Method for optimizing human coagulation factor xi and use thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to an optimization method and application of human coagulation factor XI. The optimization method of human coagulation factor XI includes codon optimization using sequence optimization tools, adding a start codon ATG to the 5' end of the human coagulation factor XI sequence, and adding a Kozak sequence before the start codon to obtain optimized human coagulation factor XI; the Kozak sequence is GCCACC. The optimization method of this invention can improve the yield and procoagulant activity of human coagulation factor XI in recombinant expression systems, providing an effective solution to the problems of human coagulation factor XI shortage and insufficient activity.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an optimization method and application of human coagulation factor XI. Background Technology

[0002] Hemophilia is a group of typical coagulation disorders that can lead to massive bleeding, requiring intervention to restore hemostasis. Hemophilia can be divided into hemophilia A, hemophilia B, and hemophilia C. Hemophilia C, also known as FXI (human coagulation factor XI) deficiency, is associated with mutations in the FXI gene. FXI is a 160 kDa glycoprotein serine protease that exists in the blood in dimer form. FXI plays an important role in the intrinsic coagulation pathway. Unlike other coagulation factors, bleeding symptoms in patients with FXI deficiency are usually mild, and spontaneous bleeding is rare, often manifesting as mild bleeding after trauma or surgery.

[0003] Unlike hemophilia A and B, there are currently very limited specific treatments for FXI deficiency. Plasma-derived FXI is the only marketed alternative treatment, but its limited availability makes it difficult to meet clinical needs. Furthermore, the development of recombinant coagulation factor XI drugs has been slow, with no recombinant protein drugs yet in clinical trials; all are in early or preclinical stages. Therefore, providing a method for preparing recombinant human coagulation factor XI to increase its yield and activity has become a pressing issue in this field. Summary of the Invention

[0004] To address the lack of existing methods for preparing recombinant human coagulation factor XI that combines high yield and activity, this invention provides an optimized method and application for human coagulation factor XI, specifically including the following technical solutions: This invention provides a method for optimizing human coagulation factor XI. The optimization method includes: using a sequence optimization tool to optimize the design of codons, adding a start codon ATG to the 5' end of the CDS sequence of human coagulation factor XI, and adding a Kozak sequence before the start codon to obtain the optimized nucleotide sequence of human coagulation factor XI; the optimized human coagulation factor XI nucleotide sequence encodes optimized human coagulation factor XI; the Kozak sequence is GCCACC; the optimized human coagulation factor XI nucleotide sequence is shown in SEQ ID NO:3.

[0005] The present invention also provides an optimized human coagulation factor XI, wherein the optimized human coagulation factor XI is encoded by the nucleotide sequence of the optimized human coagulation factor XI as described above, and the nucleotide sequence of the optimized human coagulation factor XI is shown in SEQ ID NO:3.

[0006] The present invention also provides a biological material expressing the optimized human coagulation factor XI as described above, the biological material comprising any one or more of the following: optimized human coagulation factor XI amplification primers, optimized human coagulation factor XI recombinant expression vector, optimized human coagulation factor XI recombinant expression microorganism, optimized human coagulation factor XI stably transfected cells, and optimized human coagulation factor XI monoclonal positive cell lines.

[0007] Preferably, the sequences of the amplification primers are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0008] The present invention also provides an application of the optimized human coagulation factor XI, the optimized human coagulation factor XI or biomaterial obtained by the optimization method described above, the application including improving the procoagulant activity and / or yield of recombinant human coagulation factor XI.

[0009] This invention also provides a method for preparing recombinant human coagulation factor XI, comprising: The optimized nucleotide sequence of human coagulation factor XI was inserted into a eukaryotic expression vector to obtain a recombinant expression vector; the optimized nucleotide sequence of human coagulation factor XI is shown in SEQ ID NO:3; The recombinant expression vector was transfected into eukaryotic cells to obtain recombinant expression cells; Stable transfected cells were screened from the recombinant expression cells; Single-clonal positive cell lines were screened from the stable transfected cells; The target protein was isolated and purified from the supernatant of a monoclonal positive cell line to obtain recombinant human coagulation factor XI.

[0010] Preferably, the eukaryotic expression vector includes a plasmid vector, the plasmid vector including a pcDNA3.1 vector; the eukaryotic cell includes human cells; the human cells include HEK293 cells.

[0011] Preferably, the transfection reagent includes PEI transfection reagent.

[0012] Preferably, the transfection includes transfecting the recombinant expression vector into eukaryotic cells at a ratio of 1 μg: 1 μL to 5 μL; the transfection time is 24 h to 120 h.

[0013] The present invention also provides the use of recombinant human coagulation factor XI prepared by the method described above in the preparation of a medicament for treating coagulation factor XI deficiency.

[0014] The beneficial effects of this invention are as follows: This invention provides a method for optimizing human coagulation factor XI, comprising: optimizing the codon design using a sequence optimization tool; adding a start codon ATG to the 5' end of the human coagulation factor XI sequence; and adding a Kozak sequence before the start codon to obtain optimized human coagulation factor XI; the Kozak sequence is GCCACC. The optimization method described in this invention can improve the yield and procoagulant activity of human coagulation factor XI in recombinant expression systems, providing an effective solution to the problems of human coagulation factor XI shortage and insufficient activity.

[0015] This invention also provides a method for preparing recombinant human coagulation factor XI, which can significantly improve the yield and procoagulant activity of recombinant human coagulation factor XI. In the embodiments of this invention, the detection results showed that the highest concentration of recombinant human coagulation factor XI secreted by the monoclonal cell line reached (1.260±0.045) mg / L; compared with the control group, the recombinant human coagulation factor XI expressed by the five monoclonal positive cell lines prepared by this invention can significantly shorten the coagulation time and significantly improve the procoagulant activity, reaching a maximum of (455.85±15.59)%. In summary, the preparation method provided by this invention can effectively improve the yield and procoagulant activity of recombinant human coagulation factor XI. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 This is a schematic diagram of the pcDNA-3.1-FXI recombinant expression plasmid structure in Example 1; Figure 2 This is a schematic diagram of the gene optimization design structure in Example 1; Figure 3 This is a schematic diagram of the colony PCR product verification results in Example 1; Figure 4 This is a schematic diagram of the structure verification results of the recombinant expression plasmid in Example 1; where A is the enzyme digestion verification diagram of the recombinant expression plasmid, and B is the verification diagram of the recombinant expression plasmid. Figure 5 The images shown are Western blot and SDS-PAGE images used in Example 2 to identify rhFⅪ expression; where A is the Western blot image identifying rhFⅪ expression and B is the SDS-PAGE image identifying rhFⅪ expression. Figure 6 This is a schematic diagram of the biological activity evaluation results after codon optimization in Example 3; where A is a concentration comparison diagram and B is an activity comparison diagram. Figure 7The images show the optimized Western blots for each transfection condition in Example 4; where A represents Western blots with different ratios of recombinant expression plasmid to transfection reagent; and B represents Western blots with different number of days. Figure 8 The diagram shows the results of monoclonal positive cell lines in Examples 5-8; where A represents the screening results of monoclonal positive cell lines, different colors represent different cell lines, and the arrows indicate the finally screened monoclonal positive cell lines; B represents the RT-PCR identification results of rhF11, and C represents the SDS-PAGE analysis results of rhF11. Figure 9 This is a schematic diagram of the concentration detection results of monoclonal positive cells in Example 9; where A is the standard curve plotted with BCA standard, and B is the concentration detection results of five monoclonal positive cells. Figure 10 This is a schematic diagram of the results of detecting rhFⅪ activity in cell supernatant using the APTT coagulation method in Example 10; where A is a schematic diagram of the standard curve results of FⅪ in normal reference plasma, B is the clotting time of rhFⅪ secreted by five monoclonal positive cell lines, and C is the procoagulant activity of rhFⅪ secreted by five monoclonal positive cell lines. Figure 9 and Figure 10 middle, for P <0.0001 indicates a significant difference compared to the control group. Detailed Implementation

[0018] This invention provides a method for optimizing human coagulation factor XI. The optimization method includes: using a sequence optimization tool to optimize the design of codons, adding a start codon ATG to the 5' end of the CDS sequence of the human coagulation factor XI sequence, and adding a Kozak sequence before the start codon to obtain the optimized nucleotide sequence of human coagulation factor XI; the optimized human coagulation factor XI nucleotide sequence encodes optimized human coagulation factor XI; the Kozak sequence is GCCACC; the optimized human coagulation factor XI nucleotide sequence is shown in SEQ ID NO:3.

[0019] As one implementation method, those skilled in the art will know that the classic Kozak core sequence is GCCACCAUGG (SEQ ID NO:8). The Kozak core sequence used in this invention is originally GCCACCATG, but after adding the codon ATG, the Kozak sequence is modified to GCCACC. The only difference between the Kozak core sequence of this invention and the classic Kozak sequence is the base representation. In the classic Kozak sequence, AUG is the base representation at the RNA level, while in the Kozak core sequence of this invention, ATG is the base representation at the DNA level. They are different representations of the same codon and are essentially the same. The effect of the Kozak sequence of this invention in enhancing translation initiation efficiency is consistent with the effect of the classic Kozak core sequence.

[0020] This invention adds a start codon ATG to the 5' end of the CDS sequence of human coagulation factor XI, and adds a Kozak sequence before the start codon to obtain optimized human coagulation factor XI; the nucleotide sequence of the optimized human coagulation factor XI is shown in SEQ ID NO:3. As one embodiment, the optimization method of human coagulation factor XI includes introducing a Kozak sequence into the start codon region of the CDS sequence of human coagulation factor XI. The CDS sequence of human coagulation factor XI has the NCBI accession number: GenBank: BC122863.1, as shown in SEQ ID NO:1. As one embodiment, the codons of the CDS sequence of human coagulation factor XI are optimized using artificial intelligence sequence optimization tools, and a start codon ATG is added to the 5' end of the CDS sequence of human coagulation factor XI. Furthermore, to facilitate the separation and purification of rhFXI, six purified histidine (HIS) tags are added before the stop codon.

[0021] As one implementation method, the purification tag described in this invention is an HIS tag. By adding this purification tag upstream of the stop codon of the human coagulation factor XI encoding gene, this invention achieves efficient, specific affinity purification of rhFXI, while facilitating the expression detection and qualitative analysis of the target protein without affecting its natural biological activity.

[0022] In the optimization method of this invention, adding a Kozak sequence at the start of the human coagulation factor XI gene after optimization can enhance the translation of the eukaryotic human coagulation factor XI gene. By using artificial intelligence to design and optimize codons, the translation efficiency and mRNA stability of the target gene human coagulation factor XI in HEK293T cells can be improved, laying the foundation for the efficient expression of human coagulation factor XI.

[0023] In one implementation, this invention optimizes codon design using artificial intelligence. In one implementation, the AI-optimized codon design involves using the deep learning-based sequence optimization tool RiboDecode to optimize the codon design. In one implementation, the specific optimization strategy of RiboDecode includes: input sequence, optimized sequence, parameter settings, and sequence screening. In one implementation, the input sequence includes using the wild-type coding region (CDS) sequence of the target gene as the initial input sequence. In one implementation, the sequence optimization step includes employing a multi-objective optimization strategy to simultaneously maximize translation efficiency (Ribosome Protected Fragments, RPF) and mRNA secondary structure stability (Minimum Free Energy, MFE). In one implementation, the parameter settings include: a. Cell environment: set to HEK293T to utilize the specific translation characteristics of this cell line. b. Weight balance: setting the MFE weight parameter (mfe_weight) to 0.7, prioritizing mRNA structural stability while pursuing high translation efficiency to prevent premature sequence degradation. c. Number of iterations: the optimization process involves 10 epochs. Each round generates 40,000 candidate sequences, which are evaluated and screened using a built-in neural network model. As one implementation, the sequence screening is based on a dual-criteria selection process: a. the predicted RPF value is significantly higher than that of the wild type; b. the predicted MFE value is lower (i.e., the structure is more stable). As one implementation, the CDS sequence of the wild-type human coagulation factor XI is shown in SEQ ID NO:1. As one implementation, the amino acid sequence of the human coagulation factor XI is shown in SEQ ID NO:2. For the optimized nucleotide sequence of human coagulation factor XI, the present invention selects the optimal optimized sequence generated in the 6th iteration, as shown in SEQ ID NO:3.

[0024]

[0025] SEQ ID NO.2:

[0026] The present invention also provides an optimized human coagulation factor XI, wherein the optimized human coagulation factor XI is encoded by the nucleotide sequence of the optimized human coagulation factor XI as described above, and the nucleotide sequence of the optimized human coagulation factor XI is shown in SEQ ID NO:3.

[0027] SEQ ID NO:3:5'-GCCACC ATG CACCACCACCACCACCAC TAA-3'.

[0028] Note: In SEQ ID NO:3, relative to the original human coagulation factor XI CDS sequence, the bolded portion at the 5' end is the Kozak sequence, and the underlined portion is the optimized start codon. The underlined portion at the 3' end contains 6 His tags to facilitate protein purification.

[0029] The present invention also provides biological materials expressing the optimized human coagulation factor XI as described above, said biological materials comprising any one or more of the following: optimized human coagulation factor XI amplification primers, optimized human coagulation factor XI recombinant expression vectors, optimized human coagulation factor XI recombinant expression microorganisms, optimized human coagulation factor XI stably transfected cells, and optimized human coagulation factor XI monoclonal positive cell lines.

[0030] As one implementation, the sequences of the amplification primers are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0031] The present invention also provides applications of optimized human coagulation factor XI, optimized human coagulation factor XI and / or biomaterials obtained by the optimization method described above, the applications including improving the procoagulant activity and / or yield of recombinant human coagulation factor XI.

[0032] The concentration of human coagulation factor XI prepared using the optimized human coagulation factor XI or biomaterials expressing optimized human coagulation factor XI according to this invention is 0.805±0.060~1.260±0.045 mg / L, the procoagulant activity is 149.8%±3.3%~455.9%±15.6%, and the coagulation time is 52.4±0.3~68.0±0.4 s. The optimized human coagulation factor XI or biomaterials expressing human coagulation factor XI described in this invention can achieve both high yield and coagulation activity, as well as a fast coagulation time. This invention provides an effective method for optimizing the preparation process of human coagulation factor XI.

[0033] This invention also provides a method for preparing recombinant human coagulation factor XI, comprising: inserting an optimized nucleotide sequence of human coagulation factor XI into a eukaryotic expression vector to obtain a recombinant expression vector; the optimized nucleotide sequence of human coagulation factor XI is shown in SEQ ID NO:3; transfecting the recombinant expression vector into eukaryotic cells to obtain recombinant expression cells; screening stably transfected cells from the recombinant expression cells; screening monoclonal positive cell lines from the stably transfected cells; and separating and purifying the target protein from the supernatant of the monoclonal positive cell lines to obtain recombinant human coagulation factor XI.

[0034] This invention involves inserting an optimized human coagulation factor XI sequence into a eukaryotic expression vector to obtain a recombinant expression vector. As one embodiment, the eukaryotic expression vector of this invention includes a plasmid vector. As one embodiment, the plasmid vector includes a pcDNA3.1 vector.

[0035] This invention, after obtaining the recombinant expression vector, transfects the recombinant expression vector into eukaryotic cells to obtain recombinant expression cells. In one embodiment, the eukaryotic cells include human cells. In one embodiment, the human cells include HEK293 cells. In one embodiment, the transfection is mediated by a transfection reagent to allow the recombinant expression plasmid to enter the eukaryotic cells. In one embodiment, the transfection reagent includes PEI transfection reagent. In one embodiment, the transfection ratio of the recombinant expression vector to the transfection reagent is 1 μg:1 μL~5 μL. In one embodiment, the transfection ratio of the recombinant expression vector to the transfection reagent can be any one or an intermediate value of any two of the following: 1 μg:1 μL, 1 μg:2 μL, 1 μg:3 μL, 1 μg:4 μL, and 1 μg:5 μL. In one embodiment, the preferred transfection ratio of the recombinant expression vector to the transfection reagent is 1 μg:1 μL~2 μL. In one embodiment, a more preferred transfection ratio is 1 μg:2 μL. In one embodiment, the transfection time is 24 h to 120 h. In another embodiment, the transfection time can be any one of 24 h, 48 h, 72 h, 96 h, and 120 h, or an intermediate value between any two points. In one embodiment, the transfection time is preferably 72 h to 120 h. In another embodiment, the transfection time is more preferably 96 h.

[0036] This invention, after obtaining recombinant expression cells, screens stably transfected cells from these cells. As one embodiment, the screening is performed under pressure using a medium containing G418 genimycin. As one embodiment, the medium includes MEM medium supplemented with FBS and G418 genimycin. As one embodiment, the volume percentage of FBS in the MEM medium is 10%. As one embodiment, the MEM medium containing FBS is prepared by adding 10 mL of FBS to 90 mL of MEM medium. As one embodiment, the concentration of G418 genimycin in the medium ranges from 200 to 300 μg / mL. As one embodiment, the concentration of the G418 genomycin can be any one or an intermediate value of any two of the following: 200 μg / mL, 210 μg / mL, 220 μg / mL, 230 μg / mL, 240 μg / mL, 250 μg / mL, 260 μg / mL, 270 μg / mL, 280 μg / mL, 290 μg / mL, and 300 μg / mL. As another embodiment, the preferred concentration range of the G418 genomycin is 200 μg / mL.

[0037] This invention, after obtaining stably transfected cells, screens for monoclonal positive cell lines from these cells. As one embodiment, the screening method for monoclonal positive cell lines includes limiting dilution. As one embodiment, the limiting dilution method includes the following steps: resuspending resistant cells in MEM medium containing 200 μg / mL G418 and 10% FBS, centrifuging at 1200 rpm for 3 min, collecting the cell pellet, and discarding the supernatant; adjusting the cell density to 10 cells / mL to prepare a resistant cell suspension; adding 100 μL of this cell suspension to each well of a 96-well plate, ensuring only one cell per well; subsequently, the 96-well plate is placed in a 37°C, 5% CO2 incubator for expansion culture. Once the cell confluence reaches 90%, monoclonal positive cells are screened. As one embodiment, the expansion culture involves sequentially passaged positive cells into 12-well plates, 6-well plates, T25 cell culture flasks, and T175 cell culture flasks, and then expanding the culture in the T175 cell culture flasks to ultimately establish a stable transfected cell line.

[0038] This invention, after obtaining a monoclonal positive cell line, isolates and purifies the target protein from the supernatant of the monoclonal positive cell line to obtain recombinant human coagulation factor XI. As one embodiment, the steps for isolating and purifying the target protein (recombinant human coagulation factor XI) from the supernatant are as follows: The supernatant is concentrated by ultrafiltration using a 30 kDa ultrafiltration tube, and the concentrate is collected; 40 mL of low-concentration imidazole buffer and 0.4 mL of 1% protease inhibitor are added to the concentrate, and the mixture is resuspended to prepare a protein suspension; the protein suspension is then placed on ice and incubated on a shaker at 70 rpm for 2 h to obtain a reaction mixture; the reaction mixture is then transferred to an empty affinity chromatography column, and the flow-through is collected; the chromatography column is first washed with 20 mL of medium-concentration imidazole buffer to remove unbound contaminating proteins; then, the target protein is eluted with 5 mL of high-concentration imidazole buffer and collected, finally obtaining recombinant human coagulation factor XI protein. As one embodiment, the low-concentration imidazole buffer solution has the following formulation: 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0. As another embodiment, the medium-concentration imidazole buffer solution has the following formulation: 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0. As yet another embodiment, the high-concentration imidazole buffer solution has the following formulation: 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0.

[0039] In the preparation method of recombinant human coagulation factor XI of this invention, the optimized sequence of human coagulation factor XI enhances the translation efficiency and mRNA stability of the eukaryotic human coagulation factor XI gene, laying the foundation for efficient expression of human coagulation factor XI. Simultaneously, the preparation method of this invention also systematically optimizes transfection conditions, precisely adjusting key parameters such as the mass-to-volume ratio of recombinant plasmid DNA to transfection reagent and the cell culture observation time after transfection, successfully determining the optimal transfection protocol and achieving the maximum protein expression level in the transient transfection stage. Given that the procoagulant activity of coagulation factors is directly related to their protein expression level, optimizing the expression level of coagulation factors is usually accompanied by enhanced procoagulant activity. This invention uses the activated partial thromboplastin time (APTT) coagulation method to detect the rhFXI activity in the supernatant of recombinant protein samples and control groups. The results showed that, compared with the blank control group, the rhFⅪ protein prepared by this invention could significantly promote the coagulation process of plasma lacking coagulation factor XI, with its procoagulant activity reaching up to (455.85±15.59)%, fully demonstrating the significant advantages of this preparation method in increasing the yield and procoagulant activity of rhFⅪ protein.

[0040] The present invention also provides the use of recombinant human coagulation factor XI prepared by the method described above in the preparation of a medicament for treating coagulation factor XI deficiency.

[0041] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for optimizing human coagulation factor XI and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0042] The materials used in this invention and their sources of purchase are as follows: Cell culture media: MEM medium (catalog number: PM150411) was purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; fetal bovine serum (catalog number: A5670801) and trypsin-EDTA (0.25%) (catalog number: 25200-056) were purchased from Gibco; endotoxin-free plasmid miniprep kit (catalog number: RA1508) was purchased from Baoruyi (Beijing) Biotechnology Co., Ltd. Hind III restriction endonucleases (catalog number: 1615) and BamHI. Restriction endonucleases (catalog number: 1605) were purchased from TaKaRa; containing 10× buffer and 10× Green buffer; DNA Ligation Kit Ver. 2.1 (catalog number: 6022) was purchased from TaKaRa; containing Solution I, Solution II, Solution III, and KOD FX Neo (catalog number: KFX-201) were purchased from TOYOBO; containing 2× PCR Buffer and 2 mM dNTPs; the FastPure Gel DNA Extraction Mini Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. (catalog number: DC301); Western PCR was also included. Blot consumables and G418 (catalog number: ST081) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; PEI transfection reagent was purchased from MCE Pharmaceuticals, Inc. (catalog number: HY-K2014); BCA kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (catalog number: P0010S); horseradish peroxidase-labeled goat anti-mouse IgG (catalog number: D110087) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; PAGE gel rapid preparation kit (catalog number: PG113 upgraded version) and Coomassie Brilliant Blue rapid staining solution (catalog number: PS111) were purchased from Yageo Biotechnology; One Step SuperRT-PCR Mix Kit (catalog number: T2240) and Total RNA Extraction Kit (catalog number: R1200) were purchased from Solarbio; Activated Partial Thromboplastin Time Assay Kit (catalog number: AX0502) was purchased from Wuhan Zhongtai Biotechnology Co., Ltd.; Coagulation Analyzer (BCS-6100) was purchased from Jingchuan Diagnostics Co., Ltd.; Trelief 5α competent cells (Catalog No.: TSC-C01) were purchased from Beijing Qingke Biotechnology Co., Ltd.; Factor XI deficiency plasma reagent (Catalog No.: BMC11) and normal human standard plasma (Catalog No.: NP703-2) were purchased from Shanghai Chuanfu Biotechnology Co., Ltd.

[0043] DNA sequencing, gene and primer synthesis were all completed by Beijing BGI Genomics Co., Ltd.

[0044] Example 1: Construction and Validation of Recombinant Expression Plasmid pcDNA3.1-FXI In this embodiment, the reference gene sequence of human coagulation factor XI (GenBank: BC122863.1) was retrieved from the GenBank database. Then, human protein codon optimization was performed using artificial intelligence calculations, and the corresponding gene sequence was synthesized. After optimization, the complete human coagulation factor XI gene sequence was synthesized and cloned into the gene synthesis vector pMV commonly used by BGI Genomics to construct the recombinant intermediate plasmid pMV-FXI. Using this recombinant intermediate plasmid pMV-FXI as a template, the target gene fragment of human coagulation factor XI was amplified by PCR technology and directionally cloned into the empty vector pcDNA3.1, finally constructing the recombinant expression plasmid pcDNA3.1-FXI. The plasmid structure is shown below. Figure 1 As shown in the image.

[0045] Specifically, the construction process of the recombinant expression plasmid pcDNA3.1-FⅪ is as follows: 1) Obtaining the target gene: The human coagulation factor XI gene underwent targeted optimization design, specifically as follows: Sequence optimization tools were used to optimize the codons in the coding region of the gene shown in SEQ ID NO:1, and a start codon (ATG) was added at the upstream 5' end. Furthermore, a Kozak sequence (GCCACC) was introduced at the upstream 5' end of this start codon to significantly enhance the translation efficiency of this gene in eukaryotic cells. The final result was the target gene fragment of human coagulation factor XI, i.e., the optimized nucleotide sequence of human coagulation factor XI, as shown in SEQ ID NO:3. Its structure is as follows: Figure 2 As shown.

[0046] This embodiment introduces a gene upstream of the target gene fragment using PCR technology. Hind III restriction enzyme site, introduced downstream BamH I. Restriction sites. Specific PCR amplification primers were designed using Primer 5.0 software, with the upstream primer named FⅪ- Hind III-F, its nucleotide sequence is shown in SEQ ID NO:4: the downstream primer is named FXI- Bam H I -R, whose nucleotide sequence is shown in SEQ ID NO:5: SEQ ID NO:4:5'-CCCAAGCTT GCCACC ATGATCTTTCTTTATCAGG-3'; In the upstream primer, the bolded part is... Hind III restriction site; the underlined portion is the Kozak sequence.

[0047] SEQ ID NO: 5: 5'-CGCGGATCCGACTGCCTGGGTCTTCTCCAA-3'; In the downstream primer, the bolded part is BamH I restriction site.

[0048] The PCR amplification system consisted of 1.5 μL each of upstream and downstream primers (10 μM), 1 μL template, 1 μL KOD high-fidelity enzyme, 10 μL dNTPs, 25 μL 2×PCR Buffer, and 10 μL ddH2O.

[0049] The PCR reaction program was as follows: pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 45 s, for 30 cycles, and finally extension at 72℃ for 7 min.

[0050] 2) Construct recombinant expression plasmids: The PCR amplification products were recovered and purified, and then the recovered PCR products were separately processed with the pcDNA3.1 empty vector. Hind III and BamH I. Double enzyme digestion reaction.

[0051] The enzyme digestion reaction system is prepared as follows: Hind III restriction endonucleases 3.5 μL BamH I. 3.5 μL restriction endonuclease, 20 μL target gene fragment (FⅪ) or pcDNA3.1 vector, and 3 μL 10× Green Buffer.

[0052] The enzyme digestion reaction system was placed at 37℃ for 10 min to obtain the enzyme digestion products. The enzyme digestion products were separated and identified by 1% agarose gel electrophoresis. After the target band was cut by a UV gel cutter, the target fragment was recovered and purified using a gel recovery detection kit.

[0053] The purified pcDNA3.1 vector fragment was ligated with the FXI target gene fragment according to the ligation system.

[0054] Ligation system: 2 μL of recovered pcDNA3.1 vector, 3 μL of FXI target fragment, and 5 μL of solution I.

[0055] The ligation system was incubated in a 16°C metal bath for 0.5 h to obtain the ligation product, namely the pcDNA3.1-FⅪ recombinant plasmid.

[0056] 3) Constructing monoclonal bacteria The above-mentioned ligation product was transformed into DH5α competent cells. The specific transformation procedure is as follows: ① Transformation pretreatment: Add 1 μL of solution Ⅲ to 10 μL of ligation product, followed by 40 μL of DH5α competent cells thawed on ice. Mix gently and incubate on ice for 30 min.

[0057] ② Heat shock treatment: Place the centrifuge tubes in a 42℃ metal bath for 60 seconds, then quickly transfer them to an ice bath and let them stand for 2 minutes.

[0058] ③ Resuscitation culture: Add 700 μL of antibiotic-free sterile LB liquid medium to the centrifuge tube, mix well, and then shake and revive for 60 min at 37℃ and 200 rpm. The sterile LB liquid culture medium is prepared by adding 1 g of yeast extract, 2 g of tryptone and 2 g of sodium chloride to 200 ml of ultrapure water and mixing well.

[0059] ④ Spread culture: Take 50 μL of revived bacterial solution and spread it evenly on the surface of LB solid medium containing 100 μg / mL ampicillin. Invert the culture plate and place it in a 37℃ constant temperature incubator for overnight culture.

[0060] The colony PCR verification procedure is as follows: Five single colonies were selected for colony PCR identification. The colony PCR verification results of the recombinant expression plasmid are shown below. Figure 3 As shown.

[0061] PCR reaction system: 0.5 μL upstream primer (SEQ ID NO:4), 0.5 μL downstream primer (SEQ ID NO:5), 9.5 μL 2×Rapid Taq Master Mix, and 9 μL ddH2O.

[0062] The PCR reaction program was set as follows: 95℃ pre-denaturation for 2 min; followed by 30 cycles (95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min); and finally 72℃ final extension for 7 min.

[0063] Depend on Figure 3 As can be seen, the PCR amplification product bands of the five single colonies were all located in the 1200 bp~2000 bp range, which is consistent with the theoretical size (1902 bp) of the target gene fragment (i.e. the sequence shown in SEQ ID NO:3), indicating that the recombinant expression plasmid has been successfully constructed and the target gene fragment has been correctly inserted into the vector.

[0064] Based on the colony PCR verification results, one positive single colony was selected for amplification culture: it was inoculated into LB liquid medium containing 0.1 mg / ml ampicillin (Amp) and cultured at 37℃ and 200 r / min for 12 h. A portion of the bacterial culture was sent to BGI for sequencing identification, and the sequencing results confirmed that the target gene sequence was correct; the remaining bacterial culture was used to extract the recombinant expression plasmid (pcDNA3.1-FⅪ) using an endotoxin-free plasmid extraction kit, and double enzyme digestion was performed for verification.

[0065] Double enzyme digestion reaction system: Hind III restriction endonucleases 3.5 μL BamH I. 3.5 μL restriction endonuclease, 20 μL recombinant expression plasmid, and 3 μL 10× Green Buffer.

[0066] The above double enzyme digestion reaction system was digested at 37℃ for 10 min, and the digestion products were analyzed by 1% agarose gel electrophoresis. The enzyme digestion verification results are as follows: Figure 4 As shown in Figure A, the agarose gel electrophoresis analysis results are as follows: Figure 4 As shown in B.

[0067] Depend on Figure 4 As shown in Figure A, after double enzyme digestion, the electrophoretic pattern of the recombinant plasmid shows the FXI target gene fragment band in the 1200 bp~2000 bp range, and the digested pcDNA3.1 vector fragment band above 4500 bp. Figure 4 Image B shows the agarose gel electrophoresis results of the recombinant plasmid, revealing a complete plasmid band in the 6133 bp to 8023 bp range, as expected. Combined with sequencing verification results from BGI Genomics, this confirms the successful construction of the pcDNA3.1-FⅪ recombinant expression plasmid.

[0068] Example 2: HEK293 cell culture and transient transfection of pcDNA3.1-FXI recombinant expression plasmid The culture procedure for HEK293 cells is as follows: Take the frozen HEK293 cell line out of the liquid nitrogen tank and quickly put it into a 37°C preheated constant temperature water bath. Gently shake the cryovial to accelerate thawing and ensure that the thawing is completed within 30 seconds.

[0069] After thawing, the cells were transferred to a biosafety cabinet. The cells were resuspended in PBS buffer and centrifuged at 1200 rpm for 3 min. The supernatant was discarded, and MEM medium containing 10% fetal bovine serum was used as the complete culture medium. Cells were then cultured at a concentration of 1×10⁶ cells / mL. 6Cells were seeded into T75 cell culture flasks at a density of cells / mL and incubated at 37°C with 5% CO2 for adherent culture. When the cell confluence reached 90%, 5 mL of 0.25% trypsin digestion solution was added, and the cells were digested for 1 min. The cell status was then observed under a microscope. When most cells became round and began to detach, fresh MEM medium containing 10% fetal bovine serum was immediately added to stop the digestion. The cells were then centrifuged at 1200 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in complete medium. The cells were passaged at a ratio of 1:3 and seeded into new T75 cell culture flasks and incubated statically at 37°C with 5% CO2.

[0070] The preparation and transfection procedures for HEK293 cells used for transfection are as follows: Select HEK293 cells in the logarithmic growth phase, take one T75 cell culture flask, and adjust the cell density to 1×10⁶ cells / year using MEM medium containing 10% fetal bovine serum. 6 Cells / mL were inoculated and then placed in a 37℃, 5% CO2 incubator for 24 h to adhere to the cell walls. Before transfection, the cell culture medium was changed to serum-free MEM medium, and then the transfection procedure was performed: two 2 mL EP tubes were prepared and labeled A and B respectively. 1 mL of serum-free MEM medium was added to each tube; 10 μg of pcDNA3.1-FXI recombinant expression plasmid prepared in Example 1 was added to tube A, and after thorough mixing, it was filtered through a 0.22 μm filter membrane for sterilization, and then the volume was increased to 1 mL with serum-free MEM medium for later use; 25 μL of PEI transfection reagent was added to tube B, diluted to 1 mL with serum-free MEM medium, and mixed well for later use; the plasmid dilution in tube A was slowly added to the PEI transfection reagent dilution in tube B, and the mixture was gently mixed to obtain a total volume of 2 mL. The mixture was incubated at room temperature for 30 min to allow the transfection reagent to form a stable complex with the nucleic acid. The complex was uniformly added to a culture flask containing HEK293 cells to complete transfection and obtain codon-optimized rhFXI.

[0071] The negative control group was transfected with the empty pcDNA3.1 plasmid. The procedure was the same as described above, except that the plasmid added was the empty pcDNA3.1 plasmid, resulting in the negative control transfection product.

[0072] Protein expression in the transfection reagent-nucleic acid complex sample and the negative control transfection product was identified by Western blotting and SDS-PAGE. The results are as follows: Figure 5 China A and Figure 4 As shown in B.

[0073] Depend on Figure 5As shown in Figures A and B, HEK293 cells were transfected with the pcDNA3.1-FXI recombinant expression plasmid. Western blotting and SDS-PAGE showed that specific bands were generated in the cell supernatant, with a molecular weight of approximately 61 kDa.

[0074] Example 3: Codon optimization of rhFXI and evaluation of its biological activity Following the eukaryotic cell transient transfection method described in Example 2, the successfully constructed wild-type rhFXI expression plasmid and codon-optimized rhFXI expression plasmid were transfected into HEK293 eukaryotic expression cells, respectively. Utilizing the advantages of transient transfection—no genome integration and a short exogenous gene expression cycle—efficient transient expression of the two recombinant plasmids in cells was achieved. The cell supernatant after transfection was collected, and after protein concentration and purification, the in vitro coagulation activity of wild-type and codon-optimized rhFXI proteins was quantitatively detected using the activated partial thromboplastin time (APTT) method. This provides experimental evidence to verify the regulatory effect of the codon optimization strategy on rhFXI protein expression and activity.

[0075] The expression levels and biological activities of wild-type (SEQ ID NO: 1) and optimized (SEQ ID NO: 3) rhFXI recombinant proteins expressed in transfected cells were detected, and the results are as follows: Figure 6 As shown. By Figure 6 As can be seen, the expression concentration of the optimized rhFXI recombinant protein was significantly higher than that of the wild type, and the difference between the two was statistically significant. P <0.01); In vitro coagulation activity assay results showed that the biological activity of the optimized rhFXI recombinant protein was significantly improved compared with that of the wild type ( P <0.0001).

[0076] Example 4: Optimization of conditions for transfecting HEK293 cells with recombinant expression plasmid pcDNA3.1-FXI. This embodiment systematically optimizes transfection conditions from the perspectives of the ratio of HEK293 plasmid to transfection reagent and transfection days, and uses Western blotting to detect the expression intensity of rhFⅪ under different conditions.

[0077] 1) Optimization of the ratio of recombinant expression plasmid pcDNA3.1-FXI to transfection reagent The transfection procedure was performed according to the method in Example 2, as follows: Prepare 6 T75 cell culture flasks, add 10 mL of complete culture medium containing HEK293 cells (MEM medium containing 10% fetal bovine serum) to each culture flask to resuspend the cells, adjust the cell seeding density, and ensure that the cell confluence reaches 70% after 48 h.

[0078] Five cell culture flasks were set up with five transfection ratio gradients, and transfection was performed at the inoculation ratios of recombinant expression plasmid pcDNA3.1-FⅪ to PEI transfection reagent of 1 μg:1 μL, 1 μg:2 μL, 1 μg:3 μL, 1 μg:4 μL, and 1 μg:5 μL, respectively; HEK293 cells transfected with empty pcDNA3.1 vector were set up as a negative control group.

[0079] After incubating all culture flasks at 37℃ in a 5% CO2 incubator for 48 h, the cell supernatant was collected, and the expression level of rhFⅪ protein was detected by Western blotting. The results are as follows: Figure 7 As shown in Figure A. From Figure 7 As shown in Figure A, based on the intensity and size of the bands, it can be determined that: when the ratio of plasmid to transfection reagent is 1 μg:1 μL to 1 μg:3 μL, the expression level of rhFⅪ protein is relatively high; when the ratio of plasmid to transfection reagent is 1 μg:2 μL, the expression level of rhFⅪ protein is the highest; when the ratio exceeds 1 μg:3 μL, the protein expression level decreases significantly; among them, the rhFⅪ protein expression level is the highest when transfected at a ratio of 1 μg:2 μL.

[0080] 2) Optimization of the number of days of infection The experimental setup was as follows: For five consecutive days at the same time point, HEK293 cells in the logarithmic growth phase were seeded at 70% confluence into culture flasks, with each flask containing an appropriate amount of MEM medium containing 10% fetal bovine serum. After 24 h of seeding, each group was transfected according to the optimal ratio of recombinant expression plasmid pcDNA3.1-FⅪ to PEI transfection reagent of 1 μg: 2 μL, following the method described in Example 2. HEK293 cells transfected with the empty pcDNA3.1 vector served as a negative control.

[0081] At the same time point on day 5 after the final transfection, cell supernatant from all culture flasks was collected, and rhF11 protein expression levels were detected using Western blotting. The results are as follows: Figure 7 As shown in B. From Figure 7 As shown in Figure B, the expression level of rhF11 protein was highest on day 4 after transfection.

[0082] In summary, based on the experimental results, the preferred expression conditions for rhFⅪ in this embodiment are: a ratio of plasmid DNA to transfection reagent of 1 μg: 2 μL, and a transfection time of 96 h.

[0083] Example 5: Screening of Stable Transfected Cell Lines HEK293 cells were seeded into T75 culture flasks and cultured in MEM medium containing 10% FBS. Transfection was initiated when the cell confluence reached 70%.

[0084] Take 12.5 μg of the pcDNA3.1-FXI recombinant expression plasmid prepared in Example 1, filter it through a 0.22 μm filter membrane, and then take 25 μL of PEI transfection reagent. Dilute the filtered pcDNA3.1-FXI recombinant expression plasmid and PEI transfection reagent separately with 1 mL of serum-free MEM medium. Gently mix the two dilutions and incubate at room temperature for 30 min to form a stable transfection complex (liposome complex). Slowly add the complex to a cell culture flask, and then incubate the cell culture flask at 37°C in a 5% CO2 incubator for 24 h. After incubation, the culture medium containing the complex was discarded and replaced with MEM selection medium containing 200 μg / mL G418 and 10% FBS, and cultured for another 2 days. Then, cells were treated with 0.25% trypsin digestion solution for 1 min, centrifuged at 1200 rpm for 3 min to collect the cell pellet, discarded the supernatant, and resuspended in MEM medium containing 300 μg / mL G418 and 10% FBS. The pellet was then passaged into new culture flasks and cultured for 14 days (with the MEM selection medium changed every 3 days) to obtain stable colonies of resistant cells with integrated FXI gene. The negative control group was transfected with the empty pcDNA3.1 vector, and after 24 h, the medium was replaced with complete medium containing 200 μg / mL G418 for selection. Subsequent passage, culture, and medium changes were performed identically to the experimental group.

[0085] This embodiment uses the limiting dilution method to screen for monoclonal positive cells and construct stable transfected cell lines. The specific operation procedure is as follows: Resistant cell colonies were resuspended in MEM medium containing 200 μg / mL G418 and 10% FBS, and the cell density was adjusted to 10 cells / mL to prepare a homogeneous resistant cell suspension. Two 96-well plates were used, designated as the control group (resistant cells transfected with the pcDNA3.1 empty vector) and the experimental group (resistant cells transfected with the pcDNA3.1-FXI recombinant plasmid), respectively. 100 μL of cell suspension was added to each well to ensure single-cell seeding in each well.

[0086] Two sets of 96-well plates were incubated at 37°C in a 5% CO2 incubator. After 24 h, wells containing only one cell were observed and labeled using an inverted microscope. After culturing for another 2 days, the medium containing 200 μg / mL G418 in the wells was replaced with fresh MEM medium containing 100 μg / mL G418 and 10% FBS. The medium was then replaced every 4 days thereafter. When the cell confluence in the labeled wells reached 90%, monoclonal positive cell lines were obtained. These cells were passaged stepwise in the following order: 12-well plates → 6-well plates → T25 cell culture flasks → T75 cell culture flasks, and finally expanded in T175 culture flasks. In this embodiment, 30 monoclonal cell lines were successfully obtained by limiting dilution after screening with 200 μg / mL G418 pressure, and were named A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6, C1, C2, C3, C4, C5, C6, D1, D2, D3, D4, D5, D6, E1, E2, E3, E4, E5 and E6.

[0087] The above 30 monoclonal positive cell lines were adjusted to 1×10 using serum-free cryopreservation solution. 7 The cells / mL cryopreservation density was initially set at -80°C for short-term storage, followed by long-term cryopreservation in liquid nitrogen. The expression level of recombinant human coagulation factor XI secreted by each monoclonal stable transfected cell line was assessed using a plasma coagulation time assay, and the differences in their coagulation activities were compared. The plasma coagulation time assay was performed according to the People's Republic of China Health Industry Standard WS / T 220—2021 "Technical Standard for Coagulation Factor Activity Assay," and the results are as follows: Figure 8 As shown in Figure A. Five monoclonal stable transfected cell lines with the highest coagulation activity were ultimately selected for subsequent research.

[0088] Depend on Figure 8 As shown in Figure A, no rhF1 expression was observed in the stable control group strains transfected with the empty pcDNA3.1 vector. Based on... Figure 8 As shown in the coagulation activity results, all 30 selected monoclonal cell lines have coagulation activity, and the plasma coagulation time is generally below 100 s. In this invention, five monoclonal cell lines with good growth status and short coagulation time, namely A3, B2, C1, D5 and E6, were selected for further expansion culture.

[0089] Example 6: Identification of Stable Transfected Cell Lines Total RNA was extracted from the five stable monoclonal transfected cell lines selected in Example 5 using a total RNA extraction kit. The transcriptional expression level of the human coagulation factor XI gene was then detected using a reverse One Step SuperRT-PCR Mix Kit. The specific extraction steps are as follows (using cells cultured in six-well plates as an example): Discard the cell culture supernatant from the 6-well plate in Example 5. Add 1 mL of lysis buffer to each well, gently pipette to fully lyse the cells, and incubate at room temperature for 5 min to ensure complete separation of nucleic acid and protein complexes, obtaining the sample to be tested. Transfer the sample to a 2 mL EP tube, add 0.2 mL of chloroform, tighten the cap, and shake vigorously for 15 s. Incubate at room temperature for 5 min, then centrifuge at 4°C and 12000 rpm for 10 min. Carefully aspirate the upper aqueous phase to a new 2 mL EP tube. Add 0.5 mL of wash buffer to the RNA adsorption column, incubate at room temperature for 2 min, and centrifuge at 4°C and 12000 rpm for 2 min. Discard the waste liquid in the collection tube. Add 0.2 mL of anhydrous ethanol to the upper aqueous phase collected in the previous step, mix thoroughly, and transfer to the pretreated adsorption column. Incubate at room temperature for 2 min, centrifuge at 4°C and 12000 rpm for 2 min, and discard the waste liquid. Add 0.6 mL of wash buffer to the adsorption column and centrifuge at 4°C and 12000 rpm for 2 min. Centrifuge at rpm for 2 min, discard waste liquid, and repeat this rinsing step twice; place the adsorption column in the collection tube, centrifuge at 12000 rpm for 2 min to remove residual rinsing liquid, then replace with a new collection tube and incubate at room temperature for 3 min; add 50 μL of RNase-free ddH2O to the center of the adsorption column membrane, incubate at room temperature for 5 min, then centrifuge at 12000 rpm for 2 min at room temperature to elute and obtain total RNA. The extracted total RNA was reverse transcribed into cDNA by RT-PCR and then amplified by PCR. The primers used in the PCR process were synthesized by BGI, and the primer sequences are shown below: Upstream primer P1: FⅪ-Hind Ⅲ-F (SEQ ID NO:6): 5'-TAATACGACTCACTATAGGG-3'; Downstream primer P2: FⅪ-BamH Ⅰ-R (SEQ ID NO:7): 5'-TAGAAGGCACAGTCGAGG-3'.

[0090] The RT-PCR reaction system was prepared as follows (total volume 24 μL): primers P1 / P2 1 μL, 5×SuperRT OneStep Buffer 5 μL, Enzyme Mix 1.5 μL, RNA template 1.5 μL, and DEPC-treated water 15 μL.

[0091] The RT-PCR reaction conditions were set as follows: reverse transcription at 50℃ for 15 min; pre-denaturation at 95℃ for 2.5 min; followed by 30 cycles (denaturation at 95℃ for 20 s, annealing at 65℃ for 25 s, extension at 72℃ for 78 s); and finally, a final extension at 72℃ for 10 min to end the reaction.

[0092] After the reaction was complete, 4.5 μL of PCR product was mixed with 0.5 μL of loading buffer and analyzed by 1% agarose gel electrophoresis. The results are as follows: Figure 8 As shown in B.

[0093] Depend on Figure 8 As shown in Figure B, the PCR amplification products of the stably transfected cell line exhibit a specific target band around 1200-2000 bp, indicating that the human coagulation factor XI gene has been successfully integrated into the genome of the monoclonal cell line.

[0094] Example 7 Purification of rhFⅪ Serum-free culture supernatant from the stably transfected cell line in Example 5 was collected and concentrated using a 30 kDa ultrafiltration tube. 40 mL of low-concentration imidazole buffer (lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0) and 1% protease inhibitor (PMSF) were added to the concentrated supernatant, and the mixture was thoroughly resuspended to obtain a protein suspension.

[0095] Beforehand, equilibrate HisTag Ni-NTA resin and affinity chromatography empty columns with the aforementioned low-concentration imidazole buffer. Mix the protein suspension with the equilibrated resin and incubate on an ice bath at 70 r / min for 2 h to ensure thorough binding of the target protein to the resin. Transfer the mixture to an affinity chromatography empty column. After the filtrate has completely eluted, wash the resin with 20 mL of medium-concentration imidazole buffer (washing buffer: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) to remove non-specifically bound proteins. Finally, elute the target protein with 10 mL of high-concentration imidazole buffer (elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0) and collect the eluent in a 15 mL centrifuge tube. This eluent is the purified recombinant human coagulation factor XI, denoted as the Elution fraction.

[0096] Example 8 SDS-PAGE of stably transfected cell lines SDS-PAGE electrophoresis: The Elution fraction collected in Example 7 was concentrated to 500 μL using a 30 kDa ultrafiltration concentrator. The concentrate was centrifuged at 3500 rpm for 30 min at 4°C. All liquid from the concentrator was transferred to a new EP tube as the experimental sample. 20 μL of the sample was thoroughly mixed with 5 μL of protein loading buffer and boiled at 100°C for 7 min to denature the protein, followed by centrifugation at 14000 rpm for 7 min. 10 μL of the centrifuged sample was loaded onto a polyacrylamide gel. Immediately after electrophoresis, the gel was rapidly stained with Coomassie Brilliant Blue for 30 min. After staining, the gel was destained in water. The detection results are as follows: Figure 8 As shown in C.

[0097] Depend on Figure 8 As shown in Figure C, SDS-PAGE analysis revealed that the purified Elution fraction contained a single specific band of recombinant human coagulation factor XI with a molecular weight of approximately 61 kDa, indicating that the five stable transfected cell lines screened in Example 5 could specifically express rhF XI protein.

[0098] Example 9: Determination of rhFⅪ Concentration Protein concentration determination (BCA method): After culturing the five stable monoclonal transfected cell lines from Example 8 at 37°C and 5% CO2 for 4 days, the cell supernatant was collected and the protein was purified according to the purification method in Example 7. The content of the purified recombinant human coagulation factor XI was determined using the BCA protein quantification kit. The specific operation was strictly performed according to the kit instructions, and the steps are as follows: Preparation of protein standards: Add 0.8 mL of protein standard preparation solution to a protein standard tube containing 20 mg BSA, dissolve thoroughly to prepare a 25 mg / mL stock solution, prepare immediately before use, and aliquot the remaining stock solution and store at -20℃ for long-term storage; take 20 μL of the 25 mg / mL stock solution, add 980 μL of diluent to dilute to 0.5 mg / mL, take 100 μL for later use, and aliquot the remaining diluent and store at -20℃.

[0099] Preparation of BCA working solution: Mix 5 mL of BCA reagent A with 100 μL of BCA reagent B to prepare 5.1 mL of BCA working solution. This working solution remains stable at room temperature for 24 h.

[0100] Protein concentration detection: ① Add 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL of 0.5 mg / mL protein standard to the standard wells of a 96-well plate, respectively, and bring the volume to 20 μL with standard diluent. That is, the corresponding standard concentrations are 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL; repeat each concentration in 3 wells; ② Add 20 μL of purified rhF XI samples of 5 monoclonal cells screened in Example 5 to each sample well; ③ Add 200 μL of BCA working solution to each well and incubate at 37℃ for 20-30 min; ④ Preheat the microplate reader for 15 min, set the detection wavelength to 562 nm (A562), and measure the absorbance value of each well.

[0101] Concentration Calculation: A standard curve was plotted based on the absorbance values ​​of the standards. The absorbance values ​​of the samples were then substituted into the curve equation to calculate the protein concentration of each rhF1 sample. The results are as follows: Figure 9 China A and Figure 9 As shown in B.

[0102] Depend on Figure 9 As shown in Figure A, the standard curve for the reagent kit standards is y = 0.2853x + 0.1507, R0. 2 =0.9943. The OD values ​​of rhFXI samples from five monoclonal cell lines were substituted into the standard curve to calculate the rhFXI concentration. Figure 9 As shown in Figure B, the concentrations of rhFⅪ produced by the five monoclonal cells were (0.875±0.045) mg / L, (1.260±0.045) mg / L, (0.855±0.020) mg / L, (0.885±0.035) mg / L, and (0.805±0.060) mg / L, respectively.

[0103] In summary, all five monoclonal cell lines obtained by screening in this invention can efficiently express rhFⅪ, and the protein concentrations are all at high levels. The highest expression level reached (1.260±0.045) mg / L (this concentration is the concentration after protein purification), which is significantly higher than the expression level of rhFⅪ in the existing report Kemball-Cook G, Garner I, Imanaka Y, Nishimura T, O'Brien DP, Tuddenham EG, McVey JH. High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector. Gene. 1994 Feb25;139(2):275-9. doi: 10.1016 / 0378-1119(94)90769-2. PMID: 8112618.

[0104] Example 10: Detection of the coagulant activity of rhF11 This embodiment refers to the activated partial thromboplastin time method to detect the procoagulant activity of recombinant human coagulation factor XI in cell supernatant. The specific operation steps are as follows: Preparation of standard curve: Plasma was taken from -4℃ and dissolved in 1 mL of ultrapure water. The plasma diluted 10 times was used as standard plasma. The standard plasma was then diluted with 0.05 mol / L imidazole diluent at ratios of 1 / 10, 1 / 20, 1 / 40, 1 / 80, and 1 / 160, respectively, corresponding to percentage activities of 100%, 50%, 25%, 12.5%, and 6.25%. 25 µL of the serially diluted standard plasma was mixed thoroughly with 25 µL of plasma lacking factor FXI. 50 µL of APTT reagent was added to each mixture, and the mixture was incubated for 3 min to activate the intrinsic coagulation pathway. Then, 50 µL of pre-warmed CaCl₂ was added. 2 Reagents were used to accurately record the coagulation time of each system; a standard curve was plotted with the logarithm of the percentage activity of standard plasma as the x-axis and the logarithm of the coagulation time as the y-axis, and the regression equation was obtained by curve fitting.

[0105] Sample determination: Take 25 µL of rhFXI sample diluted 10-fold or blank sample to replace normal reference plasma at a certain dilution in the standard curve preparation. Add 25 µL of FXI-deficient plasma, then add 50 µL of APTT reagent. Incubate at 37°C for 3 min on a semi-automatic coagulation analyzer. Add pre-warmed CaCl₂. 250 µL of reagent was used, and the solidification time was recorded. The solidification time of the test sample was substituted into the regression equation to calculate the percentage activity of rhFXI; all experiments were independently repeated 3 times, and the average value was taken as the final result.

[0106] The results of the rhF11 procoagulant activity assay in the supernatant of 5 monoclonal cell lines are as follows: Figure 10 As shown in Table 1.

[0107] Table 1. APTT and procoagulant activity of monoclonal positive cell lines (mean ± standard deviation)

[0108] according to Figure 10 As can be seen from A, the regression equation for normal reference plasma is y = -0.234x + 2.342, R0 2 =0.9985. The procoagulation time of the control group cell supernatant exceeded 120 s, while the procoagulation time of the monoclonal strain expressing rhFⅪ was significantly shortened ( P <0.0001)( Figure 10 Among the B strains, B2 had the shortest procoagulant time (52.4±0.3) s and a biocoagulant activity of (455.9±15.6)%. Figure 10 rhF11 obtained from monoclonal stable transfection cell lines (B and C) exhibits excellent procoagulant activity.

[0109] In summary, the rhFⅪ obtained from the monoclonal stable transfected cell line of the present invention has excellent procoagulant activity.

[0110] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for optimizing human coagulation factor XI, characterized in that, The optimization method includes: using a sequence optimization tool to optimize the design of codons, adding a start codon ATG to the 5' end of the CDS sequence of human coagulation factor XI, and adding a Kozak sequence before the start codon to obtain the optimized nucleotide sequence of human coagulation factor XI; the optimized human coagulation factor XI nucleotide sequence encodes optimized human coagulation factor XI; The Kozak sequence is GCCACC; the nucleotide sequence of the optimized human coagulation factor XI is shown in SEQ ID NO:

3.

2. An optimized human coagulation factor XI, characterized in that, The optimized human coagulation factor XI is encoded by the nucleotide sequence of the optimized human coagulation factor XI as described in claim 1, and the nucleotide sequence of the optimized human coagulation factor XI is shown in SEQ ID NO:

3.

3. A biomaterial expressing the optimized human coagulation factor XI as described in claim 2, characterized in that, The biomaterials include any one or more of the following: optimized primers for amplifying human coagulation factor XI, optimized recombinant expression vectors for human coagulation factor XI, optimized recombinant expression microorganisms for human coagulation factor XI, optimized stable transfected cells for human coagulation factor XI, and optimized monoclonal positive cell lines for human coagulation factor XI.

4. The biomaterial as described in claim 3, characterized in that, The sequences of the amplification primers are shown in SEQ ID NO:4 and SEQ ID NO:

5.

5. The application of the optimized human coagulation factor XI obtained by the optimization method of claim 1, the optimized human coagulation factor XI of claim 2, or the biomaterial of claim 3 or 4, characterized in that, The applications include increasing the procoagulant activity and / or yield of recombinant human coagulation factor XI.

6. A method for preparing recombinant human coagulation factor XI, characterized in that, include: The optimized nucleotide sequence of human coagulation factor XI was inserted into a eukaryotic expression vector to obtain a recombinant expression vector; the optimized nucleotide sequence of human coagulation factor XI is shown in SEQ ID NO:3; The recombinant expression vector was transfected into eukaryotic cells to obtain recombinant expression cells; Stable transfected cells were screened from the recombinant expression cells; Single-clonal positive cell lines were screened from the stable transfected cells; The target protein was isolated and purified from the supernatant of a monoclonal positive cell line to obtain recombinant human coagulation factor XI.

7. The preparation method according to claim 6, characterized in that, The eukaryotic expression vector includes a plasmid vector, and the plasmid vector includes the pcDNA3.1 vector; The eukaryotic cells include human cells; the human cells include HEK293 cells.

8. The preparation method according to claim 6, characterized in that, The transfection reagent includes PEI transfection reagent.

9. The preparation method according to claim 6, characterized in that, The transfection involves transfecting the recombinant expression vector into eukaryotic cells at a ratio of 1 μg: 1 μL to 5 μL; the transfection time is 24 h to 120 h.

10. The use of recombinant human coagulation factor XI prepared by the method according to any one of claims 6 to 9 in the preparation of a medicament for treating coagulation factor XI deficiency.

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