / ml.

[0069] 4. Centrifuge the cell suspension from step 3 at 2500 rpm for 3 min, remove the supernatant and collect the cell pellet. Resuspend the pellet in pre-cooled TAP + 60 mM sorbitol.

[0070] 5. Centrifuge the cell suspension from step 4 at 2500 rpm for 3 min, remove the supernatant and collect the cell pellet. Resuspend the pellet in pre-cooled TAP + 60 mM sorbitol to make the final cell volume 250 μL, and place on ice for 10 min.

[0071] 6. Gently pipette 250 μL of cell suspension with 100-200 ng of linearized plasmid DNA and pre-cool on ice for 10 min.

[0072] 7. Set the parameters of the stun gun. The stun gun model used here is BTXECM630. After turning on the stun gun by pressing "power", set the voltage to 800V, the resistance to 1575Ω, and the capacitor to 50uF.

[0073] 8. Gently wipe the moisture off the outer shell of the electrode cup with absorbent paper. Quickly place the electrode cup containing cells and DNA into the electroshock apparatus, secure it tightly with metal clamps on both sides, lower the safety cover, and press "pulse" to initiate the electroshock. Record the time; 10-20 ms is within the normal time range. Once the electroshock is complete, immediately remove the electrode cup and place it on ice for 10 minutes.

[0074] 9. Gently transfer the electrolyzed cells to a 50ml sterile centrifuge tube pre-filled with 10ml TAP + 60mM sorbitol, seal tightly, and place on a shaker at 23℃, 100rpm, under low light overnight for recovery.

[0075] Cell plating:

[0076] 1. Prepare TAP solid culture plates containing hygromycin antibiotic (1:2000).

[0077] 2. Weigh 2g of starch into a 50ml centrifuge tube, wash it once with anhydrous ethanol, centrifuge at 1000rpm for 2min and discard the supernatant, add ddH2O and repeat the washing twice, centrifuge at 1000rpm for 1min and discard the supernatant, resuspend in 70% ethanol and bring the volume to 20ml, shake well to mix and continue to centrifuge at 1000rpm for 1min and discard the supernatant.

[0078] 3. Add TAP + 60mM sorbitol to the tube, centrifuge at 1000rpm for 1min, discard the supernatant, and repeat the washing process 4 times.

[0079] 4. After overnight recovery from electrocution, centrifuge the algal solution at 2500 rpm for 3 min, discard the supernatant, add 1 ml of the starch solution washed with TAP + 60 mM sorbitol from step 3, gently mix with a pipette, and then spread it evenly onto a solid TAP culture plate containing Hygromycin antibiotic (1:2000). Invert the plate and culture at 24℃ with a photoperiod of light:dark = 16 h:8 h and a light intensity of 3000 lux. Culture under light for about 1 week and observe the germinating transformants at any time.

[0080] 5. Isolation of single-clonal algal strains: The obtained transformants were spread onto TAP plates containing the antibiotic Hygromycin. After culturing, single clones were picked and transferred to liquid TAP medium containing pre-added Hygromycin antibiotic. The plates were cultured at 24°C with shaking at 200 rpm until the plateau phase was reached.

[0081] Screening for positive transformed algal strains

[0082] 1. Spread the electrolyzed cells evenly onto TAP plates containing Hygromycin resistance and culture them under low light for transformant selection.

[0083] 2. Select successfully grown cell transformants and transfer them to TAP plates containing Hygromycin resistance for further culture. Then, select a portion of the cell transformants and transfer them to 24-well plates (containing TAP liquid medium with Hygromycin resistance). Culture at 24°C with shaking until the plateau phase.

[0084] 3. Take 1 ml of cells cultured to the plateau phase into a 1.5 ml EP tube, centrifuge at 14000 rpm for 1 min, discard the supernatant, collect the cell pellet, add 4℃ pre-cooled lysis buffer A+ (mini complete+ buffer A) and slowly pipette until the cells are fully lysed.

[0085] 4. Collect the algal solution, centrifuge and extract the protein. Use Western blot to screen the cultured Chlamydomonas reinhardtii cells. The expected size of the recombinant hirudin protein is 20.2 kDa.

[0086] 5. Screening for recombinant hirudin-containing Chlamydomonas reinhardtii strains that overexpress hirudin.

[0087] Related formulas

[0088] 1. TAP medium should be prepared in the following order:

[0089] 2. Prepare Phosphate Buffer II (for 100ml) in the following order:

[0090] 3. Solution A (for 500ml) should be prepared in the following order:

[0091] 4. Dissolve and mix the TAP trace elements (for 1L) separately in the following order:

[0092] Example 2, Inferior vena cava thrombosis model

[0093] Direct oral administration of recombinant hirudin-containing algal powder from *Chlamydomonas reinhardtii* obtained through fermentation showed that after modeling, mice administered wild-type algal powder had significantly larger thrombi, which appeared dark red to the naked eye. The thrombi of mice administered small-scale (5L) and pilot-scale (2T) fermented *Chlamydomonas reinhardtii* recombinant hirudin-containing algal powder were significantly smaller and more transparent than those of untreated mice. This indicates that administration of *Chlamydomonas reinhardtii* recombinant hirudin-containing algal powder has a significant anticoagulant effect, as shown in Figure 4.

[0094] Example 3: Testing the effect of recombinant hirudin from Chlamydomonas reinhardtii in a mouse model – thrombus weight

[0095] Figure 5 shows: A. Inferior vena cava of mice. The control group received oral administration of wild-type Chlamydomonas reinhardtii algal powder (a), and the experimental group received oral administration of Chlamydomonas reinhardtii recombinant hirudin algal powder (b). The results showed that thrombus formation occurred in the inferior vena cava of mice under ligation in the control group (a), while oral administration of Chlamydomonas reinhardtii recombinant hirudin algal powder inhibited thrombus formation in the inferior vena cava of mice under ligation (b); B. Statistical data on the weight of thrombi in the inferior vena cava of mice. The control and experimental groups were the same as in Figure A. The statistical data for mice came from three independent replicate experiments, with data for the control and experimental groups corresponding to 25 mice each. Statistical data showed a significant difference in the weight of inferior vena cava thrombi between the control and experimental groups (p<0.0001). In the control group, all mice formed thrombi (100%), while the thrombus formation rate in the experimental group was approximately 20% (and the weight of the formed thrombi was much smaller than that in the control group). Approximately 80% of the mice in the experimental group did not form observable and measurable thrombi. The statistical data further confirmed that oral administration of recombinant hirudin from Chlamydomonas reinhardtii significantly inhibited the formation of inferior vena cava ligation thrombi in mice (B).

[0096] Example 4: Comparison of adverse drug reactions of Chlamydomonas reinhardtii recombinant hirudin and its derivatives—platelet count

[0097] The comparison of thrombocytopenia in mice is shown in Figure 6. Gray bars indicate thrombocytopenia controlled within 35% or no reduction before and after drug treatment. Blue bars indicate thrombocytopenia ranges from 35% to 65% before and after drug treatment. Red bars indicate thrombocytopenia exceeding 65% before and after drug treatment. The results show that in the Chlamydomonas reinhardtii-derived recombinant hirudin group, all mice (100%) showed no or minimal thrombocytopenia before and after treatment. Heparin and bivalirudin caused significant thrombocytopenia, while Chlamydomonas reinhardtii-derived recombinant hirudin did not cause significant thrombocytopenia and had a weak effect on platelets.

[0098] Example 5: Comparison of adverse drug reactions—bleeding—of recombinant hirudin from Chlamydomonas reinhardtii and its derivatives.

[0099] The comparison of bleeding in mice is shown in Figure 7. Autopsy results showed that no bleeding occurred in the Chlamydomonas reinhardtii-derived recombinant hirudin group. The bleeding rates in the heparin and bivalirudin groups both exceeded 10% (and the bleeding was severe, including subcutaneous and visceral bleeding). The Chlamydomonas reinhardtii-derived recombinant hirudin does not activate the fibrinolytic system, and adverse bleeding reactions are rare (currently, the bleeding rate is 0%).

[0100] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0101] Sequence list information:

[0102] DTD Version: V1_3

[0103] Filename: A recombinant hirudin with tyrosine sulfate, its preparation method and its uses.

[0104] Software Name: WIPO Sequence

[0105] Software version: 2.3.0

[0106] Generation Date: 2024-11-07

[0107] Basic Information:

[0108] Current application / Intellectual Property Office: CN

[0109] Current application / applicant file name: Wuhan Yuanqi He Sheng Technologiae Societas Limitata

[0110] Applicant's Name or Company Name: Wuhan Yuanqi Hesheng Technology Co., Ltd.

[0111] Applicant's name or title / language:zh

[0112] Applicant's Name or Title / Latin Name: Wuhan Yuanqi He Sheng Technologiae Societas Limitata

[0113] Inventor's Name: Xing Junqiao

[0114] Inventor's name / language: zh

[0115] Inventor's Name / Latin Name: Xing Junqiao

[0116] Invention Title: A recombinant hirudin with tyrosine sulfate, its preparation method and its uses (zh)

[0117] Total number of sequences: 9

[0118] sequence:

[0119] Serial Number (ID): 1

[0120] Length: 267

[0121] Molecular type: DNA

[0122] Feature location / qualifier:

[0123] -source,1..267

[0124] >mol_type, other DNA

[0125] >organism, synthetic construct

[0126] residues:

[0127] Serial Number (ID): 2

[0128] Length: 909

[0129] Molecular type: DNA

[0130] Feature location / qualifier:

[0131] -source,1..909

[0132] >mol_type, other DNA

[0133] >organism, synthetic construct

[0134] residues:

[0135] Serial Number (ID): 3

[0136] Length: 72

[0137] Molecular type: DNA

[0138] Feature location / qualifier:

[0139] -source,1..72

[0140] >mol_type, other DNA

[0141] >organism, synthetic construct

[0142] residues:

[0143] Serial Number (ID): 4

[0144] Length: 90

[0145] Molecular type: AA

[0146] Feature location / qualifier:

[0147] -source,1..90

[0148] >mol_type,protein

[0149] >organism, synthetic construct

[0150] residues:

[0151] Serial Number (ID): 5

[0152] Length: 22

[0153] Molecular type: DNA

[0154] Feature location / qualifier:

[0155] -source,1..22

[0156] >mol_type, other DNA

[0157] >organism, synthetic construct

[0158] residues:

[0159] Serial Number (ID): 6

[0160] Length: 23

[0161] Molecular type: DNA

[0162] Feature location / qualifier:

[0163] -source,1.23

[0164] >mol_type, other DNA

[0165] >organism, synthetic construct

[0166] residues:

[0167] Serial Number (ID): 7

[0168] Length: 1948

[0169] Molecular type: DNA

[0170] Feature location / qualifier:

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[0172] >mol_type, other DNA

[0173] >organism, synthetic construct

[0174] residues:

[0175] Serial Number (ID): 8

[0176] Length: 330

[0177] Molecular type: DNA

[0178] Feature location / qualifier:

[0179] -source,1..330

[0180] >mol_type, other DNA

[0181] >organism, synthetic construct

[0182] residues:

[0183] Serial Number (ID): 9

[0184] Length: 145

[0185] Molecular type: DNA

[0186] Feature location / qualifier:

[0187] -source,1..145

[0188] >mol_type, other DNA

[0189] >organism, synthetic construct

[0190] residues:

[0191] END

Claims

1. A recombinant hirudin sulfated with tyrosine, characterized in that, The nucleotide sequence of the recombinant hirudin is SEQ ID No. 1, and the amino acid sequence is SEQ ID No.

4.

2. A method for preparing recombinant hirudin with tyrosine sulfate, characterized in that, The recombinant hirudin was expressed using an algal expression vector.

3. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 2, characterized in that, The algae mentioned is Chlamydomonas reinhardtii.

4. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 3, characterized in that, Includes the following steps: a) Preparation of the insert: The endogenous promoter beta2-tubulin and RBCS2 intron sequence form the key promoter element, which is linked to the Hygromycin resistance gene. A 2A peptide self-cleaving sequence is added between the recombinant hirudin gene and the Hygromycin resistance gene, and the end is attached with an HA tag. b) Plasmid preparation: The insert fragment was ligated to the Phyg3-FMDV2A plasmid vector to construct the Phyg3-FMDV2A-hirudin-HA plasmid vector. The restriction enzyme used for linearization was KpnⅠ-HF. c) Plasmid introduction into Chlamydomonas cells: Plasmid DNA was introduced into Chlamydomonas reinhardtii cells by electroporation to obtain Chlamydomonas reinhardtii-derived recombinant hirudin-expressing algal strains. d) Screening of algal strains: The obtained Chlamydomonas reinhardtii-derived recombinant hirudin-containing algal strains were screened for resistance using the antibiotic Hygromycin, thereby isolating Chlamydomonas reinhardtii cells that successfully introduced and expressed recombinant hirudin. e) Obtain a high-yielding recombinant hirudin-derived Chlamydomonas rhinelandella strain through fermentation culture.

5. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that, The nucleotide sequence of the Hygromycin resistance gene is SEQ ID No. 2; the self-cleaving sequence of the 2A peptide is SEQ ID No. 3; the sequence of the endogenous promoter beta2-tubulin is SEQ ID No. 8; and the sequence of the RBCS2 intron is SEQ ID No.

9.

6. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that, When introducing plasmid DNA into Chlamydomonas reinhardtii cells via electroporation, the concentration of Chlamydomonas reinhardtii cells used was 5 x 10⁻⁶. 7 ~3 x 10 8 per ml.

7. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that, When introducing plasmid DNA into Chlamydomonas reinhardtii cells via electroporation, the solvent used is TAP + 55–95 mM sorbitol.

8. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that, When introducing plasmid DNA into Chlamydomonas reinhardtii cells by electroporation, the electroporation device parameters are: voltage 750V-1200V, resistance 1200-1800Ω, and capacitance 50uF.

9. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that, After plasmid DNA was introduced into Chlamydomonas reinhardtii cells by electroporation, the cells were recovered by slow, low-light shaking at a temperature of 20-27°C and a rotation speed of 50-150 rpm for 6-8 hours.

10. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that, The concentration of the hygromycin antibiotic is 10-20 mg / L.

11. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that, The screening and culture conditions for the algal strains were: temperature 20-27℃, photoperiod light:dark = 16h:8h, and light intensity 3000-6000 lux.

12. The recombinant hirudin with tyrosine sulfate according to claim 1, characterized in that, Use in the preparation of medicaments for the prevention or treatment of physiological abnormalities related to thrombin; said physiological abnormalities include, but are not limited to, venous thrombosis, pulmonary embolism, thrombocytopenia, and myocardial infarction.

13. The recombinant hirudin sulfated with tyrosine according to claim 1, characterized in that, It can be administered orally.

14. The recombinant hirudin sulfated with tyrosine according to claim 1, characterized in that, It can be administered via two or more routes, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal.

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