Recombinant hirudin fusion protein, intelligent response type anticoagulant material as well as preparation method and application of intelligent response type anticoagulant material

Abstract

The invention relates to a recombinant hirudin fusion protein, an intelligent response type anticoagulant material as well as a preparation method and application of the intelligent response type anticoagulant material. The intelligent response type anticoagulant material is formed by constructing the recombinant hirudin fusion protein with a carbohydrate binding domain (CBM) and combining the recombinant hirudin fusion protein with bacterial nanocellulose or a composite material of the bacterial nanocellulose. The recombinant hirudin fusion protein disclosed by the invention can specifically respond to blood coagulation formation related enzymes so as to recover the anticoagulant activity, and can be well combined with a bacterial nanocellulose material or a composite material thereof; the intelligent response type anticoagulant material is mild in preparation condition and green and environment-friendly in technology, and has great potential in the medical application aspect of membranous anticoagulant biological materials such as heart valves and the like and tubular anticoagulant biological materials such as artificial blood vessels and the like.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and specifically relates to a recombinant hirudin fusion protein, a smart responsive anticoagulant material, its preparation method and application. Background Technology

[0002] Anticoagulant biomaterials are core components of medical devices such as artificial blood vessels, vascular stents, artificial heart valves, hemodialysis systems, membrane lung systems, and extracorporeal blood circulation systems. Their core function is to inhibit thrombus formation at the blood-contact interface. Traditional materials often lead to device failure or thromboembolic risks due to non-specific adsorption of plasma proteins and platelet activation, triggering a coagulation cascade reaction. In recent years, nanocellulose-based materials have become a research hotspot due to their unique surface properties. Their nanoscale topology can effectively reduce platelet adhesion, and by regulating surface chemistry, they can block the activation of coagulation factors, exhibiting significantly superior antithrombotic potential compared to conventional polymers. Among various nanocellulose materials, bacterial nanocellulose is considered an ideal anticoagulant substrate material due to its high crystallinity, ultrafine three-dimensional nanonetwork, excellent wet mechanical strength, and biocompatibility. However, the abundant hydroxyl groups on the surface of bacterial nanocellulose, while giving it high hydrophilicity, also lead to the following functionalization dilemmas: (1) Chemical inertness limitation: The polyhydroxy structure requires covalent modification to rely on high-energy reactions (such as sulfonation requiring concentrated sulfuric acid treatment), which easily damages the integrity of the bacterial nanofiber network; (2) Physical modification defects: Coating modification has problems such as weak interfacial bonding and easy detachment of functional layers; (3) Biosafety risks: Residual chemical crosslinking agents (such as glutaraldehyde) may cause cytotoxicity. Current research on nanocellulose anticoagulant materials mainly focuses on structural regulation (such as introducing porous structures to increase specific surface area) or simple blending (such as heparin / bacterial nanocellulose composite membranes), but there are problems such as single function and easy loss of active ingredients. In addition, existing anticoagulant drugs generally have adverse reactions such as bleeding in clinical applications, and the use of long-acting formulations may further increase the risk of bleeding. Therefore, it is urgent to develop a long-acting anticoagulant technology and material with high safety, significant efficacy and precise control of sustained release. In the field of medical materials, chitosan is often used to prepare functional composite materials by combining it with bacterial nanocellulose. Therefore, in addition to cellulose, chitosan can also be used to modify composite materials.

[0003] Carbohydrate-binding modules (CBM)-mediated protein fixation strategies should have the following unique advantages: (1) Precise positioning: Utilizing the natural affinity of CBM for carbohydrates, including cellulose or chitosan, recombinant proteins can be specifically anchored to the surface of bacterial nanofiber-based composite materials; (2) Green process: Through recombinant protein expression and mild adsorption processes, the use of toxic reagents is eliminated; Functional programmability: Fusion protein design allows for the modular design of multifunctional recombinant proteins. Hirudin (HV) is a polypeptide extracted from the salivary glands of leeches and is the strongest natural thrombin inhibitor discovered to date. It can specifically bind to thrombin to prevent thrombosis. However, hirudin has drawbacks such as a short half-life and low bioavailability in vivo, requiring frequent injections in clinical use to achieve a long-term anticoagulant effect. Taking advantage of the N-terminal modification of hirudin, which masks its activity, a smart responsive recombinant hirudin fusion protein can be constructed by introducing protein fusion tags, such as elastin-like polypeptides (ELP) and thioredoxin (TrxA), as well as responsive peptide motifs. This fusion protein specifically responds to coagulation-related enzymes: when vascular injury occurs and the coagulation cascade is initiated, key coagulation-related enzymes accumulate and are highly activated at the thrombus formation site, enabling them to specifically recognize and cleave peptide sequences in the fusion protein, releasing active hirudin or hirudin derivatives, and specifically binding to thrombin to exert an anticoagulant effect. Therefore, further fusing and expressing the recombinant hirudin protein specifically responsive to coagulation-related enzymes with a CBM-binding structure could endow cellulose-based materials with smart responsive anticoagulant properties. This means that when the material comes into contact with blood, it maintains low activity in the absence of thrombus, but at the core of thrombus formation, hirudin activity is activated in situ, thereby achieving precise and efficient inhibition of thrombus formation while minimizing the impact on systemic hemostasis. This makes it safer than heparin, a traditional clinical anticoagulant that can cause systemic bleeding. It is worth noting that currently, CBM-based anticoagulants and bacterial nanocellulose-anticoagulant composite tubular materials are still a global unexplored field. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a recombinant hirudin fusion protein, a smart responsive anticoagulant material, a preparation method thereof, and an application thereof. The recombinant hirudin fusion protein can specifically respond to coagulation-related enzymes to restore anticoagulant activity, and it can be well combined with bacterial nanocellulose materials or their composite materials.

[0005] This invention provides a recombinant hirudin fusion protein, wherein the fusion protein is a recombinant hirudin protein with a carbohydrate binding module (CBM), comprising a first part protein fusion tag, a second part hirudin or hirudin derivative, and a third part CBM; wherein the first and second parts are linked to the recognition sequence of a coagulation-related enzyme via a flexible protein linker sequence, and the second and third parts are linked via a flexible protein linker sequence.

[0006] Preferably, the first and second parts can form a fusion peptide with different protein fusion tags and coagulation-related enzyme recognition sequences, including: Trxa-coagulation-related enzyme recognition sequence-HV, ELP-coagulation-related enzyme recognition sequence-HV, SUMO-coagulation-related enzyme recognition sequence-HV, and GST-coagulation-related enzyme recognition sequence-HV. The flexible protein linker sequence is such as Gly-Ser linker or (Gly-Gly-Gly-Gly-Ser)n linker.

[0007] Preferably, the CBM includes one or more of ChBD2, ChBD3, CBM2a, CBM3, and CBDCex.

[0008] The amino acid sequence of ChBD2 is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2; the amino acid sequence of ChBD3 is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4; the amino acid sequence of CBM2a is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:6; the amino acid sequence of CBM3 is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:8; the amino acid sequence of CBDCex is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:10.

[0009] Preferably, the hirudin is recombinant hirudin, specifically including hirudin variant 1, hirudin variant 2, and hirudin variant 3; or recombinant hirudin fused with anticoagulant peptides such as human fibronectin peptide, RGD peptide, REDV peptide, or nematode anticoagulant peptide. The hirudin derivative is a hirudin analog or derivative known in the art, such as lepirudin, desirudin, and bivalirudin.

[0010] Preferably, the coagulation-related enzyme is one or more of thrombin, coagulation factor VIIa, coagulation factor IXa, coagulation factor Xa, coagulation factor XIa, coagulation factor XIIa, and coagulation factor XIIIa. More preferably, the coagulation-related enzyme is coagulation factor Xa (FXa), whose recognition sequence is Ile-Glu-Gly-Arg (IEGR), named FXa-responsivepeptide (Fr), and the corresponding gene sequence is ATTGAAGGCCGT.

[0011] More preferably, the recombinant hirudin protein with CBM is one or more of the following combinations: Trxa-Fr-hirudin-ChBD2, ELP-Fr-hirudin-ChBD2, SUMO-Fr-hirudin-ChBD2, and GST-Fr-hirudin-ChBD2. Most preferably, the recombinant hirudin protein with CBM is the Trxa-Fr-hirudin-ChBD2 recombinant protein.

[0012] Furthermore, the recombinant hirudin protein with CBM is constructed using a 3A (Three antibiotic assembly) kit, and then expressed, isolated, and purified in a microbial strain to obtain the final product.

[0013] Preferably, the basic vector in the 3A assembly toolkit includes a Bglbrick bio-brick module, a Golden Gate assembly module, a replicon replacement module, a resistance gene replacement module, and a termination sub-module.

[0014] Preferably, the basic vector resistance gene in the 3A assembly toolkit is one or more of ampicillin (Amp), tetracycline (TcR), chloramphenicol (CmR), gentamicin (GmR), and kanamycin (Kan).

[0015] Preferably, the microbial strain is a Gram-negative bacterium, such as Escherichia coli BL21(DE3) (genotype: F–ompT hsdS). B (r) B –, m B–) one or more of the following: *Gastroenteritis gal dcm (DE3)*, cellulose-producing bacteria, yeasts such as *Saccharomyces cerevisiae* BY4741 (genotype: MATa his3Δ1 leu2 met15Δ ura3-52), *Pichia pastoris* GS115, and Gram-positive bacteria such as *Corynebacterium glutamicum*. More preferably, the microbial strain is *Escherichia coli* BL21 (DE3).

[0016] The present invention also provides a smart responsive anticoagulant material, which is obtained by compounding recombinant hirudin protein with bacterial nanocellulose or its composite material.

[0017] Preferably, the bacterial nanocellulose composite material is a bacterial cellulose composite material deposited with polysaccharides (such as chitosan) that can bind to CBM, wherein the concentration of chitosan is 0.5%-2%. More preferably, the concentration of chitosan is 1%.

[0018] This invention also provides a method for preparing a smart responsive anticoagulant material, comprising the following steps:

[0019] (1) The recombinant hirudin fusion protein with carbohydrate binding domain CBM was constructed using the 3A assembly toolkit, and then expressed, isolated and purified in a microbial strain, and then prepared into a recombinant hirudin protein solution with CBM.

[0020] (2) Add a solution of recombinant hirudin protein fused with CBM to the purified bacterial nanocellulose substrate or its composite material, allow it to stand for reaction, and soak and wash with phosphate buffer to remove unbound recombinant protein to obtain a smart responsive anticoagulant material.

[0021] Preferably, the concentration of the recombinant hirudin protein solution fused with CBM in step (1) is 0.5-1 mg / mL.

[0022] Preferably, the buffer solution used in step (1) for the recombinant hirudin protein solution fused with CBM is phosphate buffer, purchased from Wuhan Saiwell Biotechnology Co., Ltd., with the main components being 10 mM sodium phosphate, 150 mM NaCl, and pH 7.2-7.4.

[0023] Preferably, the static reaction temperature in step (2) is 4 degrees Celsius, and the reaction time is 12-24 hours.

[0024] Preferably, when the bacterial nanocellulose substrate or its composite material is tubular, the recombinant hirudin protein solution is injected into the tube, and the lower end of the tube is sealed by knotting to prevent the protein solution from leaking out. After the injection, the upper end is sealed and the tube is placed in a glass petri dish containing PBS for static reaction. The bacterial cellulose tube is turned over once every 2 hours.

[0025] Preferably, when the bacterial nanocellulose substrate or its composite material is in the form of a film, the recombinant hirudin protein solution is composited with the bacterial nanocellulose substrate or its composite material by an impregnation method.

[0026] Preferably, the cleaning conditions in step (2) are to soak and clean the product in freshly prepared phosphate buffer solution for a total of 3 times, each time for 2 hours.

[0027] This invention also provides the application of a smart responsive anticoagulant material in the preparation of materials to prevent thrombosis, including artificial blood vessels, vascular stents, artificial heart valves, hemodialysis systems, extracorporeal blood circulation systems, etc.

[0028] This invention overcomes the limitations of chemical inertness during the modification of bacterial nanocellulose materials, ensuring the integrity of the bacterial nanocellulose nanonetwork is not compromised. It also avoids the problems of weak interfacial bonding and easy detachment of functional layers inherent in physical modification, while effectively mitigating the cytotoxic effects that may be caused by residual chemical crosslinking agents or metal ions, thus avoiding potential safety risks.

[0029] In addition to the advantages mentioned above, this invention also features mild reaction conditions, adjustable and controllable parameters, and minimal environmental pollution. The substrate material cellulose used in the preparation method is bacterial cellulose obtained through fermentation; all bacterial strains used in the preparation method are model strains, and all basic vector templates used are commercially available.

[0030] Beneficial effects

[0031] (1) The recombinant hirudin fusion protein of the present invention can specifically respond to coagulation-related enzymes to restore anticoagulation activity, and it can bind well to bacterial nanocellulose materials or their composite materials.

[0032] (2) This invention innovatively constructs the 3A assembly toolkit into the following modules: a Bglbrick bio-brick module, a Golden Gate assembly module, a replicon replacement module, a resistance gene replacement module, and a termination module, thereby achieving modular construction of the vector and overcoming the limitations of restriction enzyme sites in current research fields of Biobrick assembly and Golden Gate assembly, thus realizing 3A assembly. Furthermore, the convenient replicon replacement module enables plasmid replication in different hosts.

[0033] (3) Compared with the traditional oxidation and sulfation treatment in the chemical modification process, the preparation method of the intelligent responsive anticoagulant material of the present invention has milder reaction conditions and achieves the addition of functional units without destroying the integrity of the bacterial nanocellulose nanonetwork.

[0034] (4) Compared with the physical coating modification process, the intelligent responsive anticoagulant material of the present invention avoids the disadvantages of weak coating interface bonding, reduced coating activity reaction, and easy peeling of functional layer.

[0035] (5) Compared with the structural regulation and simple blending modification of traditional bacterial nanocellulose materials, the intelligent responsive anticoagulant material of the present invention avoids the problem of single function and realizes functional programmability and functional synergy.

[0036] (6) The present invention can use most of the proteins and peptides with anticoagulant effects as functional groups to bind to bacterial nanocellulose materials. Compared with chemical modification and physical coating, when the intelligent responsive anticoagulant material prepared by the present invention is applied to medical materials and devices and comes into contact with / implanted into the human body, it avoids the risks of residual chemical crosslinking agents, unstable coatings and large release of metal ions, avoids potential safety risks and avoids safety damage to the human body.

[0037] (7) The preparation of intelligent responsive anticoagulant materials by the present invention does not require the introduction of additional reagents, and the preparation method is simple and easy to implement. The preparation conditions are mild and controllable, green and environmentally friendly, and have good market application prospects.

[0038] (8) Compared with traditional processes, this invention can not only enable the material to bind more types of recombinant anticoagulants through the natural affinity between carbohydrate binding modules and bacterial nanocellulose, thereby improving functional synergy; it can also combine one or more recombinant anticoagulants with carbohydrate binding modules on the basis of anticoagulant polysaccharide-bacterial nanocellulose materials to achieve dual-effect anticoagulant performance and multifunctionality, which is beneficial to clinical applications; the reaction process is short and efficient, which is conducive to the industrial production of anticoagulant materials and devices. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the 3A assembly tool kit in Example 1.

[0040] Figure 2 This is a schematic diagram of the pBG3A series plasmids obtained in Example 1.

[0041] Figure 3 This is a schematic diagram of the pBBR1-pBG3A series plasmids obtained in Example 1.

[0042] Figure 4 This is a schematic diagram of the fluorescent protein fusion protein structure in Example 2.

[0043] Figure 5 AB is a schematic diagram of the construction of the fluorescent protein fusion protein vector in Example 2.

[0044] Figure 6 This is a diagram showing the expression of the fluorescent protein fusion protein in Example 2.

[0045] Figure 7 AB is an experimental diagram of the binding of fluorescent protein to the BNC membrane in Example 3.

[0046] Figure 8 This is a schematic diagram of the structure of the intelligent responsive recombinant hirudin-CBM fusion protein in Example 4.

[0047] Figure 9 This is a schematic diagram of the intelligent responsive recombinant hirudin-CBM fusion protein particle in Example 4.

[0048] Figure 10 This is an expression diagram of the intelligent responsive recombinant hirudin-CBM fusion protein in Example 4.

[0049] Figure 11 The results of the whole blood coagulation experiment of the intelligent responsive recombinant hirudin-CBM fusion protein in Example 4 are shown.

[0050] Figure 12 The results of the plasma recalcification experiment of the intelligent responsive recombinant hirudin-CBM fusion protein in Example 4 are shown.

[0051] Figure 13 This is a schematic diagram illustrating the construction of the intelligent responsive recombinant hirudin-CBM fusion protein in Example 5 and its binding with BNC-based materials.

[0052] Figure 14 The proliferation rate of BNC and smart-responsive anticoagulant BNC human umbilical vein endothelial cells in Example 5.

[0053] Figure 15 Activated partial thromboplastin time of BNC and smart responsive anticoagulant BNC in Example 5.

[0054] Figure 16 This is a statistical chart showing the number of platelet activations measured by the MTT assay for BNC, CS / BNC, and smart responsive anticoagulant / CS / BNC in Example 6.

[0055] Figure 17 The hemolysis rates of BNC, CS / BNC, and smart responsive anticoagulant / CS / BNC in Example 6 are given. Detailed Implementation

[0056] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0059] Example 1

[0060] Building the 3A Assembly Toolkit

[0061] To achieve modular vector construction, overcome the limitations of restriction enzyme sites in current Biobrick and Golden Gate assembly techniques, and realize 3A assembly while satisfying host variability, the vector is constructed into the following modules: a Biobrick module, a Golden Gate assembly module, a replicon replacement module, a resistance gene replacement module, and a termination module. Its structure is as follows: Figure 1 As shown.

[0062] The specific construction process of the 3A assembly toolkit:

[0063] (1) Construction of pBG3AA vector: Using pwtCas9-bacteria (Wuhan Miaoling Biotechnology Co., Ltd.) as a template, using SEQ ID NO:12 / SEQ ID NO:13 as primer pair, and using pET-15b (Wuhan Miaoling Biotechnology Co., Ltd.) as template, the backbone fragment 1 was amplified and purified; using SEQ ID NO:14 / SEQ ID NO:15 as primer pair, the T7 terminator fragment 2 was amplified and purified. Fragment 1 and fragment 2 were simultaneously digested with EcoRI and AflII restriction endonucleases, and the digestion products were purified. Then, ligation was performed, and the ligation product was transformed into E. coli DH5α (genotype: F-, φ80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17 (rK-, mK+), phoA,supE44, λ-, thi-1, The construct was validated by sequencing of competent cells (gyrA96, relA1). The successfully constructed intermediate plasmid was double-digested with EcoRI and XhoI to obtain product 3. Simultaneously, using pEcgRNA (Wuhan Miaoling Biotechnology Co., Ltd.) as a template and SEQ ID NO:16 / SEQ ID NO:17 as primers, the ccdB fragment was amplified and purified, and then double-digested with EcoRI and XhoI to obtain product 4. Products 3 and 4 were ligated to obtain a transition vector, which was then transformed into ccdBSurvival (genotype: F1). - mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (Str R (endA1 nupG fhuA::IS2) Competent cell sequencing verified the correct transition vector. Since there is a BsaⅠ restriction site in the Amp gene, the transition vector was point-mutated using SEQ ID NO:18 / SEQ ID NO:19 as primers to remove the BsaⅠ restriction site, resulting in the final vector pBG3AA, as shown in SEQ ID NO:11. The vector was then transferred into ccdB Survival for preservation.

[0064] (2) Construction of pBG3AT, pBG3AC, pBG3AG, and pBG3AK series vectors: Using pBG3AA as the backbone vector, its resistance gene module was modified. pBG3AA was double-digested with SacⅠ and AflⅡ restriction endonucleases and the target fragment 1 without the resistance gene was recovered. Using SEQ ID NO:20 / SEQ ID NO:21 as primer pair, the TcR fragment 2 in the pKD46-Tet (Wuhan Miaoling Biotechnology Co., Ltd.) vector was amplified by PCR. Using SEQ ID NO:22 / SEQ ID NO:23 as primer pair, the CmR fragment 3 in the pBAD33 (Wuhan Miaoling Biotechnology Co., Ltd.) vector was amplified by PCR. Using SEQ ID NO:24 / SEQ ID NO:25 as primer pair, the GmR fragment 4 in the pBBR1MCS-5 (Wuhan Miaoling Biotechnology Co., Ltd.) vector was amplified by PCR. Using SEQ ID NO:26 / SEQ ID Using primer pair NO:27, the Kan fragment 5 in the pUC57-Kan vector was amplified by PCR. Fragments 2, 3, 4, and 5 obtained above were double-digested with SacⅠ and AflⅡ restriction endonucleases, and then ligated with fragment 1 and transformed into ccdB Survival competent cells. The cells were plated with the corresponding antibiotic plates, and single colonies were picked for sequencing verification. After correct sequencing, the basic vectors pBG3AT-0, pBG3AC-0, pBG3AG-0, and pBG3AK were finally obtained. Because the TcR gene contains a BamHI restriction site, the GmR gene contains a BglII restriction site, and the CmR gene contains an EcoRI restriction site, the tetracycline gene in pBG3T-0 was first mutated using primers SEQ ID NO:28 / SEQ ID NO:29. Then, the chloramphenicol gene in pBG3AC-0 was mutated using primers SEQ ID NO:30 / SEQ ID NO:31. Finally, the chloramphenicol gene in pBG3AG-0 was mutated using primers SEQ ID NO:32 / SEQ ID NO:33, resulting in the pBG3AT, pBG3AC, and pBG3AG vectors. Any substitution of pBG3AA resistance with any other resistance gene besides the aforementioned resistance genes is within the scope of patent protection. A schematic diagram of the vector construction process is shown below. Figure 2 .

[0065] (3) Construction of pBBR1-pBG3AA, pBBR1-pBG3AT, pBBR1-pBG3AC, pBBR1-pBG3AG, pBBR1-pBG3AK series vectors: In order to expand the application scope of the host, pBG3AA, pBG3AT, pBG3AC, pBG3AT, and pBG3AK were used as the skeleton vectors, and their replication submodules were modified. The pBG3AA, pBG3AT, pBG3AC, pBG3AT, and pBG3AK vectors were purified by double digestion with XhoI and SpeI restriction endonucleases to obtain fragments A, T, C, G, and K. Using SEQ ID NO:34 / SEQ ID NO:35 as primers, the pBBR1 replicon fragment was amplified by PCR from the pBBR1MCS2-Tac-mCherry vector. After purification, the fragment R was obtained by double digestion with XhoI and SpeI restriction endonucleases. Fragments A, T, C, G, and K were ligated to fragment R to obtain different vectors. These vectors were transformed into ccdB Survival competent cells, plated on appropriate antibiotic plates, and single colonies were picked for sequencing verification. After successful sequencing, the final vector was obtained. Any substitution of the pBG3AA, pBG3AT, pBG3AC, pBG3AT, and pBG3AK vector replicons by any other microbial replicons is within the scope of patent protection. A schematic diagram of the vector construction process is shown below. Figure 3 .

[0066] Table 1 Primer Sequences

[0067]

[0068]

[0069] (4) Specific process of 3A assembly system and 3A-Golden Gate assembly system:

[0070] The 3A assembly utilizes three different resistances for multiple rounds of vector assembly, combined with ccdB screening, resulting in a vector assembly success rate as high as 80%. This invention utilizes the BglBrick bio-brick system, primarily based on BglII and BamHI, with their isoform BclI as a substitute restriction enzyme. The specific procedure is as follows: pBG3A vectors containing fragments 1 and 2 with different resistances are extracted, and unpurified products are obtained: EcoR1-BglII fragment 1-resistance A-BamHI, BglII-fragment 2-resistance B-BamHI-XhoI, and EcoR1-empty vector-resistance C-XhoI. These three unpurified digestion products are added to a centrifuge tube at a ratio of 3.5:3.5:1, along with 8 μL of DNA Ligation Kit.<Mighty Mix> (TAKARA) was mixed evenly and transferred into ccdBSurvival competent cells, plated on the corresponding antibiotic plate, and single colonies were picked for sequencing verification. After correct sequencing, the final vector was obtained.

[0071] However, this assembly method is only suitable for high-copy vectors. Therefore, it is relatively complex to operate on some non-type strains and host bacteria that can only use low-copy replicons such as pBBR1 or pSC101. The introduction of the Golden Gate assembly combined with the ccdB screening method simplifies the assembly of low-copy vectors. Based on the 3A assembly, the two BsaI sites in the pBBR1-pBG3A series vectors are digested with BsaI type II restriction endonuclease, and the target fragment in the 3A vector containing the BsaI site is amplified. Then, these fragments are digested and ligated directly with the unpurified digested vector without purification to obtain the correct ligation product. This invention nests the BsaI site and the ccdB gene in BglBrick, which avoids the interference of EcoRI / BglII / BamHI / BclI / XhoI and BsaI in the target gene, thus avoiding the increase in point mutations, increasing the probability of experimental success, and enabling rapid assembly of the target fragment at multiples.

[0072] Example 2

[0073] Preparation and concentration determination of red fluorescent-CBM fusion protein

[0074] To facilitate the demonstration of the fusion and recombination of the CBM-binding structure and the target protein, this embodiment uses red fluorescent protein as the model target protein. The aim is to construct a CBM binding assay vector and to induce the expression of the fusion protein by adding an inducing element to the vector. A schematic diagram of the fusion protein structure is shown below. Figure 4 As shown.

[0075] Carrier construction:

[0076] (1) Construction of pBG3AC-mCherry: Using pBBR1MCS2-Tac-mCherry as a template and SEQ ID NO:36 / SEQ ID NO:37 as primer pair, the mCherry sequence was obtained by PCR. The aforementioned sequence was inserted between the EcoRI and BamHI restriction enzyme sites of the pBG3AK vector to obtain the ligation product.

[0077] (2) pBG3AK-P T7 -TrxA: Using pET32a as a template and SEQ ID NO:38 / SEQ ID NO:39 as primers, lacI-P was obtained by PCR. T7 -lacO-TrxA, insert the aforementioned sequence between the EcoRI and XhoI restriction sites of the pBG3AK vector to obtain the ligation product.

[0078] (3) pBG3AA-P T7 Construction of the TrxA-mcherry vector: The vector pBG3AK-P was prepared using EcoRI and BamHI. T7 -TrxA was double-digested, and the vector pBG3AC-mCherry was double-digested with BglⅡ and XhoⅠ. The vector pBG3AA was double-digested with EcoRI and BamHI. The vector backbone fragment and lacI-P were recovered and purified. T7 The -lacO-TrxA fragment and the mcherry fragment were ligated to obtain the ligation product.

[0079] (4) pBG3AK-P T7 -TrxA-mcherry-T T7 Vector construction: Using pBG3AA-PT7-mcherry vector as a template, lacI-P was amplified using SEQ ID NO: 40 / SEQ ID NO: 41 primers. T7 The -lacO-TrxA-mcherry sequence, utilizing the original terminator T of the pBG3AK vector. T7 The amplified fragment was inserted between the XhoⅠ and EcoRI restriction sites of the pBG3AK vector to obtain the complete expression vector. Figure 5 A).

[0080] (5) Construction of pUC57-ChBD2: The gene sequence encoding ChBD2 was optimized and synthesized, and the aforementioned sequence was inserted between the SalⅠ and EcoRⅤ restriction sites of the pUC57 vector.

[0081] (6) Construction of pBG3AK-Linker-ChBD2 vector: Using pUC57-ChBD2 vector as template, the Linker-ChBD2 sequence was amplified using SEQ ID NO:42 / SEQ ID NO:43 as primers (Table 1). 6×His and the stop codon TGA were added to the 3′ end of the sequence and inserted between BglⅡ and XhoⅠ of pBG3AK through enzyme digestion and ligation reaction to obtain the ligation product.

[0082] (7) Construction of pBG3AG-mcherry-Linker-ChBD2 vector: pBG3AC-mcherry vector was double-digested with EcoRI and BamHI, pBG3AG vector was double-digested with BglII and XhoI, and pBG3AG vector was double-digested with EcoRI and XhoI. The purified vector backbone fragment, mcherry fragment and Linker-ChBD2 fragment were ligated to obtain the ligation product.

[0083] (8) pBG3AA-P T7 Construction of the TrxA-mcherry-Linker-ChBD2 vector: The vector pBG3AK-P was prepared using EcoRI and BamHI. T7 -TrxA was double-digested, and the vector pBG3AG-mcherry-Linker-ChBD2 was double-digested with BglⅡ and XhoⅠ. The vector pBG3AA was then double-digested with EcoRI and XhoⅠ. The resulting fragments were purified to obtain the vector backbone and lacI-P. T7 The -lacO-TrxA fragment and the mcherry-Linker-ChBD2 fragment were ligated to obtain the ligation product.

[0084] (9) pBG3AK-P T7 -TrxA-mcherry-Linker-ChBD2-T T7 Vector construction: using pBG3AA-P T7 Using the -mcherry-Linker-ChBD2 vector as a template, and primers SEQ ID NO:44 / SEQ ID NO:45 as primers, lacI-P was amplified. T7 The -lacO-TrxA-mcherry-Linker-ChBD2 sequence, utilizing the original terminator T of the pBG3AK vector. T7 The amplified fragment was inserted between the BamHI and EcoRI restriction sites of the pBG3AK vector to obtain the complete expression vector. Figure 5 B).

[0085] (10) pBG3AK-P T7 -Construction of Protein Of Interest (POI) vectors: To improve the expression level of target proteins, the TrxA fusion tag can be replaced with protein fusion tags such as GST, ELP, and SUMO through seamless cloning (Table 2) to increase protein expression levels.

[0086] Table 2. Amino acid and nucleotide sequences corresponding to protein fusion tags

[0087]

[0088] Expression of the target protein:

[0089] (1) Obtaining the expression strain: Take 10 μL of the extracted pBG3AK-P T7 -TrxA-mcherry-T T7 and pBG3AK-P T7 -Trx-A-mcherry-Linker-ChBD2-T T7 The plasmid was transformed into BL21(DE3) competent cells by chemical transformation and cultured at 37°C with shaking on LB agar medium containing 50 mg / L kanamycin for 12-16 h. Single clones were selected for sequencing verification, and clones with correct sequencing results were preserved in glycerol.

[0090] (2) Activation of bacterial strains: BL21(DE3) bacteria stored at -80℃ were inoculated into 10mL LB, with an additional 50 mg / L kanamycin added, and shaken overnight at 37℃.

[0091] (3) Scale-up culture and protein expression: The bacterial culture that had been cultured overnight was inoculated twice into 500 mL of LB medium at a ratio of 1:100 for scale-up culture, and cultured at 37 ℃ with shaking until OD. 600 The concentration was approximately 0.6. IPTG was added to initiate the expression of the fusion protein. The protein was induced overnight at 16℃, 37℃, and 180 rpm. After induction, the bacterial pellet was collected by centrifugation at 4℃ and 4000 rpm for 20 min.

[0092] Purification of the target protein:

[0093] (1) Add lysis buffer at a ratio of 4 mL per gram of bacterial sludge. Add lysis buffer to the collected bacterial pellet and resuspend the bacterial cells thoroughly (the resuspension process is performed on ice). Place the resuspended bacterial solution in an ice-water bath for ultrasonic disruption (5 s sonication, 7 s interval, 99 cycles). Repeat this process 3 times to achieve complete lysis of bacterial cells. Centrifuge the disrupted sample at 12,000 rpm for 30 minutes at 4°C and collect the supernatant for later use.

[0094] (2) Place Ni-NTA agarose gel into an affinity chromatography purification column. First, rinse the column bed with ultrapure water, and then equilibrate the column with lysis buffer. Add the supernatant obtained in step (1) to the equilibrated purification column and incubate on ice for 1 hour to allow the target protein to fully bind with Ni-NTA agarose. After incubation, wash the column multiple times with wash buffer to remove unbound contaminants; then elute the column with elution buffer four times to fully wash off the target protein bound to the column. After elution, wash the column three times with ultrapure water and equilibrate the column again with lysis buffer for subsequent use or storage.

[0095] Target protein concentration and concentration determination:

[0096] (1) Add the collected target protein to the ultrafiltration column and centrifuge multiple times. Replace the protein buffer with 1×PBS solution to concentrate the target protein.

[0097] (2) The final concentration of the target protein obtained is determined by the BCA method or the concentration and purity of the purified protein are determined by an ultra-micro spectrophotometer.

[0098] SDS-PAGE results are as follows Figure 6 As shown, after IPTG-induced expression, high-purity fusion proteins TrxA-mCherry and TrxA-mCherry-Linker-ChBD2 with single bands were obtained, and their actual molecular weights were basically consistent with their theoretical molecular weights of 39.05 kDa and 51.5 kDa, respectively. Protein concentration measurements showed that the concentrations of the obtained mCherry and mCherry-ChBD2 fusion proteins were 1.3 mg / mL and 1.2 mg / mL, respectively.

[0099] Example 3

[0100] Preparation of bacterial cellulose nanomaterials containing CBM-red fluorescent protein

[0101] To verify the stable composite of recombinant protein and bacterial nanocellulose material, the following work was carried out in this embodiment:

[0102] (1) Using Komagataeibacter xylinus ATCC 23770, BNC tubes or BNC membranes with an inner diameter of 3 mm, an outer diameter of 8 mm, and a length of 15 cm were prepared by static culture. Then, they were immersed in 0.1 M NaOH solution at 80°C and washed repeatedly 4-6 times until the BNC tubes or BNC membranes turned milky white. Then, they were immersed in deionized water until neutral to obtain purified BNC tubes or BNC membranes.

[0103] (2) The bacterial cellulose membrane was immersed in the TrxA-mCherry-ChBD2 fusion protein solution obtained in Example 2 for 24 h at room temperature. At the same time, the TrxA-mCherry protein solution was set as the control group. The initial concentration of the protein solution was uniformly 1 mg / mL.

[0104] (3) Fluorescent protein membrane binding experiment

[0105] After soaking and binding, the bacterial cellulose membrane was removed and immersed in 5 mL of PBS solution (pH=7.4) at 4℃ for 12 h to remove non-specific adsorption. After removing the surface solution, the membrane was photographed using a fluorescence imager, ensuring that parameters such as depth of field, gain, and exposure time remained constant during the imaging process. The average fluorescence intensity of the sample was then analyzed using ImageJ. The procedure was as follows: ① Extract a single channel; ② Adjust the threshold to select an appropriate region; ③ Set the parameters to be measured; ④ Detect. Average fluorescence intensity = sum of fluorescence intensities in the region / area of ​​the region. Each sample was tested in triplicate. The experimental results are as follows: Figure 7 As shown in Figure A, the color of the BNC membrane in the mCherry / BNC group changed from purple to light purple upon visual observation after PBS immersion, while the color change of the BNC membrane in the mCherry-ChBD2 / BNC group was not significantly different before and after PBS immersion. The average fluorescence intensity is shown in Figure A. Figure 7 As shown in Figure B, the fluorescence intensity of the BNC membrane in the mCherry / BNC group decreased significantly after PBS immersion, while there was no significant difference in the average fluorescence intensity of the BNC membrane in the mCherry-ChBD2 / BNC group. These membrane binding experiment results demonstrate that ChBD2 endows the fusion protein with strong BNC membrane adsorption, and ChBD2 has great potential to be used as a tool for BNC biomodification.

[0106] Example 4

[0107] Obtaining and Assessing the Anticoagulant Activity of a Smart-Responsive Recombinant Hirudin-CBM Fusion Protein

[0108] Example 3 above demonstrated that CBM can endow the fusion protein with strong BNC membrane adsorption. Therefore, this example aims to obtain a CBM-containing smart-responsive recombinant hirudin fusion protein. The hirudin in this example is hirudin variant I (HV). The overall protein structure diagram is shown below. Figure 8 As shown.

[0109] 1. Construction of the carrier:

[0110] (4) Construction of the pBG3AK-Fr-HV vector: The gene sequence encoding hirudin was optimized and synthesized (Table 3). Then, a coagulation factor Xa cleavage site was introduced at the N-terminus of the hirudin protein. Its amino acid recognition sequence is Ile-Glu-Gly-Arg (IEGR), named FXa-responsive peptide (Fr), and the corresponding gene sequence is ATTGAAGGCCGT. The aforementioned Fr-HV sequence was inserted between BglⅡ and BamHⅠ in the pBG3AK vector.

[0111] (5) Construction of pBG3G-Fr-HV-Linker-ChBD2 vector: pBG3AK-Fr-HV vector was double-digested with EcoRI and BamHI, pBG3K-Linker-ChBD2 vector was double-digested with BglII and XhoI, and pBG3AG vector was double-digested with EcoRI and XhoI. The recovered and purified vector backbone fragment, Fr-HV fragment and Linker-ChBD2 fragment were ligated to obtain the ligation product.

[0112] Table 3. Amino acid and nucleotide sequences corresponding to hirudin proteins

[0113]

[0114] (6) pBG3AC-P T7 Construction of the -TrxA-Fr-HV-Linker-ChBD2 vector: The promoter of vector pBG3AK-T7-TrxA was double-digested with EcoRI and BamHI, vector pBG3G-Fr-HV-Linker-ChBD2 was double-digested with BglII and XhoI, and vector pBG3AC was double-digested with EcoRI and XhoI. The vector backbone fragment and promoter P were recovered and purified. T7 The fragment and the HV-Linker-ChBD2 fragment were ligated to obtain the ligation product.

[0115] (7) pBG3AK-P T7 -TrxA-Fr-HV-Linker- ChBD2-T T7Construction: using pBG3AC-P T7 Using the TrxA-Fr-HV-Linker-ChBD2 vector as a template, and primers SEQ ID NO: 44 / SEQ ID NO: 45 as primers, P was amplified. T7 The -TrxA-Fr-HV-Linker-ChBD2 sequence, utilizing the original terminator T of the pBG3K vector. T7 The amplified fragment was inserted between the BamHI and EcoRI restriction sites of the pBG3AK vector to obtain the complete expression vector. Figure 9 ).

[0116] (8) pBG3AK-P T7 -Protein fusion tag-Fr-HV-Linker-ChBD2-T T7 Vector construction: To enhance the expression level of hirudin, the TrxA fusion tag opposite the N-terminal coagulation factor Xa cleavage site of hirudin can be replaced with protein fusion tags such as GST, ELP, and SUMO through seamless cloning to increase protein expression.

[0117] (9) Take 10 μL of the extracted pBG3AK-P T7 -TrxA-Fr-HV-Linker- ChBD2-T T7 The plasmid was transformed into BL21(DE3) competent cells by chemical transformation and cultured at 37°C with shaking on LB agar medium containing 50 mg / L kanamycin for 12-16 h. Single clones were selected for sequencing verification, and clones with correct sequencing results were preserved in glycerol.

[0118] 2. Expression, purification, and concentration determination of intelligent responsive recombinant hirudin-CBM fusion protein

[0119] The expression, purification, and concentration determination of the target protein were performed according to the methods described in Example 2. Figure 10 The SDS-PAGE results shown indicate that, after IPTG-induced expression, a single, high-purity fusion protein, TrxA-Fr-HV-Linker-ChBD2, was obtained, and its actual molecular weight is basically consistent with the theoretical molecular weight of 33 kDa. Protein concentration measurements showed that the obtained TrxA-Fr-HV-Linker-ChBD2 fusion protein concentration was 1.2 mg / mL.

[0120] 3. Determination of the anticoagulant activity of intelligent responsive recombinant hirudin-CBM fusion protein

[0121] (1) Take 50 μL of 1 g / ml recombinant protein, add 1 μL of Fxa (New England Biolabs) to it, and react the reaction system at room temperature for 4 h to obtain the cleaved protein sample.

[0122] (2) Prepare 0.5% bovine fibrinogen (Shanghai Maclean Biochemical Technology Co., Ltd.) and working concentration recombinant thrombin (Shanghai Yuanye Biotechnology Co., Ltd.) solutions with 50 mM Tris-HCl buffer (containing 50 mM NaCl, pH 7.4): 40 U / mL and 100 U / mL.

[0123] (3) Add 200 μL of a solution containing 0.5% bovine fibrinogen to a 2 mL EP tube, then add 100 μL of the protein solution to be tested to the tube, shake slowly, and incubate in a metal bath at 37°C for 5 min.

[0124] (4) Add thrombin solution (40 U / mL, 5 μL per minute) dropwise while gently shaking until coagulation (or coagulation occurs), record the volume of thrombin solution consumed, and repeat the measurement 3 times.

[0125] (5) Use an equal volume of physiological saline to replace the protein solution and thrombin as a blank control in the experiment. Other conditions remain unchanged and the same procedure is followed.

[0126] (6) Calculate the antithrombin activity according to the formula: U = C1V1 / C2V2;

[0127] U represents the antithrombin activity units per 1 g of sample, U / g; C1 represents the concentration of the thrombin solution, U / mL; C2 represents the concentration of the test solution, g / mL; V1 represents the volume of thrombin solution consumed, μL; V2 represents the amount of test solution added, μL.

[0128] The activity of recombinant hirudin was determined to be 506 U / mg.

[0129] 4. Intelligent responsive recombinant hirudin-CBM fusion protein whole blood coagulation assay

[0130] Whole blood clotting time test

[0131] (1) Use a 10 mL syringe to draw 5 mL of fresh rabbit whole blood using the method of heart blood collection, and inject it into a blood collection tube containing anticoagulant.

[0132] (2) Inject 500 μL of CaCl2 (0.1 M) solution into 5 mL of fresh rabbit anticoagulated whole blood to activate the coagulation reaction.

[0133] (3) Add a BNC membrane containing recombinant hirudin-CBM fusion protein (0.96 mg / mL) to a 24-well plate. Inject 50 μL of activated blood evenly onto the sample surface in each well of the 24-well plate and incubate at 37°C. At 5, 15, 25, 35, 45, and 55 min, add 2 mL of ultrapure water along the wall into the well and gently mix. Incubate for another 5 min, then aspirate 200 μL into three replicates in a 96-well plate to determine A. 540 The concentration of hemoglobin was assessed, and thus the coagulation effect of each group was evaluated. Fresh rabbit whole blood contains coagulation-related enzymes, and CaCl₂ was added. 2， Therefore, theoretically, during blood clotting, coagulation factor X (Fx) is activated into coagulation factor Xa (Fxa). At this time, because the recombinant hirudin-CBM fusion protein contains an Fxa response site, the Ile-Glu-Gly-Arg tetrapeptide in the recombinant hirudin-CBM fusion protein is recognized and cleaved by Fxa, releasing active recombinant hirudin, which binds to thrombin to exert an anticoagulant effect. Experimental results show that, compared with the control group saline, the TrxA-Fr-HV-ChBD2 fusion protein slows down the blood clotting rate. Figure 11 This demonstrates that the fusion protein possesses intelligently responsive anticoagulant properties. Furthermore, increasing the binding amount of the recombinant hirudin-CBM fusion protein further slows down blood clotting.

[0134] 5. Plasma recalcification time test

[0135] (1) Place the sample in a 48-well plate, add 500 μL of anemic platelet plasma (PPP), place it on a constant temperature shaker at 37˚C, and shake at 100 rpm for 1 h. The control group is an empty plate with 500 μL of PPP added, and the incubation conditions are the same as the sample.

[0136] (2) Take 100 μL of incubated plasma and transfer it to a 96-well plate. Each sample is in triplicate. Quickly add 100 μL of CaCl2 aqueous solution (0.025 M) to each well. The negative control is 100 μL of PPP incubated in the well plate plus 100 μL of physiological saline. The positive control is 100 μL of PPP incubated in the well plate plus 100 μL of ultrapure water.

[0137] (3) Immediately place the sample in an ELISA reader and incubate at 37°C. Set up a dynamic testing program and measure the absorbance at 405 nm every 30 seconds for 45 minutes to obtain the plasma recalcification kinetic curve. The time to reach half of the maximum absorbance value is the plasma recalcification time (HM time). Figure 12 ).

[0138] Example 5

[0139] Preparation of Smart-Response Anticoagulant Bacterial Nanocellulose Materials by Combining Recombinant Proteins and Bacterial Nanocellulose Materials

[0140] (1) Using Komagataeibacter xylinus ATCC 23770, BNC tubes or BNC membranes with an inner diameter of 3 mm, an outer diameter of 8 mm, and a length of 15 cm were prepared by static culture. Then, they were immersed in 0.1 M NaOH solution at 80°C and washed repeatedly 4-6 times until the BNC tubes or BNC membranes turned milky white. Then, they were immersed in deionized water until neutral to obtain purified BNC tubes or BNC membranes.

[0141] (2) The recombinant hirudin fusion protein solution containing CBM obtained in Example 1 was added to the BNC tube or BNC membrane obtained in step (1), and the reaction was allowed to proceed at 4°C for 12 hours. Subsequently, the material was immersed and washed in PBS buffer for a total of 3 times, 2 hours each time, to wash away the unbound recombinant protein, and the anticoagulant bacterial nanocellulose composite material was obtained (the preparation principle and process are as follows). Figure 13 ).

[0142] (3) In vitro cell compatibility

[0143] Ethylene oxide-sterilized BNC and TrxA-Fr-HV-ChBD2 / BNC were placed in 24-well plates, with 500 μL added to each well, for a total volume of 10. 4 Human umbilical vein endothelial cells were cultured in fresh medium every other day. Cell proliferation was assessed using a CCK-8 assay kit at 1, 3, and 5 days of culture. Figure 14 As shown, with the extension of culture time, TrxA-Fr-HV-ChBD2 / BNC significantly increased the cell proliferation rate of BNC and had the effect of promoting endothelial cell proliferation.

[0144] (4) In vitro blood compatibility

[0145] Blood was drawn from the marginal ear vein of rabbits using disposable vacuum sodium citrate (9:1) anticoagulant tubes. The blood was gently mixed and centrifuged to collect the supernatant plasma (3000 rpm, 30 min). Ethylene oxide-sterilized BNC and TrxA-Fr-HV-ChBD2 / BNC were placed in sterile centrifuge tubes. After adding plasma, the mixture was incubated at 37ºC for 5 min. An activated partial thromboplastin time (APTT) kit was then added, and the mixture was tested. Figure 15 As shown, the recombinant hirudin fusion protein significantly improved the APTT of BNC, indicating that TrxA-Fr-HV-ChBD2 / BNC has good anticoagulant properties.

[0146] Example 6

[0147] Smart responsive anticoagulant chitosan / bacterial nanocellulose materials

[0148] (1) Chitosan / bacterial nanocellulose composite material

[0149] Chitosan was dissolved in a 1% glacial acetic acid aqueous solution to prepare a 1% chitosan solution. The purified BNC tubes or membranes were immersed in the 1% chitosan solution and equilibrated in a water bath at 30 °C and 100 r / min for 24 h. The composite material was then removed, and the chitosan on the tube surface was rinsed with deionized water. It was then placed in a prepared 0.1 mol / L sodium hydroxide aqueous solution and allowed to stand for 24 h to fix the chitosan through alkaline precipitation (or, sodium sulfate, sodium pyrophosphate, or other ionotropic gelation methods could be used to form microspheres of CS for fixation). After fixation, the resulting CS / BNC composite material was removed, rinsed repeatedly with ultrapure water (pH=7.0), sterilized at 121 °C for 20 min, and then stored at 4 °C for later use.

[0150] (2) Intelligent responsive recombinant hirudin-CBM fusion egg and chitosan / bacterial nanocellulose material composite

[0151] The recombinant hirudin protein solution containing CBM obtained in Example 5 was added to the CS / BNC tube or CS / BNC membrane obtained in step (1), and allowed to stand for 12 hours at 4°C. Then the material was soaked and washed in PBS buffer for a total of 3 times, each time for 2 hours, to wash away the unbound recombinant protein and obtain the anticoagulant bacterial nanocellulose composite material.

[0152] (3) In vitro blood compatibility

[0153] ① Platelet activation assay: The material was evenly spread in a 24-well plate. 400 μL of the supernatant obtained from centrifugation (2000 rpm, 10 min) was added to the surface of the material, and incubated at 37ºC for 2 h. Then, the plasma was removed, and the platelets were washed three times with physiological saline to remove any unadhered platelets. 500 μL of MTT diluent (working concentration 0.5 mg / mL) was added, and the plate was incubated at 37ºC for 4 h. After incubation, the solution in the wells was aspirated, 1 mL of DMSO solution was added, and the plate was shaken at 37ºC for 30 min. Then, 200 μL of the DMSO reaction solution was aspirated into a 96-well plate, and the absorbance was measured at 492 nm using a microplate reader. Each sample was tested in triplicate. Figure 16As shown, CS / BNC significantly increased platelet activation on the BNC surface. This may be related to the presence of amino and hydroxyl groups in the chitosan structure. These groups can specifically bind to glycoprotein receptors on the platelet surface, thereby triggering platelet activation signaling pathways, leading to platelet activation and aggregation. The platelet activation level of TrxA-Fr-HV-ChBD / CS / BNC was significantly lower than that of CS / BNC and lower than that of BNC. This indicates that the addition of recombinant hirudin effectively reduced the platelet activation level on the surfaces of CS / BNC and BNC, suggesting that TrxA-Fr-HV-ChBD / CS / BNC is a material with excellent anticoagulant properties.

[0154] ② Hemolysis rate: The materials were cut into equal sizes and placed in 24-well plates, then immersed in 2 mL of physiological saline. A material-free group containing 2 mL of physiological saline was used as a negative control, and a material-free group containing 2 mL of ultrapure water was used as a positive control. All samples were incubated at 37ºC for 2 h, and then 1.5 × 10⁻⁶ ml of ultrapure water was added to each well. 8 Collect 100 red blood cells and incubate them at 37ºC for 1 h. Centrifuge the red blood cell suspension at 750 × g for 5 min, transfer 200 μL of the supernatant to a 96-well plate, and measure the absorbance at 540 nm. The hemolysis rate (R0) is then determined. h The calculation is as follows:

[0155] R h = (ODtest – Odneg) / (ODpos – Odneg) × 100%.

[0156] like Figure 17 As shown, the hemolysis rate of all BNC-based materials is less than 1%, which is lower than the hemolysis rate standard for Class III medical devices (<5%). This means that none of the three materials mentioned above will cause hemolysis, meeting the blood compatibility requirements for implantable blood contact medical devices.

Claims

1. A recombinant hirudin fusion protein, characterized in that: The fusion protein is a recombinant hirudin protein with a carbohydrate binding module (CBM), comprising a first part protein fusion tag, a second part hirudin or hirudin derivative, and a third part CBM; wherein the first and second parts are linked to the recognition sequence of coagulation-related enzymes via a flexible protein linker sequence, and the second and third parts are linked via a flexible protein linker sequence.

2. The recombinant hirudin fusion protein according to claim 1, characterized in that: The CBM includes one or more of ChBD2, ChBD3, CBM2a, CBM3, and CBDCex; the hirudin is recombinant hirudin, specifically including hirudin variant 1, hirudin variant 2, or hirudin variant 3, or recombinant hirudin fused with human fibronectin peptide, RGD peptide, REDV peptide, or nematode anticoagulant peptide; the coagulation-related enzyme is one or more of thrombin, coagulation factor VIIa, coagulation factor IXa, coagulation factor Xa, coagulation factor XIa, coagulation factor XIIa, and coagulation factor XIIIa.

3. The recombinant hirudin fusion protein according to claim 1, characterized in that: The recombinant hirudin protein containing CBM was constructed using the 3A assembly toolkit, and then expressed, isolated, and purified in a microbial strain.

4. The recombinant hirudin fusion protein according to claim 3, characterized in that: The basic vector in the 3A assembly toolkit includes a Bglbrick bio-brick module, a Golden Gate assembly module, a replicon replacement module, a resistance gene replacement module, and a termination submodule; the resistance gene in the basic vector of the 3A assembly toolkit is one or more of ampicillin, tetracycline, chloramphenicol, gentamicin, and kanamycin.

5. The recombinant hirudin fusion protein according to claim 3, characterized in that: The microbial strain is one or more of Escherichia coli, yeast, Corynebacterium glutamicum, and cellulose-producing bacteria.

6. A smart responsive anticoagulant material, characterized in that: It is obtained by combining the recombinant hirudin protein as described in claim 1 with bacterial nanocellulose or a composite material thereof.

7. The intelligent responsive anticoagulant material according to claim 6, characterized in that: The bacterial nanocellulose composite material is a bacterial cellulose composite material with polysaccharides deposited that can bind to CBM.

8. A method for preparing the smart responsive anticoagulant material as described in claim 6, comprising the following steps: (1) The recombinant hirudin protein fused with CBM was constructed using the 3A assembly toolkit, then expressed, isolated and purified in a microbial strain, and then prepared into a recombinant hirudin protein solution fused with CBM; (2) Add a solution of recombinant hirudin protein fused with CBM to the purified bacterial nanocellulose substrate or its composite material, allow it to stand for reaction, and soak and wash with phosphate buffer to remove unbound recombinant protein to obtain a smart responsive anticoagulant material.

9. The preparation method according to claim 8, characterized in that: The concentration of the recombinant hirudin protein solution fused with CBM in step (1) is 0.5-1 mg / mL.

10. The use of a smart responsive anticoagulant material as described in any one of claims 6 in the preparation of materials for preventing thrombosis.

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