Recombinant hirudin fusion protein, coding gene, prokaryotic expression method of recombinant hirudin fusion protein and application of recombinant hirudin fusion protein

Abstract

The invention relates to a recombinant hirudin fusion protein, a coding gene and a prokaryotic expression method and application of the recombinant hirudin fusion protein. The recombinant hirudin fusion protein comprises an HMg variant amino acid sequence and a fusion tag. The recombinant hirudin fusion protein has the advantages that the hirudin variant in poecilobdella manillensis is adopted to construct an efficient prokaryotic expression system, the hirudin yield and activity are improved under the condition that post-translational modification is not needed, the anticoagulant efficiency and the production process of the recombinant hirudin fusion protein provide an important basis for development of a new generation of antithrombotic drugs, and the recombinant hirudin fusion protein has wide application prospects. The progress of cardiovascular disease treatment and hirudin production is expected to be promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a recombinant hirudin fusion protein, a coding gene and a prokaryotic expression method and application thereof. BACKGROUND

[0002] Thrombosis can occur in the arterial or venous circulation system, which can aggravate the development of ischemic heart disease, ischemic stroke and venous thromboembolism, etc. Thrombin amplifies the coagulation signal through the endogenous and exogenous coagulation pathways, is the core enzyme of thrombosis, and is also the main target of anticoagulant drugs. Traditional anticoagulant drugs such as heparin are widely used, but have limitations such as high risk of bleeding, drug resistance, and the need for frequent monitoring. In contrast, hirudin can specifically bind to the active site and substrate binding site of thrombin to form a 1:1 irreversible complex, thereby blocking its catalytic function and reducing thrombosis, and has the advantages of strong anticoagulant effect, clear target of action, and small side effects of bleeding, etc. and is considered as an ideal candidate for a new generation of anticoagulant drugs.

[0003] Hirudin is a 7000 Da polypeptide composed of 64-66 amino acids, derived from the salivary glands of the genus Hirudo. Haycraft first described hirudin in 1884. Hirudin can be divided into natural hirudin and its derivatives. Natural hirudin is mainly isolated and purified from the salivary glands of leeches, and its derivatives are obtained by genetic engineering modification of amino acid sequences, which retain the core sequence and have considerable physiological activity. So far, more than ten different hirudin variants (HVs) have been found in leeches. These peptides have high stability and activity. As an effective natural thrombin inhibitor, hirudin shows a series of beneficial properties, including anticoagulant and antithrombotic, anti-fibrosis, anti-atherosclerosis, anti-angiogenesis and anti-tumor, and anti-fibrosis. Many clinical trials have shown the efficacy of hirudin in preventing and treating several complex diseases including cardiovascular and cerebrovascular diseases.

[0004] Natural hirudin has been widely concerned due to its anticoagulant and pharmacological properties. Although the leech contains the highest concentration of natural hirudin, the resource is limited, and the traditional extraction method can greatly reduce the yield of hirudin in the extraction and purification process, while the advanced method is high in cost and complex in operation. A variety of recombinant hirudin has been developed and approved by the US Food and Drug Administration for anticoagulant therapy, such as lepirudin, bivalirudin and desirudin. The development of genetic engineering technology provides a new way for the acquisition and optimization of hirudin. By designing or directed evolution of amino acid sequence, the expression efficiency, thermal stability and anticoagulant activity can be improved. However, existing studies have shown that the Tyr63 sulfation of natural hirudin is the core structural feature of the stable salt bridge formed by the electrostatic interaction of Exosite I basic residues (such as Lys81) of thrombin, however, this post-translational modification cannot be realized in prokaryotic expression systems such as Escherichia coli, which seriously restricts the activity expression of recombinant hirudin. In view of this technical bottleneck, Yan Sun team found through molecular dynamics simulation that without relying on Tyr63 sulfation, some acidic amino acid mutations of hirudin can achieve breakthrough optimization of inhibitory activity and binding energy.

[0005] Studies have shown that the hirudin derived from the Asian species Hirudinaria manillensis has better antithrombotic properties than the commonly used leech species due to its unique molecular structure, and has broad research prospects. SUMMARY

[0006] Thrombotic diseases seriously threaten human health, and traditional anticoagulants have defects such as high risk of bleeding, frequent monitoring, etc. Hirudin, as a direct thrombin inhibitor, has the advantages of high efficiency, strong targeting, small side effects, etc., but natural extraction has problems such as limited resources and high cost, and genetic engineering technology provides a new way for the production of hirudin. Prokaryotic expression is low in cost, high in yield and convenient to operate, but it lacks post-translational modification, resulting in low activity of hirudin.

[0007] In order to overcome the problems existing in the prior art, the present application provides the following technical scheme:

[0008] The first aspect of the present application provides a recombinant hirudin fusion protein, which comprises an HMg variant amino acid sequence and a fusion tag, and the HMg variant amino acid sequence is any one of the following (a1)-(a3):

[0009] (a1) the amino acid sequence shown in SEQ ID NO: 1;

[0010] (a2) an amino acid sequence that has more than 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and has the same or similar biological activity;

[0011] (a3) an amino acid sequence represented by (a1) or (a2) having one or more amino acid residues substituted and / or deleted and / or added, and having the same or similar biological activity;

[0012] The fusion tag comprises a sequence of a His tag or a Trx tag.

[0013] In the present invention, "recombinant hirudin fusion protein" may also be expressed as "recombinant hirudin protein", "recombinant hirudin", "rHMg", "recombinant hirudin rHMg", "recombinant hirudin protein rHMg" or "recombinant hirudin fusion protein rHMg".

[0014] In the present invention, SEQ ID No. 1 is as follows:

[0015] VSYTGCTESGQNYCLCVGSDICGDGKHCEMDGSENKCVDGEGTPKRQTS GPSDFEEFSLDDIEQK.

[0016] In some preferred embodiments, the recombinant hirudin fusion protein comprises an HMg variant amino acid sequence and a fusion tag, wherein the HMg variant amino acid sequence is the amino acid sequence shown in SEQ ID NO: 1; and the fusion tag comprises 6-10 histidines.

[0017] The second aspect of the present invention provides a nucleic acid molecule encoding a recombinant hirudin fusion protein, as shown in any one of Formula 1, Formula 2, Formula 3, and Formula 4, wherein:

[0018] Restriction site 1-first fusion tag-nucleotide encoding HMg variant-restriction site 2

[0019] (Formula 1)

[0020] Restriction site 1'-nucleotide encoding HMg variant-second fusion tag-restriction site 2'

[0021] (Formula 2)

[0022] Restriction site 1-nucleotide encoding HMg variant-restriction site 2-third fusion tag

[0023] (Formula 3)

[0024] Fourth fusion tag-enzyme cleavage site 1'-nucleotide encoding HMg variant-enzyme cleavage site 2'

[0025] (Formula 4)

[0026] Wherein, Formula 1, Formula 2, Formula 3 or Formula 4 is suitable for expression in a prokaryotic system;

[0027] The first fusion tag, the second fusion tag, the third fusion tag, and the fourth fusion tag are sequences comprising a His tag or a Trx tag respectively;

[0028] The nucleotide encoding the HMg variant is any one of the following (b1)-(b3):

[0029] (b1) the nucleotide sequence shown in SEQ ID NO: 2;

[0030] (b2) a nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 2 and has the same or similar biological activity;

[0031] (b3) A nucleotide sequence composed of the nucleotide sequence shown in (b1) or (b2) by substitution and / or deletion and / or addition of one or several nucleotides, and having the same or similar biological activity; the restriction enzyme cleavage site 1 is Nde I or BamHI; the restriction enzyme cleavage site 1' is Nde I or BamH I; the restriction enzyme cleavage site 2 is XhoⅠ or EcoR I; the restriction enzyme cleavage site 2' is XhoⅠ or EcoR I.

[0032] It should be noted that Formula 1, Formula 2, Formula 3 or Formula 4 is preferred, but does not limit the structure of the fusion protein.

[0033] In some preferred embodiments, the fusion tag is a His tag, which comprises an amino acid sequence of 6-10 histidines.

[0034] In the present invention, SEQ ID NO: 2 is as follows:

[0035] GTTTCTTACACTGGTTGTACTGAATCTGGTCAAAACTACTGTTTGTGTGTTGGTTCTGATATTTGTGGTGACGGTAAACATTGTGAAATGGATGGTTCTGAAAATAAGTGTGTTGATGGTGAAGGTACTCCAAAGAGACAAACTTCTGGTCCATCTGATTTCGAAGAATTTTCTTTGGATGATATTGAACAAAAG.

[0036] In some preferred embodiments, the enzyme cleavage site 1 is Nde I (SEQ ID NO: 3: CATATG) or BamH I (SEQ ID NO: 4: GGATCC). In some more preferred embodiments, the enzyme cleavage site 1 is Nde I (SEQ ID NO: 4: CATAG).

[0037] In some preferred embodiments, the restriction enzyme cleavage site 1' is Nde I (SEQ ID NO: 3: CATATG) or BamH I (SEQ ID NO: 4: GGATCC). In some more preferred embodiments, the restriction enzyme cleavage site 1' is Nde I (SEQ ID NO: 4: CATAG).

[0038] In some preferred embodiments, the restriction enzyme cleavage site 2 is XhoI (SEQ ID NO: 5: CTCGAG) or EcoR I (SEQ ID NO: 6: GAATTC). In some more preferred embodiments, the restriction enzyme cleavage site 2 is XhoI (SEQ ID NO: 5: CTCGAG).

[0039] In some preferred embodiments, the restriction enzyme cleavage site 2' is Xho I (SEQ ID NO: 5: CTCGAG) or EcoR I (SEQ ID NO: 6: GAATTC). In some more preferred embodiments, the restriction enzyme cleavage site 2' is Xho I (SEQ ID NO: 5: CTCGAG).

[0040] In some preferred embodiments, the nucleic acid molecule encoding the recombinant hirudin fusion protein is represented by Formula 3, and the nucleotide encoding the HMg variant is the amino acid sequence shown in SEQ ID NO: 2.

[0041] The third aspect of the present invention provides an expression vector or expression cassette comprising the above-mentioned nucleic acid molecule encoding the recombinant hirudin fusion protein.

[0042] The fourth aspect of the present invention provides a host cell comprising the above-mentioned expression vector.

[0043] In the present invention, the host cell includes a recombinant microorganism (such as a recombinant bacterial strain) or a genetically engineered cell line.

[0044] A fifth aspect of the present invention provides a prokaryotic expression method for a recombinant hirudin fusion protein, comprising the following steps:

[0045] 1) The constructed pET-HMg recombinant plasmid vector was transformed into the Escherichia coli expression strain BL21 or RosettapaLysS using the heat shock method;

[0046] 2) picking a single clone of the E. coli expression strain and culturing it in a first liquid culture medium at 35-38° C. with shaking overnight for 12-16 hours;

[0047] 3) Transfer the overnight cultured bacteria into the second liquid culture medium at a volume ratio of 1:80-120 and culture at 35-38°C with shaking until the OD 600 =0.8-1.2; then add IPTG dropwise or slowly to a final concentration of 0.5-1.5 mM, culture at a temperature of 16-18°C with shaking, and induce expression at 160-200 rpm for 16-24 hours;

[0048] 4) centrifuging and collecting the E. coli pellet; suspending the cells in PBS or Tris-HCl buffer, and ultrasonically disrupting until the solution is clear, wherein the ultrasonic disruption conditions include a cycle of 2-5 seconds of disruption and 7 seconds of rest, a total ultrasonic disruption time of 90-180 minutes, and an ultrasonic power of 80%-90%;

[0049] 5) centrifuging the solution obtained after the ultrasonic disruption, collecting the supernatant, and then purifying the supernatant using nickel column affinity chromatography; gradient elution is performed using a buffer containing 15-25 mM, 150-300 mM, and 500-600 mM imidazole, and the target recombinant hirudin fusion protein is present in the 150-300 mM imidazole buffer.

[0050] In some preferred embodiments, the E. coli expression strain in step 1) is BL21(DE3).

[0051] In some preferred embodiments, step 2) comprises picking a single clone of the E. coli expression strain and culturing it in liquid culture medium overnight at 37° C. with shaking for 12-16 hours.

[0052] In some preferred embodiments, the liquid culture medium in the prokaryotic expression method of the recombinant hirudin fusion protein is LB liquid culture medium.

[0053] In some preferred embodiments, the E. coli expression strain in step 2) is BL21 (DE3).

[0054] In some preferred embodiments, step 3) includes inducing expression at a final IPTG concentration of 1 mM, a temperature of 18° C., and a speed of 170 rpm for 24 h.

[0055] In some preferred embodiments, the centrifugation condition in step 4) is centrifugation at 3000-5000 rpm for 20-40 min. In some more preferred embodiments, the centrifugation condition in step 4) is centrifugation at 4000 rpm for 30 min.

[0056] In some preferred embodiments, the Tris-HCl in step 4) contains 20 mM Tris, 200 mM NaCl, and pH=8.0.

[0057] In the present invention, the interval in step 4) can be understood as an intermediate stop or pause. The time of ultrasonic fragmentation includes the time of the interval.

[0058] In some preferred embodiments, the centrifugation condition in step 5) is centrifugation at 10,000-15,000 rpm for 5-15 min. In some more preferred embodiments, the centrifugation condition in step 5) is centrifugation at 12,000 rpm for 10 min.

[0059] In some preferred embodiments, step 5) comprises performing gradient elution using a buffer solution containing 20 mM, 200 mM and 500 mM imidazole, and the target recombinant hirudin fusion protein is present in a buffer solution containing 200 mM imidazole.

[0060] In some preferred embodiments, a prokaryotic expression method of a recombinant hirudin fusion protein comprises the following steps:

[0061] 1) The constructed pET-HMg recombinant plasmid vector was transformed into the Escherichia coli expression strain BL21 (DE3) using the heat shock method;

[0062] 2) picking a single clone of the E. coli expression strain and placing it in a first LB liquid medium, and culturing it overnight at 37°C with shaking for 12-16 hours;

[0063] 3) The bacterial solution obtained from the overnight culture was transferred into a second LB liquid medium at a volume ratio of 1:100 and cultured at 37°C with shaking until the OD 600 =1.0; then IPTG was added dropwise or slowly to a final concentration of 1 mM, cultured at 18°C ​​with shaking, and induced for 24 h at 170 rpm;

[0064] 4) centrifuging at 4000 rpm for 30 minutes and collecting the E. coli pellet; suspending the cells in a Tris-HCl or PBS buffer and sonicating until the solution is clear; the sonication conditions include a 3-second break cycle followed by a 7-second pause, for a total of 120 minutes, and an ultrasonic power of 80%-90%; the Tris-HCl buffer contains 20 mM Tris, 200 mM NaCl, and has a pH of 8.0;

[0065] 5) The solution obtained after the ultrasonic disruption was centrifuged at 12000 rpm for 10 minutes, the supernatant was collected, and then the supernatant was purified by nickel column affinity chromatography; gradient elution was performed using a buffer containing 20 mM, 200 mM, and 500 mM imidazole, and the target recombinant hirudin fusion protein was present in the 200 mM imidazole buffer.

[0066] The sixth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned recombinant hirudin fusion protein and pharmaceutical excipients.

[0067] In the present invention, a "pharmaceutical composition" refers to a formulation of a compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to animals (including humans and livestock). The medium includes pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0068] In the present invention, "pharmaceutical excipients" include but are not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that is approved by the relevant government regulatory authorities for use in drugs.

[0069] As used herein, the term "pharmaceutically acceptable" refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the invention and is relatively non-toxic, i.e., the substance can be administered to a subject without causing an adverse biological response or interacting in an adverse manner with any of the components included in the composition.

[0070] The anticoagulant activity of the recombinant hirudin fusion protein of the present invention is concentration-dependent. The effective concentration range obtained by measuring the thrombin inhibition rate using a chromogenic substrate is 2.8-76 nM (the inhibition rate can reach 50%-90%).

[0071] The seventh aspect of the present invention provides the use of the above-mentioned recombinant hirudin fusion protein and the above-mentioned pharmaceutical composition in the preparation of drugs for anticoagulation, treatment of thrombosis or prevention of thrombosis.

[0072] In some embodiments, the effects of the recombinant hirudin fusion protein include directly inhibiting thrombin activity, indirectly affecting the intrinsic and extrinsic coagulation pathways, significantly prolonging the whole blood coagulation time, reducing the whole blood coagulation rate, and affecting platelet aggregation (PF).

[0073] The beneficial effects of the present invention include at least:

[0074] This study utilizes a hirudin variant from the leech Hirudo philadelphica to construct an efficient prokaryotic expression system, increasing hirudin yield and activity without the need for post-translational modification. Combining bioinformatics prediction with multimodal activity assays, the study comprehensively analyzes the structural properties and anticoagulant efficacy of rHMg, comparing it with the clinical drug bivalirudin, providing a basis for the development of novel anticoagulant drugs.

[0075] This paper focuses on the hirudin variant HMg from leech Hirudo, and explores its preparation, anticoagulant mechanism and clinical application potential through bioinformatics analysis, genetic engineering optimization and multimodal activity evaluation. The prokaryotic expression method of the recombinant hirudin fusion protein of the present invention is based on the pET-HMg (BL21) prokaryotic expression system to achieve soluble and efficient expression of the recombinant hirudin fusion protein (i.e., rHMg). However, when induced expression conditions outside the scope of protection of the present invention (such as 35-38° C. and excessively high IPTG concentration) are used, a large number of inclusion bodies will be generated, seriously affecting subsequent purification and activity. Through multi-index detection, the antithrombin activity of rHMg is 9573 ATU / mg, and it can significantly prolong the whole blood clotting time in a dose-dependent manner, and significantly prolong the APTT, PT, and TT times. The chromogenic substrate method confirms that the thrombin inhibitory activity of rHMg is more than 135 times that of bivalirudin, and the binding affinity of rHMg is more than 542 times that of bivalirudin. Combined with bioinformatics analysis, the anticoagulant mechanism of rHMg is further revealed. The prokaryotic expression system successfully established in the present invention can efficiently produce the recombinant hirudin fusion protein rHMg without the need for post-translational modification. The activity significantly exceeds that of most reported recombinant products. The excellent anticoagulant efficacy and production process of rHMg provide an important basis for the development of a new generation of antithrombotic drugs, and are expected to promote the treatment of cardiovascular diseases and the progress of hirudin production.

[0076] The features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 Shown is a sequence alignment diagram of the HMg variants of the embodiments of the present invention.

[0078] Figure 2 A phylogenetic tree showing HMg variants according to embodiments of the present invention is shown.

[0079] Figure 3 The electrophoresis diagram shows the recombinant hirudin fusion protein rHMg according to an embodiment of the present invention.

[0080] Figure 4 The figure shows the results of denaturing mass spectrometry molecular weight determination of the recombinant hirudin fusion protein according to the embodiment of the present invention.

[0081] Figure 5 The physical map of the recombinant plasmid vector constructed in the examples of the present invention is shown.

[0082] Figure 6 Show the effect of rHMg on APTT, PT, and TT.

[0083] Figure 7 Shows the binding and inhibitory effects of bivalirudin on thrombin.

[0084] Figure 8 This shows the binding and inhibitory effects of the recombinant hirudin fusion protein of the embodiment of the present invention on thrombin.

[0085] Figure 9 The IC values ​​of the recombinant hirudin fusion protein and bivalirudin on thrombin activity were determined by the chromogenic substrate method. 50 . DETAILED DESCRIPTION

[0086] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, intended to be used for explaining the present invention, and do not constitute a limitation of the present invention.

[0087] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article. In the description of this application, unless otherwise stated, the meaning of similar terms such as "multiple / multiple" is two or more.

[0088] As used herein, when a specific numerical value is mentioned, it is intended that the numerical value may vary within a range of ±5%.As used herein, the term "comprising" or "including" may be open, semi-closed, or closed.

[0089] In the description of the present invention, it should be noted that, if specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used, if the manufacturer is not specified, are all commonly used materials and reagents that can be purchased commercially.

[0090] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Please note that these are intended to explain rather than limit the present invention.

[0091] Example 1

[0092] 1 Materials and Methods

[0093] 1.1 Materials

[0094] Restriction endonucleases Nde I, Xho I, T4 DNA ligase, gel recovery kit, PCR purification kit, and plasmid extraction kit were all purchased from Shanghai Shenggong Bioengineering Co., Ltd.; bovine fibrinogen, bovine thrombin, and dialysis bags were purchased from Solebol; human thrombin was purchased from Shanghai Yika Biotechnology Co., Ltd.; the chromogenic substrate S2238 was purchased from Nanjing Dulai Biotechnology Co., Ltd.; APTT, PT, and TT coagulation kits were purchased from Shanghai Sun Biotechnology Co., Ltd.; and the coagulation and platelet function test kit (viscoelastic method) was purchased from Century Yikang (Tianjin) Medical Technology Development Co., Ltd. Fresh whole blood and plasma were donated by volunteers from Jinggangshan University (who signed informed consent forms).

[0095] 1.2 Experimental methods

[0096] 1.2.1 Bioinformatics analysis

[0097] The protein sequence of the hirudin HMg involved in this study is a variant of the hirudin from the leech Phenytrium spp., namely:

[0098] SEQ ID No. 1:

[0099] VSYTGCTESGQNYCLCVGSDICGDGKHCEMDGSENKCVDGEGTPK RQTSGPSDFEEFSLDDIEQK

[0100] Sequence alignment was performed using the BLAST tool of NCBI. Several homologous sequences from different leeches were selected and aligned using MEGA software. A phylogenetic tree was constructed using the neighbor-joining (NJ) method, and 1000 bootstrap tests were performed.

[0101] 1.2.2 Construction of pET-HMg prokaryotic expression vector

[0102] Using SnapGene software, upstream and downstream primers containing the restriction enzyme sites Nde I and Xho I were designed based on the multiple cloning site sequence of the pET vector and the HMg gene sequence to amplify the target gene HMg. Through double restriction enzyme digestion, gel recovery, T4 DNA ligase ligation, transformation into Escherichia coli Top10, colony PCR, and sequencing, the recombinant expression vector pET-HMg containing six histidine tags was obtained.

[0103] PCR primers:

[0104] FP (SEQ ID NO: 7):

[0105] 5'-GGAATTCCATATGGTTTCTTACACTGGTTGTACTGA-3';

[0106] RP (SEQ ID NO: 8):

[0107] 5'-CCGCTCGAGCTTTTGTTCAATATCATCCAAAGAAAT-3'

[0108] 1.2.3 Expression, purification and detection of recombinant hirudin protein (rHMg)

[0109] The constructed recombinant plasmid vector pET-HMg (such as Figure 5 coli expression strain BL21 (DE3) was transformed using the heat shock method; a single colony was picked and placed in 20 mL of the first LB liquid medium, and cultured overnight at 37°C with shaking for 12-16 hours. The bacterial solution obtained by the overnight culture was transferred to the second LB liquid medium according to a volume ratio of 1:100 for expansion culture, and the culture was expanded to OD 600 When the pH value was 1.0, IPTG was slowly added to a final concentration of 1 mM. The cells were cultured at 18°C ​​with shaking and induced for 24 hours at 170 rpm. The cells were centrifuged at 4000 rpm for 30 minutes, and the E. coli BL21 pellet was collected. The cells were suspended in PBS and sonicated at 90% power for a total of 120 minutes, with a 3-second ultrasonication cycle followed by a 7-second interval, until the solution was clear. The sonicated solution was centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected. The supernatant was then purified using nickel affinity chromatography using a gradient elution using a buffer containing 20 mM, 200 mM, and 500 mM imidazole. The target recombinant hirudin fusion protein was present in the 200 mM imidazole buffer. The purified recombinant hirudin fusion protein was dialyzed against PBS to remove the imidazole and stored at -80°C. The expression of the recombinant hirudin fusion protein samples at each stage was analyzed by SDS-PAGE.

[0110] The molecular weight of the purified recombinant hirudin fusion protein was determined by denaturing mass spectrometry. ① 20 μL of a 1 mg / mL sample was placed in a 1.5 mL EP tube and centrifuged at 14,000 g for 5 minutes. ② 18 μL of the supernatant was transferred to a sample vial. ③ 1 μL of the sample was loaded. ④ Data acquisition was performed using a Vanquish UHPLC-Q Exactive Plus Biopharm high-resolution LC / MS system. ⑤ Mobile phase A consisted of 0.1% formic acid in water; mobile phase B consisted of 0.1% formic acid in acetonitrile. The gradient settings were: 0-3 min, 2% B; 3-10 min, 5%-95% B; 10-13 min, 95% B; 13-13.1 min, 95%-2% B; 13.1-15 min, 2% B. The flow rate was 0.3 mL / min. Chromatographic column: ACQUITY UPLC Protein BEH C4, 300A, 1.7 μm, 2.1 × 100 mm. Mass spectrometry acquisition range: 400–2500 m / z. ⑥ Data were analyzed using the molecular weight analysis function of BioPharmaFinder software.

[0111] 1.2.4 Detection of recombinant hirudin rHMg activity by thrombin titration

[0112] (1) Solution preparation: Dilute the sample to 0.0039 mg / mL with PBS. Dilute thrombin to 40 U / mL with saline. Dissolve fibrinogen in saline to 5 mg / mL.

[0113] (2) Immerse the white porcelain plate in a 37°C water bath. Add 200 μL of fibrinogen solution and 100 μL of sample solution to the wells, mix thoroughly, and incubate for 5 minutes. Add 5 μL of 40 U / mL thrombin solution dropwise, stir, and observe within 1 minute whether the solution in the wells forms stringy lines. If not, add 5 μL of thrombin dropwise every minute until signs of coagulation appear. Record the amount of thrombin added. Use PBS as a negative control.

[0114] (3) Calculation: U = C1V1 / C2V2, where U (unit ATU / mg) represents the thrombin activity units per 1 mg of recombinant hirudin rHMg, C1 is the thrombin concentration, V1 is the thrombin volume, C2 is the sample concentration, and V2 is the sample volume.

[0115] 1.2.5 Detection of rHMg activity using viscoelastic coagulometer

[0116] According to the instructions of the Century Yikang coagulation analyzer, the anticoagulant effect of recombinant hirudin rHMg on whole blood was detected: ① Preheat the instrument, install the probe and reagent cup; ② Incubate 400μL of whole blood with 100μL of recombinant hirudin rHMg at 37℃ for 5 minutes; ③ Take 20uL of 0.25M calcium chloride and inject it into the anticoagulant tube containing 500uL of anticoagulant blood, and quickly invert it upside down 3 times to mix; ④ Inject 360uL of treated blood into the reagent cup and start the test immediately; ⑤ Read the values: ACT is the activated whole blood clotting time, CR is the coagulation rate, and PT is the platelet function.

[0117] 1.2.6 APTT, PT, and TT assays for rHMg activity and EC 50

[0118] (1) rHMg was diluted to 50-7500 nM in a gradient, and mixed at a ratio of recombinant hirudin rHMg: plasma = 1:4 to obtain the plasma sample to be tested. PBS was used as a negative control.

[0119] (2) APTT method: Add 100 μL of the sample to be tested to 100 μL of APTT reagent and incubate at 37°C for 5 min. Add 100 μL of 0.025 M CaCl2 preheated at 37°C and record the clotting time.

[0120] PT method: Incubate 100 μL of the sample at 37°C for 5 minutes. Add 200 μL of PT reagent pre-warmed at 37°C and record the clotting time.

[0121] TT method: Incubate 200 μL of the sample at 37°C for 5 min. Add 200 μL of TT reagent and record the clotting time.

[0122] (3) Data analysis: GraphPad Prism software was used to nonlinearly fit the EC values ​​of rHMg for APTT, PT, and TT. 50 , simple linear regression generates a histogram

[0123] 1.2.7 Chromogenic substrate assay for the inhibition constant Ki and IC of recombinant hirudin rHMg against thrombin 50

[0124] (1) The chromogenic substrate S2238 was diluted in Tris-HCl buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% PEG6000, and 0.1% BSA, pH 8.0) to concentrations of 0, 100, 200, 400, 600, 800, and 1000 μM. Recombinant hirudin rHMg was diluted in PBS to final concentrations ranging from 0.00003 to 4.9 μM. The final concentration of the positive control bivalirudin ranged from 0.023 to 11.466 μM.

[0125] PBS served as a negative control.

[0126] (2) Incubate 10 μL of 40 U / mL thrombin with 10 μL of recombinant hirudin rHMg at 37°C for 10 min. Add 180 μL of S-2238 to a total volume of 200 μL. Measure the absorbance at 405 nm every 20 seconds for 5 minutes on a microplate reader to detect the release of 4-nitroaniline.

[0127] (3) Inhibition constant Ki and half-maximal inhibitory concentration IC 50 Nonlinear regression analysis was performed using GraphPad Prism software. The Ki was determined by analyzing the absorbance changes at different substrate and inhibitor concentrations. The IC was determined by analyzing the inhibition rate of different inhibitor concentrations at 1000 μM S2238. 50 The significance was analyzed using SPSS software.

[0128] 2 Results

[0129] 2.1 Bioinformatics analysis results

[0130] After performing a BLAST search on the HMg amino acid sequence in NCBI, a total of 20 homologous sequences from leeches including Hirudinaria manillensis, Hirudo medicinalis, Hirudo verbana, Hirudotroctina, Hirudo orientalis, and Whitmania pigra were selected.

[0131] Homologous sequences were aligned using MEGA software and visualized using GeneDoc software. Figure 1 , from which we can see that these sequences have high homology, the genes are relatively conserved and the functions are stable.

[0132] The evolutionary tree was constructed using the neighbor-joining method on MEGA software. Figure 2 It can be seen that the HMg variant is most closely related to Hirullin-P18 in Hirudo philippinosus, with a similarity of up to 90.62%.

[0133] 2.2 rHMg protein expression and purification results

[0134] Sequencing confirmed that the recombinant plasmid vector pET-HMg was successfully constructed, encoding 74 amino acids:

[0135] SEQ ID NO:9:

[0136] MVSYTGCTESGQNYCLCVGSDICGDGKHCEMDGSENKCVDGEGTPKRQTSGPSDFEEFSLDDIEQKLEHHHHHH

[0137] The molecular weight is about 8kDa, including the enzyme cleavage site sequence and 6 histidines. According to the SDS-PAGE results, the size of the induced expressed protein is about 8-13kDa. After purification, a high-purity protein can be obtained. Due to the degree of gel denaturation and the accuracy of the standard protein marker, the specific molecular weight cannot be determined. Figure 4 ) It can be seen that the molecular weight of the fusion protein is 8042.31Da, which is basically consistent with the theoretical molecular weight, indicating that the obtained protein is a His-HMg fusion protein.

[0138] Figure 3 The electrophoresis diagram of the recombinant hirudin fusion protein rHMg is shown, wherein the numbers in the diagram represent the following meanings: 1: protein marker, 2-7: purified rHMg at different concentrations.

[0139] 3.3 Preliminary identification of the anticoagulant activity of recombinant hirudin rHMg

[0140] The anticoagulant activity of rHMg was determined using the white porcelain plate thrombin titration method. The calculated anticoagulant activity of rHMg was 9573±296.1 ATU / mg.

[0141] The effects of rHMg on whole blood were assessed using a coagulometer. The principle of this kit and coagulometer is to measure the viscoelastic properties of blood using a probe to reflect whole blood clotting time and platelet function. Table 1 shows that rHMg significantly prolonged whole blood clotting time (ACT), decreased clotting rate (CR), and affected platelet aggregation (PF) in a dose-dependent manner.

[0142] Table 1 Effect of recombinant hirudin rHMg on whole blood coagulation

[0143] rHMg(nM) ACT(s) CR(s) PF(s) 0 135 22.2 5.6 100 179 19.8 4.2 500 257 13.8 3.5 2500 781 10.8 0.3

[0144] 3.4 Recombinant hirudin rHMg prolongs APTT, PT, and TT

[0145] The effects of recombinant hirudin rHMg on the intrinsic and extrinsic coagulation pathways of plasma were determined by APTT, PT, and TT coagulation kits. HMg can significantly prolong the APTT, PT, and TT of plasma (e.g. Figure 6 shown), median effective concentration (EC 50) were 79.25±7.00nM, 1048±176.70nM, and 0.09±0.12nM, respectively. The greatest effect was on prolonging the thrombin time (TT), followed by the activated partial thromboplastin time (APTT), and finally the thromboplastin time (PT). These data suggest that recombinant hirudin rHMg achieves its anticoagulant effect by acting on thrombin, intrinsic coagulation pathways, and extrinsic coagulation pathways.

[0146] 3.5 Inhibition constant Ki and IC 50

[0147] Bivalirudin is a direct thrombin inhibitor currently used in the pharmaceutical market for the treatment of cardiovascular diseases and other conditions. The chromogenic substrate S2238 (HD-Phe-Pip-Arg-pNA-2HCl) specifically binds to thrombin and hydrolyzes to release 4-nitroaniline. Using a chromogenic substrate assay, we compared the binding and inhibitory effects of recombinant hirudin rHMg and bivalirudin on thrombin to determine the direct inhibitory effect of recombinant hirudin on thrombin and its potential application.

[0148] Depend on Figure 7 and Figure 8 It can be seen (p<0.001) that the Ki value of recombinant hirudin rHMg (0.323±0.144nM) is smaller than that of bivalirudin (175.1±65.4nM), and its binding affinity to thrombin is higher.

[0149] Depend on Figure 9 It can be seen (p<0.001) that the IC 50 The IC value was 2.8±0.03nM, which was lower than that of the positive control bivalirudin. 50 The inhibitory effect of rHMg on thrombin activity was better than that of rHMg (376.0±23.64nM), which was 135 times lower than that of rHMg (376.0±23.64nM). Therefore, rHMg had a better inhibitory effect on thrombin activity and had a promising development prospect.

[0150] 3 Results, Discussion and Conclusion

[0151] Natural hirudin resources are limited, the extraction and purification methods are complex, and the yield is low, so its large-scale application is severely restricted. Based on this, genetic engineering recombinant technology has become the mainstream strategy for hirudin production, but the low-cost and simple prokaryotic expression still faces key challenges such as inclusion body formation and lack of post-translational modification (such as tyrosine sulfation). Tyr63 sulfation of natural hirudin is crucial to the activity of hirudin, however, this post-translational modification cannot be achieved in prokaryotic expression systems such as Escherichia coli. It is worth noting that the prokaryotically expressed rHMg of the present invention can exhibit ultra-high anticoagulant activity without relying on Tyr63 sulfation.

[0152] The present invention revealed through multiple sequence alignment and phylogenetic analysis that the HMg variant shares a high degree of homology (93.55% similarity) with Hirulin-P18 from the leech of the Philippine leech. Key residues in its active center are highly conserved, suggesting that it inhibits thrombin via a "two-site binding" mechanism: the N-terminal domain occupies the thrombin active site, while the C-terminal acidic residues directly bind to the catalytic site through electrostatic complementarity. Despite the lack of Tyr63 sulfation, the C-terminal enriched negatively charged cluster effectively anchors the basic pocket of thrombin Exosite I, thereby compensating for the effects of the lack of post-translational modification.

[0153] The present invention successfully expresses rHMg in Escherichia coli BL21 using a prokaryotic expression vector. Soluble expression is increased by induction at low temperatures (16-18°C) and prolonged induction time (16-24 hours). The purification method is simple, yielding approximately 36 mg of high-purity rHMg per 1 L of culture fluid (or a second liquid culture medium). Thrombin titration assays determined the activity of rHMg to be 9573 ATU / mg (i.e., 345 ATU per mL of culture fluid), significantly higher than most reported prokaryotically expressed recombinant hirudin. For example, the activity of rHV3 prokaryotically expressed in Corynebacterium glutamicum by Yali Wang et al. was only 3.187 ATU / mL, while the activity of the recombinant hirudin obtained by HKongwei et al. was only 114 ATU / mg.

[0154] rHMg was used for real blood anticoagulation. The anticoagulant effect of rHMg on whole blood was evaluated by viscoelastic coagulometer, and it was found that it could prolong the activated whole blood clotting time (ACT) in a dose-dependent manner: 2.5μM rHMg prolonged the ACT from the baseline of 135s to 781s, far exceeding the clinical threshold (100-240s), and significantly inhibited the coagulation rate and platelet aggregation. The regulation of the coagulation system involves endogenous, exogenous and common pathways. The three coagulation tests further revealed that rHMg had the most significant effect on prolonging the thrombin time (TT) (EC 50 =0.09 nM), followed by activated partial thromboplastin time (APTT, EC 50 =79.25 nM) and prothrombin time (PT, EC 50 =1048nM), which is superior to most reported natural and recombinant hirudin. This also confirms that rHMg hirudin can achieve a highly effective anticoagulant effect by directly inhibiting thrombin activity and indirectly regulating the intrinsic and extrinsic coagulation pathways, suggesting its potential application in extracorporeal circulation or blood preservation, and also laying the foundation for subsequent research on anticoagulant mechanisms.

[0155] Bivalirudin (a hirudin derivative) is a synthetic polypeptide with a molecular weight of 2180 Da and has been approved by the FDA as a direct thrombin inhibitor (DTI) for percutaneous coronary intervention. The present invention uses kinetic analysis to show that under the same experimental conditions, the inhibition constant of rHMg on thrombin (Ki = 0.323 nM) is 542 times lower than that of bivalirudin (Ki = 175.1 nM), and its half-maximal inhibitory concentration (IC 50 =2.8nM) was also significantly superior to bivalirudin (IC 50 =376nM, a 135-fold increase in activity). This performance surpasses that of recombinant hirudin reported in the literature, such as the two derivative peptides studied by Boyle et al. (IC 50 The Ki values ​​for rHMg were 0.140±0.040μM and 2.40±0.30μM, respectively, with Ki values ​​of 0.29±0.07μM and 0.054±0.09μM. Cho Yeow Koh's team previously measured a Ki of 1.78nM for bivalirudin, while the present invention achieved a higher Ki (175.1nM). This discrepancy may be due to the effect of experimental conditions (such as ionic strength and temperature fluctuations) on the sensitivity of the chromogenic substrate assay (S2238). Despite this, the Ki value for rHMg was still lower than all control groups, highlighting its potent thrombin-binding ability.

[0156] Although hirudin has problems such as poor stability in vivo, susceptibility to proteases, low bioavailability, immunogenicity, and limited natural hirudin resources, these problems will be improved by methods such as protein modification, drug delivery system research, and optimization of expression conditions. The present invention is based on a prokaryotic expression system that can efficiently produce recombinant hirudin rHMg without the need for post-translational modification. Its specific activity significantly surpasses most reported recombinant products, and its activity is more than 135 times higher than that of the clinical drug bivalirudin. As the most potent direct thrombin inhibitor currently available for anticoagulation, hirudin has shown great potential in the fields of cardiovascular disease, extracorporeal anticoagulation, and blood preservation. The rHMg of the present invention, with its high activity, low cost, and simplified process, provides a new candidate molecule for the clinical transformation of a new generation of antithrombotic drugs, which can promote the development of anticoagulant therapy.

[0157] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solutions of the present invention may be subjected to various simple modifications, including combining the various technical features in any other appropriate manner. These simple modifications and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A recombinant hirudin fusion protein, characterized in that: The recombinant hirudin fusion protein comprises an HMg variant amino acid sequence and a fusion tag, wherein the HMg variant amino acid sequence is any one of the following (a1)-(a3): (a1) the amino acid sequence shown in SEQ ID NO: 1; (a2) an amino acid sequence that has more than 90% identity with the amino acid sequence shown in SEQ ID NO: 1 and has the same or similar biological activity; (a3) an amino acid sequence represented by (a1) or (a2) having one or more amino acid residues substituted and / or deleted and / or added, and having the same or similar biological activity; The fusion tag comprises a sequence of a His tag or a Trx tag.

2. The recombinant hirudin fusion protein according to claim 1, characterized in that The recombinant hirudin fusion protein comprises an HMg variant amino acid sequence and a fusion tag, wherein the HMg variant amino acid sequence is the amino acid sequence shown in SEQ ID NO: 1; and the fusion tag comprises 6-10 histidines.

3. A nucleic acid molecule encoding a recombinant hirudin fusion protein, characterized in that: As shown in any one of Formula 1, Formula 2, Formula 3, and Formula 4, wherein: Restriction site 1-first fusion tag-nucleotide encoding HMg variant-restriction site 2 (Formula 1) Restriction site 1'-nucleotide encoding HMg variant-second fusion tag-restriction site 2' (Equation 2) Restriction site 1-nucleotide encoding HMg variant-restriction site 2-third fusion tag (Equation 3) Fourth fusion tag-enzyme cleavage site 1'-nucleotide encoding HMg variant-enzyme cleavage site 2' (Formula 4) Wherein, Formula 1, Formula 2, Formula 3 or Formula 4 is suitable for expression in a prokaryotic system; The first fusion tag, the second fusion tag, the third fusion tag, and the fourth fusion tag are sequences comprising a His tag or a Trx tag respectively; The nucleotide encoding the HMg variant is any one of the following (b1)-(b3): (b1) the nucleotide sequence shown in SEQ ID NO: 2; (b2) a nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 2 and has the same or similar biological activity; (b3) a nucleotide sequence obtained by substituting and / or deleting and / or adding one or more nucleotides of the nucleotide sequence shown in (b1) or (b2), and having the same or similar biological activity; The enzyme cutting site 1 is N de I or B H I; The enzyme cutting site 1' is N de I or B H I; The enzyme cutting site 2 is X ho I or E coR I; The enzyme cleavage site 2' is X ho I or E coR I.

4. The nucleic acid molecule encoding the recombinant hirudin fusion protein according to claim 3, characterized in that: The nucleic acid molecule encoding the recombinant hirudin fusion protein is represented by Formula 3, and the nucleotide encoding the HMg variant is the amino acid sequence shown in SEQ ID NO:

2.

5. An expression vector or expression cassette, characterized in that: A nucleic acid molecule encoding a recombinant hirudin fusion protein according to claim 3.

6. A host cell, characterized in that Comprising the expression vector according to claim 5.

7. A prokaryotic expression method for a recombinant hirudin fusion protein, characterized in that: The following steps are involved: 1) The constructed pET-HMg recombinant plasmid vector was transformed into the Escherichia coli expression strain BL21 or Rosetta pLysS using the heat shock method; 2) picking a single clone of the E. coli expression strain and culturing it in a first liquid culture medium at 35-38°C with shaking overnight for 12-16 hours; 3) Transfer the overnight cultured bacteria into the second liquid culture medium at a volume ratio of 1:80-120 and culture at 35-38°C with shaking until the OD 600 =0.8-1.2; then add IPTG dropwise or slowly to a final concentration of 0.5-1.5 mM, culture at 16-18°C with shaking, and induce expression at 160-200 rpm for 16-24 h; 4) centrifuging and collecting the E. coli pellet; suspending the cells in PBS or Tris-HCl buffer and sonicating until the solution is clear, wherein the sonication conditions include a cycle of sonication for 2-5 seconds, with an interval of 5-10 seconds, for a total of 90-180 minutes, and an ultrasonic power of 80%-90%; 5) Centrifuging the solution obtained after the ultrasonic disruption, collecting the supernatant, and then purifying the supernatant using nickel column affinity chromatography; gradient elution is performed using a buffer containing 15-25 mM, 150-300 mM, and 500-600 mM imidazole, where the target recombinant hirudin fusion protein is present in the 150-300 mM imidazole buffer.

8. A pharmaceutical composition, characterized in that The invention comprises the recombinant hirudin fusion protein according to claim 1 or 2, the recombinant hirudin fusion protein expressed by the nucleic acid molecule encoding the recombinant hirudin fusion protein according to claim 3 or 4, the recombinant hirudin fusion protein expressed by the expression vector or expression cassette according to claim 5, the recombinant hirudin fusion protein expressed by the host cell according to claim 6, or the recombinant hirudin fusion protein obtained by the prokaryotic expression method of the recombinant hirudin fusion protein according to claim 7, and pharmaceutical excipients.

9. Use of the recombinant hirudin fusion protein according to claim 1 or 2 or the pharmaceutical composition according to claim 7 in the preparation of drugs for anticoagulation, treatment of thrombosis or prevention of thrombosis.

10. The use according to claim 9, characterized in that The effects of the recombinant hirudin fusion protein include being able to directly inhibit thrombin activity, indirectly affect the internal and external coagulation pathways, significantly prolong the whole blood coagulation time, reduce the whole blood coagulation rate and affect platelet aggregation.

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