Recombinant bifunctional hirudin variant 2 fusion protein as well as preparation method and application thereof

Abstract

The invention provides a recombinant bifunctional hirudin variant 2 fusion protein as well as a preparation method and application thereof, and belongs to the technical field of biotechnology and protein engineering. The recombinant bifunctional hirudin variant 2 fusion protein is obtained by introducing an RGD sequence into a mutation site in a hirudin variant 2, and introducing a His tag, a protease cleavage site and an FXa recognition sequence. After being activated, the recombinant bifunctional hirudin variant 2 fusion protein has obvious antithrombin effect, targeting characteristic and anti-platelet aggregation activity, and can achieve good effect of inhibiting and resisting platelet aggregation under the condition of low concentration. Besides, the expression quantity of the recombinant bifunctional hirudin variant 2 fusion protein is remarkably improved by overexpressing at least one of molecular chaperone genes, antioxidant and stress reaction genes, secretion pathway related genes and bZIP transcription factor genes, and the recombinant bifunctional hirudin variant 2 fusion protein is stable in production process, high in efficiency and suitable for large-scale industrial production and application.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and protein engineering technology, specifically to a recombinant bifunctional hirudin variant 2 fusion protein, its preparation method, and its applications. Background Technology

[0002] Thrombotic diseases are a group of illnesses caused by the abnormal coagulation of blood clots within blood vessels, leading to blood flow obstruction. Common examples include venous thromboembolism (deep vein thrombosis and pulmonary embolism), arterial thrombosis (such as myocardial infarction and stroke), and atrial fibrillation-related thrombosis. These diseases have extremely high morbidity and mortality rates worldwide. It is estimated that venous thromboembolism affects approximately 10 million people annually, while arterial thrombosis-related diseases cause approximately 17 million deaths annually, accounting for 31% of global deaths. These diseases not only seriously threaten patients' lives but also place a heavy burden on society and healthcare systems, making anticoagulation and antithrombotic therapy an important area of ​​research.

[0003] Hirudin is a natural anticoagulant peptide isolated from the saliva of medical leeches. It has a molecular weight of approximately 7 kDa and is composed of 65 amino acids. It blocks thrombin activity by forming a non-covalent reversible complex with the inactive substrate recognition site and active site of thrombin in a 1:1 ratio, making it one of the most potent known direct thrombin inhibitors. Compared to traditional anticoagulants such as heparin and vitamin K antagonists, hirudin has several advantages: ① its ability to inhibit thrombin activity is significantly stronger than traditional drugs; ② it can directly inhibit thrombin activity without the need for antithrombin III and heparin cofactors; ③ it has no interfering effect on other coagulation factors; ④ its reversible binding to thrombin allows for flexible control during anticoagulation therapy. These characteristics give hirudin unique advantages in precise anticoagulation and reducing the risk of bleeding. However, traditional methods of producing hirudin have some problems, including high cost and low yield from extraction from natural leeches, limited expression efficiency of recombinant expression systems, and insufficient protein activity and stability, which limit its large-scale application and further optimization.

[0004] During blood coagulation, coagulation factor FXa is a key intersection of intrinsic and extrinsic coagulation pathways. It directly participates in fibrin formation by catalyzing the conversion of prothrombin to thrombin, driving thrombus formation. FXa has weak activity in its free state, but its activity is significantly enhanced at the thrombus site, specifically cleaving the Ile-Glu-Gly-Arg sequence. If the FXa recognition sequence is added to hirudin protein, this sequence blocks the anticoagulant function of hirudin at non-thrombus sites; however, at the thrombus site, activated FXa can cleave the recognition sequence, activating the anticoagulant activity of hirudin. This design achieves targeted release of hirudin at the thrombus site, effectively reducing the risk of off-target bleeding.

[0005] In platelet aggregation, GPIIb and GPIIIa are key glycoprotein complexes on the platelet membrane. They mediate platelet adhesion and aggregation by binding to fibrinogen or fibronectin, which is a crucial step in arterial thrombosis. RGD (Arg-Gly-Asp) is a functional tripeptide sequence derived from an extracellular matrix protein that can efficiently bind to the GPIIb / IIIa complex and prevent its binding to fibrinogen, thereby inhibiting platelet aggregation. Introducing the RGD sequence into antiplatelet drugs can significantly enhance their antithrombotic effects, making it an important means of treating thrombotic diseases.

[0006] Pichia pastoris is a widely used recombinant protein expression system in industrial production, possessing the ability to efficiently express exogenous proteins and making it suitable for large-scale industrial applications. Compared to other expression systems, Pichia pastoris has significant advantages, including high-level expression through the strong promoter AOX1, protein secretion guided by the α-factor signal peptide, reducing cell disruption and purification steps, and its post-translational modification capabilities (such as disulfide bond formation and glycosylation) closely resembling those of native proteins, enhancing the activity and stability of recombinant proteins. Furthermore, Pichia pastoris has low cultivation costs and high fermentation efficiency, making it economically viable for industrial production. Therefore, Pichia pastoris has become an important choice for preparing hirudin proteins. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides a recombinant bifunctional hirudin variant 2 (RDH2) fusion protein with targeted antithrombin activity and antiplatelet aggregation activity. After activation, the RDH2 fusion protein exhibits significant antithrombin activity, targeted characteristics, and antiplatelet aggregation activity, achieving good antiplatelet aggregation effects even at low concentrations. Furthermore, this invention significantly increases the expression level of the RDH2 fusion protein through gene editing technology, and the production process is stable, efficient, and suitable for large-scale industrial applications.

[0008] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a recombinant bifunctional hirudin variant 2 fusion protein, which is obtained by introducing an RGD sequence at a mutation site in hirudin variant 2 (HV2), and introducing a His tag, a protease cleavage site and an FXa recognition sequence; wherein the amino acid sequence of hirudin variant 2 is shown in SEQ ID NO: 1.

[0010] The recombinant bifunctional hirudin variant 2 fusion protein of the present invention comprises three main parts: First, the introduced FXa recognition sequence (Ile-Glu-Gly-Arg, isoleucine-glutamic acid-glycine-arginine), the addition of which significantly enhances the targeting of the recombinant bifunctional hirudin variant 2 fusion protein and effectively reduces bleeding side effects; Second, it contains bifunctional hirudin variant 2 with antithrombin activity and antiplatelet aggregation activity, making the monomer size of the recombinant bifunctional hirudin variant 2 fusion protein of the present invention approximately 6 kDa~15 kDa. The introduction of the RGD tripeptide sequence on this basis enhances the antiplatelet aggregation effect while maintaining antithrombin activity; Third, to facilitate the separation and purification of the recombinant bifunctional hirudin variant 2 fusion protein, the present invention introduces a 6×His tag in the fusion protein for affinity chromatography purification; simultaneously, a protease cleavage site is set between this tag and the FXa recognition sequence to remove the His tag retained after affinity chromatography.

[0011] Furthermore, an RGD sequence is introduced at any three consecutive mutation sites at positions 24-43 of the amino acid sequence of hirudin variant 2. For example, an RGD sequence is introduced at mutation sites 24-26, 27-29, 30-32, 33-35, 36-38, 39-41, or 41-43 of the amino acid sequence of hirudin variant 2.

[0012] Furthermore, the amino acid sequence after introducing the RGD sequence at the mutation sites of positions 33-35 of the hirudin variant 2 is shown in SEQ ID NO: 2.

[0013] Furthermore, the amino acid sequence of the recombinant bifunctional hirudin variant 2 fusion protein is shown in SEQ ID NO: 3.

[0014] Furthermore, proteases capable of recognizing the protease cleavage site include, but are not limited to, enterokinase (EK), arginase, TEV protease, intestinal peptidase, or SUMO protease.

[0015] In a second aspect, the present invention provides a gene encoding the recombinant bifunctional hirudin variant 2 fusion protein, the sequence of which is shown in SEQ ID NO: 4.

[0016] Thirdly, the present invention provides a recombinant expression vector containing the gene sequence encoding the recombinant bifunctional hirudin variant 2 fusion protein.

[0017] Furthermore, the recombinant expression vector includes, but is not limited to, plasmids.

[0018] Furthermore, the plasmid includes, but is not limited to, pPIC.

[0019] Fourthly, the present invention provides a host comprising the recombinant expression vector.

[0020] Furthermore, the host includes, but is not limited to, Pichia pastoris, bacteria, or mammalian cells.

[0021] Furthermore, the host is Pichia pastoris.

[0022] The recombinant expression vector of the present invention can be expressed using hosts such as Pichia pastoris, bacteria, or mammalian cells. Compared to bacteria or mammalian cells, Pichia pastoris not only supports efficient eukaryotic expression but also exhibits good secretion performance. To optimize the expression system, the present invention designs coding gene sequences based on the codon preference of Pichia pastoris and inserts these genes into the expression vector pPIC (e.g., pPIC9K) series of plasmids. Using these gene sequences, the target gene is synthesized using Overlap PCR technology, and then recombinant plasmids are formed with the expression vector pPIC series of plasmids using Gibson Assembly technology.

[0023] Fifthly, the present invention provides a method for increasing the expression level of the recombinant bifunctional hirudin variant 2 fusion protein by overexpressing at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes, thereby significantly increasing the expression level of the recombinant bifunctional hirudin variant 2 fusion protein.

[0024] To address the high production cost of recombinant hirudin products in existing technologies, this invention modifies and optimizes the recombinant host through genetic engineering to increase the expression level of the recombinant bifunctional hirudin variant 2 fusion protein. Specifically, this invention constructs an overexpression host by introducing at least one of the following genes: molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes. This significantly increases the expression level of the recombinant bifunctional hirudin variant 2 fusion protein, achieving a 6-fold increase, reaching a level suitable for industrial production. Therefore, the host provided by this invention, in addition to containing the recombinant expression vector, also contains at least one of the following genes: molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes. Using the recombinant host of this invention to produce the recombinant bifunctional hirudin variant 2 fusion protein results in high protein yield and significantly reduced production costs, fundamentally overcoming the key bottleneck restricting the industrialization of recombinant hirudin products, and possessing significant application prospects and market value.

[0025] Furthermore, the molecular chaperone genes include, but are not limited to, at least one of BIP, PDI1, ERO1, CPR5, CNE1, and HAC1; the antioxidant and stress response genes include, but are not limited to, at least one of YAP1, SOD1, TRX1, and CTA1; the secretion pathway-related genes include, but are not limited to, at least one of SEC1, SEC53, VPS10, SAR1, and SSO1; and the bZIP-type transcription factor genes include, but are not limited to, at least one of HAC1, YAP1, and GCN4. In one example of the present invention, by co-transforming the engineered strain GS115-RDH2 containing the recombinant bifunctional hirudin variant 2 fusion protein gene into the overexpression vector pGAPZ A (see SEQ ID NO: 5) containing the HAC1 gene, the correct folding and expression of the recombinant bifunctional hirudin variant 2 fusion protein were effectively promoted, and the protein yield was approximately 6 times that of the control group (without overexpression of the target gene).

[0026] Furthermore, a method for increasing the expression level of the recombinant bifunctional hirudin variant 2 fusion protein includes the following steps:

[0027] Construct a recombinant expression vector containing a gene encoding a recombinant bifunctional hirudin variant 2 fusion protein;

[0028] The recombinant expression vector was introduced into the host, and high-expression Mut was obtained through resistance selection and MM / MD plate phenotype selection. + (Fast methanol utilization) phenotype host;

[0029] Construct an overexpression plasmid containing an overexpression target gene; the overexpression target gene includes at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes.

[0030] Overexpression plasmid was introduced into Mut + Phenotypic host;

[0031] Overexpressing hosts were obtained through Zeocin resistance screening;

[0032] Overexpressing hosts were cultured, secreted expression was performed, and the protein was purified to obtain the recombinant bifunctional hirudin variant 2 fusion protein.

[0033] Purification of the secreted recombinant bifunctional hirudin variant 2 fusion protein can be performed using various conventional techniques, including but not limited to crude extraction, dialysis, ultrafiltration, and liquid chromatography. Liquid chromatography techniques include, but are not limited to, ion exchange, affinity chromatography, and hydrophobic chromatography. In one example of this invention, a His-tag affinity chromatography column is used for protein purification.

[0034] Specifically, the method for increasing the expression level of the recombinant bifunctional hirudin variant 2 fusion protein includes the following steps:

[0035] (1) Construct an expression plasmid pPIC9K-RDH2 containing the DNA sequence of the recombinant bifunctional hirudin variant 2 fusion protein;

[0036] (2) pPIC9K-RDH2 was introduced into the expression plasmid of strain GS115 by electroporation. Mut was obtained by resistance selection and MM / MD plate phenotypic selection. + (Fast methanol utilization) phenotypic engineered strain GS115-RDH2;

[0037] (3) Extract the target gene for overexpression from the genome of GS115 strain; the target gene for overexpression includes at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes;

[0038] (4) Construct an overexpression plasmid containing the overexpressed target gene;

[0039] (5) The overexpression plasmid containing the overexpression target gene was introduced into the engineered strain GS115-RDH2 by electroporation, and the overexpression engineered strain was obtained by Zeocin resistance screening.

[0040] (6) The overexpressing engineered strain was cultured to secrete and express the recombinant bifunctional hirudin variant 2 fusion protein;

[0041] (7) After purification, a single recombinant bifunctional hirudin variant 2 fusion protein was obtained.

[0042] Sixthly, this invention provides the application of the recombinant bifunctional hirudin variant 2 fusion protein in the preparation of drugs for the prevention / treatment of thrombosis. The recombinant bifunctional hirudin variant 2 fusion protein of this invention can be widely used in the prevention and treatment of thrombosis-related diseases, including venous thrombosis, acute myocardial infarction, angina pectoris, disseminated intravascular coagulation, heparin-induced thrombocytopenia, and reembolism after thrombolysis.

[0043] Compared with the prior art, the advantages of the present invention are:

[0044] The recombinant bifunctional hirudin variant 2 fusion protein of the present invention has several significant advantages and demonstrates its broad application potential, specifically:

[0045] 1. Compared with natural hirudin, the recombinant bifunctional hirudin variant 2 fusion protein of the present invention, due to the introduction of the FXa recognition sequence, can specifically activate anticoagulant activity at the thrombus site, thereby significantly enhancing the targeting of the recombinant bifunctional hirudin variant 2 fusion protein, reducing the risk of nonspecific bleeding, and greatly improving the safety of treatment. In contrast, natural hirudin, due to its lack of targeting, is prone to causing systemic bleeding side effects and has poor safety.

[0046] 2. The recombinant bifunctional hirudin variant 2 fusion protein of the present invention not only retains antithrombin activity comparable to that of natural hirudin, but also introduces the RGD tripeptide sequence into its structure, thereby endowing the recombinant bifunctional hirudin variant 2 fusion protein with antiplatelet aggregation function, forming a dual-function synergistic effect. In contrast, natural hirudin only possesses antithrombin activity, while its antiplatelet aggregation effect is limited.

[0047] 3. The recombinant bifunctional hirudin variant 2 fusion protein of the present invention contains a 6×His tag, which can be efficiently separated by affinity chromatography, simplifying the purification process and providing convenience for industrial production.

[0048] 4. Regarding the production process, this invention significantly increases the yield of recombinant bifunctional hirudin variant 2 fusion protein by overexpressing at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP-type transcription factor genes. In the laboratory shake-flask culture stage, the expression level of recombinant bifunctional hirudin variant 2 fusion protein has exceeded 3 g / L, and it is expected to be further increased to 5-10 times in the fermenter, significantly reducing production costs.

[0049] 5. The recombinant bifunctional hirudin variant 2 fusion protein of the present invention has strong clinical applicability and can be used to treat a variety of diseases such as deep vein thrombosis, acute myocardial infarction, cerebrovascular disease, and tumor-related thrombosis. Based on the high-density culture and secretory expression characteristics of hosts such as Pichia pastoris, the production process of the recombinant bifunctional hirudin variant 2 fusion protein of the present invention is stable and efficient, suitable for large-scale industrial production and application, providing a safe, efficient, and economical new option for the treatment of thrombotic diseases. Attached Figure Description

[0050] Figure 1 This is a PCR identification diagram of the pPIC9K-RDH2 recombinant plasmid;

[0051] Figure 2 PCR identification diagram of the GS115-RDH2 engineered strain colony;

[0052] Figure 3 PCR identification diagram of the HAC1-pGAPZ A overexpression plasmid construction;

[0053] Figure 4 PCR identification of colonies of the engineered strain oeHAC1-GS115-RDH2;

[0054] Figure 5 SDS-PAGE image of purified RDH2 fusion protein;

[0055] Figure 6 Western blot identification of the RDH2 fusion protein;

[0056] Figure 7 A comparison of the effects of HAC1 gene overexpression on protein expression levels;

[0057] Figure 8 A comparison of the antithrombin activities of different hirudin variants;

[0058] Figure 9 The protein expression levels of recombinant strains constructed using different overexpression target genes;

[0059] Figure 10 The antithrombin activity of fusion proteins expressed by recombinant strains overexpressing different target genes was measured. Detailed Implementation

[0060] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] This invention provides a recombinant bifunctional hirudin variant 2 fusion protein, which is obtained by introducing an RGD sequence at a mutation site in hirudin variant 2 (HV2), and introducing a His tag, a protease cleavage site and an FXa recognition sequence; wherein, the amino acid sequence of hirudin variant 2 is shown in SEQ ID NO: 1.

[0062] In some examples, an RGD sequence is introduced at any three consecutive mutation sites between positions 24 and 43 of the hirudin variant 2 amino acid sequence. In the following specific embodiments, the amino acid sequence after introducing the RGD sequence at mutation sites between positions 33 and 35 of the hirudin variant 2 amino acid sequence is shown in SEQ ID NO: 2.

[0063] In some examples, proteases capable of recognizing the protease cleavage site include, but are not limited to, enterokinase (EK), arginase, TEV protease, intestinal peptidase, or SUMO protease. In the following specific examples, the protease capable of recognizing the protease cleavage site is enterokinase.

[0064] In some examples, the expression level of the recombinant bifunctional hirudin variant 2 fusion protein is significantly increased by overexpressing at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes, specifically including the following steps:

[0065] Construct a recombinant expression vector containing a gene encoding a recombinant bifunctional hirudin variant 2 fusion protein;

[0066] The recombinant expression vector was introduced into the host, and high-expression Mut was obtained through resistance selection and MM / MD plate phenotype selection. + (Fast methanol utilization) phenotype host;

[0067] Construct an overexpression plasmid containing an overexpression target gene; the overexpression target gene includes at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes.

[0068] Overexpression plasmid was introduced into Mut + Phenotypic host;

[0069] Overexpressing hosts were obtained through Zeocin resistance screening;

[0070] Overexpressing hosts were cultured, secreted expression was performed, and the protein was purified to obtain the recombinant bifunctional hirudin variant 2 fusion protein.

[0071] In the following specific embodiments, the amino acid sequence of the recombinant bifunctional hirudin variant 2 fusion protein is shown in SEQ ID NO: 3, and the gene sequence encoding the recombinant bifunctional hirudin variant 2 fusion protein is shown in SEQ ID NO: 4.

[0072] Example 1: A recombinant bifunctional hirudin variant 2 (RDH2) fusion protein

[0073] 1. Cloning of the RDH2 fusion protein gene

[0074] Based on the DNA sequence of the RDH2 fusion protein and the codon bias of yeast, six primers were designed to amplify the RDH2 fusion protein gene fragment using overlap PCR. The base sequences of the six primers used are shown below:

[0075]

[0076] The PCR reaction system is shown below:

[0077]

[0078] The PCR product was gel-cleaved, purified, and the gene for the RDH2 fusion protein was obtained.

[0079] 2. Construction of pPIC9K-RDH2 recombinant plasmid

[0080] The RDH2 fusion protein gene was ligated into the expression vector pPIC9K using Gibson Assembly technology. Primers were designed to introduce 20-40 bp homologous sequences overlapping the ends of the pPIC9K vector at the 5' and 3' ends of the RDH2 fusion protein gene. The non-homologous portions of the primers were used to amplify the target gene, while the homologous portions were used to achieve seamless splicing in the subsequent Gibson Assembly reaction. The designed primer sequences are shown below:

[0081]

[0082] Using the RDH2 fusion protein gene as a template, the target gene containing homologous fragments was amplified at its 5' and 3' ends using PCR. The pPIC9K vector was linearized by double digestion with EcoRI and NotI. The digestion products were purified by agarose gel electrophoresis and used for Gibson Assembly reactions.

[0083] The recovered linearized vector fragment and the amplified RDH2 fusion protein gene fragment were mixed at a molar ratio of 1:3 and added to Gibson Assembly Master Mix for seamless cloning. The Gibson Assembly reaction product was transformed into competent E. coli DH5α, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated at 37 °C for 16–18 h. Single clones were selected for colony PCR. PCR identification results are shown below. Figure 1 As shown, a band of approximately 250 bp was observed in 1% agarose gel electrophoresis, and sequencing confirmed the accuracy of the splicing. The constructed recombinant plasmid was named pPIC9K-RDH2.

[0084] 3. Construction of the GS115-RDH2 engineered strain

[0085] The pPIC9K-RDH2 plasmid constructed in step 2 was transformed into Pichia pastoris GS115 using electroporation, and high-copy Mut cells were obtained through G418 resistance selection and MM / MD plate phenotypic selection. + Phenotypic engineered strains.

[0086] First, GS115 competent cells were prepared. Under aseptic conditions, the GS115 strain was activated on YPD plates and cultured at 28°C for 2 days until single colonies grew. A single colony was picked and transferred to a 100 mL Erlenmeyer flask containing 20 mL of YPD, and cultured overnight at 28°C on a shaker until OD500. 600 =Approximately 3, then transfer to a 1 L Erlenmeyer flask containing 100 mL of YPD, to achieve the initial OD. 600 The concentration was 0.3, and the culture was carried out at 28°C; when the OD... 600 When the concentration is 1.3-1.5, centrifuge at 4000 rpm for 5 min; remove the supernatant, resuspend the cells in 30 mL of pre-chilled ultrapure water, centrifuge at 4000 rpm for 5 min, and remove the supernatant; resuspend the cells again in 30 mL of pre-chilled ultrapure water, centrifuge at 4000 rpm for 5 min; remove the supernatant, resuspend the cells in 8 mL of SB solution, and incubate at 28 ℃ and 220 rpm for 30 min; centrifuge to remove the supernatant, resuspend the cells three times in 1 mL of pre-chilled 1 M sorbitol solution, and then resuspend the cells again in 1 mL of pre-chilled 1 M sorbitol solution; aliquot into pre-chilled lyophilized tubes to obtain GS115 competent cells.

[0087] During electroporation, 80 µL of GS115 competent cells were mixed thoroughly with 1–5 µg of linearized pPIC9K-RDH2 plasmid and transferred to a pre-chilled 0.2 cm electroporation cuvette. Electroporation was performed using an electroporator set to 1.1 kV and a pulse duration of 5 ms. Immediately after electroporation, 300 µL of pre-chilled 1 M sorbitol solution and 300 µL of pre-chilled MD solution were added to the electroporation cuvette, mixed well, and transferred to a sterile centrifuge tube. The cells were incubated at 28 °C for 2 h to promote cell repair. A suitable amount was then plated onto MD plates, which were inverted in a 28 °C incubator until single colonies appeared, approximately 2–3 days later.

[0088] During the high-copy screening process, transformants grown on MD plates were spot-inoculated onto YPD plates containing 0.5 mg / mL G418 using a pipette tip and incubated at 28 ℃ for 3-4 days. After single colonies grew on the plates, they were inoculated onto plates with different G418 concentrations (1.0, 2.0, 4.0, 10.0 mg / mL) to screen for transformants with the highest G418 resistance, which were then identified as high-copy strains.

[0089] During phenotypic screening, the high-copy strains selected were inoculated onto MD and MM plates, respectively, and incubated at 28°C for 2-3 days. Growth was observed, and transformants showing minimal difference in growth on MD and MM plates were designated as Mut transformants. + Conversely, transformants that grow rapidly on MD but very slowly on MM are classified as Muts. High-copy Muts are obtained through screening. + Phenotypic strains.

[0090] For the obtained high copy of Mut + Colony PCR was performed on the phenotypic strains. The upstream primer was located in the AOX1 promoter, and the downstream primer was located in the AOX1 terminator. The specific sequences are shown below:

[0091]

[0092] PCR identification results as follows Figure 2 As shown, 1% agarose gel electrophoresis revealed bands of approximately 730 bp and 2100 bp in size, which were confirmed by sequencing. This indicates that the pPIC9K-RDH2 expression vector was successfully integrated into the GS115 genome, and the strain phenotype is Mut. + The constructed engineered strain was named GS115-RDH2.

[0093] 4. Construction of HAC1-pGAPZ A overexpression plasmid

[0094] The HAC1 gene was extracted from the genome of Pichia pastoris GS115, cloned into the pGAPZ A expression vector, and a recombinant plasmid HAC1-pGAPZ A for overexpression was constructed. First, genomic DNA was extracted from GS115 using a yeast genomic DNA extraction kit, and the extracted genomic DNA was used as a template for HAC1 gene amplification. The HAC1 gene was amplified using specific primers, with 20-40 bp homologous fragments designed at the 5' and 3' ends of the primers to overlap with the linearized region of the vector, providing a template for subsequent Gibson assembly. The primer sequences are shown below:

[0095]

[0096] The PCR amplification reaction system included genomic DNA template, specific primers, high-fidelity DNA polymerase, a dNTP mixture, and reaction buffer. The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3 min; 35 cycles (95 ℃ for 15 s, 57 ℃ for 15 s, 72 ℃ for 1 min); and final extension at 72 ℃ for 5 min. The amplification products were detected by 1% agarose gel electrophoresis, and the target band was recovered and purified by gel excision.

[0097] The pGAPZ A vector was linearized by double digestion with Xho1 and Not1 enzymes. The digestion products were separated and recovered by agarose gel electrophoresis. The Gibson Assembly reaction system consisted of the linearized pGAPZ A vector, the HAC1 gene fragment, and Gibson Assembly Master Mix. The reaction conditions were 50 °C for 1 h. The resulting splice product was directly used for the transformation of competent E. coli DH5α.

[0098] The ligation product was transformed into DH5α competent cells and plated on LB agar plates containing 50 µg / mL Zeocin. Single clones were selected after incubation at 37°C for 16 h. Positive clones were verified by colony PCR. PCR identification results are shown below. Figure 3 As shown, a band of approximately 1100 bp was observed in 1% agarose gel electrophoresis, which was confirmed by sequencing, indicating that the HAC1 gene was successfully cloned into the pGAPZ A vector. The plasmid was extracted. The constructed overexpression vector was named HAC1-pGAPZ A.

[0099] 5. Construction of the engineered strain oeHAC1-GS115-RDH2

[0100] The HAC1-pGAPZ A overexpression plasmid constructed in step 4 was transformed into the GS115-RDH2 engineered strain in step 3 by electroporation, and the oeHAC1-GS115-RDH2 engineered strain overexpressing HAC1 was obtained by Zeocin resistance screening.

[0101] First, GS115-RDH2 competent cells were prepared. Under aseptic conditions, the GS115-RDH2 engineered strain was activated on YPD plates and cultured at 28 ℃ for 2 days until single colonies appeared. Single colonies were then picked and transferred to 100 mL Erlenmeyer flasks containing 20 mL of YPD medium and cultured overnight at 28 ℃ on a shaker until OD reached [value missing]. 600 =Approximately 3. Subsequently, the bacterial culture was transferred to a 1 L Erlenmeyer flask containing 100 mL of YPD medium, allowing the initial OD to reach approximately 3. 600 The concentration was 0.3, and the culture was carried out at 28 °C; when the OD... 600 When the bacterial concentration reaches 1.3-1.5, centrifuge at 4000 rpm for 5 min and remove the supernatant. Resuspend the cells twice in 30 mL of pre-chilled sterile ultrapure water, discarding the supernatant after each centrifugation. Then, resuspend the cells in 8 mL of SB solution and incubate at 28 ℃ and 220 rpm for 30 min. After centrifugation to remove the supernatant, wash three times with 1 mL of pre-chilled 1M sterile sorbitol solution, and finally resuspend the cells in 1 mL of pre-chilled 1M sterile sorbitol. Aliquot the bacterial solution into lyophilized tubes to obtain GS115-RDH2 competent cells.

[0102] During electroporation, 80 µL of GS115-RDH2 competent cells were mixed thoroughly with 1–5 µg of linearized HAC1-pGAPZA plasmid and transferred to a pre-chilled 0.2 cm electroporation cuvette. The electroporation conditions were 1.5 kV voltage and 5 ms pulse duration. Immediately after electroporation, 300 µL of pre-chilled 1M sorbitol solution and 300 µL of pre-chilled YPD medium were added, mixed well, and transferred to a sterile centrifuge tube. The mixture was incubated at 28 °C for 2 h to promote cell repair. An appropriate amount of the bacterial culture was spread onto a YPD plate containing 200 µg / mL Zeocin and incubated upside down at 28 °C until single colonies appeared (approximately 2–3 days).

[0103] In the process of developing resistant high-copy-count strains, 20 well-grown single colonies were picked from Zeocin plates and sequentially inoculated into YPD liquid medium, BMGY liquid medium, and BMMY liquid medium for protein expression. After 48 h, the supernatant was collected for protein concentration determination using the BCA method. Based on the results, the strain with the highest protein expression level was selected for subsequent experiments.

[0104] Colony PCR was performed to validate the high-copy-count strains. The specific primer sequences are shown below:

[0105]

[0106] PCR identification results as follows Figure 4 As shown, a specific band of approximately 900 bp was detected in 1% agarose gel electrophoresis, and this was confirmed by sequencing, indicating that the HAC1 gene was successfully integrated into the GS115-RDH2 engineered strain. The resulting engineered strain was named oeHAC1-GS115-RDH2.

[0107] 6. Cultivation of oeHAC1-GS115-RDH2 engineered strain and expression and purification of RDH2 fusion protein

[0108] First, the engineered strain oeHAC1-GS115-RDH2 obtained in step 5 was taken from the -80 ℃ glycerol storage and inoculated onto YPD plates for activation. A single well-grown strain was then inoculated into 25 mL of BMGY liquid medium and cultured at 30 ℃ and 220 rpm for 24 h until the bacterial culture reached OD. 600 The target concentration was reached at 6-8. After the bacterial cells reached the target concentration, they were centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the cells were transferred to 50 mL of BMMY liquid medium to begin inducing the expression of the RDH2 fusion protein. During the induction period, methanol was added to the medium every 24 h to a final concentration of 0.5%, and induction continued for 120 h. Samples were taken at 24 h, 48 h, 72 h, 96 h, and 120 h to compare the protein expression levels of the oeHAC1-GS115-RDH2 engineered strain and the GS115-RDH2 engineered strain. The results are as follows: Figure 7 As shown in the figure, HAC1 represents the oeHAC1-GS115-RDH2 engineered strain; CG represents the control group, i.e., the GS115-RDH2 engineered strain. Figure 7 The results showed that the protein expression level of the oeHAC1-GS115-RDH2 engineered strain was significantly higher than that of the GS115-RDH2 engineered strain, and the expression level of the oeHAC1-GS115-RDH2 strain reached the highest level compared with the control group at 72 h.

[0109] After induction culture, the culture supernatant was collected by centrifugation at 4000 rpm for 10 min. The supernatant was filtered through a 0.45 µm microporous membrane to remove suspended bacterial cells and impurities; the supernatant was the crude extract containing the RDH2 fusion protein. The crude extract was then concentrated by ultrafiltration, reducing 50 mL of supernatant to 1 mL and removing culture medium components. The RDH2 fusion protein was purified using a His-tag affinity chromatography kit (P2226, Beyotime), yielding a purified RDH2 fusion protein solution. The SDS-PAGE identification image of the purified RDH2 fusion protein is shown below. Figure 5 As shown.

[0110] 7. Identification of RDH2 fusion protein

[0111] To further verify the presence and specificity of the His tag expression in the RDH2 fusion protein, Western blot was used for identification. First, the protein, after SDS-PAGE electrophoresis, was transferred to a PVDF membrane (0.22 µm pore size) and transferred at 100 mA for 30 min. After transfer, the PVDF membrane was placed in TBS blocking buffer containing 5% skim milk and blocked at room temperature for 2 h to reduce non-specific binding. After blocking, the PVDF membrane was incubated overnight at 4 °C with 6× His-tagged monoclonal antibody (primary antibody, mouse-derived, 1:5000 diluted in TBST). The next day, it was incubated at room temperature for 2 h with HRP-labeled anti-mouse IgG secondary antibody (1:10000 diluted in TBST). The PVDF membrane was then washed three times with TBST for 10 min each time. Finally, ECL chemiluminescence reagent was added, and the luminescence signal was observed using a gel imaging system. The Western blot results are shown below. Figure 6 As shown, a clear, specific band was observed at approximately 15 kDa, consistent with the theoretical molecular weight of the RDH2 fusion protein. Western blot results validated the specificity of the His tag in the RDH2 fusion protein. Combined with SDS-PAGE detection results, the successful expression and purification of the RDH2 fusion protein of this invention can be confirmed.

[0112] Example 2: Antithrombin activity of RDH2 fusion protein

[0113] The antithrombin activity of the RDH2 fusion protein prepared in Example 1 was detected, and the results are shown in Table 1. Sample A was the control group, i.e., plasma; Sample B consisted of RDH2 fusion protein with a 6×his-tag added to plasma, with a final concentration of 1 µg / mL; Sample C consisted of RDH2 fusion protein with a 6×his-tag added to plasma, with a final concentration of 2.5 µg / mL; Sample D consisted of RDH2 fusion protein with a 6×his-tag added to plasma, with a final concentration of 5 µg / mL; Sample E consisted of RDH2 fusion protein cleaved by FXa added to plasma, with a final concentration of 1 µg / mL; Sample F consisted of RDH2 fusion protein cleaved by FXa added to plasma, with a final concentration of 2.5 µg / mL; Sample G consisted of RDH2 fusion protein cleaved by FXa added to plasma, with a final concentration of 5 µg / mL; TT represents thrombin time; APTT represents activated partial thromboplastin time.

[0114] Table 1: Results of antithrombin activity assay of RDH2 fusion protein

[0115]

[0116] Table 1 shows that the RDH2 fusion protein containing the 6×His, EK, and FXa recognition sequences exhibits virtually no anticoagulant activity. However, upon complete cleavage with the FXa protease, the released hirudin displays anticoagulant activity comparable to that of natural hirudin. These results indicate that the purified RDH2 fusion protein possesses high anticoagulant activity after enzymatic cleavage. In in vitro experiments, the RDH2 fusion protein activated by FXa cleavage exhibits targeting properties, which helps reduce anticoagulant activity at non-target sites, thereby mitigating the potential bleeding side effects of hirudin.

[0117] In anticoagulation experiments, the RDH2 fusion protein activated by FXa cleavage significantly prolonged both APTT (activated partial thromboplastin time) and TT (thrombin time) in the four coagulation parameters. The prolongation of APTT indicates that the RDH2 fusion protein exerts its effect by inhibiting coagulation factors (such as IXa, VIIIa, or XIa) in the intrinsic coagulation pathway, effectively blocking the intrinsic coagulation cascade. The significant prolongation of TT further demonstrates that the activated RDH2 fusion protein can directly inhibit thrombin activity, preventing fibrin formation from fibrinogen, thereby achieving an anticoagulant effect.

[0118] Example 3: Antiplatelet aggregation activity of RDH2 fusion protein

[0119] The antiplatelet aggregation activity of the RDH2 fusion protein prepared in Example 1 was tested, and the results are shown in Table 2. Among them, sample A was the control group, i.e., platelet-rich plasma (PRP); sample B was PRP with 5 µL of physiological saline added; sample C was PRP with RDH2 fusion protein containing 6×His-tag added to make its final concentration 8 µg / mL; sample D was PRP with RDH2 fusion protein cleaved by FXa added to make its final concentration 8 µg / mL.

[0120] Table 2: Results of antiplatelet aggregation activity assay of RDH2 fusion protein

[0121]

[0122] Experimental results showed that, using physiological saline as a control, the antiplatelet aggregation activity of the RDH2 fusion protein reached 40.4% when the concentration was 8 µg / mL. This result indicates that the RDH2 fusion protein of the present invention can exhibit good antiplatelet aggregation effects even under low concentration conditions.

[0123] As described above, this invention first constructs the gene of the RDH2 fusion protein, and successfully amplifies and obtains the target gene fragment through optimized primer design and overlap PCR technology. Then, using Gibson Assembly technology, the RDH2 fusion protein gene is seamlessly cloned into the expression vector pPIC9K, and a recombinant plasmid is constructed. The pPIC9K-RDH2 plasmid is then introduced into Pichia pastoris GS115 using electroporation. High-copy-count screening and phenotypic identification yielded a highly efficient GS115-RDH2 engineered strain. The HAC1 gene is extracted, and the HAC1-pGAPZ A overexpression vector is constructed, laying the foundation for further increasing the expression level of the RDH2 fusion protein. Subsequently, the HAC1-pGAPZ A plasmid is transformed into the GS115-RDH2 engineered strain, successfully constructing the oeHAC1-GS115-RDH2 engineered strain. High-expression strains are obtained through protein concentration determination screening. Furthermore, this invention also demonstrates the optimization of culture conditions for the oeHAC1-GS115-RDH2 engineered strain and the purification process of the RDH2 fusion protein. The specificity of the His tag in the RDH2 fusion protein was verified by Western blot. It was also verified that the prepared RDH2 fusion protein, after activation, has significant antithrombin activity, targeting characteristics, and antiplatelet aggregation activity, and can achieve good antiplatelet aggregation effect even at low concentrations.

[0124] Comparative Example 1: Activity Comparison of Different Hirudin Variant

[0125] To verify the superiority of hirudin variant 2 selected in this invention in terms of anticoagulant activity, pPIC9K vectors containing the gene sequences of hirudin variant 1 (amino acid sequence see SEQ ID NO: 6), variant 2 and variant 3 (amino acid sequence see SEQ ID NO: 7) were constructed and electroporated into GS115 strain. The specific construction method was the same as in Example 1, and protein expression was performed under the same conditions.

[0126] After fermentation expression, Ni was used 2+ The proteins obtained by affinity chromatography were activated by FXa restriction enzyme digestion, and the antithrombin activities of the three target proteins were detected by antithrombin titration. The results are as follows: Figure 8 As shown in the figure, among the three recombinant hirudin variants, recombinant hirudin variant 2 has the highest antithrombin activity, with a specific activity of 10100 ATU / mg, which is significantly higher than that of recombinant hirudin variant 1 and recombinant hirudin variant 3.

[0127] The above results indicate that hirudin variant 2 exhibits superior anticoagulant activity in recombinant expression compared to other common natural hirudin variants. Therefore, this invention preferentially selects hirudin variant 2 as the functional core for constructing a targeted anticoagulant protein.

[0128] Comparative Example 2: Effects of overexpression of different regulatory genes on expression yield

[0129] To verify the effectiveness of the overexpression of the target gene HAC1 used in this invention in improving the expression level of the target protein, recombinant strains that overexpress regulatory genes such as HAC1, PDI, ERO1, BIP, YAP1, GCN4, SEC1 and SEC53 were constructed. The construction methods of the overexpression vector and the recombinant strains are described in Example 1. The above recombinant strains were induced to express under the same fermentation conditions.

[0130] After fermentation, the fermentation supernatant was collected, and the expression of the target protein was detected by SDS-PAGE. ImageJ software was used to quantitatively analyze the band gray values ​​to assess the protein expression level. The experimental results are shown below. Figure 9 Subsequently, through Ni 2+ The target protein was purified by affinity chromatography and its functional activity was activated by FXa enzyme digestion. The antithrombin activity of the protein was then detected by antithrombin titration. The experimental results are shown below. Figure 10 .

[0131] Depend on Figure 9-10It can be seen that, in terms of protein expression level, all regulatory genes can increase the expression level to varying degrees, but the target protein expression level in the HAC1 overexpression strain of this invention is the highest, reaching 3.16 g / L, which is about 6 times that of the control group (without overexpression of the target gene). In terms of protein anticoagulant activity, the target protein activity expressed by the PDI, ERO1, BIP, HAC1 and YAP1 overexpression strains is significantly higher than that of the control group. Considering both protein expression level and biological activity, the HAC1 overexpression strain was determined to be the optimal engineered strain.

[0132] In summary, the RDH2 fusion protein provided by this invention exhibits a dual-function synergistic effect in antithrombin and antiplatelet aggregation. Furthermore, this invention significantly enhances the expression level of the recombinant bifunctional hirudin variant 2 fusion protein by overexpressing at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes, providing strong support for its large-scale production and clinical application in thrombotic diseases.

[0133] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A recombinant bifunctional hirudin variant 2 fusion protein, characterized in that, By introducing the RGD sequence at the mutation site in hirudin variant 2, and introducing the His tag, protease cleavage site and FXa recognition sequence, a recombinant bifunctional hirudin variant 2 fusion protein was obtained; wherein, the amino acid sequence of hirudin variant 2 is shown in SEQ ID NO:

1.

2. The recombinant bifunctional hirudin variant 2 fusion protein according to claim 1, characterized in that, An RGD sequence is introduced at any three consecutive mutation sites between positions 24 and 43 of the amino acid sequence of hirudin variant 2.

3. The recombinant bifunctional hirudin variant 2 fusion protein according to claim 2, characterized in that, An RGD sequence was introduced at the 33rd to 35th mutation sites of the amino acid sequence of hirudin variant 2. The amino acid sequence after the introduction of the RGD sequence is shown in SEQ ID NO:

2.

4. The recombinant bifunctional hirudin variant 2 fusion protein according to claim 3, characterized in that, The amino acid sequence of the recombinant bifunctional hirudin variant 2 fusion protein is shown in SEQ ID NO:

3.

5. A gene encoding the recombinant bifunctional hirudin variant 2 fusion protein according to any one of claims 1-4, characterized in that, The sequence of the gene is shown in SEQ ID NO:

4.

6. A recombinant expression vector, characterized in that, It includes the gene encoding the recombinant bifunctional hirudin variant 2 fusion protein as described in claim 5.

7. A host, characterized in that, It includes the recombinant expression vector of claim 6.

8. A method for increasing the expression level of the recombinant bifunctional hirudin variant 2 fusion protein according to any one of claims 1-4, characterized in that, The expression level of the recombinant bifunctional hirudin variant 2 fusion protein was significantly increased by overexpressing at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes.

9. The method for increasing the expression level of recombinant bifunctional hirudin variant 2 fusion protein according to claim 8, characterized in that, Includes the following steps: Construct a recombinant expression vector containing a gene encoding a recombinant bifunctional hirudin variant 2 fusion protein; The recombinant expression vector was introduced into the host, and high expression of Mut was obtained through resistance selection and phenotypic selection. + Phenotypic host; Construct an overexpression plasmid containing an overexpression target gene; the overexpression target gene includes at least one of molecular chaperone genes, antioxidant and stress response genes, secretion pathway-related genes, and bZIP transcription factor genes. Overexpression plasmid was introduced into Mut + Phenotypic host; Overexpressing hosts were obtained through resistance selection; Overexpressing host was cultured, secreted, and purified to obtain recombinant bifunctional hirudin variant 2 fusion protein.

10. Use of the recombinant bifunctional hirudin variant 2 fusion protein according to any one of claims 1-4 in the preparation of a medicament for the prevention / treatment of thrombosis.

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