Tyrosine sulfated recombinant hirudin as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to tyrosine sulfated recombinant hirudin as well as a preparation method and application thereof. In order to improve the safety and synthesis yield of the recombinant hirudin and reduce the production cost, the tyrosine sulfated recombinant hirudin is successfully prepared through an algae expression vector.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a tyrosine-sulfated recombinant hirudin, a preparation method and application thereof. Background Art

[0002] Hirudin is a polypeptide extracted from the salivary glands of leeches. It can specifically bind to thrombin to prevent thrombosis and has been developed as an effective anticoagulant. The FDA has approved recombinant hirudin and its derivatives, such as bivalirudin, lepirudin, desirudin, etc., which are mainly used to prevent complications of thromboembolic diseases, prevent arterial thrombosis after surgery, and prevent the formation of thrombi after thrombus dissolution or vascular regeneration. However, as a polypeptide biological drug, ordinary recombinant hirudin and its derivatives have a short half-life in the body after gastrointestinal injection. If clinical patients use frequent injections to achieve the anticoagulant effect, there will be a risk of infection; the clinical application of anticoagulants generally has the side effect of bleeding, so the development of a long-acting preparation is extremely important.

[0003] Hirudin is the most effective natural thrombin inhibitor known and is widely used in 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. The efficacy of recombinant hirudin currently available on the market is only one-tenth of that of natural hirudin. The main reason for this difference is that natural hirudin (Sulfo hirudin) is a short peptide composed of 63 to 65 amino acids, with three disulfide-bonded functional domains at its N-terminus. The tyrosine at position 63 at the C-terminus is modified by tyrosylprotein sulfotransferase (TPST) in the Golgi apparatus of the cell to form sulfonated tyrosine. The occurrence of tyrosine sulfation modification is very important for the activity of hirudin. Without this modification, most bioengineering hosts cannot complete the post-translational modification reaction of hirudin, which makes the activity of recombinant hirudin lacking sulfonation significantly reduced compared with natural hirudin. In addition, since similar recombinant hirudin products on the market are mostly derived from E. coli or yeast engineering bacteria, they lack the function of post-translational modification. Microalgae, as a new type of chassis organism, has this function and undergoes sulfation modification at 87Tyr, making its activity higher than other recombinant hirudin products derived from yeast, E. coli, etc.

[0004] Studies have shown that TPST only exists in higher plants and animals and microalgae. Traditional plant and microbial production systems each have their own limitations: although the plant system has the ability of post-translational modification, it is greatly affected by the planting environment, has poor gene expression stability, and has a long growth cycle and occupies arable land; the microbial system has problems such as endotoxins and inclusion bodies, and most microorganisms lack the ability of post-translational modification, resulting in limited synthetic protein categories. In contrast, microalgae have the advantages of rapid growth, clear genetic background, easy transformation and expression of foreign genes, and contain TPST enzymes, which are suitable for the expression and production of recombinant hirudin. At present, in the expression and production of recombinant hirudin, there is no technical route that uses algae as a host, genetic engineering and metabolic engineering as a technical basis, and introduces foreign genes to induce secretion and expression of heterologous target proteins. The recombinant hirudin generated based on this technical route has the characteristics of tyrosine sulfation at the 63rd position of the C-terminus of natural hirudin, and has great application potential and economic value. Summary of the invention

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

[0006] The first aspect of the present invention provides a tyrosine-sulfated recombinant hirudin.

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

[0008] The third aspect of the present invention provides the use of the above-mentioned tyrosine-sulfated recombinant hirudin in the preparation of a drug for preventing or treating physiological abnormalities associated with thrombin.

[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 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 tyrosine-sulfated recombinant hirudin according to the second aspect of the present invention is characterized in that the recombinant hirudin is expressed by an algae 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 insert fragments: The endogenous promoter beta2-tubulin and RBCS2 intron sequence constitute the key promoter element, which is linked to the hygromycin resistance gene. A 2A peptide self-cleavage sequence is added between the recombinant hirudin gene and the hygromycin resistance gene, and the HA tag is connected to the end.

[0015] b) Preparation of plasmid: The inserted fragment was connected to the Phyg3-FMDV2A plasmid vector to construct the Phyg3-FMDV2A-hirudin-HA plasmid vector, and the endonuclease used for linearization was KpnⅠ-HF.

[0016] c) Introduction of plasmid into Chlamydomonas cells: Plasmid DNA is introduced into Chlamydomonas reinhardtii cells by electroporation, thereby obtaining a Chlamydomonas reinhardtii-derived recombinant hirudin strain expressing recombinant hirudin.

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

[0018] e) A high-yield recombinant hirudin algae strain derived from Chlamydomonas reinhardtii is obtained by a fermentation culture method.

[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 2A peptide self-cleavage sequence 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 present invention, when the plasmid DNA is introduced into Chlamydomonas reinhardtii cells by electroporation, the concentration of Chlamydomonas reinhardtii cells used is 5 x 10 7 ~3x 10 8 Pieces / ml.

[0021] In some embodiments of the second aspect of the present invention, when the plasmid DNA is introduced 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 the plasmid DNA is introduced into Chlamydomonas reinhardtii cells by electroporation, the parameters of the electroporator are voltage 750V-1200V, resistance 1200-1800Ω, and capacitance 50uF.

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

[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 of the algae strains 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 the present invention discloses the use of tyrosine-sulfated recombinant hirudin in the preparation of drugs for preventing or treating physiological abnormalities associated with thrombin; the physiological abnormalities include but are not limited to venous thrombosis, pulmonary embolism, thrombocytopenia, and myocardial infarction.

[0027] The fourth aspect of the present invention discloses an oral administration route of tyrosine-sulfated recombinant hirudin.

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

[0029] Beneficial effects:

[0030] By conducting in vivo tests on the recombinant hirudin protein of Chlamydomonas reinhardtii, we obtained the following beneficial results: the recombinant hirudin expression system based on Chlamydomonas reinhardtii as the chassis cell showed significant advantages in production cost, biosafety, production efficiency, ease of genetic manipulation, product activity and in vivo testing, opening up a new path for the research and development and production of recombinant protein drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 1. Figure 1 Western blot detection of protein expression of recombinant hirudin strain from Chlamydomonas reinhardtii

[0032] 2. Figure 2 Sulfation identification of recombinant hirudin from Chlamydomonas reinhardtii using western blot

[0033] 3. Figure 3 Comparison of enzyme activity of other similar recombinant hirudin expression products

[0034] 4. Figure 4 Anticoagulant effect of 5L (A) and 2T (B) fermented algae powder after oral administration in a mouse model

[0035] 5. Figure 5 Mouse model testing the effect of recombinant hirudin of Chlamydomonas reinhardtii - thrombus weight; A. Inferior vena cava of mice, the control group was orally administered with wild-type Chlamydomonas reinhardtii (a), and the experimental group was orally administered with recombinant hirudin Chlamydomonas reinhardtii (b); B. Statistical data on thrombus weight after ligation of the inferior vena cava of mice (the control group and the experimental group are the same as in Figure A)

[0036] 6. Figure 6 Comparison of adverse drug reactions of recombinant hirudin from Chlamydomonas reinhardtii and its derivatives - platelets

[0037] 7. Figure 7 Comparison of adverse drug reactions of Chlamydomonas reinhardtii recombinant hirudin and its derivatives - bleeding

[0038] The terms used in the present invention, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

[0039] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0040] The reagents used in the following examples were purchased from common commercial sources, and the experimental operations and experimental conditions not specified were referred to the conventional operations and conventional conditions in the art.

[0041] Example 1

[0042] pHyg3-FMDV2A-CrHirudin-HA construction and amplification of Hirudin target fragment

[0043] 1. Using the artificially synthesized CrHirudin fragment plasmid SEQ ID No.1 as a template, PCR amplification was performed using the high-fidelity enzyme (Cat. No.: P505) of Nanjing Novozyme Biotechnology Co., Ltd. The amplification primer sequence Primer-S is SEQ ID No.5, Primer-AS is SEQ ID No.6, and the fragment length is 267bp;

[0044] 2. Add the PCR amplification system in the following order:

[0045]

[0046] 3. The PCR program was set as follows:

[0047]

[0048] 4. After 1% agarose gel electrophoresis, cut out the gel block of target size and use the gel recovery kit (B518131-0100) of Sangon Biotechnology (Shanghai) Co., Ltd. for gel recovery.

[0049] Homologous recombination to obtain expression vector

[0050] according to Figure 2 As shown, using vector pHyg3 as a template, the fragment was inserted into the vector by homologous recombination, located 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, and the first generation sequencing was performed for confirmation.

[0051] 1. Linearize the vector using restriction endonucleases from Takara and add the following reagents in order:

[0052]

[0053] 2. After 2% agarose gel electrophoresis, the gel block with a target size of 4.3 kb was cut out and gel recovery was performed using the gel recovery kit (B518131-0100) of Sangon Biotechnology (Shanghai) Co., Ltd.

[0054] 3. Perform homologous recombination using the Nanjing Novozyme Biotech Co., Ltd. kit (C112-02) and add the following reagents in the following order:

[0055]

[0056] Maintain a constant temperature of 4.37℃ for 30 minutes.

[0057] 5. Take 10uL of the recombinant product and add it to the E. coli competent cells DH5α, tap the tube wall gently, and let it stand on ice for 30 minutes.

[0058] 6.42.0℃ water bath for 45s, incubate on ice for 2min, then add SOC, mix well and incubate at 37℃, 200rpm for 1h.

[0059] 7. After 1 hour, spread the liquid in the centrifuge tube onto a solid culture plate containing 100 ng / uL Amp+, blow dry, and culture inverted in a 37°C incubator for 8 hours.

[0060] 8. Pick the positive monoclonal colonies for liquid culture, shake and mix, and then perform first-generation sequencing to confirm.

[0061] Preparation of expression vector DNA

[0062] 1. Inoculate an appropriate amount of bacterial solution into a medium containing 100 ng / mL Amp+ and culture at 37°C, 200 rpm for 12 h.

[0063] 2. Centrifuge at 8000xg for 2 min to collect the bacteria.

[0064] 3. Use the column plasmid extraction kit (B515109-0100) of Sangon Biotech (Shanghai) Co., Ltd., add 250uL Buffer P1 suspended bacterial solution, then add 250uL Buffer P2, let stand at room temperature for 4 minutes, and finally add 300uL Buffer P3, invert 5-10 times while adding, centrifuge at 12000xg for 5-10 minutes, and pour the supernatant into the adsorption column.

[0065] 4. Add 500uL wash buffer, centrifuge at 9000xg for 30s, and repeat once.

[0066] 5. Centrifuge the empty adsorption column at 9000xg for 1 min.

[0067] 6. Place the adsorption column in a 1.5mL centrifuge tube, add 50-100uL ddH20, let stand for 1min, and centrifuge for 1min.

[0068] 7. Linearize with KpnⅠ-HF endonuclease (R3142L) from NEB and add reagents in the following order:

[0069]

[0070] 8. The linearized plasmid was further subjected to 1% agarose electrophoresis, and the target fragment was purified by gel recovery product (Biological Engineering Product Gel Recovery Kit, Catalog No.: B110092; Biological Engineering Product Purification Kit, Catalog No.: B110093), and the purified product was stored at -20°.

[0071] Algal transformation

[0072] 1. Cultivate 21 gr of wild-type Chlamydomonas reinhardtii in a blowing bottle filled with TAP medium until the logarithmic phase, then transfer to a 200 mL triangular shake flask and place it on a shaker at 200 rpm for 8-16 hours with continuous light until the cell concentration reaches 4x10 6 cells / ml.

[0073] 2. In the clean bench, wash the electrode cup with anhydrous ethanol for 3 times, and then wash it with TAP + 60mM sorbitol for 3 times, and then place the electric shock cup in the refrigerator at -20° for 30 minutes for use.

[0074] 3. Centrifuge at 2500 rpm for 3 min to collect the cells in step 1 and resuspend the cells to 2 x 10 8 cells / ml.

[0075] 4. Centrifuge the cell suspension in step 3 at 2500 rpm for 3 min, remove the supernatant and collect the cell pellet, and resuspend it in pre-cooled TAP + 60 mM sorbitol.

[0076] 5. Centrifuge the cell suspension in step 4 at 2500 rpm for 3 min, remove the supernatant and collect the cell pellet, resuspend it with pre-cooled TAP + 60 mM sorbitol to make the final cell volume 250 ul, and place it on ice for 10 min.

[0077] 6. Mix 250 μL of cell suspension and 100-200 ng of linearized plasmid DNA by gently pipetting and place on ice for 10 minutes to pre-cool.

[0078] 7. Set the defibrillator parameters. The defibrillator model used here is BTXECM630. After turning on the defibrillator by pressing "power", set the voltage to 800v, ​​the resistance to 1575Ω, and the capacitance to 50uF.

[0079] 8. Gently wipe off the moisture on the outer shell of the electrode cup with absorbent paper, quickly place the electric shock cup containing cells and DNA in the electric shock instrument, clamp the metal on both sides tightly, lower the safety cover, press "pulse" to perform electric shock, and record the time. 10-20ms is the normal time range. After the electric shock is completed, immediately take out the electric shock cup and place it on ice for 10 minutes.

[0080] 9. Use a pipette to gently transfer the electroporated cells into a 50 ml sterile centrifuge tube pre-added with 10 ml TAP + 60 mM sorbitol. Cover tightly and place on a shaker at 23°C, 100 rpm, slow and weak light overnight to recover.

[0081] Cell plating:

[0082] 1. Prepare TAP solid culture plates containing Hygromycin antibiotic (1:2000).

[0083] 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, repeat the washing twice with ddH2O, centrifuge at 1000rpm for 1min and discard the supernatant, resuspend it in 70% ethanol to 20ml, shake it thoroughly to mix, and continue centrifuging at 1000rpm for 1min and discard the supernatant.

[0084] 3. Add TAP + 60mM sorbitol into the tube, centrifuge at 1000rpm for 1min, discard the supernatant, and repeat the washing for 4 times.

[0085] 4. Centrifuge the algae solution after overnight recovery from electroshock at 2500rpm for 3min, discard the supernatant, add 1ml of the starch solution washed with TAP+60mM sorbitol in step 3, gently blow with a pipette to mix, and then spread it on a solid TAP culture plate containing Hygromycin antibiotics (1:2000) and culture it upside down. The culture temperature is 24℃, the photoperiod is light:dark=16h:8h, the light intensity is 3000lux, and the culture is carried out under light for about 1 week, and the germinated transformants are observed at any time.

[0086] 5. Isolation of monoclonal algae strains: Spread the obtained transformants onto TAP plates containing Hygromycin antibiotics. Pick out single clones after cultivation and transfer them to liquid TAP medium containing Hygromycin antibiotics pre-added. Cultivate with shaking at 24°C and keep the rotation speed at 200 rpm until the stationary phase.

[0087] Screening of positive transformed algae strains

[0088] 1. Spread the electroporated cells evenly onto TAP plates containing Hygromycin resistance and culture under weak light for transformant screening.

[0089] 2. Pick the successfully grown cell transformants to continue culturing on TAP plates containing Hygromycin resistance, then pick some of the cell transformants into 24-well plates (TAP liquid culture medium with Hygromycin resistance), and culture at 24°C with shaking until the stationary phase.

[0090] 3. Take 1 ml of cells cultured to the stationary phase into a 1.5 ml EP tube, centrifuge at 14000 rpm for 1 min, discard the supernatant, collect the cell pellet, add 4°C pre-cooled lysis buffer bufferA+ (mini complete+bufferA), and slowly pipette until the cells are fully lysed.

[0091] 4. Collect the algae liquid, extract the protein after centrifugation, and use western blot to screen the cultured Chlamydomonas reinhardtii cells. The expected size of the recombinant hirudin protein is 20.2 kDa.

[0092] 5. Screen out recombinant hirudin algae strains derived from Chlamydomonas reinhardtii that overexpress hirudin.

[0093] Related recipes

[0094] 1. Prepare TAP medium in the following order:

[0095]

[0096] 2.Phosphate Buffer Ⅱ (for 100ml) is prepared in the following order:

[0097]

[0098] 3. Solution A (for 500ml) is prepared in the following order:

[0099]

[0100] 4. Dissolve TAP trace elements (for 1L) in the following order and mix well:

[0101]

[0102] Example 2, Inferior vena cava thrombosis model

[0103] The results showed that after modeling, the thrombus of mice taking wild-type algae powder was significantly enlarged, and dark red in naked eye observation. The thrombus of mice taking small-scale (5L) and pilot-scale (2T) fermented Chlamydomonas reinhardtii recombinant hirudin algae powder was significantly smaller and more transparent than that of mice not taking the drug. This shows that taking Chlamydomonas reinhardtii recombinant hirudin algae powder has a significant anticoagulant effect. Figure 4 shown.

[0104] Example 3, Mouse Model Test of the Effect of Recombinant Hirudin from Chlamydomonas reinhardtii - Thrombus Weight

[0105] like Figure 5 As shown, A. Inferior vena cava of mice. The control group was orally administered with wild-type Chlamydomonas reinhardtii powder (a), and the experimental group was orally administered with recombinant hirudin powder of Chlamydomonas reinhardtii (b). The results showed that thrombosis was formed in the inferior vena cava of mice in the control group (a), and oral administration of recombinant hirudin powder of Chlamydomonas reinhardtii had an inhibitory effect on thrombosis in the inferior vena cava of mice (b); B. Statistical data on the weight of thrombus in the inferior vena cava of mice. The control group and the experimental group are the same as those in Figure A. The statistical data of mice are from 3 independent repeated experiments, and the data of the control group and the experimental group correspond to 25 mice respectively. Statistical data showed that there was a significant difference in the weight of thrombus in the inferior vena cava between the control group and the experimental group (p<0.0001), and all mice in the control group formed thrombus (100%), while the thrombus formation rate of mice in the experimental group was about 20% (and the weight of thrombus formation was much smaller than that in the control group), and about 80% of the mice in the experimental group did not form observable and measurable thrombus. The statistical data further confirmed that oral administration of recombinant hirudin Chlamydomonas reinhardtii had a significant inhibitory effect on thrombus formation in the inferior vena cava ligation of mice (B).

[0106] Example 4, Comparison of adverse drug reactions of Chlamydomonas reinhardtii recombinant hirudin and its derivatives - platelets

[0107] Comparison of thrombocytopenia in mice, such as Figure 6As shown. The gray bar graph indicates that the range of thrombocytopenia before and after drug treatment is controlled within 35%, or there is no reduction. The blue bar graph indicates that the range of thrombocytopenia before and after drug treatment is 35-65%. The red bar graph indicates that the range of thrombocytopenia before and after drug treatment exceeds 65%. The test results show that the platelets of all mice (100%) in the group of recombinant hirudin from Chlamydomonas reinhardtii were not reduced or the reduction was controlled within 35% before and after treatment. Heparin and bivalirudin can cause significant thrombocytopenia, and recombinant hirudin from Chlamydomonas reinhardtii basically does not cause significant thrombocytopenia and has a weak effect on platelets.

[0108] Example 5: Comparison of adverse drug reactions of Chlamydomonas reinhardtii recombinant hirudin and its derivatives - bleeding

[0109] Comparison of bleeding in mice, such as Figure 7 As shown, the results of autopsy showed that no bleeding was observed in the group treated with recombinant hirudin from Chlamydomonas reinhardtii. The bleeding rates in the heparin and bivalirudin groups were both over 10% (and the bleeding was severe, including subcutaneous and organ bleeding). Recombinant hirudin from Chlamydomonas reinhardtii does not activate the fibrinolytic system, and the adverse reaction of bleeding is rare (the current statistical data shows that the bleeding rate is 0).

[0110] 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A tyrosine-sulfated recombinant hirudin, 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 tyrosine-sulfated recombinant hirudin, characterized in that: The recombinant hirudin is expressed by an algae expression vector.

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

4. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 3, characterized in that: The following steps are involved: a) Preparation of insert fragments: The endogenous promoter beta2-tubulin and RBCS2 intron sequence constitute the key promoter element, which is linked to the hygromycin resistance gene. A 2A peptide self-cleavage sequence is added between the recombinant hirudin gene and the hygromycin resistance gene, and the HA tag is connected to the end. b) Preparation of plasmid: The inserted fragment was connected to the Phyg3-FMDV2A plasmid vector to construct the Phyg3-FMDV2A-hirudin-HA plasmid vector, and the endonuclease used for linearization was KpnⅠ-HF. c) Introduction of plasmid into Chlamydomonas cells: Plasmid DNA is introduced into Chlamydomonas reinhardtii cells by electroporation, thereby obtaining a Chlamydomonas reinhardtii-derived recombinant hirudin strain expressing recombinant hirudin. d) Screening of algal strains: The obtained Chlamydomonas reinhardtii-derived recombinant hirudin algal strains were screened for resistance using the hygromycin antibiotic, thereby isolating the Chlamydomonas reinhardtii cells that have been successfully introduced and express recombinant hirudin. e) A high-yield recombinant hirudin algae strain derived from Chlamydomonas reinhardtii is obtained by a fermentation culture method.

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 2A peptide self-cleavage sequence 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.

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

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

8. The method for preparing tyrosine-sulfated recombinant hirudin according to claim 4, characterized in that: When plasmid DNA is introduced into Chlamydomonas reinhardtii cells by electroporation, the parameters of the electroporator 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 the plasmid DNA was introduced into the Chlamydomonas reinhardtii cells by electroporation, the Chlamydomonas reinhardtii 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 h.

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 culturing conditions of the algae strains are as follows: a temperature of 20-27° C., a photoperiod of light:dark=16h:8h, and a light intensity of 3000-6000 lux.

12. The tyrosine-sulfated recombinant hirudin according to claim 1, characterized in that: Use in the preparation of drugs for preventing or treating physiological abnormalities associated with thrombin; the physiological abnormalities include but are not limited to venous thrombosis, pulmonary embolism, thrombocytopenia, and myocardial infarction.

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

14. The tyrosine-sulfated recombinant hirudin according to claim 1, characterized in that: Administration may be by two or more routes, including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and intranasal.

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