A recombinant tissue factor rhTF mutant and its application

Abstract

The present invention belongs to the field of biotechnology engineering and provides a recombinant tissue factor (rhTF) mutant and its application. The recombinant tissue factor mutant is: rhTF mutant A, whose amino acid sequence is shown in SEQ ID NO.3 and whose mutation sites are simultaneously mutated: T17F, I22R, S47R, T112W, S188R, and V207R, and whose nucleotide sequence is shown in SEQ ID NO.4; or rhTF mutant B, whose amino acid sequence is shown in SEQ ID NO.5 and whose mutation sites are simultaneously mutated: T17F, I22R, S47F, T112W, S188K, and V207K, and whose nucleotide sequence is shown in SEQ ID NO.6. These mutants have high procoagulant activity and good thermal stability and are used in biomedicine, clinical prothrombin time determination, and wound hemostasis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology engineering, and in particular relates to a recombinant tissue factor rhTF mutant and an application thereof. Background Art

[0002] Tissue factor (TF), also known as coagulation factor III, CD142, is a transmembrane glycoprotein with a molecular weight of 47 kDa. The mature TF molecule consists of 263 amino acid residues, including an extracellular region of 219 amino acids, a hydrophobic transmembrane region of 23 amino acid residues, and an intracytoplasmic region of 21 amino acid residues. The extracellular region is the functional domain of TF.

[0003] Tissue factor is a receptor and cofactor for coagulation factor VII and is the initiator of the extrinsic coagulation pathway. Under normal physiological conditions, tissue factor is present in extravascular tissues, including the adventitial cells of the vascular wall, the fibroblasts surrounding blood vessels, the fibrous capsule of organs such as the liver, spleen, and kidneys, as well as epidermal cells in the outer layer of the skin, glomerular epithelial cells, cerebral cortex, myocardial cells, alveolar macrophages, the gastrointestinal tract, parts of the genitourinary tract, and endometrial stromal cells. Tissue factor is isolated from circulating blood. When a blood vessel is damaged, the integrity of the vascular wall is compromised, exposing tissue factor to the circulating blood. It binds to coagulation factor VII in the blood, forming a complex, activating coagulation factor X, which further activates thrombin, and ultimately activates fibrin to form a coagulation clot, achieving physiological hemostasis.

[0004] As the initiator of the extrinsic coagulation system, tissue factor not only initiates the blood coagulation cascade by binding to factor VII / VIIa but also acts as an anchor through its tight binding to cell membranes, confining physiological coagulation to the site of injury. Furthermore, tissue factor plays a crucial role in wound healing, angiogenesis, tissue remodeling, and inflammatory responses. Therefore, as a crucial active protein, tissue factor has significant applications in biomedicine, clinical prothrombin time determination, and wound hemostasis.

[0005] Natural tissue factor is primarily found on cell membranes, making it difficult to extract and prepare large quantities from tissue cells. For applications in prothrombin time (PT) assays and hemostatic materials, chemical extraction and isolation of tissue factor from human brain, human placenta, and rabbit brain powder are used to prepare prothrombin time (PT) assay reagents. However, due to varying tissue sources and the varying sensitivity of tissue factor from different species to coagulation factors, chemically extracted tissue factor exhibits low sensitivity and poor stability. With the development of modern biotechnology, genetic engineering has enabled the recombinant expression of tissue factor in Escherichia coli and eukaryotic cells, resulting in superior recombinant tissue factor and addressing the tissue factor source issue. This recombinant tissue factor has been used in the production of clinical prothrombin time assay reagents, effectively improving their sensitivity and stability. It has also been applied to composite hemostatic materials such as silica-based collagen hydrogels, starch microparticles, and chitosan, achieving rapid local hemostasis.

[0006] However, in practical applications, the activity and stability of recombinant tissue factor are crucial indicators. In the production of prothrombin time (PT) assay reagents, recombinant tissue factor is the primary component, and its coagulation activity directly reflects the reagent's sensitivity. Highly active recombinant tissue factor accurately reflects PT levels. Furthermore, during the PT assay, the reagent is preheated at 37°C for a prolonged period, making the thermal stability of recombinant tissue factor a crucial indicator of reagent stability. Furthermore, as a hemostatic material for trauma, recombinant tissue factor possesses excellent hemostatic activity and a long shelf life. Since it directly participates in the entire blood coagulation process, it holds great promise for application in these applications. However, its inherent activity and stability remain key challenges. Therefore, developing a recombinant tissue factor with both high coagulation activity and excellent thermal stability could effectively address the bottlenecks in tissue factor application. Summary of the Invention

[0007] The purpose of the present invention is to provide a recombinant tissue factor rhTF mutant and its application, to obtain a recombinant tissue factor mutant with high coagulant activity and good thermal stability, to better apply and promote it, to improve the sensitivity and stability of prothrombin time determination reagents, and to improve the hemostatic effect and shelf life of composite hemostatic materials.

[0008] The present invention is achieved by the following technical solution: a recombinant tissue factor rhTF mutant, wherein the recombinant tissue factor mutant is: rhTF mutant A, whose amino acid sequence is shown in SEQ ID NO.3 and whose nucleotide sequence is shown in SEQ ID NO.4; or rhTF mutant B, whose amino acid sequence is shown in SEQ ID NO.5 and whose nucleotide sequence is shown in SEQ ID NO.6.

[0009] The recombinant tissue factor rhTF is obtained by adding a purification tag 6HIS to the N-terminus of TF derived from human placenta, wherein the amino acid sequence of TF from human placenta is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0010] The rhTF mutant is a mutant in which amino acids at positions 17, 22, 47, 112, 188, and 207 in the rhTF amino acid sequence as shown in SEQ ID NO.1 are simultaneously mutated at multiple sites. The mutation sites are numbered according to the rhTF as shown in SEQ ID NO.1 after removing the N-terminal 6HIS purification tag.

[0011] The rhTF mutant is any one of the following mutations a or b:

[0012] a. The six positions of the recombinant tissue factor rhTF of the amino acid sequence shown in SEQ ID NO.1 were mutated simultaneously, namely, threonine at position 17 was mutated to phenylalanine, which was named T17F; isoleucine at position 22 was mutated to arginine, which was named I22R; serine at position 47 was mutated to arginine, which was named S47R; threonine at position 112 was mutated to tryptophan, which was named T112W; serine at position 188 was mutated to arginine, which was named S188R; and valine at position 207 was mutated to arginine, which was named V207R; this mutant was named recombinant tissue factor rhTF mutant A;

[0013] b. The six positions of the recombinant tissue factor rhTF with the amino acid sequence shown in SEQ ID NO.1 were mutated simultaneously, namely, the threonine at position 17 was mutated to phenylalanine, which was named T17F; the isoleucine at position 22 was mutated to arginine, which was named I22R; the serine at position 47 was mutated to phenylalanine, which was named S47F; the threonine at position 112 was mutated to tryptophan, which was named T112W; the serine at position 188 was mutated to lysine, which was named S188K; and the valine at position 207 was mutated to lysine, which was named V207K; this mutant was named recombinant tissue factor rhTF mutant B.

[0014] The nucleotide sequence of the recombinant tissue factor rhTF mutant A is shown in SEQ ID NO. 4; the nucleotide sequence of the recombinant tissue factor rhTF mutant B is shown in SEQ ID NO. 6.

[0015] The recombinant tissue factor rhTF mutant is used in preparing clinical prothrombin time determination reagents and preparing traumatic wound hemostasis drugs.

[0016] The method for preparing the recombinant tissue factor rhTF mutant is based on the crystal structure of the TF and factor VIIa complex, and molecular simulation docking and mutation screening are performed using Discovery Studio (2017R1). According to the energy calculation results, the lowest energy mutant A: T17F, I22R, S47R, T112W, S188R, V207R, and mutant B: T17F, I22R, S47F, T112W, S188K, V207K are obtained; the recombinant tissue factor mutant is obtained by structural analysis and intermolecular interaction analysis.

[0017] The present invention provides recombinant tissue factor rhTF and its mutants. The expression of recombinant tissue factor rhTF and its mutants in Escherichia coli is achieved. A prothrombin time assay method for measuring the activity of recombinant tissue factor rhTF and its mutants is established, and activity and thermal stability analyses and comparisons are conducted. The activity of the recombinant tissue factor rhTF mutant of the present invention is significantly improved compared to that of recombinant tissue factor rhTF. In the prothrombin time assay, the PT value of the mutant protein at a concentration of 6.25 ng / ml is comparable to that of the recombinant protein at a concentration of 12.5 ng / ml. The thermal stability of the recombinant tissue factor rhTF mutant is significantly improved compared to that of recombinant tissue factor rhTF. At 37°C, the prothrombin time of the mutant protein remains within the normal range on day 10, while the prothrombin time of the recombinant protein is prolonged to over 13 seconds on day 8. At 60°C, the prothrombin time of the recombinant protein is prolonged beyond the normal range on day 4, while the prothrombin time of the mutant protein is prolonged to over 13 seconds on day 8, demonstrating significantly better stability than the recombinant protein.

[0018] Among the mutation sites in recombinant tissue factor rhTF mutant A, four sites have been mutated to arginine, a positively charged, basic amino acid. This improves the surface charge of the recombinant protein and enhances the thermal stability of the rhTF mutant. I22R and V207R mutate the isoleucine (I) at position 22 to arginine, and the valine (V) at position 207 to arginine, transforming hydrophobic side chains into hydrophilic, basic, positively charged arginines. S47R and S188R also optimize the amino acid mutations, transforming the polar, neutral side chain serine into the positively charged, larger side chain arginine, fostering charge interactions. This invention optimizes charge interactions through targeted amino acid mutations, successfully enhancing the thermal stability of the rhTF mutant.

[0019] Among the mutation sites of recombinant tissue factor rhTF mutant B, I22R is the mutation of isoleucine (I) at position 22 to arginine, and the hydrophobic side chain amino acid mutates to arginine, a hydrophilic basic positively charged side chain amino acid; S188K is the mutation of serine (S) at position 118 to lysine, a basic positively charged amino acid; V207K is the mutation of valine (V) at position 207 to lysine, a basic positively charged amino acid, which improves the surface charge of the recombinant protein, forms charge interactions, and improves the thermal stability of the recombinant tissue factor rhTF mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the plasmid map;

[0021] Figure 2 The electrophoresis diagram of the purification of three proteins; in the figure: M is a marker; 1 is the recombinant protein rhTF; 2 is mutant A; 3 is mutant B;

[0022] Figure 3 Figure 2 is a structural diagram of mutants A and B. In the figure: A is mutant A; B is mutant B;

[0023] Figure 4 The relationship between the concentration of recombinant tissue factor and its mutants and the prothrombin time (PT) time;

[0024] Figure 5 The results of prothrombin time determination at 37°C;

[0025] Figure 6 The results of prothrombin time determination at 60℃ are shown. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0028] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0029] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0030] The plasmid pET-28a (Cat. No. B540183) and BL21(DE3) competent cells (Cat. No. B528414) used in the following examples were obtained from Sangon Biotech (Shanghai) Co., Ltd. The formulas for the culture medium, phospholipid solution, and assay solution are as follows:

[0031] LB medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, sterilized at 121°C for 20 min.

[0032] Phospholipid solution: 20 mM Tris-HCl, 0.3% sodium deoxycholate, 5 mg / ml phospholipid (Avanti, Cat No 790413, Birmingham AL), pH 7.5.

[0033] Assay solution: 100 mM Hepes, 3 mg / ml BSA, 100 mM NaCl, 10 mM CaCl2 pH 7.0.

[0034] Disruption buffer: 100 mM Tris-HCl, 1% Triton X-100, pH 8.0).

[0035] Elution buffer B: 20 ​​mM Tris, 150 mM NaCl, 50 mM imidazole, pH 7.5.

[0036] Elution buffer II: 20 mM Tris, 150 mM NaCl, 200 mM imidazole, pH 7.5.

[0037] Storage solution: 100 mmol / L Tris-HCl, 150 mM NaCl, 0.2 mmol / L PMSF, pH 7.5.

[0038] Normal quality control plasma involved in the following examples: Shanghai Sun Biotechnology Co., Ltd. Coagulation non-fixed value quality control product (normal), item number 182. Coagulometer involved: Shanghai Sun Biotechnology Co., Ltd. fully automatic coagulation analyzer UP series. Protein content determination involved: According to the BCA method, the kit is BCA protein concentration determination kit, Shanghai Biyuntian Biotechnology Co., Ltd., item number P0012S. Ni Sepharose HP affinity chromatography filler involved: GE HisTrap 5ml, item number 17524801. Sequence synthesis involved: commissioned by Beijing BGI Protein Research and Development Center Co., Ltd. for synthesis. Endonuclease involved: Thermo Scientific™ FastDigestNdeI item number FD0584, Thermo Scientific™ FastDigestXhoI item number FD0694. The reagents involved (sodium deoxycholate, Triton X-100, imidazole, PMSF, IPTG, kanamycin, Tris-HCl Hepes, BSA, NaCl, and CaCl2) were obtained from Sangon Biotech (Shanghai) Co., Ltd.

[0039] 1. Sequence design and synthesis of recombinant proteins and mutants: With the advancement of experimental structural biology, computational modeling and protein design methods have become increasingly powerful, providing rational approaches to solving highly complex problems in protein engineering.

[0040] Based on the mechanism by which TF and Factor VIIa interact to form a complex, which is the key to their physiological coagulation activity, and based on the theoretical foundation of the crystal structure of the TF / Factor VIIa complex, the present invention identifies the key amino acid residues involved in the interaction between TF and Factor VIIa: Thr17, Lys20, Ile22, Glu24, Gln37, Asp44, Lys46, Lys48, Asp58, Thr60, Phe76, Tyr78, Gln110, Leu133, Arg135, Phe140, Val207, and their adjacent amino acids. The interaction and efficient binding of these amino acids, and those adjacent to them, with Factor VIIa directly influence the activity and stability of the TF / Factor VIIa complex.

[0041] Therefore, based on the crystal structure of the complex of TF and Factor VIIa, the present invention used DiscoveryStudio (2017R1) for molecular simulation docking and mutation screening. According to the energy calculation results, the lowest energy mutant A (T17F, I22R, S47R, T112W, S188R, V207R) and the lower energy mutant B (T17F, I22R, S47F, T112W, S188K, V207K) were obtained. Figure 3 The present invention utilizes structural analysis and intermolecular interaction analysis to obtain recombinant human tissue factor (rhTF) mutants.

[0042] 2. Vector construction, expression and purification of recombinant tissue factor rhTF: The present invention also provides recombinant expression vectors and host bacteria for recombinant tissue factor rhTF and its mutants, as well as fermentation expression and purification methods, wherein the expression vector is PET-28a and the host bacteria is recombinant Escherichia coli BL21 (DE3).

[0043] 1. Vector construction: artificially synthesize the nucleotide sequence according to the sequence of SEQ ID NO. 2, add the 6HIS sequence at the N-terminus, the HIS sequence is the sequence shown in SEQ ID NO. 8, and design NdeI, XhoI Enzyme cutting site. NdeI, XhoI Enzyme digestion, enzyme digestion products and the same endonuclease NdeI, XhoI The enzyme-cut PET28a vector was connected, and the ligation product was transformed into BL21 (DE3) competent cells to obtain positive clones and construct recombinant tissue factor rhTF and its mutants. pET28a Expression vectors, such as Figure 1 The specific reaction system is shown in Table 1.

[0044] Table 1: 50ul reaction system

[0045]

[0046] The specific operation steps are as follows: add 1 μg DNA, 5 μl 10× enzyme reaction buffer, NdI 1ul enzyme, XOt2ul of the enzyme digestion solution was added, followed by redistilled water to a total volume of 50μl. After mixing the reaction system, the EP tube was placed in a 37°C water bath for 1 hour to allow for the enzyme digestion reaction. 2μl of 0.1mol / L EDTA (pH 8.0) was added to the EP tube and mixed to stop the reaction. 100μl of competent BL21(DE3) cells were thawed on ice in an EP tube. 10μl of the enzyme digestion solution was added, mixed, and placed on ice for 30 minutes. After incubating the EP tube in a 42°C water bath for 60 seconds, the tube was quickly transferred to an ice bath and shaken. After 3 minutes, 500μl of LB medium was added and the cells were incubated at 37°C at 200 rpm for 1 hour. 100μl of the above solution was spread onto a kanamycin-resistant LB plate and incubated overnight at 37°C for 12-16 hours to obtain positive clones.

[0047] 2. Fermentation expression:

[0048] a. Pick multiple single colonies and clone them into a tube containing 5 ml of LB liquid medium with kanamycin resistance. The final concentration of kanamycin in LB medium is 50 μg / ml. Incubate at 250 rpm and shake at 37°C overnight for 16 hours.

[0049] b. Inoculate 1 / 100 volume of the culture into 20 ml of kanamycin-resistant LB medium in a 250 ml Erlenmeyer shake flask. The final kanamycin concentration in the LB medium is 50 μg / ml. Incubate the culture at 37°C with a shaker at 250 rpm for 2.5-4 hours until the OD600 of the culture is between 0.4-0.6. Add IPTG (final concentration 2 mM) and induce the culture at 37°C with a shaker at 250 rpm for 10 hours. Centrifuge 1-2 ml of the induced culture at 8000 g / min, discard the supernatant, and collect the precipitate for SDS-PAGE electrophoresis to screen for strains that clearly express the target protein.

[0050] c. First, inoculate the selected strain into a 250ml Erlenmeyer shake flask containing 20ml of kanamycin-resistant LB medium at a final kanamycin concentration of 50μg / ml. Incubate the strain overnight at 37°C with shaking for 16 hours. Then, inoculate 1 / 100th of the bacterial volume into a 3000ml Erlenmeyer shake flask containing 1000ml of kanamycin-resistant LB medium at a final kanamycin concentration of 50μg / ml. Incubate the strain at 37°C with shaking at 250 rpm for 2.5-4 hours until the OD600 reaches 0.4-0.6. Then, add IPTG (final concentration 2mM) and induce the strain at 250 rpm at 37°C for 10 hours. Harvest the strains by centrifugation at 8000g / min.

[0051] 3. Purification of recombinant tissue factor rhTF:

[0052] a. Solution preparation: Disruption buffer: 100 mM Tris-HCl, 1% Triton X-100, pH 8.0); Elution buffer I: 20 mM Tris, 150 mM NaCl, 50 mM imidazole, pH 7.5; Elution buffer II: 20 mM Tris, 150 mM NaCl, 200 mM imidazole, pH 7.5; Storage solution: 100 mmol / L Tris-HCl, 150 mM NaCl, 0.2 mmol / L PMSF, pH 7.5.

[0053] b. Weigh the cells collected during fermentation expression and evenly suspend them in the disruption solution. Ultrasonic disruption is performed at a ratio of 1 g of wet cells to 5 mL of solution until the liquid is clear. Centrifuge at 20,000 g for 15 min, and collect the supernatant. Filter the supernatant through a 0.22 µM filter membrane and separate and purify using a Ni Sepharose HP affinity chromatography column: First, equilibrate the affinity column with 5 column volumes of the disruption solution, then adsorb on a Ni Sepharose HP affinity chromatography column. Then, elute impurities with elution buffer І and the recombinant protein with elution buffer II, and collect the target protein peak.

[0054] c. The collected target protein is exchanged with the storage solution, concentrated by ultrafiltration into the storage solution and stored at -20°C to obtain the recombinant tissue factor rhTF protein. Figure 2 shown.

[0055] 3. Vector Construction, Expression and Purification of Recombinant Tissue Factor (rhTF) Mutant A

[0056] 1. Vector construction: artificially synthesize the nucleotide sequence according to SEQ ID NO.4, add the 6HIS sequence at the N-terminus (according to SEQ ID NO 8), and design NdI 、 XOt Enzyme cutting site. NdI 、 XOt Enzyme digestion, enzyme digestion products and the same endonuclease NdI 、 XOt The enzyme-digested PET28a vector was ligated, and the ligation product was transformed into BL21(DE3) competent cells to obtain positive clones (the specific method is the same as the vector construction of recombinant tissue factor rhTF).

[0057] 2. Fermentation expression: The specific method is the same as that of fermentation expression of recombinant tissue factor rhTF.

[0058] 3. Purification of recombinant tissue factor rhTF mutant A: The specific method is the same as the purification of recombinant tissue factor rhTF, mutant A, such as Figure 2 shown.

[0059] IV. Vector construction and expression purification of recombinant tissue factor rhTF mutant B: Vector construction: artificially synthesize the nucleotide sequence according to the sequence of SEQ ID NO 6, add the 6HIS sequence at the N-terminus (according to SEQ ID NO 8), and design NdI 、 XOt Enzyme cutting site. NdI 、 XOt Enzyme digestion, digestion products and the same endonuclease enzyme NdI 、 XOt The digested PET28a vector was ligated and the ligation product was transformed into BL21(DE3) competent cells to obtain positive clones (the specific method is the same as the vector construction of recombinant tissue factor rhTF). The specific method of fermentation expression is the same as the fermentation expression of recombinant tissue factor rhTF. The specific method of purification of recombinant tissue factor rhTF mutant B is the same as the purification of recombinant tissue factor rhTF. Mutant B, such as Figure 2 shown.

[0060] 5. Activity Analysis of Recombinant Tissue Factor (rhTF) and Its Mutants

[0061] The present invention also provides a method for determining the activity of recombinant tissue factor (rhTF) and its mutants. In the presence of calcium ions, a quantitative amount of recombinant tissue factor (rhTF) and its mutants are added to a phospholipid solution, incubated at 37°C for 30 minutes, and the prothrombin time (PT) of normal human plasma is measured using a coagulometer. Under normal circumstances, the prothrombin time is 10-13 seconds. A shorter clotting time indicates a stronger binding affinity and structural stability of the TF-Factor VIIa complex, and thus, better activity of the recombinant protein. A clotting time exceeding 13 seconds indicates reduced coagulation factor levels, but if normal human plasma remains unchanged, it actually reflects lower tissue factor activity in the reagent.

[0062] The specific method is as follows:

[0063] 1. Solution preparation: Buffer: Prepare 100mM Tris-HCl pH 7.5 buffer; Phospholipid solution: Prepare a solution of 20mM Tris-HCl, 0.3% sodium deoxycholate, and 5mg / ml phospholipids at pH 7.5, and stir thoroughly at 37°C to dissolve to obtain a clear liquid; Assay solution: Prepare a solution of 100mM Hepes, 3mg / ml BSA, 100mM NaCl, and 10mM CaCl2 at pH 7.0, and stir thoroughly to dissolve to obtain a clear liquid;

[0064] 2. Incubation of recombinant tissue factor rhTF at 37°C:

[0065] (1) Measure 50 μg, 25 μg, 12.5 μg, 6.25 μg, 3.125 μg, 1.56 μg, 0.78 μg, and 0.39 μg of recombinant tissue factor (rhTF) respectively and dilute to 150 μL in a 2 ml EP tube.

[0066] (2) Add 150 μL of phospholipid solution to each EP tube.

[0067] (3) Add 700 μL of buffer solution to each EP tube, mix thoroughly, and keep warm in a 37°C constant temperature water bath for 30 minutes to obtain incubation solution.

[0068] 3. Incubation of recombinant tissue factor rhTF mutant A at 37°C: Measure 50 μg, 25 μg, 12.5 μg, 6.25 μg, 3.125 μg, 1.56 μg, 0.78 μg, and 0.39 μg of recombinant tissue factor rhTF mutant A, respectively, and dilute to 150 μL in a 2 ml EP tube. The remaining steps are the same as those for the 37°C incubation of recombinant tissue factor rhTF.

[0069] 4. Incubation of recombinant tissue factor rhTF mutant B at 37°C: Measure 50 μg, 25 μg, 12.5 μg, 6.25 μg, 3.125 μg, 1.56 μg, 0.78 μg, and 0.39 μg of recombinant tissue factor rhTF mutant B, respectively, and dilute to 150 μL in a 2 ml EP tube. The remaining steps are the same as those for the 37°C incubation of recombinant tissue factor rhTF.

[0070] 5. Determination of prothrombin time:

[0071] (1) Measure 1 μL of incubation solution of recombinant tissue factor rhTF, rhTF mutant A, and rhTF mutant B at different concentrations and add them to 1 ml of assay solution (1:1000 dilution).

[0072] (2) Keep the above solution in a constant temperature water bath at 37°C. After 30 minutes, follow the operation of the coagulometer to measure the PT time of the solution in each EP tube.

[0073] 6. PT time and protein activity: In the prothrombin time assay, the different amounts of recombinant tissue factor rhTF or recombinant tissue factor rhTF mutant added to the incubation medium resulted in different prothrombin times, reflecting the difference in activity between recombinant tissue factor rhTF and its mutants. The activity of the recombinant tissue factor rhTF mutant of the present invention was significantly improved compared to that of recombinant tissue factor rhTF. In the prothrombin time assay, the PT values ​​at a mutant protein concentration of 6.25 ng / ml and a recombinant protein concentration of 12.5 ng / ml were comparable. The results are shown in Table 2. Figure 4The plasma used was normal quality control plasma.

[0074] Table 2: Activities of recombinant tissue factor and its mutants

[0075]

[0076] VI. Thermal stability of recombinant tissue factor (rhTF) and its mutants

[0077] Based on the activity determination, the present invention conducted stability tests at 37°C and 60°C. The prothrombin time was measured at different temperatures and for different time periods, and the thermal stability of recombinant tissue factor rhTF and its mutants was determined by the change in time.

[0078] Take 10 μl of the incubation solution of recombinant tissue factor rhTF and its mutant at a concentration of 50 μg / ml and add it to 10 ml of the assay solution (1:1000 dilution). Keep 5 ml of each of the two solutions warm in a 37°C constant temperature water bath and a 60°C constant temperature water bath respectively. Take samples every day to measure the prothrombin time and record the PT value.

[0079] The results of prothrombin time determination are shown in Table 3. Figure 5 、 Figure 6 The results showed that the thermal stability of the rhTF mutant was significantly improved compared to that of the recombinant rhTF. At 37°C, the mutant's prothrombin time remained within the normal range on day 10, while the recombinant protein's prothrombin time was prolonged to over 13 seconds on day 8. At 60°C, the mutant's prothrombin time was prolonged to over 13 seconds on day 8, while the recombinant protein's prothrombin time was already extended beyond the normal range on day 4. Clearly, the 60°C stability of the rhTF mutant was twice that of the recombinant protein.

[0080] Table 3: Prothrombin time measurement results

[0081]

[0082] Among the mutation sites in recombinant tissue factor rhTF mutant A, four sites have been mutated to arginine, a positively charged, basic amino acid. This improves the surface charge of the recombinant protein and enhances the thermal stability of the rhTF mutant. I22R and V207R mutate the isoleucine (I) at position 22 to arginine, and the valine (V) at position 207 to arginine, transforming hydrophobic side chains into hydrophilic, basic, positively charged arginines. S47R and S188R also optimize the amino acid mutations, transforming the polar, neutral side chain serine into the positively charged, larger side chain arginine, fostering charge interactions. This invention optimizes charge interactions through targeted amino acid mutations, successfully enhancing the thermal stability of the rhTF mutant.

[0083] Among the mutation sites of recombinant tissue factor rhTF mutant B, I22R is the mutation of isoleucine (I) at position 22 to arginine, and the hydrophobic side chain amino acid mutates to arginine, a hydrophilic basic positively charged side chain amino acid; S188K is the mutation of serine (S) at position 118 to lysine, a basic positively charged amino acid; V207K is the mutation of valine (V) at position 207 to lysine, a basic positively charged amino acid, which improves the surface charge of the recombinant protein, forms charge interactions, and improves the thermal stability of the recombinant tissue factor rhTF mutant.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant tissue factor (rhTF) mutant, characterized in that: The recombinant tissue factor mutant is: rhTF mutant A, whose amino acid sequence is shown in SEQ ID NO.3 and nucleotide sequence is shown in SEQ ID NO.4; or rhTF mutant B, whose amino acid sequence is shown in SEQ ID NO.5 and nucleotide sequence is shown in SEQ ID NO.

6.

2. The nucleic acid encoding a recombinant tissue factor rhTF mutant according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid encoding the recombinant tissue factor rhTF mutant A is shown in SEQ ID NO. 4; the nucleotide sequence of the nucleic acid encoding the recombinant tissue factor rhTF mutant B is shown in SEQ ID NO.

6.

3. Use of the recombinant tissue factor (rhTF) mutant according to claim 1 in the preparation of a clinical prothrombin time assay reagent and a hemostatic drug for traumatic wounds.

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