A codon optimization method for human coagulation factor IX and a recombinant human coagulation factor IX encoding gene and application thereof

Abstract

This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to a method for codon optimization of human coagulation factor IX and the recombinant human coagulation factor IX encoding gene and its applications. Based on RiboDecode, this invention optimizes relevant parameters to improve the codons of the target gene, thereby increasing the expression level and activity of the target gene in the host. The optimized FIX gene mRNA molecule exhibits good stability and high translation efficiency; further addition of GCCACC upstream of the start codon further enhances expression efficiency. This invention optimizes transfection-related operations, identifies optimal transfection conditions, and achieves maximum expression levels through transient transfection, effectively improving the yield of recombinant human coagulation factor IX. The results of the examples show that the yield of recombinant human coagulation factor IX can reach 1.58±0.10 mg / L, and the coagulation activity can reach 66.8±0.51%, with significant optimization of all key indicators.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to a method for optimizing human coagulation factor IX codons and the recombinant human coagulation factor IX encoding gene and its applications. Background Technology

[0002] Hemophilia B is an X-linked recessive inherited bleeding disorder caused by a deficiency or dysfunction of coagulation factor IX (FIX). Patients experience reduced FIX activity in their blood, leading to impaired clotting and persistent bleeding, whether spontaneous or after minor trauma. Repeated bleeding in joints and muscles can cause disability, and severe internal organ or intracranial hemorrhage can be life-threatening. Currently, the standard alternative therapy for this disease is regular infusion of exogenous FIX preparations to prevent and control bleeding. However, traditional plasma-derived FIX preparations have limitations, including limited supply, potential viral contamination risks, and the potential to induce the production of inhibitors (neutralizing antibodies).

[0003] FIX is a structurally complex glycoprotein with highly post-translational modifications. Its maturation requires several key steps, including vitamin K-dependent γ-carboxylation, hydroxylation, glycosylation, and proteoproteasome activation. γ-carboxylation, in particular, is crucial for its calcium-dependent membrane-binding and procoagulant activities. This complex processing places extremely high demands on the host cell. Many expression systems cannot complete these modifications, resulting in inactive expression products. While early eukaryotic cell lines could perform partial modifications, their γ-carboxylation efficiency was often low, limiting the yield and specific activity of recombinant human coagulation factor IX (rhFIX). Therefore, providing a method for preparing human coagulation factor IX to increase its yield and activity has become a pressing problem in this field. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for codon optimization of human coagulation factor IX and a recombinant human coagulation factor IX encoding gene and its application. This method enables efficient expression of recombinant human coagulation factor IX in eukaryotic cells, especially HEK293 cells, thereby increasing the yield and specific activity of recombinant human coagulation factor IX, reducing the coagulation time of recombinant human coagulation factor IX, and improving coagulation activity.

[0005] This invention provides a codon optimization method to improve the expression level and activity of a target gene in a host. The wild-type coding region sequence of the target gene is input into RiboDecode, a deep learning-based sequence optimization tool. The wild-type coding region sequence is iteratively optimized through an optimization module to obtain an optimized sequence. The optimized sequence is then evaluated and screened. The optimization module optimizes the wild-type coding region sequence based on the specific translation characteristics of eukaryotic cells, and sets the MFE weight parameter to 0.7.

[0006] Preferably, the iteration optimization is performed 8 to 10 times, generating 40,000 candidate sequences each time; The evaluation and screening include: predicting the RPF value of the optimized sequence, selecting optimized sequences with RPF values ​​significantly higher than wild-type sequences to obtain a first screening set; predicting the MFE value of the optimized sequences in the first screening set, and selecting the optimized sequence with the lowest MFE value, which is the target gene codon optimized sequence.

[0007] Preferably, the target gene includes human coagulation factor IX, and the nucleotide sequence of the wild-type coding region of human coagulation factor IX is shown in SEQ ID NO:1; the eukaryotic cells include HEK293 cells.

[0008] The present invention provides a recombinant human coagulation factor IX encoding gene obtained by the codon optimization method described above, comprising the nucleotide sequence shown in SEQ ID NO:2.

[0009] Preferably, the nucleotide sequence shown in SEQ ID NO:2 has GCCACC tandemly upstream of the start codon; A purification tag is added upstream of the stop codon of the nucleotide sequence shown in SEQ ID NO:2.

[0010] This invention provides a biological material expressing recombinant human coagulation factor IX, the biological material comprising a recombinant expression vector and / or recombinant cells, the recombinant expression vector comprising a base vector and the recombinant human coagulation factor encoding gene described in the above-mentioned technical solution inserted into the base vector; The recombinant cells include eukaryotic cells and the recombinant human coagulation factor encoding gene or the recombinant expression vector described in the above-mentioned technical solution introduced into the eukaryotic cells.

[0011] This invention provides the application of the recombinant human coagulation factor encoding gene or the biomaterial described in the above-mentioned technical solutions in one or more of the following: (1) Improve the expression efficiency of recombinant human coagulation factor IX in eukaryotic cells; (2) Enhance the procoagulant activity of recombinant human coagulation factor IX expressed in eukaryotic cells; (3) Increase the production of recombinant human coagulation factor IX expressed in eukaryotic cells; (4) Prepare drugs for treating hemophilia B.

[0012] This invention provides a method for efficiently expressing recombinant human coagulation factor IX in eukaryotic cells, comprising the following steps: The recombinant human coagulation factor encoding gene described in the above technical solution is transfected into eukaryotic cells, and after obtaining stably transfected cells, monoclonal cells are obtained through screening. The monoclonal cells were cultured in a large-scale manner, the supernatant was collected, and the target protein was extracted from the supernatant to obtain recombinant human coagulation factor IX.

[0013] Preferably, the transfection uses PEI transfection reagent; the mass-to-volume ratio of the human coagulation factor IX recombinant expression vector to the PEI transfection reagent is 1 μg: 2 μL, and the transfection time is 3 days; The recombinant expression vector of human coagulation factor IX was transfected into eukaryotic cells, and cells were selected under pressure to obtain stably transfected cells. After obtaining stably transfected cells, limiting dilution screening was used to obtain monoclonal cells.

[0014] Preferably, the step of extracting the target protein from the supernatant includes: The supernatant was concentrated by ultrafiltration to obtain a concentrated solution, which was then resuspended to obtain a protein suspension. After incubation, a mixture was formed, which was then subjected to chromatographic filtration and washing to obtain recombinant human coagulation factor IX.

[0015] Beneficial effects: This invention inputs the wild-type coding region sequence of the target gene into RiboDecode, a deep learning-based sequence optimization tool. Based on the specific translation characteristics of eukaryotic cells, the wild-type coding region sequence is optimized. An MFE weight parameter of 0.7 is set for evaluation and screening, maximizing codon optimization of the target gene and improving its expression level and activity in the host. Specifically, this invention optimizes the human coagulation factor IX coding gene using this codon optimization method, enhancing mRNA molecule stability and improving translation efficiency.

[0016] Furthermore, this invention adds a GCCACC tandem upstream of the start codon and a purification tag upstream of the stop codon in the nucleotide sequence shown in SEQ ID NO:2, which can further improve the expression efficiency of this factor in eukaryotic cells and increase the yield and activity of recombinant human coagulation factor IX.

[0017] This invention, through optimizing transfection-related operations, successfully identified the optimal transfection conditions for recombinant human coagulation factor IX expression in eukaryotic cells, thereby achieving maximum expression levels during transient transfection and effectively improving the yield of recombinant human coagulation factor IX. The results of the examples show that the yield of recombinant human coagulation factor IX in this invention can reach 1.58±0.10 mg / L, and the coagulation activity can reach 66.8±0.51%. All key indicators have been significantly optimized. This not only solves the problem of raw material shortage in hemophilia B treatment but also avoids the blood-borne infection risk of traditional human products due to the use of a serum-free cell culture system, laying a solid foundation for the drug development of rhFIX. Attached Figure Description

[0018] 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.

[0019] Figure 1 This is a schematic diagram of the recombinant expression vector in Example 1; Figure 2 This is a schematic diagram of gene design in Example 1; Figure 3 This is a diagram illustrating the validation of pcDNA3.1-FIX recombinant expression in Example 1. Figure 4 This is a diagram showing the restriction enzyme digestion verification of pcDNA3.1-FIX recombinant expression in Example 1. Figure 5 This is the colony PCR verification result from Example 1; Figure 6 This is a graph showing the results of SDS-PAGE analysis to identify rhFIX in Example 2; Figure 7 This is a graph showing the results of Western blot analysis to identify rhFIX in Example 2; Figure 8 The results of protein concentration detection after codon optimization in Example 3; Figure 9 The results of protein activity detection after codon optimization in Example 3; Figure 10 This is a schematic diagram of the coagulation time detection results of the monoclonal cell line in Example 4; Figure 11 This is a diagram showing the RT-PCR identification results of rhFIX in the monoclonal positive cell line of Example 5; Figure 12 This is a schematic diagram of SDS-PAGE analysis of the monoclonal positive cell line in Example 6; Figure 13 Standard curves were plotted for the FIX standard in Example 7; Figure 14This is a graph showing the concentration detection results of monoclonal cells in Example 7; Figure 15 This is the standard curve of FIX procoagulant activity in normal reference plasma in Example 8; Figure 16 The coagulation time of the monoclonal positive cell line in Example 8; Figure 17 This represents the logarithm of the percentage activity of the monoclonal positive cell line in Example 8. express P <0.01, express P <0.0001. Detailed Implementation

[0020] This invention provides a codon optimization method to improve the expression level and activity of a target gene in the host. The wild-type coding region sequence of the target gene is input into RiboDecode, a deep learning-based sequence optimization tool. The wild-type coding region sequence is iteratively optimized by the optimization module to obtain an optimized sequence. The optimized sequence is then evaluated and screened. The optimization module optimizes the wild-type coding region sequence based on the specific translation characteristics of eukaryotic cells, with the MFE weight parameter set to 0.7.

[0021] In one implementation method, the iterative optimization in this invention involves 8 to 10 iterations, generating 40,000 candidate sequences each time. In this invention, after 8 to 10 iterations, the average gain from further increasing the number of iterations is limited. However, excessively large iterations can lead to overfitting / accumulation of errors in the surrogate model, increasing the risk of "improved predictions but limited actual improvements." This invention limits the number of iterations to avoid the accumulation of model errors and overfitting risks caused by excessive iterations, thereby improving the stability and generalization of candidate sequences and offering the advantage of improved optimization reliability.

[0022] As one implementation method, the evaluation and screening of the present invention includes: predicting the RPF value of the optimized sequence, selecting optimized sequences with RPF values ​​significantly higher than those of the wild type to obtain a first screening set; predicting the MFE value of the optimized sequences in the first screening set, and selecting the optimized sequence with the lowest MFE value, which is the target gene codon optimized sequence. As one implementation method, the evaluation and screening of the present invention utilizes the neural network model built into RiboDecode.

[0023]

[0024] In one embodiment, the eukaryotic cells described in this invention include HEK293 cells. The optimized recombinant human coagulation factor IX encoding gene of this invention can efficiently express recombinant human coagulation factor IX in eukaryotic cells, especially HEK293 cells, increasing the yield and specific activity of recombinant human coagulation factor IX, reducing the clotting time of recombinant human coagulation factor IX, and improving coagulation activity.

[0025]

[0026] In one embodiment, GCCACC is added before the start codon of the nucleotide sequence shown in SEQ ID NO:2 of the present invention. GCCACC and the start codon ATG form a Kozak sequence, which can enhance the translation efficiency of the human coagulation factor IX encoding gene. In another embodiment, a purification tag is added upstream of the stop codon of the nucleotide sequence shown in SEQ ID NO:2 of the present invention. Adding a purification tag upstream of the stop codon of the human coagulation factor IX encoding gene in this invention achieves efficient, specific affinity purification of rhFIX, facilitates the detection and qualitative analysis of the target protein expression, and does not affect its natural biological activity.

[0027] As one embodiment, the nucleotide sequence of the recombinant human coagulation factor IX encoding gene of the present invention is shown in SEQ ID NO:3, specifically: GCCACCATG

[0028] This invention provides a biological material expressing recombinant human coagulation factor IX, the biological material comprising a recombinant expression vector and / or recombinant cells, the recombinant expression vector comprising a base vector and a recombinant human coagulation factor encoding gene as described in the above-mentioned technical solution inserted into the base vector; the recombinant cells comprising eukaryotic cells and a recombinant human coagulation factor encoding gene as described in the above-mentioned technical solution or the recombinant expression vector introduced into the eukaryotic cells.

[0029] In one embodiment, the basic vector of the present invention is a eukaryotic expression vector. In another embodiment, the eukaryotic expression vector of the present invention is a pcDNA3.1 plasmid. In yet another embodiment, the present invention inserts the human coagulation factor IX encoding gene or the human coagulation factor IX expression element described in the above-described technical solution into the pcDNA3.1 plasmid. Hind III and BamH Between the I restriction sites. This invention uses a eukaryotic expression vector as the basic vector to enable rhFIX expression in eukaryotic cells. The pcDNA3.1 expression vector used in this invention contains the NeoR resistance selection marker. The NeoR gene is an anti-G418 selection gene inherent in the pcDNA3.1 vector and is a commonly used selection element in site-specific recombination technology, which can significantly improve selection efficiency and help obtain stable expression cell lines.

[0030] The present invention provides the application of the recombinant human coagulation factor encoding gene or the biological material described in the above technical solution in one or more of the following: (1) improving the expression efficiency of recombinant human coagulation factor IX in eukaryotic cells; (2) improving the procoagulant activity of recombinant human coagulation factor IX expressed in eukaryotic cells; (3) improving the yield of recombinant human coagulation factor IX expressed in eukaryotic cells; (4) preparing a drug for treating hemophilia B.

[0031] In one embodiment, the eukaryotic cells described in this invention include HEK293 cells. HEK293 cells are human embryonic kidney cells, which are easily cultured in high-density suspension, have high transfection efficiency, and can achieve high-level transient expression. As a human cell line, HEK293 cells possess a nearly complete human protein post-translational modification mechanism, including a highly efficient γ-carboxylation system. This invention compared the expression of the recombinant human coagulation factor IX encoding gene described in the above-described technical solution and the wild-type human coagulation factor IX encoding gene in HEK293 cells. The results showed that the expression level of the optimized recombinant human coagulation factor IX encoding gene described in the above-described technical solution in HEK293 cells was significantly higher than that of the wild-type human coagulation factor IX encoding gene.

[0032] This invention provides a method for efficiently expressing recombinant human coagulation factor IX in eukaryotic cells, comprising the following steps: The recombinant expression vector of human coagulation factor IX described in the above technical solution was transfected into eukaryotic cells, and after obtaining stably transfected cells, monoclonal cells were obtained through screening. The monoclonal cells were cultured in a large-scale manner, the supernatant was collected, and the target protein was extracted from the supernatant to obtain recombinant human coagulation factor IX.

[0033] In one implementation method, the present invention transfects the human coagulation factor IX recombinant expression vector described in the above technical solution into eukaryotic cells, followed by pressure selection to obtain stably transfected cells. The present invention does not have strict requirements on the specific method of pressure selection; based on the resistance selection marker inherent in the basic vector, pressure selection can be performed to obtain stable expression cell lines. For example, using pcDNA3.1 as the basic vector, the pcDNA3.1 expression vector contains the NeoR resistance selection marker. After successful transfection of cells with the pcDNA3.1-FIX recombinant vector, the cells will acquire resistance to G418. Pressure selection using G418 will allow stably transfected FIX cells to grow normally in a medium containing G418, while cells that have not integrated the target gene will undergo apoptosis during the selection process. In one implementation method, the pressure concentration of G418 in the present invention is 200~300 µg / mL.

[0034] In one embodiment, the eukaryotic cells described in this invention include HEK293 cells.

[0035] In one embodiment, the transfection described in this invention uses PEI transfection reagent. In another embodiment, the recombinant expression vector is transfected into eukaryotic cells with a density of 70%. In one embodiment, the mass-to-volume ratio of the human coagulation factor IX recombinant expression vector to the PEI transfection reagent is 1 μg:(1~5) μL; in another embodiment, the mass-to-volume ratio is 1 μg:2 μL. In one embodiment, the transfection time is 1~5 days; in another embodiment, the transfection time is 3 days. This invention, through systematic optimization of transfection conditions, adjusting key parameters such as cell seeding quantity, plasmid DNA to transfection reagent ratio, and post-transfection observation time, ultimately determines the optimal transfection protocol to maximize the instantaneous transfection expression level. When the ratio of transfection reagent to plasmid is too low, the number of transfection complex particles formed is insufficient, and the unbound plasmids, due to their negative charge, will interfere with the transfection effect; while when the ratio is too high, it will aggravate cytotoxicity and increase the risk of protein degradation, ultimately leading to a decrease in expression efficiency.

[0036] After obtaining the stably transfected cells, the present invention performs screening to obtain monoclonal cells.

[0037] In one embodiment, the screening method described in this invention is limiting dilution screening. As one embodiment, the limiting dilution method of this invention includes: dispersing cells into single cells through serial dilution, seeding them in a 96-well plate, culturing them, and then allowing the single cells to proliferate to form uniform clones, from which target monoclonal cell lines are screened. This invention utilizes limiting dilution to screen positive clones; this method is simple to operate and highly practical.

[0038] After obtaining the monoclonal cells, the present invention expands the culture of the monoclonal cells, collects the supernatant, extracts the target protein from the supernatant, and obtains recombinant human coagulation factor IX.

[0039] In one embodiment, the expanded culture described in this invention uses MEM medium containing 10% FBS and is cultured in an incubator at 37 ℃ and 5% CO2. In another embodiment, the step of extracting the target protein from the supernatant according to this invention includes: ultrafiltration and concentration of the supernatant to obtain a concentrated solution, resuspending it to obtain a protein suspension, incubating it to form a mixture, and then performing chromatographic filtration and washing to obtain recombinant human coagulation factor IX.

[0040] Using the method of this invention, recombinant human coagulation factor IX can be efficiently expressed in eukaryotic cells. The highest expression concentration of recombinant human coagulation factor IX, as detected by the BCA method, reached 1.58 ± 0.10 mg / L. Protein expression level was positively correlated with coagulation activity. APTT analysis showed that, compared with the control group, the recombinant human coagulation factor IX prepared by this invention significantly promoted the coagulation of factor IX-deficient plasma, with a coagulation activity reaching up to 102.5%.

[0041] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for optimizing the codons of human coagulation factor IX, the recombinant human coagulation factor IX encoding gene, and their applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the present invention has no additional requirements for the raw materials used in the preparation, and conventional commercially available products in the art can meet the requirements.

[0043] The reagents and consumables used in the embodiments of this invention are as follows: MEM basal culture medium: Wuhan Pronosai Life Science Technology Co., Ltd., product number: PM150411; 0.25% trypsin: Gibco, catalog number: A5670801; Fetal bovine serum: Gibco, catalog number: 25200-056; PEI transfection reagent: MCE, catalog number: HY-K2014; Hind III restriction endonuclease: TaKaRa, catalog number: 1615, contains 10× buffer; BamHI restriction endonuclease: TaKaRa, catalog number: 1605, contains 10× Green buffer; DNA Ligation Kit Ver.2.1: TaKaRa, Catalog No.: 6022, containing Solution I, Solution II, and Solution III; PVDF membrane: Shanghai Beyotime Biotechnology Co., Ltd., Product No.: FFP32; G418 (Geneticin, or Neomycin): Shanghai Beyotime Biotechnology Co., Ltd., Product No.: ST081; BCA protein concentration assay kit: Shanghai Beyotime Biotechnology Co., Ltd., catalog number P0010S; Mouse anti-6×His Tag monoclonal antibody: Shanghai Sangon Life Sciences Co., Ltd., catalog number; Horseradish peroxidase-labeled goat anti-mouse IgG: Shanghai Sangon Life Science Co., Ltd., Product No.: D110087; One Step SuperRT-PCR Mix Kit: Solarbio, Catalog No.: T2240; Total RNA Extraction Kit: Solarbio, Catalog No.: R1200; Rapid purification gel DNA extraction kit: Nanjing Novizan Biotechnology Co., Ltd., catalog number: DC301). Endotoxin-free plasmid small-scale rapid extraction kit: Beijing Baoruyi Biotechnology Co., Ltd., Product No.: RA1508; Coomassie Brilliant Blue Rapid Stain Solution: Shanghai Yamei Biomedical Technology Co., Ltd., Product No.: PS111; TBST Instant Granules: Beijing Lanbolide Trading Co., Ltd., Item No.: T7209B; APTT kit: Wuhan Zhongtai Biotechnology Co., Ltd., catalog number: JC202; Trelief 5α competent cells: Qingke Biotechnology, catalog number: TSC-C01; KOD FX Neo: TOYOBO, KFX-201, containing 2×PCR Buffer, 2 mM dNTPs; Standard plasma: Shanghai Chuanfu Biotechnology Co., Ltd., Product No.: BMS09 Coagulation analyzer: Jingchuan Diagnostics Technology Co., Ltd., BCS-6100; Factor IX-deficient plasma: Shanghai Chuanfu Biotechnology Co., Ltd., Product No.: BMC09; DNA sequencing, gene and primer synthesis were all completed at Beijing BGI Genomics Co., Ltd.

[0044] Example 1 Construction and validation of pcDNA3.1-FIX recombinant expression vector 1. The human coagulation factor IX gene sequence (NC_000023.11) was retrieved from the GenBank database and codon optimization was performed to ensure its compatibility with the HEK293 cell expression system. The specific optimization strategy is as follows: (1) Input sequence: The wild-type coding region sequence (CDS) of the target gene was used as the initial input. (2) Optimization objective: A multi-objective optimization strategy was adopted to maximize translation efficiency (Ribosome Protected Fragments, RPF) and mRNA secondary structure stability (Minimum Free Energy, MFE). (3) Parameter settings: a. Cell environment: Set to HEK293T to take advantage of the specific translation characteristics of this cell line. b. Weight balance: The MFE weight parameter (mfe_weight) was set to 0.7. While pursuing high translation efficiency, a higher priority was given to mRNA structural stability to prevent premature sequence degradation. c. Number of iterations: The optimization process was carried out for 10 epochs. 40,000 candidate sequences were generated in each epoch and evaluated and screened by the built-in neural network model. (4) Sequence selection: The final candidate sequence is selected based on two criteria: a. The predicted RPF value is significantly higher than that of the wild type; based on condition a, the sequence with the lowest predicted MFE value (i.e., more stable structure) is selected. For Factor IX, the optimal sequence generated in the 5th iteration is selected.

[0045] 2. To achieve the expression of recombinant human coagulation factor IX (rhFIX) gene in a eukaryotic vector, the optimized FIX gene (SEQ ID NO:3) was cloned into the pcDNA3.1 vector using PCR technology, ultimately constructing the recombinant expression vector (rhFIX). Figure 1 Specifically: (1) GCCACC was added upstream of the start codon of the optimized FIX gene to improve the translation efficiency of eukaryotic genes; at the same time, PCR technology was used to introduce GCCACC upstream of the gene fragment. Hind III restriction site, downstream setting BamHThe PCR product obtained by PCR amplification after the I restriction site is the human coagulation factor IX gene fragment containing the Kozak sequence, abbreviated as FIX fragment. Specifically, Primer 5.0 software was used to design PCR amplification primers; the upstream primer was named FIX-Hind III-F (SEQ ID NO:4): 5'-CCCAAGCTT GCCACCATG CAGCGAGTCAATATGATC-3', the bolded part is Hind III restriction site; the underlined portion is the Kozak sequence; the downstream primer is named FIX-BamH IR (SEQ ID NO:5): 5'-CGCGGATCCGGTCAATTTAGTCTTCTCTTTAATCCAGT-3', the bolded portion is... BamH For I restriction site and gene design, see [link to gene design documentation]. Figure 2 .

[0046] PCR amplification system: 1.5 μL each of upstream and downstream primers, 1 μL of synthesized human coagulation factor IX gene sequence, 1 μL of KOD high-fidelity enzyme, 10 μL of dNTPs, 10 μL of 2×PCR Buffer, and 25 μL of ddH2O.

[0047] The PCR reaction program was as follows: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; 72℃ final extension for 7 min.

[0048] (2) Adopt Hind III and BamH I. Restriction endonucleases were used to double-digest the FIX fragment and the empty pcDNA3.1 vector, respectively. The digestion system was prepared as follows: 3.5 μL of each restriction enzyme, 20 μL of either the FIX fragment or the pcDNA3.1 vector, and 3 μL of 10× Green Buffer. After digestion at 37°C for 10 min, the digestion products were separated by 1% agarose gel electrophoresis. After excising the target band, the DNA was recovered using a rapid purification gel DNA extraction kit.

[0049] (3) The recovered FIX fragment and pcDNA3.1 vector were ligated in a 16℃ metal bath for 0.5 h to successfully construct the pcDNA3.1-FIX recombinant expression vector. The ligation system consisted of 3 μL of recovered FIX fragment, 2 μL of recovered pcDNA3.1 vector and 5 μL of solution I.

[0050] (4) The transformation of pcDNA3.1-FIX recombinant expression vector using Trelief 5α competent cells was carried out as follows: ① Take 10 μL of pcDNA3.1-FIX recombinant expression vector, add 1 μL of solution III, and then add 40 μL of competent cells (TSC-E01, Qingke Biotechnology) thawed on ice. Mix slowly and then incubate on ice for 30 min. ② After heat shock in a 42℃ metal bath for 60 s, quickly transfer to an ice bath and incubate for 2 min. ③ Add 700 μL of sterile LB liquid medium without antibiotics to the centrifuge tube, shake well, and then revive at 37℃ and 200 rpm for 60 min. ④ Take 50 μL of the revive bacterial solution and spread it evenly on LB solid medium containing 100 μg / mL ampicillin. Invert the plate and incubate overnight in a 37℃ incubator.

[0051] 3. Enzyme digestion verification of the pcDNA3.1-FIX recombinant expression vector Recombinant plasmids were obtained using an endotoxin-free plasmid extraction kit, and the plasmids were verified by enzyme digestion. Hind III and BamH The recombinant expression vector was double-digested with two restriction endonucleases. The digestion system was prepared as follows: Hind III. BamH 3.5 μL each of restriction endonucleases, 20 μL of pcDNA3.1-FIX recombinant expression vector, and 3 μL of 10× Green Buffer were added. After digestion at 37℃ for 10 min, the digestion products were detected by 1% agarose gel electrophoresis. Results are as follows: Figure 3 and Figure 4 As shown, the target band exists in the range of 6133 bp to 8023 bp. Figure 3 );use Hind III and BamH After double digestion of the recombinant expression vector, electrophoresis results showed a FIX target band at 1200 bp to 2000 bp, and the digested pcDNA3.1 vector plasmid fragment was present above 4500 bp. Figure 4 This confirms the successful construction of the pcDNA3.1-FIX recombinant expression vector. Based on this verification result, the plasmid was sequenced by BGI Genomics and confirmed to be free of abnormalities.

[0052] 4. Colony PCR validation of the pcDNA3.1-FIX recombinant expression vector Five independent single colonies were selected for colony PCR detection. The PCR reaction system was prepared as follows: 2.75 μL each of forward and reverse primers, 55 μL of 2×Rapid Taq Master Mix, and 49.5 μL of ddH2O. The reaction program was set as follows: 95℃ pre-denaturation for 2 min; followed by 35 cycles (95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min); and finally, 72℃ final extension for 7 min. The colony PCR validation results are shown below. Figure 5 As shown, agarose gel electrophoresis analysis revealed that the amplified bands of all five pcDNA3.1-FIX single colonies were located in the 1200 bp-2000 bp range, consistent with the size of the 1423 bp target protein, indicating that all five colonies contained the successfully constructed recombinant expression vector. Four positive colonies were selected and cultured in a constant temperature shaker at 37℃ and 200 r / min for 12 h. A portion of the bacterial culture was sent to BGI Genomics for sequencing, and the results were confirmed to be accurate.

[0053] Example 2 HEK293 cell culture and transient transfection of pcDNA3.1-FIX recombinant expression vector 1. Transient transfection: HEK293 cells were grown in MEM basal medium supplemented with 10% fetal bovine serum and cultured at 37°C and 5% CO2. When the cells reached 80% confluence, they were treated with 0.25% trypsin for 1 min, followed by centrifugation at 1200 rpm for 3 min. HEK293 cells were selected as the target cell line for transfection. The cells were plated one day before transfection using the pcDNA3.1-FIX recombinant expression vector prepared in Example 1. Using a T75 culture flask as an example, the culture medium was replaced with serum-free MEM basal medium before transfection. 10 μg of pcDNA3.1-FIX recombinant expression vector was placed in a 2 mL EP tube, and 1 mL of serum-free MEM basal medium was added and gently mixed to prepare the recombinant vector mixture. Separately, 25 μL of PEI transfection reagent was placed in a new 2 mL EP tube, and 1 mL of serum-free MEM basal medium was added and mixed to obtain the PEI mixture. Transfer the recombinant vector mixture to an EP tube containing PEI mixture, gently mix to a total volume of 2 mL, and incubate at room temperature for 30 min to form the transfection reagent-nucleic acid complex. Add this complex evenly to a culture flask containing HEK293 cells. A negative control was prepared using the pcDNA3.1 empty plasmid.

[0054] 2. Protein Concentration and Purification: Cell supernatant was collected 48 h post-transfection and concentrated using a 30 kDa ultrafiltration tube at 4°C, with the supernatant replaced three times with PBS buffer. The concentrate was mixed with His Tag Ni-NTA resin (Roche) pre-equilibrated with Lysis Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), and then incubated on an ice bath at 65 r / min for 2 h. The mixture was then transferred to a chromatography column, filtered, and washed with Washing Buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) to remove unbound proteins. Finally, rhFIX was eluted with Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0) and collected, then concentrated through a 30 kDa ultrafiltration tube. 20 µL of the concentrated sample was mixed with 5 µL of protein loading buffer and boiled for 5 min. SDS-PAGE electrophoresis parameters were set to 130 V for 50 min. After electrophoresis, the separating gel was immersed in Coomassie Brilliant Blue rapid staining solution for 40 min. Proteins from the gel were transferred to a PVDF membrane using a semi-dry electroporation technique at 350 mA for 80 min. The membrane was washed with TBST for 30 s at room temperature, blocked with 5% skim milk powder for 2 h at room temperature, incubated overnight at 4°C with slow shaking after adding primary antibody, and washed with TBST with rapid shaking for 10 min (repeated 3 times). Secondary antibody was added and incubated at room temperature with slow shaking for 1 h, followed by TBST washing with rapid shaking for 10 min (repeated 3 times). The membrane was then exposed and developed in an imaging system. Western blot and SDS-PAGE techniques were used to analyze and identify the collected Elution eluent, with the pcDNA3.1 empty vector used as a blank control. Results are as follows: Figure 6 and Figure 7 As shown, both detection methods showed a specific band at the 50 kDa position; however, this specific band was not detected in the cell supernatant transfected with the pcDNA3.1 empty plasmid, indicating that the rhFIX enzyme has been successfully expressed in HEK293 cells.

[0055] Example 3 Codon optimization and biological activity evaluation of rhFIX Referring to the transient transfection method in eukaryotic cells in Example 2, the successfully constructed wild-type rhFIX expression plasmid and the codon-optimized rhFIX expression plasmid were transfected into HEK293 cells, respectively. Taking advantage of the characteristics of transient transfection—no genome integration and short exogenous gene expression cycle—efficient transient expression of the two recombinant plasmids in cells was achieved. Cell supernatants were collected after transfection, and after protein concentration and purification, the in vitro coagulation activity of wild-type and codon-optimized rhFIX proteins was quantitatively detected using the activated partial thromboplastin time (APTT) method. The differences in biological activity between the two recombinant proteins were systematically compared, providing experimental evidence to verify the regulatory effect of codon optimization strategies on rhFIX protein expression and activity.

[0056] The expression levels and biological activity assays of wild-type and optimized recombinant proteins after transfection are as follows: Figure 8 and Figure 9 As shown, the expression concentration of the optimized recombinant protein was significantly higher than that of the wild type, and the difference was statistically significant. P <0.01); the biological activity of the optimized recombinant protein was significantly improved compared with that of the wild type ( P <0.0001), and the activity showed a positive correlation with the protein expression concentration.

[0057] Example 4 Screening of stable transfected cell lines 1. Seed HEK293 cells into T75 culture flasks and cultured in MEM medium containing 10% FBS. Transfection was started when the cell density reached 70%.

[0058] 2. Take 10 μg of recombinant expression plasmid and perform bacterial filtration treatment. Then, dilute the plasmid and 20 μL of PEI transfection reagent in 1 mL of MEM medium. Mix the two dilutions thoroughly and let stand at room temperature for 30 min to complete the preparation of liposome complex.

[0059] 3. Gently add the liposome complex dropwise to a cell culture flask and incubate at 37°C with 5% CO2 for 48 hours. After incubation, discard the MEM medium containing the liposomes and replace it with MEM medium supplemented with 250 μg / mL G418 and 10% FBS, and continue culturing for 2 days. After culturing, digest the cells with 0.25% trypsin for 1 min, centrifuge at 1200 rpm for 3 min, collect the cell pellet and discard the supernatant. Then resuspend the pellet in MEM medium containing 250 μg / mL G418 and 10% FBS, passage it to a new culture flask and continue culturing for 20 days, changing the medium every 4 days, until resistant cell colonies carrying the FIX gene are obtained.

[0060] 4. The empty pcDNA3.1 vector was used as a negative control. After 48 h of transfection, the vector was transferred to complete medium containing 250 μg / mL G418 for screening. The subsequent procedures were the same as described above.

[0061] 5. Treat resistant cell colonies using the limiting dilution method, transfer them to 96-well plates to screen for single-clone positive cells, and construct stable transfected cell lines. The specific procedures are as follows: (1) Resuspend the resistant cells in MEM medium containing 250 μg / mL G418 and 10% FBS, and adjust the cell density to 10 cells / mL to prepare a resistant cell suspension. Add 100 µL of the cell suspension to each well of a 96-well plate to ensure that each well contains only 1 cell. Set up two experimental groups: the control group is resistant cells transfected with the empty pcDNA3.1 vector, and the experimental group is resistant cells transfected with the pcDNA3.1-FIX recombinant plasmid. Each group uses one 96-well plate. Incubate the 96-well plate at 37℃ and 5% CO2. After 24 h, observe under an inverted microscope, select wells containing only 1 cell and mark them, and continue to incubate for 2 days. Then replace the medium in the 96-well plate with fresh MEM medium containing 250 μg / mL G418 and 10% FBS. After replacement, put the plate back into the incubator and continue to incubate. Change the medium every 3 days. When the cell density in the labeled wells reached 90%, the resulting cell lines were considered monoclonal positive cell lines. These were then sequentially passaged into 12-well plates, 6-well plates, T25 culture flasks, and T75 culture flasks. After transfection into T75 culture flasks, expansion culture was initiated. In this example, 21 monoclonal cell lines were successfully obtained under a selection pressure of 250 µg / mL G418, all of which successfully expressed rhFIX. No rhFIX expression was detected in stable transfected cells transfected with the empty pcDNA3.1 vector. Based on clotting time (… Figure 10 Five monoclonal cell lines with excellent growth status and short clotting time (FIX-17, FIX-28, FIX-35, FIX-57, and FIX-62) were selected from these cells and their culture was expanded continuously.

[0062] Example 5 PCR identification of stable transfected cell lines Total RNA was extracted from the five monoclonal positive cell lines screened in Example 3 using a total RNA extraction kit, and the expression of the FIX gene was detected by a reverse transcription RT-PCR kit.

[0063] 1. Total RNA Extraction: Taking a six-well plate culture system as an example, take the monoclonal positive cell line cultured in the plate, aspirate the supernatant with a 1 mL pipette, add 1 mL of lysis buffer to lyse the cells, mix by pipetting and aspirating, and let stand at room temperature for 5 min to completely separate the nucleic acid and protein complex, preparing the sample to be tested. Transfer the sample to be tested to a 2 mL EP tube, add 0.2 mL of chloroform, tighten the cap, shake vigorously for 15 s, let stand at room temperature for 5 min, then centrifuge at 4℃ and 12000 rpm for 10 min, carefully transfer the upper aqueous phase to a new 2 mL EP tube. Add 0.5 mL of column wash buffer to the adsorption column, let stand at room temperature for 2 min, centrifuge at 4℃ and 12000 rpm for 2 min, and discard the waste liquid. Add 0.2 mL of anhydrous ethanol to the collected upper aqueous phase, mix thoroughly to obtain a mixture, add the mixture to the adsorption column and let stand for 2 min, centrifuge at 4℃ and 12000 rpm for 2 min, and discard the waste liquid. Add 0.6 mL of wash buffer to the adsorption column, centrifuge at 12000 rpm for 2 min at 4 °C, and discard the waste liquid. Repeat this step twice. Centrifuge the adsorption column empty at 12000 rpm for 2 min, discard the original collection tube, place the adsorption column in a new collection tube, and let it stand at room temperature for 3 min. Add 50 µL of RNase-free ddH2O to the center of the adsorption column membrane, let it stand at room temperature for 5 min, and then centrifuge at 12000 rpm for 2 min at room temperature to obtain RNA from the monoclonal positive cell line.

[0064] 2. RNA was reverse transcribed into cDNA by RT-PCR and then amplified by PCR; the primers used were synthesized by BGI Genomics, and their specific sequences are as follows: Upstream primer P1: FIX-Hind III-F: (SEQ ID NO:6): 5'-TAATACGACTCACTATAGGG-3'; Downstream primer P2: FIX-BamH IR (SEQ ID NO:7): 5'-TAGAAGGCACAGTCGAGG-3'.

[0065] RT-PCR system: primers P1 / P2 1 µL, 5×SuperRT OneStep Buffer 5 µL, Enzyme Mix 1.5 µL, RNA Template 1.5 µL, DEPC-treated water 15 µL.

[0066] PCR reaction conditions: reverse transcription 50℃, 15 min; pre-denaturation 95℃, 2.5 min; denaturation 95℃, 20 s; annealing 65℃, 25 s; extension 72℃, 78 s; 30 cycles; extension at 72℃ for 10 min to finish.

[0067] After the reaction was complete, 4.5 µL of the reaction product was mixed with 0.5 µL of loading buffer and subjected to 1% agarose gel electrophoresis. The results are as follows: Figure 11 As shown, the PCR products of the monoclonal positive cell line were analyzed by 1% agarose gel, and the target fragment was located at about 1400 bp, which indicates that the FIX gene has been integrated into the genome of the monoclonal cell line.

[0068] Example 6 SDS-PAGE electrophoresis analysis of stable transfected cell lines 1. Purification of rhFIX (1) Collect the supernatant of the FBS-free stable transfected cell line cultured in Example 4, concentrate it using a 30 kDa ultrafiltration tube, and then add 40 mL of low-concentration imidazole buffer and 1% protease inhibitor (PMSF) to the concentrated supernatant. After resuspending, obtain a protein suspension.

[0069] (2) Equilibrate the nickel column in advance with low-concentration imidazole buffer (lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). Mix the protein suspension with the pre-equilibrated HisTag Ni-NTA resin, and then incubate on ice at 65 rpm for 2 h on a shaker to form a mixture. Transfer the mixture to an empty affinity chromatography column. After the filtrate has been completely filtered, wash the column 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 unbound contaminating proteins.

[0070] (3) Finally, 5 mL of high-concentration imidazole buffer (elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0) was used to elute the target protein and collect it into a 15 mL centrifuge tube. The collected eluent is the purified rhFIX, named Elution.

[0071] 2. SDS-PAGE detection The collected Elution was concentrated to 500 µL using a 30 kDa ultrafiltration tube concentration method. The concentrate was centrifuged at 3500 rpm for 25 min at 4 °C, and all liquid was transferred to a new EP tube to obtain Sample 1. 20 µL of Sample 1 was mixed with 5 µL of protein loading buffer, boiled at 100 °C for 5 min, and then centrifuged at 14000 rpm for 5 min. 10 µL of the centrifuged sample was loaded onto the plate. After polyacrylamide gel electrophoresis, the sample was immediately stained with Coomassie Brilliant Blue for 30 min, and then destained in distilled water. The detection results are as follows: Figure 12 As shown, the purified Elution cell lines all exhibited specific bands of rhFIX, with a molecular weight of approximately 50 kDa, indicating that all five stable cell lines could specifically express rhFIX.

[0072] Example 7 Determination of rhFIX protein concentration 1. Strictly follow the operating procedures in the BCA kit instructions. The protein standard preparation steps are as follows: Add 0.8 mL of protein standard preparation solution to a tube containing 20 mg BSA protein standard, and dissolve thoroughly to prepare a 25 mg / mL protein standard solution. Use this solution immediately after preparation; aliquot the remaining solution and store it at -20°C for long-term storage. Take an appropriate amount of the 25 mg / mL protein standard solution and dilute to a final concentration of 0.5 mg / mL. Specifically, pipette 20 μL of the 25 mg / mL protein standard and add 980 μL of diluent, then mix well. Reserve 100 μL of the diluted 0.5 mg / mL protein standard; aliquot the remaining solution and store it at -20°C for long-term storage.

[0073] 2. 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. The working solution remains stable at room temperature for 24 h.

[0074] 3. Protein Concentration Assay: Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of standard to the standard wells of a 96-well plate, respectively, and bring the volume to 20 μL with standard diluent. The corresponding standard concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Take 20 μL of purified rhFIX samples from each of the five monoclonal cell lines and add them to the sample wells of the 96-well plate. Add 200 μL of BCA working solution to each well and incubate at 37°C for 20–30 min. Preheat the microplate reader 15 min beforehand, set the detection wavelength to A562, and measure the rhFIX concentration. Plot a standard curve and calculate the protein concentration of the sample by substituting the OD value of the sample into the standard curve. The results are as follows: Figure 13 and Figure 14 As shown, a standard curve was plotted for the reagent kit standards: y = 0.2356x + 0.1634, R0. 2 =0.9952, the rhFIX concentrations produced by the five monoclonal cell lines were: FIX-17: 1.15±0.05 mg / L, FIX-28: 1.47±0.04 mg / L, FIX-35: 1.58±0.10 mg / L, FIX-57: 0.95±0.03 mg / L, and FIX-62: 1.34±0.04 mg / L. The recombinant protein extracted in this invention represents a significant improvement over similar products described in the prior art (Wang Xin, Wang Linhong, Xie Yanyan, et al. Adenovirus-mediated expression of human coagulation factor IX gene in mouse adipose-derived stem cells [J]), in which the expression yield of adenovirus-mediated human coagulation factor IX gene in mouse adipose-derived stem cells (ADSC) was 1.07±0.20 mg / L.

[0075] Example 8 rhFIX coagulant activity assay The procoagulant activity of rhFIX was determined according to the method for determining coagulation factor IX (one-stage coagulation method) in the 2020 edition of the Chinese Pharmacopoeia. The specific steps are as follows: Standard plasma was diluted to 1 / 10, 1 / 20, 1 / 40, 1 / 80, and 1 / 160 times with imidazole diluent, corresponding to percentage activities of 100%, 50%, 25%, 12.5%, and 6.25%, respectively. The recombinant protein to be tested and standard plasma of known concentrations were diluted with buffer and then mixed with FIX-deficient plasma. APTT reagent was then added and incubated for 3 min to activate the intrinsic coagulation pathway. Finally, calcium solution was added, and the coagulation time of each mixture was accurately recorded. A standard curve was plotted with the logarithm of the percentage activity of standard plasma on the x-axis and the logarithm of the coagulation time on the y-axis. The FIX activity value of the test sample could be calculated from this curve. The coagulation time of the test sample was measured using the same method, and the result was substituted into the regression equation to obtain the percentage activity of rhFIX in the sample. Three independent replicate experiments were conducted to analyze the procoagulant activity of rhFIX in the supernatant of five monoclonal positive cell lines. The results are as follows: Figures 15-17 As shown in Table 1.

[0076] Table 1. Results of rhFIX procoagulant activity assay in monoclonal positive cell lines

[0077] according to Figures 15-17 As shown in Table 1, the regression equation for normal reference plasma is y = -0.2264x + 2.304, R0 2=0.9856. The coagulation time of the control group's blank cell supernatant was greater than 120 s. rhFIX expressed in 5 monoclonal positive cells significantly shortened the activated partial thromboplastin time. The coagulation time of the experimental group's rhFIX was significantly different from that of the control group's blank supernatant. P <0.0001), the shortest coagulation time of FIX-57 was 77.8 s, and the highest activity reached 66.8%. Compared with similar proteins reported in the prior art (Wang Xin, Wang Linhong, Xie Yanyan, et al. Adenovirus-mediated expression of human coagulation factor IX gene in mouse adipose-derived stem cells [J]), the activity index was significantly improved. In the prior art, the expression yield of adenovirus-mediated human coagulation factor IX gene in mouse adipose-derived stem cells (ADSC) was 1.07±0.20 mg / L, and the activity was 8.5%.

[0078] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A codon optimization method for improving the expression level and activity of a target gene in a host, characterized in that, The wild-type coding region sequence of the target gene is input into RiboDecode, a deep learning-based sequence optimization tool. The optimization module iteratively optimizes the wild-type coding region sequence to obtain the optimized sequence. The optimized sequences are evaluated and screened; The optimization module optimizes the wild-type coding region sequence based on the specific translation characteristics of eukaryotic cells, and sets the MFE weight parameter to 0.

7.

2. The codon optimization method according to claim 1, characterized in that, The iteration optimization is performed 8 to 10 times, generating 40,000 candidate sequences each time; The evaluation and screening include: predicting the RPF value of the optimized sequence, selecting optimized sequences with RPF values ​​significantly higher than wild-type sequences to obtain a first screening set; predicting the MFE value of the optimized sequences in the first screening set, and selecting the optimized sequence with the lowest MFE value, which is the target gene codon optimized sequence.

3. The codon optimization method according to claim 1 or 2, characterized in that, The target gene includes human coagulation factor IX, and the nucleotide sequence of the wild-type coding region of human coagulation factor IX is shown in SEQ ID NO:1; the eukaryotic cells include HEK293 cells.

4. A recombinant human coagulation factor IX encoding gene obtained based on the codon optimization method according to any one of claims 1 to 3, characterized in that, Includes the nucleotide sequence shown in SEQ ID NO:

2.

5. The recombinant human coagulation factor IX encoding gene according to claim 4, characterized in that, The nucleotide sequence shown in SEQ ID NO:2 has GCCACC tandemly upstream of the start codon; A purification tag is added upstream of the stop codon of the nucleotide sequence shown in SEQ ID NO:

2.

6. A biomaterial expressing recombinant human coagulation factor IX, said biomaterial comprising a recombinant expression vector and / or recombinant cells, characterized in that, The recombinant expression vector includes a base vector and the recombinant human coagulation factor encoding gene as described in claim 4 or 5 inserted into the base vector; The recombinant cells include eukaryotic cells and the recombinant human coagulation factor encoding gene of claim 4 or 5 introduced into the eukaryotic cells or the recombinant expression vector.

7. The use of the recombinant human coagulation factor encoding gene of claim 4 or 5 or the biomaterial of claim 6 in one or more of the following: (1) Improve the expression efficiency of recombinant human coagulation factor IX in eukaryotic cells; (2) Enhance the procoagulant activity of recombinant human coagulation factor IX expressed in eukaryotic cells; (3) Increase the production of recombinant human coagulation factor IX expressed in eukaryotic cells; (4) Prepare drugs for treating hemophilia B.

8. A method for efficiently expressing recombinant human coagulation factor IX in eukaryotic cells, characterized in that, Includes the following steps: The recombinant human coagulation factor encoding gene as described in claim 4 or 5 is transfected into eukaryotic cells, and then monoclonal cells are obtained by screening after obtaining stably transfected cells. The monoclonal cells were cultured in a large-scale manner, the supernatant was collected, and the target protein was extracted from the supernatant to obtain recombinant human coagulation factor IX.

9. The method according to claim 8, characterized in that, The transfection was performed using PEI transfection reagent; the mass-to-volume ratio of the human coagulation factor IX recombinant expression vector to the PEI transfection reagent was 1 μg: 2 μL, and the transfection time was 3 days. The recombinant expression vector of human coagulation factor IX was transfected into eukaryotic cells, and cells were selected under pressure to obtain stably transfected cells. After obtaining stably transfected cells, limiting dilution screening was used to obtain monoclonal cells.

10. The method according to claim 8, characterized in that, The step of extracting the target protein from the supernatant includes: The supernatant was concentrated by ultrafiltration to obtain a concentrated solution, which was then resuspended to obtain a protein suspension. After incubation, a mixture was formed, which was then subjected to chromatographic filtration and washing to obtain recombinant human coagulation factor IX.

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