Reconstructed hirudin fusion protein and application thereof

Abstract

The invention relates to a reconstructed hirudin fusion protein and application thereof, and the reconstructed hirudin fusion protein comprises a first part of thrombus targeting peptide and a second part of hirudin or hirudin derivative mutant, wherein the first part of thrombus targeting peptide is connected with the second part of hirudin or hirudin derivative through a recognition sequence of thrombus site related enzyme. The anticoagulant is different from some traditional anticoagulants such as heparin and hirudin, and has important significance in preventing and treating thrombus and reducing side effects generated in prevention and treatment of thrombus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a reconstructed hirudin fusion protein and its applications. Background Technology

[0002] Hirudin is a low-molecular-weight protein found in the salivary glands of the medicinal leech (Hirudo medicinalis). It typically consists of 65-66 amino acid residues and is a specific inhibitor of thrombin, directly inhibiting thrombin activity without the need for other factors in the blood. Hirudin has a high affinity for thrombin, forming a 1:1 molar complex with it. Therefore, hirudin inhibits the proteolytic activity of thrombin and its binding to endothelial opsonins. Simultaneously, it inhibits the vasoconstrictive function of thrombin during thrombus formation, thus exerting a significant anticoagulant effect.

[0003] The traditional method for obtaining natural hirudin is extraction from medicinal leeches. However, this method yields only 20 μg per leech, resulting in very low production. Furthermore, the limited availability and high cost of natural leeches hinder large-scale production of natural hirudin for clinical application as an anticoagulant. Since scientists cloned the cDNA of hirudin in 1986, research on reconstructed hirudin has made significant progress. This has led to the production of reconstructed hirudin based on various host organisms, such as Escherichia coli, Lactococcus lactis, Bacillus subtilis, yeast, and eukaryotic cells, making large-scale production of reconstructed hirudin possible.

[0004] Currently, various reconstituted hirudins have been approved for marketing in Europe, the United States, Japan, and other countries. Clinically, they are mainly used to treat thrombotic diseases such as heparin-induced type II thrombocytopenia (HAT II) and acute coronary syndrome (ACS). In addition, hirudin analogues and reconstituted hirudin drugs, such as bivalirudin, have been approved for percutaneous coronary intervention in non-high-risk patients, and its application in coronary artery bypass grafting and extracorporeal membrane oxygenation (ECMO) is currently being explored. Desirudin is indicated for reducing deep vein thrombosis and pulmonary embolism after elective hip replacement surgery. Lepirudin can significantly reduce the risk of new thrombosis in HIT patients and reduce the amputation rate due to thrombosis.

[0005] Compared with traditional thrombotic drugs such as heparin, aspirin, and t-PA, hirudin has the following advantages in thrombotic therapy: it has high specificity, directly inhibiting thrombin activity without the need for other factors; hirudin is a small molecule protein with almost no toxicity or antigenicity; and it has stable and long-lasting antithrombotic and anticoagulant effects. Hirudin is the most potent natural thrombin-specific inhibitor discovered to date. However, some problems with hirudin affect its clinical application: ① If real-time monitoring of the dosage is lacking during thrombotic treatment, systemic bleeding may occur, and there are currently no suitable antidotes; ② Hirudin's function in thrombotic therapy is relatively limited, specifically, it can only effectively prevent and treat venous thrombosis, while its ability to prevent and treat arterial thrombosis is limited; ③ Hirudin has a short half-life in the body, and its concentration in the blood drops rapidly within a short period of time. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a reconstructed hirudin fusion protein and its application.

[0007] This invention provides a reconstructed hirudin fusion protein, which comprises a first thrombosis-targeting peptide and a second hirudin or a hirudin derivative mutant, wherein the first thrombosis-targeting peptide and the second hirudin or hirudin derivative are linked by a recognition sequence of a thrombosis site-related enzyme.

[0008] The mutant is based on the amino acid sequence shown in SEQ ID NO.2, with the S at position 32 mutated to R, to construct the RDG sequence.

[0009] SEQ ID NO.2: VVYTDCTESG QNLCLCEGSN VCGQGNKCIL GSDGEKNQCV TGEGTPKPQS ENDGDFEEIP EEYLQ;

[0010] The reconstructed hirudin fusion protein is sequentially linked from the N-terminus to the C-terminus with a thrombus-targeting peptide, a thrombus site-related enzyme recognition sequence, and a hirudin mutant (the hirudin cyclic flexible region is mutated into small peptides such as RGD).

[0011] Preferably, the thrombosis-targeting peptide includes one or more of the following peptides: CREKA, GPRP, RGD, KYGCRGDWPC, GNQEQVSPLTLLKC, LEKNSTY, CQQHHLGGAKQAGDV, GPRPVTSEIHLK, NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR.

[0012] Preferably, the thrombus site-associated enzyme includes one or more of activated coagulation factor VII (FVIIa), coagulation factor IX (FIXa), coagulation factor Xa (FXa), coagulation factor XI (FXIa), coagulation factor XII (FXIIa), coagulation factor XIII (FXIIIa), and thrombin.

[0013] Furthermore, the thrombus site-related enzyme is coagulation factor Xa.

[0014] Preferably, the protein contains an amino acid residue sequence as shown in SEQ ID NO.1.

[0015] The present invention provides a nucleic acid molecule encoding the reconstructed hirudin fusion protein.

[0016] The present invention provides an expression vector containing the encoded nucleic acid, including but not limited to the pET series (such as pET-15b, pET-28a, pET-32a, etc.), pET-SUMO, pET-MBP, pGEX, pMAL, pBAD, pRha, and the pCold series.

[0017] The present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium comprises any of the recombinant host bacteria encoding the reconstructed hirudin fusion protein gene.

[0018] The host bacteria include Escherichia coli.

[0019] This invention provides a method for preparing genetically engineered bacteria, comprising: transferring a plasmid into a host bacterium and culturing it.

[0020] Preferably, the plasmid includes pET32a-fluorescent protein-HV and pET32a-TRXa-nHV, where n=1~20.

[0021] HV is the protein with the amino acid sequence shown in SEQ ID NO.1.

[0022] Furthermore, the fluorescent protein includes, but is not limited to, fluorescent proteins such as mCherry eGFP.

[0023] This invention provides a method for preparing reconstructed hirudin fusion protein, comprising: transferring a plasmid into a host bacterium to induce expression, and then collecting and purifying the plasmid to obtain the reconstructed hirudin fusion protein.

[0024] The plasmids include pET32a-fluorescent protein-HV and pET32a-TRXa-nHV, where n = 1~20.

[0025] Furthermore, the fluorescent protein includes mCherry eGFP.

[0026] The purification process includes affinity chromatography using a nickel column containing Ni-NTA agarose.

[0027] This invention provides a visual fusion protein consisting of a 6× His tag, a fluorescent protein, and an amino acid sequence as shown in SEQ ID NO.1.

[0028] This invention provides a tandem protein, in which proteins are expressed in tandem within the same reading frame. The proteins are linked by a protease recognition sequence. After expression, the protease cleaves the purification tag and the solubilization tag together to obtain multiple proteins containing the amino acid sequence shown in SEQ ID NO. 1.

[0029] This invention provides a method for preparing a recombinant hirudin fusion protein with targeted antithrombin and antiplatelet aggregation properties, comprising the following steps: chemically synthesizing the base sequence of the protein described in claim 1, inserting the coding gene into a vector to prepare a recombinant vector, transforming host cells with the constructed recombinant vector, culturing the host cells, recovering and purifying the bifunctional recombinant hirudin fusion protein, and obtaining the bifunctional recombinant hirudin by enterokinase digestion, wherein the host cell is Escherichia coli.

[0030] This invention provides the application of the reconstructed hirudin fusion protein and the genetically engineered bacteria in the preparation of thrombosis prevention drugs, antithrombin and antiplatelet aggregation drugs, biocompatible materials, health products, and functional foods.

[0031] This invention, while retaining the specific thrombin-inhibiting effect of hirudin, utilizes a thrombus-targeting peptide to block the active N-terminus of hirudin, thereby reducing the bleeding side effects of this hirudin derivative at non-thrombolytic sites. It also enables the reconstituted hirudin to accumulate at the thrombus site. When the hirudin derivative is located at the thrombus site, the enzymes present at the thrombus site specifically cleave the N-terminal blocking peptide of the hirudin derivative, releasing the antithrombin activity of hirudin and exerting a specific anticoagulant effect at the thrombus site. Furthermore, the flexible ring region is mutated into cyclic peptides such as RGD, which are suitable for functioning in ring regions and inhibiting platelet aggregation.

[0032] This invention provides a reconstructed hirudin that targets thrombus sites and allows for controlled blocking and release of bioactive substances, as well as a tandem production mode of the reconstructed hirudin protein in Escherichia coli within the same reading frame and a visualized production mode of the small molecule protein using fluorescent protein as an indicator tag. In particular, it relates to an Escherichia coli genetically engineered strain that produces the reconstructed hirudin fusion protein.

[0033] Beneficial effects

[0034] This invention combines seamless cloning and Golden Gate techniques to construct recombinant plasmids using synthetic biology methods, achieving efficient and stable expression of reconstructed hirudin. Compared to naturally extracted hirudin, the yield is significantly increased, which is expected to provide a novel drug for the treatment of thrombotic diseases and also provides ideas for the development of new therapeutic drugs for thrombotic diseases.

[0035] This invention overcomes the limitations of existing technologies that involve low extraction yield and high cost of hirudin from natural leeches through exogenous expression by microorganisms. By designing the amino acid sequence, hirudin is endowed with a targeting effect, and its activity can be effectively blocked before reaching the target site, reducing the side effects of hirudin. This invention also solves the problem that it is difficult to ensure the correctness of the higher-order structure of hirudin through chemical synthesis. Attached Figure Description

[0036] Figure 1 The structure of the fusion protein constructed in all instances was predicted via alphafold;

[0037] Figure 2 SDS-PAGE image of the fusion protein purification results;

[0038] Figure 3 The results are from the thrombin titration activity verification method;

[0039] Figure 4 This is the result of a whole blood coagulation test;

[0040] Figure 5 The experimental results were used to explore the blood calcium resuscitation time.

[0041] Figure 6 This is a schematic diagram of the protein structure described in Example 2;

[0042] Figure 7 This is a schematic diagram of the protein structure described in Example 3. Detailed Implementation

[0043] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0044] Example 1

[0045] Design and gene synthesis of reconstructed hirudin fusion protein:

[0046] Based on in-depth research on the structure-activity relationship and thrombosis mechanism of natural hirudin, while retaining the specific inhibitory effect of natural hirudin on thrombin activity, a high-affinity sequence (GPRP) with fibrinogen D domain and D-dimer was added to the N-terminus of hirudin. This blocked the active N-terminus of the hirudin-like substance before cleavage, preventing it from exerting its effect in non-thrombosis sites. At the same time, the reconstructed hirudin was enriched at the thrombosis site. An activated coagulation factor X (FXa) recognition sequence was inserted after the GPRP. After being specifically recognized and cleaved, it can release the active N-terminus of hirudin to exert a specific inhibitory effect on thrombin. Using the amino acid sequence shown as VVYTDCTESG QNLCLCEGSN VCGQGNKCIL GSDGEKNQCV TGEGTPKPQS ENDGDFEEIPEE YLQ, the S at position 32 was mutated to R to construct the RDG sequence; this enhances its antiplatelet aggregation activity, thus constructing a reconstructed hirudin molecule composed of 73 amino acids, as shown in SEQ ID NO.1:

[0047] Gly -Pro -Arg -Pro -Ile -Glu -Gly -Arg -Val -Val -Tyr -Thr -Asp -Cys-Thr -Glu -Ser -Gly -Gln -Asn -Leu -Cys -Leu -Cys -Glu -Gly -Ser -Asn -Val -Cys -Gly -Gln -Gly -Asn -Lys -Cys -Ile -Leu -Gly -Arg -Gly -Asp -Glu -Lys -Asn -Gln -Cys -Val -Thr -Gly -Glu -Gly -Thr -Pro -Glu -Pro -Gln -Ser -His -Asn -Asp -Gly -Asp -Phe -Glu -Glu -Ile -Pro -Glu -Glu -Tyr -Leu -Gln.

[0048] Example 2

[0049] To facilitate the isolation, purification, and identification of the bifunctional reconstructed hirudin, a 6×His tag and a red fluorescent protein mCherry were sequentially inserted at its N-terminus to achieve nickel column affinity chromatography purification and visual monitoring of bacterial growth / protein expression. Subsequently, an enterokinase (EK) cleavage site DDDDK was added to facilitate the removal of the His-mCherry tag after purification, thereby obtaining the target protein as described in claim 1. The structure designed and constructed based on the above reconstructed hirudin molecule is shown in Figure 6.

[0050] (1) PCR amplification of the target gene

[0051] The synthetic plasmid pET32a-HV was obtained by plasmid extraction, and the mCherry-containing plasmid in the laboratory was used as a template for subsequent PCR.

[0052]

[0053] Primers were designed based on the pET32a-HV sequence and the mCherry plasmid sequence. While amplifying the mCherry gene, homologous arms were inserted at both ends of the insertion site on the pET32a plasmid to obtain the vector fragment and insert fragment required for constructing the recombinant plasmid. These were then purified and recovered, and the recombinant plasmid pET32a-mCherry-HV was subsequently obtained using a seamless cloning kit. After treatment with the recommended conditions of the homologous recombination kit, the recombination reaction system was immediately used to transform competent cells. To obtain higher cloning efficiency and subsequent expression strains, the recombinant product was transformed into E. coli DH5α. After heat shock transformation, the cells were evenly spread on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12 h to obtain positive clones carrying the recombinant plasmid.

[0054] Example 3

[0055] To facilitate the exploration of the expression of small molecule proteins such as hirudin in biological systems, a tandem expression system for reconstructed hirudin was designed, the structure of which is shown in Figure 7 (n=1 to 20 in the figure).

[0056] (1) Construction of Pet32a-TRXa-HV

[0057] Primers were designed based on the pET32a-mCherry-HV plasmid sequence, and a seamless cloning kit was used to construct the primers.

[0058]

[0059] (2) Construction of Pet32a-TRXa-2HV

[0060] Primers were designed based on the pET32a-TRXa-HV plasmid sequence.

[0061]

[0062] Based on the ET32a-TRXa-HV plasmid, PCR was performed using the primers described above. The PCR-obtained fragment was digested with BSA1 enzyme according to the manufacturer's instructions for 30 min. Ligation was then performed using T4 ligase at 16℃.

[0063] (3) Construction of Pet32a-TRXa-4HV

[0064] Primers were designed based on the pET32a-TRXa-HV plasmid sequence.

[0065]

[0066] Based on the ET32a-TRXa-HV plasmid, PCR was performed using the primers described above. After recovering the fragment from the gel, a total volume of 20 μL was obtained and PCR was performed using a PCR instrument at ① 37℃-2 min, ② 16℃-5 min for 20 cycles. The amount of fragment added was calculated according to the following formula, and the remaining volume was made up with ddH2O.

[0067]

[0068]

[0069] m i : The quality (ng) of the i-th inserted segment;

[0070] m v : The mass of the carrier (ng);

[0071] L i L v : Lengths (bp) of the inserted fragment and the vector, respectively.

[0072] (4) Construction of pET-32a-TRXa-6HV

[0073] Primers were designed based on the pET32a-TRXa-HV plasmid sequence.

[0074]

[0075] Using pET-32a-TRXa-HV plasmid as a template, PCR amplification was performed using the primers described above. After recovering the target fragment, a total reaction volume of 20 μL was prepared and the recombination reaction was carried out on a PCR instrument under the following conditions: ① 37 ℃ for 2 min, ② 16 ℃ for 5 min, for a total of 20 cycles. The amount of insert fragment added was determined according to the calculation formula given in (3), and the remaining volume was made up with ddH2O.

[0076] (5) Construction of Pet32a-TRXa-8HV

[0077] Using pET-32a-TRXa-HV plasmid as a template, PCR was performed using the primers mentioned above, with the remaining primers being ZT3.0f, ZT3.0r, PD3.1f, PD3.1r, PD3.2f, PD3.2r, PD3.3f, PD3.4r, PD3.5f, and PD3.5r from (4). After recovering the fragment from the gel, 20 μL of fragment was collected and PCR was performed using a PCR instrument at ① 37℃ for 2 min and ② 16℃ for 5 min for 20 cycles. The amount of fragment added was calculated according to the formula mentioned in (3), and the remaining volume was made up with ddH2O.

[0078]

[0079] (6) Plasmid transformation

[0080] After the above treatment, the recombinant reaction system was immediately used to transform competent cells. To obtain higher cloning efficiency and subsequent expression strains, the recombinant products were transformed into E. coli DH5α. After heat shock transformation, the cells were evenly spread on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12 h to obtain positive clones carrying the recombinant plasmid.

[0081] Example 4

[0082] (1) Homologous recombination and transformation of the reconstructed hirudin fusion protein gene

[0083] After extracting the plasmids constructed in steps 2 and 3 and confirming the sequencing results were correct, they were transformed into E. coli BL21(DE3) competent cells. The cells were then evenly spread on LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37°C for 12 h to obtain positive clones carrying the recombinant plasmid.

[0084] (2) Induced expression of reconstructed hirudin fusion protein in recombinant Escherichia coli

[0085] The constructed E. coli BL21(DE3) containing the expression plasmid was picked and inoculated into LB liquid medium. It was cultured at 37 ℃ and 180 rpm for about 10 h as seed culture. Then, it was inoculated into fresh LB at a 1% inoculum and cultured under the same conditions for 3–3.5 h. When the OD was about 0.4, IPTG (final concentration 0.3 mM) was added and expression was induced for 16 h at 25 ℃ and 180 rpm.

[0086] (3) Collect and purify the reconstructed hirudin fusion protein

[0087] Since the fusion protein is expressed within the bacteria, the cells were first collected by centrifugation, resuspended in lysis buffer, and then disrupted using an ultrasonic homogenizer. Due to the protein's thermostability, it was heated at 75°C for 15 min, followed by centrifugation at 12000 rpm for 30 min. The design incorporates a 6×His tag at the N-terminus; therefore, the supernatant was collected and purified by Ni-NTAagarose nickel affinity chromatography.

[0088] The lysis buffer used in affinity chromatography consisted of 20 mL of 1M Tris, 17.54 g of NaCl, 800 mL of deionized water, and pH 8.0.

[0089] Wash buffer: 20 mL 1M Tris, 17.54 g NaCl, 0.136 g imidazole, 800 mL deionized water, pH 8.0.

[0090] Elution buffer: 20 mL 1M Tris, 17.54 g NaCl, 3.4 g imidazole, 800 mL deionized water, pH 8.0.

[0091] The results are as follows Figure 2 As shown, the actual molecular weight is consistent with the theoretical molecular weight.

[0092] Example 5

[0093] Assay of reconstituted hirudin antithrombin activity

[0094] (1) Enzymatic cleavage removes the tag of the fusion protein and the GPRP that plays a blocking role.

[0095] Prepare the replacement buffer by mixing the eluent obtained from nickel column affinity chromatography and transferring it to a 15 mL 3 kDa MiliPore ultrafiltration centrifuge tube. Centrifuge at 4000 rpm for 30 min at 4°C. Add replacement buffer to the concentrate to a volume of 10 mL. Repeat centrifugation and replenish the volume with replacement buffer to achieve a total concentration of over 1000 times, thus removing imidazole and sodium chloride from the reconstituted hirudin fusion protein solution for subsequent enzymatic digestion.

[0096] The purified reconstituted hirudin fusion protein was digested with enterokinase to remove the His tag and mCherry, thus obtaining reconstituted hirudin. Enterokinase was diluted with 25 mM Tris-HCl (pH 8.0) to a concentration of approximately 1.1335 U / mL. The amount of enzyme needed was determined by estimating that 1 U of enterokinase could digest 0.5 mg of fusion protein. The diluted enterokinase was added to the reconstituted hirudin fusion protein solution obtained after solution replacement, and the solution was incubated at 25°C for digestion. Samples were collected after 16 hours of digestion for FXa digestion.

[0097] The reconstituted hirudin obtained after enterokinase digestion was digested with FXa to remove the blocking effect of GPRP peptide on the N-terminus of the reconstituted hirudin activity. The amount of enzyme used was determined by estimating that 1 μg of FXa could digest 50 μg of fusion protein. The protein solution was prepared by digestion with 5 μL of 10× buffer and 40 μL of EK, and 50 μL of FXa and ultrapure water were prepared as a FXa digestion reaction system. The system was incubated in a water bath at 23℃ for 6 h for subsequent determination of the reconstituted hirudin activity.

[0098] The 10× buffer (100 mL) consists of 20 mL of 1M Tris-HCl, 5.84 g of NaCl, and 0.22 g of CaCl2, diluted to 100 mL with ultrapure water and stored at room temperature.

[0099] (2) Thrombin titration method for determining the activity of reconstituted hirudin

[0100] Accurately measure 50 μL of the sample to be tested and place it in a 1.5 mL EP tube. Add 100 μL of tris(hydroxymethyl)aminohydrochloride buffer containing 0.5% fibrinogen and mix well. Incubate in a water bath at 37°C for 5 min. Add thrombin solution dropwise. First, prepare thrombin solutions of 1000 U / mL, 500 U / mL, 200 U / mL, 100 U / mL, and 40 U / mL. For the first titration, use the 1000 U / mL solution. Add 2 μL each time and incubate at 37°C for 1 min, observing for coagulation or the appearance of clumps. Record the thrombin activity consumed in this titration. For the second titration, near the first coagulation point, use the 500 U / mL thrombin solution. Continue this process, recording the volume of thrombin solution consumed in the final titration. Calculate the antithrombin activity of reconstituted hirudin using the following formula: [Formula omitted for brevity]. U represents the antithrombin activity units per 1 g of sample, U / g; C1 represents the thrombin solution concentration, U / mL; C2 represents the concentration of the sample to be tested, g / mL; V1 represents the volume of thrombin solution added, μL; V2 represents the volume of the sample to be tested added, μL.

[0101] The thrombin solution (40 U / mL) is prepared by dissolving 1000 U of thrombin reagent in 1 mL of physiological saline, mixing thoroughly, aliquoting, and storing at -20℃ for later use. It should be diluted with physiological saline before use.

[0102] Tris-HCl buffer (5 mL) containing 0.5% fibrinogen (based on coagulation): Dissolve 25 mg of fibrinogen in 5 mL of Tris-HCl (pH 7.4), mix well, aliquot into 1 mL tubes, and store at -20°C for later use.

[0103] The results are as follows Figure 3 As shown, the N-terminal targeting peptide effectively blocks the activity of the small peptide, and the peptide produced using the tandem method has better activity.

[0104] (3) Whole blood coagulation test

[0105] Take 5 mL of rabbit whole blood into a 50 mL centrifuge tube, add 500 μL of 0.025 M CaCl2 solution (10% of the whole blood volume), and immediately mix gently. During the sample addition process, slowly add the CaCl2 solution along the wall of the centrifuge tube while gently rotating the tube to ensure thorough mixing.

[0106] Then, add blood samples in volumes of 100 μL per well. The order of blood addition should be in ascending order of incubation time to ensure consistency of sample processing at each time point. After adding the samples, immediately start the timer and incubate the samples at a constant temperature of 37°C.

[0107] At incubation times of 5, 15, 25, 35, 45, 55, 60, 65, 70, and 75 min, 2 mL of ultrapure water was slowly added to the well wall and gently mixed by pipetting. After incubation for another 5 min, 200 μL of the supernatant was aliquoted into three replicates of a 96-well plate, and absorbance (A) was measured. 540 Using the saline group as a control, the experimental group had 100 μL of 0.1 mg / mL protein solution added per 1 mL of blood.

[0108] The results are as follows Figure 4 The experimental group shown maintained its anticoagulation activity throughout the period from the start of coagulation to complete coagulation, as seen in the control group.

[0109] (4) Blood calcium recovery time

[0110] (1) Take 500 μL of plasma sample (PPP, platelet-poor plasma) and incubate it at 37 ℃ for 1 h to ensure that the system is in physiological temperature equilibrium.

[0111] (2) After incubation, the plasma samples were divided into the following three groups:

[0112] ① Sample group (test group):

[0113] Add 100 μL of the incubated PPP to a 96-well plate, and then add 100 μL of 0.01 M CaCl2 solution and 100 μL of 0.1 mg / mL protein solution to each well.

[0114] ② Negative control group: 100 μL of incubated PPP was added to a 96-well plate, and 100 μL of physiological saline was added to each well.

[0115] ③ Positive control group: Add 100 μL of incubated PPP to a 96-well plate, and then add 100 μL of 0.01 M CaCl2 solution to each well.

[0116] When adding samples, perform two replicates for every 500 μL PPP. First, add the physiological saline required for the negative control, then quickly add 0.01 M CaCl2 solution using a multichannel pipette. To ensure the parallelism of the experiment, all parallel samples should be added along the same vertical line.

[0117] (3) Detection conditions

[0118] After sample addition, the 96-well plate was placed in a microplate reader for kinetic analysis at a wavelength of 405 nm. The assay program was set to measure absorbance (A) every 30 seconds. 405 ), continuously monitor for 45 minutes. Before detection, the kinetic cycle program must be set, and then the absorbance parameters must be set.

[0119] A was analyzed in the sample group, negative control group, and positive control group. 405 Comparison of kinetic curves reveals a significant inhibitory effect of this peptide on plasma coagulation activity, as shown in the following results. Figure 5 As shown.

Claims

1. A reconstructed hirudin fusion protein, characterized in that, The reconstructed hirudin fusion protein includes a first part thrombosis-targeting peptide and a second part hirudin or a hirudin derivative mutant, wherein the first part thrombosis-targeting peptide and the second part hirudin or hirudin derivative are linked by a recognition sequence of a thrombosis site-related enzyme.

2. The reconstructed hirudin fusion protein according to claim 1, characterized in that, The mutant is based on the amino acid sequence shown in SEQ ID NO.2, with the S at position 32 mutated to R to construct the RDG sequence; The thrombosis-targeting peptides include one or more of the following: CREKA, GPRP, RGD, KYGCRGDWPC, GNQEQVSPLTLLKC, LEKNSTY, CQQHHLGGAKQAGDV, GPRPVTSEIHLK, NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR peptides. The thrombus site-related enzymes include one or more of activated coagulation factors VII, IX, Xa, XI, XII, XIII, and thrombin.

3. The reconstructed hirudin fusion protein according to claim 1, characterized in that, The protein contains the amino acid residue sequence shown in SEQ ID NO.

1.

4. A nucleic acid molecule encoding the reconstructed hirudin fusion protein of claims 1-3.

5. An expression vector containing the nucleic acid encoding as described in claim 4.

6. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria include the recombinant host bacteria encoding the reconstructed hirudin fusion protein gene as described in any one of claims 1-3.

7. A method for preparing a genetically engineered bacterium, comprising: The plasmid was obtained by transferring it into a host bacterium and culturing it.

8. The preparation method according to claim 7, characterized in that, The plasmid includes at least one of pET32a-fluorescent protein-HV and pET32a-TRXa-nHV, wherein n=1~20.

9. A method for preparing a reconstructed hirudin fusion protein, comprising: The plasmid was transferred into the host bacteria to induce expression, and then collected and purified to obtain the reconstructed hirudin fusion protein.

10. The use of the reconstructed hirudin fusion protein of claim 1 and the genetically engineered bacteria of claim 7 in the preparation of thrombosis prevention drugs, the preparation of antithrombin and antiplatelet aggregation drugs, biocompatible materials, health products, and functional foods.

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