Recombinant hirudin fusion protein with collagen affinity and preparation method and application thereof

Abstract

This invention relates to a recombinant hirudin fusion protein with collagen affinity, its preparation method, and its applications. The fusion protein is a recombinant hirudin fusion protein incorporating a collagen-binding domain (CBD). The fusion protein of this invention possesses affinity for type I and type III collagen, and can inhibit thrombin activity to exert an anticoagulant effect. Furthermore, it can bind well to collagen-based composite materials, making it suitable for use in cardiovascular implants such as heart valves, cardiac occluders, dialysis membranes, oxygenation membranes, artificial blood vessels, and vascular stents, as well as in extracorporeal circulation and blood purification devices, enhancing anticoagulant and antithrombotic effects.

Description

Technical Field

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

[0002] Collagen is one of the most abundant proteins in living organisms and a major component of the extracellular matrix of vertebrates. Collagen is classified into various categories according to its structure and function, but all of them have a unique triple helix structure, which gives them good mechanical properties. In addition, due to its good biocompatibility and wide availability (the main sources of natural collagen are relatively abundant poultry, livestock and marine animal resources, etc.), it has been increasingly widely used in biomedical materials. Collagen-based medical materials are mainly divided into the following categories: (1) Bone graft scaffolds: Since the protein component in human bones is mainly collagen, the ability of exogenous natural collagen to repair bone defects and regenerate bone, and to supplement the inorganic components (hydroxyapatite) necessary for bones, can serve as a good cartilage substitute and bone scaffold. (2) Blood contact materials and wound dressings: Due to its good biocompatibility and special porous structure, collagen is widely used in wound dressings and hemostatic materials. Studies have shown that collagen can activate platelets and promote the production of clotting factors, which is the basis for the hemostasis of collagen materials; on the other hand, collagen may also contain a large number of growth factors, which can promote cell proliferation and accelerate the healing process during wound healing. (3) Periodontal regeneration and mucosal tissue healing in oral medicine: Collagen not only has a certain hemostatic ability, but also has the function of promoting tissue regeneration, which is very important for the application of this material in oral medicine. Collagen sponge can not only be used for trauma management after tooth extraction, but also as a barrier membrane for the repair of periodontal bone defects, playing a role in guiding the regeneration of periodontal tissue. (4) Heart valves and tissue engineering: Collagen-based heart valves have natural biocompatibility, reduce the risk of immune rejection, and have good cell adhesion and proliferation support capabilities, and can be personalized through 3D printing and other technologies. (5) Artificial blood vessels and vascular stents: Large-diameter blood vessel substitutes need to undergo a pre-coagulation process in the early stage of the experiment, while collagen, as a coating of vascular grafts, can block the pores of textile blood vessels, replacing the traditional pre-coagulation process or other special operations, thereby achieving the effect of immediate hemostasis. For small-diameter artificial blood vessels, collagen also has certain practical application value due to its good biocompatibility and its role in promoting endothelialization.

[0003] While collagen possesses good biocompatibility and promotes endothelialization, its potential procoagulant effect cannot be ignored. Therefore, there is an urgent need for anticoagulant functionality in collagen-based medical materials. The anticoagulant functionalization of natural collagen typically requires physical or chemical modification, or simply compounding with other materials. However, in physical or chemical modifications and compounding with multiple materials, simple material layering can lead to unstable bonding and easy detachment, while the introduction of cross-linking agents such as glutaraldehyde and EDC can cause residual contamination and enhanced cytotoxicity. Furthermore, clinically used bio-based heart valves, made from animal tissues / organs such as bovine pericardium and porcine aortic valves through decellularization, are essentially composed of collagen fibers, and these materials and devices also require better anticoagulant functionality. Therefore, a green solution for the anticoagulant functionalization of collagen is urgently needed.

[0004] Hirudin (HV) is a polypeptide extracted from the salivary glands of leeches. It is the most potent natural thrombin inhibitor discovered to date, specifically binding to thrombin to prevent thrombus formation. However, hirudin has drawbacks such as a short half-life and low bioavailability, requiring frequent injections in clinical use to achieve a long-term anticoagulant effect. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant hirudin fusion protein with collagen affinity, its preparation method and application. The collagen binding domain is fused and expressed at the C-terminus of hirudin through genetic engineering, and then formed with collagen-based bacterial cellulose or other collagen-based materials to form a medical material with dual functions of anticoagulation and collagen binding strength and resistance to blood flushing.

[0006] Collagen-binding domain (CBD) mediated protein fixation strategy should have the following unique advantages: (1) Precise positioning: By utilizing the natural affinity of CBD with different types of collagen, recombinant proteins can be specifically anchored to the surface of collagen-based composite materials; (2) Green process: By using recombinant protein expression and mild adsorption process, the use of toxic reagents is eliminated; Functional programmability: Fusion protein design allows for the modular design of multifunctional recombinant proteins.

[0007] Taking advantage of the fact that the activity of hirudin is masked after its N-terminus is modified, a smart responsive recombinant hirudin fusion protein with coagulation-related enzyme responsiveness 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 can specifically respond 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, thereby specifically recognizing and cleaving peptide sequences in the fusion protein, releasing active hirudin or hirudin derivatives, and specifically binding to thrombin to exert an anticoagulant effect.

[0008] Therefore, if recombinant hirudin protein, which specifically responds to enzymes related to the coagulation process, can be further fused and expressed with a CBD-binding structure, it will be possible to endow collagen-based materials with intelligent responsive anticoagulant properties. That is, after the material comes into contact with blood, it maintains low activity when there is no 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 is safer than heparin, a traditional clinical anticoagulant that can cause systemic bleeding. It is worth noting that the technology of combining CBD-based anticoagulant fusion proteins with collagen-based materials is currently a blank area globally.

[0009] This invention provides a recombinant hirudin fusion protein with collagen affinity. The fusion protein is a recombinant hirudin fusion protein fused with a collagen-binding domain (CBD), comprising a first fusion protein tag TrxA, a second hirudin or a hirudin derivative, and a third CBD. The first and second parts are linked by a flexible protein linker sequence Linker-recognition sequence of coagulation-related enzymes, and the second and third parts are linked by a Linker sequence.

[0010] 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 the Gly-Ser linker or (Gly-Gly-Gly-Gly-Ser)n linker.

[0011] Preferably, the CBD is a polypeptide or protein that can specifically bind to collagen, including one or more of the following: an engineered heptapeptide derived from mammalian collagenase, collagenase colG or colH derived from Clostridium histolyticum, a metalloproteinase from Vibrio mimicus, or the A3 region of vasomotor hemophilia factor vWF.

[0012] The amino acid sequence of the collagenase Co1G is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2; the amino acid sequence of the collagenase Co1H is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4; the amino acid sequence of the engineered heptapeptide is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:6; the amino acid sequence of the Vibrio mimicry metalloproteinase is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:8; the amino acid sequence of vWF-A3 is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:10.

[0013] Table 1. Amino acid and nucleotide sequences corresponding to collagen-binding domains

[0014]

[0015] Preferably, the hirudin derivative includes hirudin variant 1, hirudin variant 2, and hirudin variant 3; or it is a recombinant hirudin fused with anticoagulant peptides such as human fibronectin peptide, RGD peptide, REDV peptide, and nematode anticoagulant peptide. The hirudin derivative is a hirudin analog or derivative known in the art, such as lepirudin, desirudin, and bivalirudin.

[0016] 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.

[0017] More preferably, the CBD-fused recombinant hirudin protein is one or more of the following combinations: Trxa-Fr-hirudin-heptacapeptide, ELP-Fr-hirudin-heptacapeptide, SUMO-Fr-hirudin-heptacapeptide, and GST-Fr-hirudin-heptacapeptide. Most preferably, the CBD-fused recombinant hirudin protein is the Trxa-Fr-hirudin-heptacapeptide recombinant protein.

[0018] Preferably, the recombinant hirudin fusion protein is constructed using a 3A (Three antibiotic assembly) assembly toolkit, then transferred to an expression host, and the target protein is obtained through expression, separation, and purification.

[0019] 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.

[0020] 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).

[0021] Preferably, the microbial strain is one or more of the following: Gram-negative bacteria, such as *Escherichia coli* BL21(DE3) (genotype: F-ompThsdSB (rB-, mB-) 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).

[0022] Preferably, the expression host includes one or more of Escherichia coli, yeast, Corynebacterium glutamicum, cellulose-producing bacteria, and mammalian cells.

[0023] The present invention also provides a smart responsive collagen-based anticoagulant material, which is obtained by compounding the recombinant hirudin fusion protein with collagen-based bacterial cellulose or other collagen-based materials.

[0024] Preferably, the composite method includes the following steps:

[0025] (1) The recombinant hirudin fusion protein was expressed, isolated and purified in a microbial strain, and then prepared into a recombinant hirudin protein solution;

[0026] (2) The purified bacterial nanocellulose substrate or other collagen-based material is placed in an acetic acid solution containing porcine collagen and shaken on a shaker. After the sample is removed, it is shaken in a genipin solution to fully soak and crosslink. After crosslinking is completed, it is rinsed with distilled water until the residual glutaraldehyde is completely removed, and the collagen / bacterial nanocellulose substrate or other collagen-based composite material is obtained.

[0027] (3) The prepared collagen / bacterial nanocellulose substrate or other collagen-based composite material is added to the recombinant hirudin protein solution, allowed to stand for reaction, and then soaked and washed with phosphate buffer to remove unbound recombinant protein, thus obtaining a smart responsive collagen-based anticoagulant material.

[0028] Preferably, the concentration of porcine skin collagen in step (2) is 0.25%-1%.

[0029] Preferably, the concentration of acetic acid in step (2) is 0.1%.

[0030] Preferably, the concentration of genipin in step (2) is 1-5 mM / L, more preferably, the concentration is 3 mM / L.

[0031] Preferably, the phosphate buffer in step (3) is purchased from Wuhan Saiweier Biotechnology Co., Ltd., and its main components are 10 mM sodium phosphate, 150 mM NaCl, and pH 7.2-7.4.

[0032] Preferably, the static reaction temperature in step (3) is 4 degrees Celsius, and the reaction time is 12-24 hours.

[0033] Preferably, when the collagen-based bacterial cellulose substrate or other collagen-based composite material is tubular, the recombinant hirudin protein solution is injected into the tube, and the lower end of the tube is sealed by tying a knot to prevent the protein solution from leaking out. After the injection is completed, 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.

[0034] Preferably, when the collagen-based bacterial nanocellulose substrate or other collagen-based composite material is in the form of a film, the recombinant hirudin protein solution is impregnated with the collagen-based bacterial nanocellulose substrate or other collagen-based composite material.

[0035] Preferably, the cleaning conditions in step (3) are to soak and clean the product in the freshly prepared phosphate buffer solution for a total of 3 times, each time for 2 hours.

[0036] The present invention also provides an application of the aforementioned intelligent responsive collagen-based anticoagulant material in the preparation of materials for preventing thrombosis, including cardiovascular implants, such as artificial blood vessels, vascular grafts, vascular stents, artificial heart valves, heart occluders, etc., and extracorporeal blood circulation and purification systems, such as hemodialysis systems, hemoperfusion devices, extracorporeal membrane oxygenation (ECMO) consumables, extracorporeal circulation tubing, etc.

[0037] This invention overcomes the difficulties in modifying collagen-based materials, avoiding damage to the integrity and functional activity of the composite material's structural network; it also avoids the problems of weak interfacial bonding and easy detachment of functional layers that exist in physical modification, and effectively avoids the cytotoxic effects that may be caused by residual chemical crosslinking agents or metal ions, thus avoiding potential safety risks.

[0038] In addition to the advantages mentioned above, this invention also features mild reaction conditions, adjustable and controllable parameters, and minimal environmental pollution. The collagen used in the preparation method can be derived from animal sources, recombinant collagen, decellularized matrix, tissue engineering materials, etc.

[0039] The cellulose matrix material that is combined with collagen is bacterial cellulose obtained through fermentation.

[0040] All strains used in the preparation method are model strains, and all basic vector templates used are commercially available.

[0041] Beneficial effects

[0042] (1) The fusion protein of the present invention has affinity for type I and type III collagen, and can inhibit the action of thrombin to exert anticoagulant effect. It can specifically respond to coagulation-related enzymes to restore anticoagulant activity. It can also bind well to collagen-based bacterial cellulose materials and other collagen-based materials. It is suitable for use in cardiovascular implants such as heart valves, heart occluders, dialysis membranes, blood oxygen membranes, artificial blood vessels, and vascular stents, as well as in extracorporeal circulation and blood purification devices. It can enhance anticoagulant and antithrombotic effects.

[0043] (2) This invention can not only enable the material to bind more types of recombinant anticoagulants through the natural affinity between the collagen binding module and the collagen-based composite material, thereby improving functional synergy; it can also combine one or more recombinant anticoagulants with collagen binding modules on the basis of anticoagulant polysaccharide-collagen-based composite material to achieve dual-effect anticoagulant performance and multifunctionality, which is beneficial to clinical application; the reaction process is short and efficient, which is conducive to the industrial production of anticoagulant materials and devices.

[0044] (3) This invention innovatively constructs the 3A assembly toolkit into the following modules: Bglbrick biobrick module, Golden Gate assembly module, replicon replacement module, resistance gene replacement module, and termination module, thereby realizing the modular construction of the vector and overcoming the limitations of restriction sites in the current research field of Biobrick assembly and Golden Gate assembly, thus realizing 3A assembly. Furthermore, the plasmid can be replicated in different hosts through the convenient replicon replacement module.

[0045] (4) Compared with the traditional oxidation and sulfation treatment in the chemical modification process, the preparation method of the intelligent responsive collagen-based anticoagulant material of the present invention has milder reaction conditions, and achieves the improvement of anticoagulant activity while ensuring the structural integrity and performance of the material.

[0046] (5) Compared with the physical coating modification process, the intelligent responsive collagen-based anticoagulant material of the present invention avoids the disadvantages of weak coating interface bonding, reduced coating activity reaction, and easy peeling of functional layer.

[0047] (6) Compared with the structural regulation and simple blending modification of traditional collagen-based composite materials, the intelligent responsive collagen-based anticoagulant material of the present invention avoids the problem of single function and realizes functional programmability and functional synergy.

[0048] (7) The present invention can use most of the proteins and peptides with anticoagulant effects as functional groups to bind to collagen-based composite 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.

[0049] (8) The preparation of intelligent responsive collagen-based anticoagulant material 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. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the 3A assembly tool kit in Example 1.

[0051] Figure 2 This is a schematic diagram of the pBG3A series plasmids obtained in Example 1.

[0052] Figure 3 This is a schematic diagram of the pBBR1-pBG3A series plasmids obtained in Example 1.

[0053] Figure 4This is a schematic diagram of the fluorescent protein fusion protein structure in Example 2.

[0054] Figure 5 AC is a schematic diagram of the construction of the fluorescent protein fusion protein vector in Example 2.

[0055] Figure 6 AC is the expression diagram of the fluorescent protein fusion protein in Example 2.

[0056] Figure 7 This is a diagram of the fluorescent protein binding to the Col / BNC membrane in Example 3.

[0057] Figure 8 This is a schematic diagram of the structure of the intelligent responsive recombinant hirudin-CBD fusion protein in Example 4.

[0058] Figure 9 This is a schematic diagram of the intelligent responsive recombinant hirudin-CBD fusion protein particle in Example 4.

[0059] Figure 10 This is an expression diagram of the intelligent responsive recombinant hirudin-CBD fusion protein in Example 4.

[0060] Figure 11 AB represents the whole blood coagulation test results of the intelligent responsive recombinant hirudin-CBD fusion protein in Example 4.

[0061] Figure 12 This is a schematic diagram illustrating the construction of the intelligent responsive recombinant hirudin-CBD fusion protein in Example 5 and its binding with collagen-based BNC materials.

[0062] Figure 13 The results of the experiment on the proliferation rate of human umbilical vein endothelial cells in the intelligent responsive anticoagulation collagen-based bacterial nanocellulose composite material in Example 5 are shown. Detailed Implementation

[0063] 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.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0065] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0066] Example 1

[0067] Building the 3A Assembly Toolkit

[0068] 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.

[0069] The specific construction process of the 3A assembly toolkit:

[0070] (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.

[0071] (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 .

[0072] (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 .

[0073] Table 2 Primer Sequences

[0074]

[0075]

[0076] (4) Specific process of 3A assembly system and 3A-Golden Gate assembly system:

[0077] 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.

[0078] 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 Golden Gate assembly combined with ccdB screening makes the assembly of low-copy vectors simple and easy to operate. It only requires digesting the two BsaI sites in the pBBR1-pBG3A series vectors with BsaI type II restriction endonuclease based on 3A assembly, and amplifying the target fragment in the 3A vector with BsaI sites. Then, these fragments can be directly ligated with unpurified digested vectors without purification to obtain the correct ligation product. This invention nests BsaI sites and ccdB genes in BglBrick, which can avoid the interference of EcoRI / BglII / BamHI / BclI / XhoI and BsaI in the target gene, thus avoiding the increase in the number of point mutations, increasing the probability of experimental success, and enabling the rapid assembly of the target fragment at multiple times.

[0079] Example 2

[0080] Preparation and concentration determination of red fluorescent-CBD fusion protein

[0081] To facilitate the demonstration that collagen CBD can be fused with the target protein for expression, this embodiment uses red fluorescent protein as the model target protein. The aim is to construct a target protein-CBD 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.

[0082] Carrier construction:

[0083] (1) Construction of pBG3AC-mCherry-His: Using pBBR1MCS2-Tac-mCherry as a template and SEQ ID NO:36 / SEQ ID NO:37 as primer pair, the mCherry-His sequence was obtained by PCR. The aforementioned sequence was inserted between the EcoRI and BamHI restriction sites of the pBG3AC vector to obtain the ligation product.

[0084] (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.

[0085] (3) pBG3AA-P T7 Construction of the TrxA-mcherry-His 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.

[0086] (4) pBG3AK-P T7 -TrxA-mcherry-His-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 BamHI and EcoRI restriction sites of the pBG3AK vector to obtain the complete expression vector. Figure 5 A).

[0087] (5) Construction of pBG3AC-mCherry-CBD (heptacapeptide)-His: Using pBBR1MCS2-Tac-mCherry as a template and SEQ ID NO:42 / SEQ ID NO:43 as primer pair, the mCherry-CBD (heptacapeptide)-His sequence was obtained by PCR. The aforementioned sequence was inserted between the EcoRI and BamHI restriction enzyme sites of the pBG3AC vector to obtain the ligation product.

[0088] (6) pBG3AA-P T7 Construction of the -TrxA-mCherry-Linker-CBD (heptacapeptide)-His vector: The vector pBG3AK-P was prepared using EcoRI and BamHI. T7 -TrxA was double-digested, and the vector pBG3AC-mCherry-Linker-CBD-His 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-CBD (heptacapeptide)-His fragment were ligated to obtain the ligation product.

[0089] (7) pBG3AK-P T7 -TrxA-mcherry-Linker-CBD (Heptapeptide)-His-T T7 Vector construction: using pBG3AA-P T7 Using the -mCherry-Linker-CBD-His 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-CBD (heptacapeptide)-His sequence was used, utilizing the existing 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).

[0090] (8) Construction of pBG3AC-mCherry: Using pBBR1MCS2-Tac-mCherry as a template and SEQ ID NO:46 / SEQ ID NO:47 as primer pair, the mCherry sequence was obtained by PCR. The aforementioned sequence was inserted between the EcoRI and BamHI restriction sites of the pBG3AC vector to obtain the ligation product.

[0091] (9) Construction of pUC57-CBD(vWF)-His: The gene sequence encoding CBD(vWF) was optimized and synthesized, and the aforementioned sequence was inserted between the SalⅠ and EcoRⅤ restriction sites of the pUC57 vector.

[0092] (10) Construction of pBG3AK-Linker-CBD(vWF)-His vector: Using pUC57-CBD(vWF) vector as template, the Linker-CBD(vWF) sequence was amplified using SEQ ID NO:48 / SEQ ID NO:49 as primer pair. 6×His and the stop codon TGA were added to the 3′ end of the sequence and inserted between BglⅡ and XhoⅠ of pBG3AK by enzyme digestion and ligation reaction to obtain the ligation product.

[0093] (11) Construction of pBG3AG-mcherry-Linker-CBD(vWF)-His 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-CBD(vWF) fragment were ligated to obtain the ligation product.

[0094] (12) pBG3AA-P T7 Construction of the -TrxA-mcherry-Linker-CBD-His(vWF)-His vector: The vector pBG3AK-P was prepared using EcoRI and BamHI. T7 -TrxA was double-digested, and the vector pBG3AG-mcherry-Linker-CBD(vWF) was double-digested with BglⅡ and XhoⅠ. The vector pBG3AA was double-digested with EcoRI and XhoⅠ. The vector backbone fragment and lacI-P were recovered and purified. T7 The -lacO-TrxA fragment and the mcherry-Linker-CBD (vWF) fragment were ligated to obtain the ligation product.

[0095] (13) pBG3AK-P T7 -TrxA-mcherry-Linker-CBD(vWF)-His-T T7 Vector construction: using pBG3AA-P T7Using the -mcherry-Linker-CBD(vWF) vector as a template, lacI-P was amplified using SEQ ID NO:50 / SEQ ID NO:51 as primers. T7 The -lacO-TrxA-mcherry-Linker-CBD(vWF) sequence utilizes 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 C).

[0096] (14) 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 3) to increase protein expression levels.

[0097] Table 3. Amino acid and nucleotide sequences corresponding to protein fusion tags

[0098]

[0099] Expression of the target protein:

[0100] (1) Obtaining the expression strain: Take 10 μL of the extracted pBG3AK-P T7 -TrxA-mcherry-His-T T7 and pBG3AK-P T7 -TrxA-mcherry-Linker-CBD-His-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.

[0101] (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℃.

[0102] (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. 600The concentration was approximately 0.6. 0.3 mM 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.

[0103] Purification of the target protein:

[0104] (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.

[0105] (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.

[0106] Target protein concentration and concentration determination:

[0107] (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.

[0108] (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.

[0109] SDS-PAGE results are as follows Figure 6 As shown, after IPTG-induced expression, all three experiments yielded a single, high-purity fusion protein: TrxA-mCherry (…). Figure 6 A) TrxA-mCherry-Linker-CBD (Heptapeptide) Figure 6 B) and TrxA-mCherry-Linker-CBD (vWF) Figure 6C), and the actual molecular weights of the three are basically consistent with their theoretical molecular weights of 39.05 kDa, 40.5 kDa, and 60.9 kDa, respectively. The protein concentration determination results show that the concentrations of the obtained mCherry, mCherry-CBD (heptacapeptide), and mCherry-CBD (vWF) fusion proteins are 1.3 mg / mL, 1.2 mg / mL, and 1.2 mg / mL, respectively.

[0110] Example 3

[0111] Preparation of collagen-based bacterial nanocellulose composites containing CBD-red fluorescent protein

[0112] To verify the stable composite of recombinant protein and collagen-based bacterial cellulose nanoparticles, this embodiment used red fluorescent protein as a protein model to carry out the following work:

[0113] (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.

[0114] (2) Dissolve porcine collagen powder in 0.1% acetic acid to prepare collagen solutions with concentrations of 0%, 0.25%, 0.5% and 1.0%, respectively. Place the purified BNC membrane or BNC tube in the collagen solution and shake it at 160 rpm and 20°C for 24 h. Then take out the sample and soak it in 3 mM / L genipin solution for crosslinking. After 6 h, take out the sample and wash it with distilled water until there is no residue, thus obtaining Col / BNC tube or Col / BNC membrane.

[0115] (3) Four composite materials, namely 0%-Col / BNC, 0.25%-Col / BNC, 0.5%-Col / BNC, and 1.0%-Col / BNC, were prepared and respectively incubated with the TrxA-mCherry-CBD (heptacapeptide) fusion protein solution obtained in Example 2 for 24 hours to test whether CBD (heptacapeptide) could bind to Col-based BNC. In addition, to eliminate the interference of non-specific adsorption of protein solutions, 0.5%-Col / BNC that had been soaked in the TrxA-mCherry fusion protein solution lacking CBD (heptacapeptide) was set as the TrxA-mCherry / 0.5%-Col / BNC control group. To ensure the comparability of the experiments, the initial concentration of the fusion protein solutions involved above was uniformly 1 mg / mL.

[0116] (4) Binding experiment of fluorescent protein to Col / BNC membrane

[0117] After the immersion binding was completed, the Col / BNC membrane was removed and immersed in 5 mL of PBS solution (pH=7.4) at 4℃ for 3 days to remove non-specific adsorption. After removing the solution from the surface, 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.

[0118] Experimental results are as follows Figure 7 As shown, after PBS immersion, the BNC membrane in the collagen-free TrxA-mCherry-CBD (heptacapeptide) / 0%-Col / BNC experimental group changed from purple to transparent under naked-eye observation. However, the BNC membrane in the mCherry-CBD (heptacapeptide) / Col / BNC groups containing 0.25%, 0.5%, and 1.0% collagen concentrations changed from purple to light purple after PBS immersion. Furthermore, the fluorescence intensity of the BNC membrane in the CBD-free TrxA-mCherry / 0.5%-Col / BNC group showed a significant difference in average fluorescence intensity compared to the CBD-containing TrxA-mCherry-CBD (heptacapeptide) / 0.5%-Col / BNC group after PBS immersion. These membrane binding experiment results demonstrate that CBD endows the fusion protein with a certain degree of collagen adsorption, and CBD has great potential as a tool for the biomodification of collagen-based composite materials.

[0119] Example 4

[0120] Obtaining and Assessing the Anticoagulant Activity of a Smart-Responsive Recombinant Hirudin-CBD Fusion Protein

[0121] Example 3 above demonstrated that CBD (heptapeptide) can endow the fusion protein with certain Col / BNC membrane adsorption properties. Therefore, this example aims to prepare a smart responsive recombinant hirudin fusion protein containing CBD. The hirudin in this example is hirudin variant I (HV). The overall protein structure diagram is shown below. Figure 8 As shown.

[0122] 1. Construction of the carrier:

[0123] (4) Construction of the pBG3AK-Fr-HV-Linker-CDB-His vector: The gene sequence encoding hirudin was optimized and synthesized (Table 4). 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. A Linker-CBD (heptacapeptide) and a histidine tag were introduced at the N-terminus. The aforementioned Fr-HV-Linker-CDB-His sequence was inserted between BglⅡ and BamHⅠ in the pBG3AK vector.

[0124] Table 4. Amino acid and nucleotide sequences corresponding to hirudin proteins

[0125]

[0126] (6) Construction of pBG3AC-PT7-TrxA-Fr-HV-Linker-CBD-His vector: The promoter of vector pBG3AK-T7-TrxA was double-digested with EcoRI and BamHI, the promoter of vector pBG3AK-Fr-HV-Linker-CDB-His was double-digested with BglII and XhoI, and the promoter of vector pBG3AC was double-digested with EcoRI and XhoI. The purified vector backbone fragment, promoter PT7 fragment and HV-Linker-CBD fragment were ligated to obtain the ligation product.

[0127] (7) pBG3AK-PT7-TrxA-Fr-HV-Linker-CBD-His-T T7 Construction: Using the pBG3AC-PT7-TrxA-Fr-HV-Linker-CBD-His vector as a template, the PT7-TrxA-Fr-HV-Linker-CBD-His sequence was amplified using SEQ ID NO: 62 / SEQ ID NO: 63 as primers. The amplified fragment was then inserted between the BamHI and EcoRI restriction sites of the pBG3AK vector using the existing terminator TT7 of the pBG3K vector to obtain the complete expression vector. Figure 9 ).

[0128] Table 5 Primer sequences from Example 4

[0129]

[0130] (8) Construction of pBG3AK-PT7-protein fusion tag-Fr-HV-Linker-CBD-TT7 vector: In order to improve the expression level of hirudin, the TrxA fusion tag in front of the N-terminal coagulation factor Xa cleavage site of hirudin can be replaced by GST, ELP, SUMO and other protein fusion tags to improve the protein expression level.

[0131] (9) Take 10 μL of the extracted pBG3AK-PT7-TrxA-Fr-HV-Linker-CBD-His-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.

[0132] 2. Expression, purification, and concentration determination of intelligent responsive recombinant hirudin-CBD fusion protein

[0133] 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-CBD, was obtained, and its actual molecular weight is basically consistent with the theoretical molecular weight of 22 kDa. Protein concentration determination results show that the concentration of the obtained TrxA-Fr-HV-Linker-CBD fusion protein is 2.8 mg / mL.

[0134] 3. Determination of the anticoagulant activity of intelligent responsive recombinant hirudin-CBD fusion protein

[0135] (1) Take 50 μL of 1 mg / 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.

[0136] (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.

[0137] (3) Add 100 μL of a solution containing 0.5% bovine fibrinogen to a 2 mL EP tube, then add 50 μl of the 0.2 mg / mL protein solution to be tested to the tube, shake slowly, and incubate in a metal bath at 37°C for 5 min.

[0138] (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.

[0139] (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.

[0140] (6) Calculate the antithrombin activity according to the formula: U = C1V1 / C2V2;

[0141] 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.

[0142] Experimental results showed that white flocculent material formed upon adding 5 μL to the control group, while the test protein coagulated after adding 490 μL. Figure 11 A), the recombinant hirudin activity was calculated to be 490 U / mg.

[0143] 4. Intelligent responsive recombinant hirudin-CBD fusion protein whole blood coagulation assay

[0144] Whole blood clotting time test:

[0145] (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.

[0146] (2) Inject 500 μL of CaCl2 (0.1 M) solution into 5 mL of fresh rabbit anticoagulated whole blood to activate the coagulation reaction.

[0147] (3) Add a BNC membrane containing recombinant hirudin-CBD fusion protein (0.96 mg / mL) to a 24-well plate. Take 50 μL of activated blood and inject it evenly into the sample surface of the 24-well plate. Incubate at 37°C. Add 2 mL of ultrapure water along the wall into the well at 5, 15, 25, 35, 45 and 55 min and gently blow to mix. Incubate for another 5 min. Aspirate 200 μL into 3 replicates in a 96-well plate to measure A540 and evaluate the concentration of hemoglobin. Then evaluate the coagulation effect of each group. Since fresh rabbit whole blood contains coagulation-related enzymes and CaCl2 was added, theoretically, during blood coagulation, coagulation factor X (Fx) would be activated into coagulation factor Xa (Fxa). At this point, because the recombinant hirudin-CBD fusion protein contains an Fxa response site, the Ile-Glu-Gly-Arg tetrapeptide in the recombinant hirudin-CBD fusion protein is recognized and cleaved by Fxa, releasing active recombinant hirudin, which binds to thrombin to exert an anticoagulant effect. Experimental results showed that, compared with the control group saline, the TrxA-Fr-HV-CBD fusion protein slowed down the blood coagulation rate. Figure 11 (B) demonstrates that the fusion protein possesses intelligently responsive anticoagulant properties.

[0148] Example 5

[0149] Preparation and performance testing of the intelligent responsive anticoagulant collagen-based bacterial nanocellulose composite material TrxA-Fr-HV-CBD / Col / BNC

[0150] (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.

[0151] (2) The TrxA-mCherry-CBD (heptacapeptide) fusion protein solution obtained in Example 2 was added to the Col / BNC tube or Col / BNC membrane obtained in Example 3 and allowed to stand at 4°C for 24 hours. Then, 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 composite material was obtained (the preparation principle and process are as follows). Figure 12 ).

[0152] (3) In vitro cell compatibility:

[0153] Sterilized BNC and TrxA-Fr-HV-CBD / Col / 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 13 As shown, with the extension of culture time, TrxA-Fr-HV-CBD / Col / BNC significantly increased the cell proliferation rate of BNC, and had the effect of promoting endothelial cell proliferation.

Claims

1. A recombinant hirudin fusion protein with collagen affinity, characterized in that, The fusion protein is a recombinant hirudin fusion protein fused with a collagen-binding domain (CBD), comprising a first fusion protein tag TrxA, a second hirudin or a hirudin derivative, and a third CBD; wherein the first and second parts are linked by a flexible protein linker sequence Linker-recognition sequence of coagulation-related enzymes, and the second and third parts are linked by a Linker sequence.

2. The recombinant hirudin fusion protein according to claim 1, characterized in that, The CBD is a polypeptide or protein that can specifically bind to collagen, including one or more of the following: engineered heptapeptides derived from mammalian collagenase, collagenase colG or colH derived from Clostridium histolyticum, metalloproteinases from Vibrio mimicus, or the A3 region of vasomotor hemophilia factor vWF.

3. The recombinant hirudin fusion protein according to claim 1, characterized in that, The hirudin derivatives include hirudin variant 1, hirudin variant 2, or hirudin variant 3.

4. The recombinant hirudin fusion protein according to claim 1, characterized in that, The recombinant hirudin fusion protein was constructed using the 3A assembly toolkit, then transferred to an expression host, and the target protein was obtained through expression, separation, and purification.

5. The recombinant hirudin fusion protein according to claim 4, 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.

6. The recombinant hirudin fusion protein according to claim 4, characterized in that: The expression hosts include one or more of the following: Escherichia coli, yeast, Corynebacterium glutamicum, cellulose-producing bacteria, and mammalian cells.

7. A smart responsive collagen-based anticoagulant material, characterized in that, It is obtained by compounding the recombinant hirudin fusion protein as described in claim 1 with collagen-based bacterial cellulose or other collagen-based materials.

8. The intelligent responsive collagen-based anticoagulant material according to claim 7, characterized in that, The composite method includes the following steps: (1) The recombinant hirudin fusion protein was expressed, isolated and purified in a microbial strain, and then prepared into a recombinant hirudin protein solution; (2) Place the purified bacterial nanocellulose substrate or other collagen-based material in an acetic acid solution containing porcine collagen, shake on a shaker, remove the sample and then shake it in a genipin solution to fully soak and crosslink it; After cross-linking is completed, rinse with distilled water until residual glutaraldehyde is completely removed to obtain collagen / bacterial nanocellulose matrix or other collagen-based composite materials. (3) The prepared collagen / bacterial nanocellulose substrate or other collagen-based composite material is added to the recombinant hirudin protein solution, allowed to stand for reaction, and then soaked and washed with phosphate buffer to remove unbound recombinant protein, thus obtaining a smart responsive collagen-based anticoagulant material.

9. The application of the smart responsive collagen-based anticoagulant material as described in claim 7 in the preparation of materials for preventing thrombosis.

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