Method for improving expression of recombinant human coagulation factor X in HEK293 cell and application

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a method for improving expression of a recombinant human blood coagulation factor X in HEK293 cells and application of the recombinant human blood coagulation factor X. The method comprises the following steps: inserting a human blood coagulation factor X gene segment containing a Kozak sequence into an eukaryotic expression vector to obtain a recombinant expression vector; and transforming the recombinant expression vector into an HEK293 cell, so as to obtain the HEK293 cell with high expression of the recombinant human coagulation factor X. When the recombinant HEK293 cell is used for production, detection shows that the concentration of the recombinant human coagulation factor X (rhFX) can reach 5.2 ng / mL and is increased by more than 4 times compared with the prior art, and the rhFX obtained by the invention has good biological activity.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for improving the expression of recombinant human coagulation factor X in HEK293 cells and its application. Background Art

[0002] Factor X deficiency is a rare autosomal recessive bleeding disease with an incidence of approximately 1:500,000 to 1:1,000,000; clinical manifestations include hemarthrosis, umbilical cord stump, gastrointestinal tract and central nervous system bleeding. Currently, the treatments for factor X deficiency include factor X concentrate, fresh frozen plasma (FFP) transfusion and prothrombin complex concentrate (PCC). However, these methods have some obvious disadvantages, including infection risk, allergic reactions, and thrombosis and disseminated intravascular coagulation (DIC) that may be caused by PCC. Therefore, how to effectively treat factor X deficiency remains a problem that needs to be solved urgently.

[0003] Bleeding disorders are an important class of diseases involving multiple clinical disciplines. The first-generation products for the treatment of bleeding disorders are blood-derived coagulation factors, but they have limitations: the raw materials for production are difficult to obtain, the amount of plasma collected upstream determines the output of the drug; and human products have the possibility of blood-borne infection. The development of recombinant technology has freed coagulation factors from dependence on plasma, and product supply is no longer restricted by the amount of plasma collected, avoiding the possibility of infection. Congenital bleeding disorders are mostly caused by molecular gene mutations in the coagulation cascade pathway. Coagulation factor X is a key factor in the coagulation cascade and plays a vital role in the hemostasis process. However, since patients with coagulation factor X deficiency are relatively rare and the number is far lower than that of hemophilia A and hemophilia B, they are often not taken seriously, resulting in slow progress in related research and development. At present, there is no recombinant human coagulation factor X (rhFX) drug on the market, and only the plasma concentrate Coagadex has been approved by the FDA for marketing. In terms of scientific research, researchers tried to express rhFX in Escherichia coli, and the final protein existed in the form of inclusion bodies, which hindered further optimization and development. Some scholars have also used HEK293 cells to express rhFX, and the expression yield was 1.317±0.146ng / ml, which still has room for improvement. Therefore, it is particularly important to provide a method for increasing the yield of recombinant human coagulation factor X. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application for increasing the expression of recombinant human coagulation factor X in HEK293 cells, wherein the method can significantly increase the yield of recombinant human coagulation factor X, and the recombinant human coagulation factor X has coagulation biological activity.

[0005] The present invention provides a method for increasing the expression of recombinant human coagulation factor X in HEK293 cells, comprising the following steps:

[0006] Inserting a human coagulation factor X gene fragment containing a Kozak sequence into a eukaryotic expression vector to obtain a recombinant expression vector;

[0007] The recombinant expression vector is transformed into HEK293 cells to obtain recombinant HEK293 cells that highly express recombinant human coagulation factor X.

[0008] As a preferred embodiment, the nucleotide sequence of the human coagulation factor X gene fragment containing the Kozak sequence is shown as SEQ ID NO.1.

[0009] As a preferred embodiment, the amplification primers for the human coagulation factor X gene fragment containing the Kozak sequence include a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0010] As a preferred embodiment, the eukaryotic expression vector includes a pcDNA3.1 plasmid.

[0011] As a preferred embodiment, the transformation method is to mix the recombinant expression vector with PEI to obtain a transfection reagent-nucleic acid complex, and use the transfection reagent-nucleic acid complex to transfect HEK293 cells.

[0012] As a preferred embodiment, the mass volume ratio of the recombinant expression vector to PEI is 1 μg:1-5 μL.

[0013] As a preferred embodiment, after the recombinant expression vector is mixed with PEI, the method further comprises incubating the recombinant expression vector and PEI at room temperature, wherein the incubation time is 20 to 40 minutes.

[0014] As a preferred solution, the density of the HEK293 cells is 50% to 100%.

[0015] As a preferred embodiment, the transfection time is 1 to 7 days.

[0016] The present invention also provides the use of the recombinant human coagulation factor X in the preparation of a drug for treating coagulation factor X deficiency.

[0017] Beneficial effects:

[0018] The present invention provides a method for increasing the expression of recombinant human coagulation factor X in HEK293 cells, the method comprising the following steps: inserting a human coagulation factor X gene fragment containing a Kozak sequence into a eukaryotic expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into HEK293 cells to obtain recombinant HEK293 cells that highly express recombinant human coagulation factor X. The recombinant HEK293 cells of the present invention are used for production, and the concentration of recombinant human coagulation factor X (rhFX) can reach 5.2 ng / mL after detection, which is more than 4 times higher than that of the prior art, and the rhFX obtained by the present invention has good activity. The rhFX obtained by using the recombinant HEK293 cells of the present invention for production can solve the problem that raw materials for the production of hemorrhagic diseases are difficult to obtain, and the method can obtain rhFX by culturing cells, thereby avoiding the problem of blood-borne infection in human products. Therefore, the present invention obtains rhFX with coagulation biological activity, which lays a foundation for further promoting the research and development of rhFX drugs.

[0019] In order to improve the expression level of rhFX, the present invention adds a Kozak sequence at the beginning of the target gene to enhance the translation efficiency of the eukaryotic gene. Western blot analysis and SDS-PAGE analysis detected a single band in the supernatant with a molecular weight of about 56kD, proving that rhFX was successfully expressed using recombinant HEK293 cells. The present invention systematically adjusts the HEK293 cell density, the ratio of the recombinant expression vector to the transfection reagent, and the transfection time. The results show that the rhFX expression amount gradually increases from the 1st to the 3rd day, and the protein expression amount gradually decreases from the 4th to the 6th day, indicating that the rhFX protein expression amount is the highest on the 3rd day after transfection. When the HEK293 cell density is 80%, the recombinant expression vector and PEI are transfected at a mass volume ratio of 1μg:2μL, the rhFX protein expression amount is the highest, and the optimal expression conditions of rhFX are successfully determined. The human coagulation factor X (F10) enzyme-linked immunosorbent assay kit detects the rhFX concentration to be 5.2ng / mL, and the rhFX concentration is more than 4 times higher than that of the prior art. The PT coagulation method was used to detect the activity of rhFX protein and the supernatant of the empty vector of the control group pcDNA3.1. The results showed significant differences (P<0.0001). The rhFX protein promoted the coagulation of factor X-deficient plasma. When the chromogenic substrate method was used to detect the activity of rhFX enzyme, the enzyme titration of rhFX was performed. At 2nM, rhFX had a large window and showed a linear relationship below 5nM, indicating that the sample had high activity. The chromogenic substrate method was used to verify the positive drug of rhFX. The results showed that the IC of Edoxaban was 50 It is 2.415 nM, which is close to 2.3 nM reported in the prior art, proving that rhFX has good activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 Schematic diagram of the recombinant expression vector and gene design in Example 1, wherein (A) is a schematic diagram of the recombinant expression vector, and (B) is a schematic diagram of the gene design;

[0022] Figure 2 The pcDNA3.1-FX recombinant expression vector in Example 1 is verified, wherein (A) is a diagram of the restriction enzyme digestion verification of the pcDNA3.1-FX recombinant expression vector, and (B) is a diagram of the verification of the pcDNA3.1-FX recombinant expression vector;

[0023] Figure 3 The colony PCR product in Example 1 was verified;

[0024] Figure 4 The Western blot and SDS-PAGE analysis and identification of rhFX expression in Example 2, wherein (A) is a Western blot identification diagram, and (B) is a SDS-PAGE analysis and identification diagram;

[0025] Figure 5 Western blot analysis of rhFX expression under different conditions in Example 3, wherein (A) is sampling at different transfection days, (B) is transfection at different ratios of the recombinant expression vector and PEI, and (C) is transfection at different densities of HEK293 cells;

[0026] Figure 6 The concentration of rhFX in the supernatant was detected by ELISA in Example 4;

[0027] Figure 7 The PT coagulation method in Example 5 was used to detect the activity of rhFX in the cell supernatant;

[0028] Figure 8 The chromogenic substrate method was used to determine the activity of rhFX enzyme in Example 6, wherein (A) is the titration result of rhFX enzyme, and (B) is the verification diagram of the chromogenic substrate method for rhFX as a positive drug. DETAILED DESCRIPTION

[0029] The present invention provides a method for increasing the expression of recombinant human coagulation factor X in HEK293 cells, comprising the following steps:

[0030] Inserting a human coagulation factor X gene fragment containing a Kozak sequence into a eukaryotic expression vector to obtain a recombinant expression vector;

[0031] The recombinant expression vector is transformed into HEK293 cells to obtain recombinant HEK293 cells that highly express recombinant human coagulation factor X.

[0032]

[0033] As a specific embodiment, the human coagulation factor X gene fragment containing the Kozak sequence is specifically designed to add a start codon (ATG) before the sequence of the human coagulation factor X gene (GenBank: M57285.1), and a Kozak sequence (GCCACC) is added before the start codon to obtain the human coagulation factor X gene fragment containing the Kozak sequence. In the present invention, adding a Kozak sequence at the beginning of the human coagulation factor X gene can enhance the translation efficiency of the eukaryotic human coagulation factor X gene.

[0034] The amplification primers for the human coagulation factor X gene fragment containing the Kozak sequence of the present invention include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0035] The sequences of the primers of the present invention are as follows:

[0036] Forward primer (SEQ ID NO.2): 5'-CCCAAGCTT GCCACC ATGGGAAGACCC CTGCATCTGGT-3', the bold part is the Hind III restriction site; the underlined part is the Kozak sequence;

[0037] Reverse primer (SEQ ID NO. 3): 5'-CGCGGATCCTCAATGATGGTGGTGGTG GTGCTTCAGGG-3', the bold part is the BamH I restriction site.

[0038] The preparation method of the human coagulation factor X gene fragment containing the Kozak sequence of the present invention can be: searching the gene sequence of human coagulation factor X (M57285.1) in GenBank, optimizing and synthesizing the human protein codon by Beijing Liuhe BGI Gene Technology Co., Ltd., obtaining the gene sequence of synthetic human coagulation factor X, designing primers containing the Kozak sequence (GCCACC), obtaining amplification primers, the nucleotide sequence of the forward primer is shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3, and the synthetic human coagulation factor X gene sequence is amplified by the amplification primers to obtain the human coagulation factor X gene fragment containing the Kozak sequence.

[0039] In order to express rhFX in eukaryotic cells, the present invention can use a eukaryotic expression vector when constructing a recombinant expression vector. In a specific embodiment, the eukaryotic expression vector is a pcDNA3.1 plasmid.

[0040] The transformation method of the present invention is to mix the recombinant expression vector with PEI to obtain a transfection reagent-nucleic acid complex, and use the transfection reagent-nucleic acid complex to transfect HEK293 cells.

[0041] The mass volume ratio of the recombinant expression vector to PEI of the present invention can be 1 μg: 1 to 5 μL; in a specific embodiment, the mass volume ratio can be 1 μg: 1 μL, 1 μg: 2 μL, 1 μg: 3 μL, 1 μg: 4 μL, or 1 μg: 5 μL; as a specific embodiment, when the mass volume ratio of the recombinant expression vector to PEI is 1 μg: 2 μL, the expression level of rhFX is the highest; the amino acid sequence of the recombinant human coagulation factor X (rhFX) is shown in SEQ ID NO.4. As a specific embodiment, the amino acid sequence of the recombinant human coagulation factor X (rhFX) is specifically: SEQ ID NO.4: MGRPL HLVLLSASLAGLLLLGESLFIRREQANNILARVTRANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEFWNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKNCELFTR KLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADNGKACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITWKPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVGGQECKDGECP WQALLINEENEGFCGGTILSEFYILTAAHCLYQAKRFKVRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYDFDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGIVSGFGRTH EKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQNMFCAGYDTKQEDACQGDSGGPHVTRFKDTYFVTGIVSWGEGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPEVITSSPLK.

[0042] After the recombinant expression vector of the present invention is mixed with PEI, the recombinant expression vector and PEI are incubated at room temperature, and the incubation time can be 20 to 40 minutes; in a specific embodiment, the incubation time can be 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes or 40 minutes.

[0043] The density of the HEK293 cells of the present invention can be 50% to 100%; in a specific embodiment, the density can be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; as a specific embodiment, when the density of the HEK293 cells is 80%, the expression level of the rhFX is the highest.

[0044] The transfection time of the present invention can be 1 to 7 days; in a specific embodiment, the time can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days; as a specific implementation method, the expression level of rhFX enzyme gradually increases from 1 to 3 days after transfection, and the expression level of rhFX enzyme begins to gradually decrease from 3 to 6 days, indicating that the expression level of rhFX enzyme is the highest on the 3rd day after transfection.

[0045] The present invention also provides the use of the recombinant human coagulation factor X in the preparation of a drug for treating coagulation factor X deficiency.

[0046] The recombinant HEK293 cells of the present invention were used for production, and the concentration of recombinant human coagulation factor X (rhFX) was tested to be 5.2 ng / mL, which was more than 4 times higher than that of the prior art. The activity of rhFX protein and the supernatant of the empty vector of the control group pcDNA3.1 were detected by the PT coagulation method, and the results showed that rhFX significantly promoted the coagulation of X-deficient plasma. When the rhFX enzyme activity was detected by the chromogenic substrate method, rhFX was titrated. At 2 nM, rhFX had a large window and showed a linear relationship below 5 nM, indicating that the rhFX enzyme of the present invention has high activity. The positive drug verification of rhFX by the chromogenic substrate method showed that the IC of Edoxaban was 50 It is 2.415 nM, which is close to 2.3 nM reported in the prior art, proving that rhFX has good activity.

[0047] The rhFX obtained by using the recombinant HEK293 cells of the present invention can solve the problem of difficulty in obtaining raw materials for the production of hemorrhagic diseases. At the same time, the method obtains rhFX by culturing cells, which can avoid the problem of blood-borne infection in human products. Therefore, the present invention obtains rhFX with coagulation biological activity, laying a foundation for further promoting the research and development of rhFX drugs.

[0048] To further illustrate the present invention, a method for increasing the expression of recombinant human coagulation factor X in HEK293 cells and its application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products known to those skilled in the art can be used.

[0050] MEM basal medium (Wuhan Punosai Life Science Technology Co., Ltd., catalog number: PM150411); fetal bovine serum, 0.25% trypsin-EDTA (Gibco, catalog number: 10091148, 25200056); endotoxin-free plasmid small-scale rapid extraction kit (Baoruyi (Beijing) Biotechnology Co., Ltd., catalog number: RA1508); HindⅢ restriction endonuclease, BamH I restriction endonuclease (TaKaRa, catalog number: 1615, 1605), containing 10x buffer, 10x Greenbuffer; ligation kit DNA Ligation Kit Ver. 2.1 (TaKaRa, catalog number: 6022), containing SolutionⅠ, SolutionⅡ, SolutionⅢ. Rapid purification gel DNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd., catalog number: DC301); Trelief5α competent cells (Qingke Bio, catalog number: TSC-C01). KOD FX Neo (TOYOBO, KFX-201), containing 2x PCR Buffer, 2mM dNTPs; polyetherimide (PEI), Edoxaban (MCE, Cat. No.: HY-K2014, HY-10264); mouse anti-6X His Tag monoclonal antibody, HRP-labeled goat anti-mouse IgG (Sangon, catalog number: D191001-0025, D110087-0100); PVDF membrane (Biyuntian, catalog number: FFP32); human coagulation factor X (F10) enzyme-linked immunosorbent assay kit (ELKBiotechnology, catalog number: ELK1557); Russell's viper venom factor X activator (RVV) (Pentapharm, catalog number: 121-06); S2765 (Beijing Asrel Biotechnology Co., Ltd., catalog number: AS00-0102); coagulation analyzer (Jingchuan Diagnostic Technology Co., Ltd., BCS-6100) and prothrombin time assay kit (Wuhan Zhongtai Biotechnology Co., Ltd., catalog number: PX1201); DNA sequencing, gene and primer synthesis were all completed by Beijing Liuhe BGI Genomics Co., Ltd.

[0051] Example 1 Construction and verification of the recombinant expression vector pcDNA3.1-FX

[0052] The gene sequence of human coagulation factor X (M57285.1) was retrieved from GenBank, and the human protein codon was optimized and synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd. to make it suitable for HEK293 cell expression. After the gene sequence of human coagulation factor X was synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd., in order to express the recombinant human coagulation factor X (rhFX) gene in a eukaryotic vector, the FX gene was constructed into the pcDNA3.1 vector using the PCR method, and the following was constructed: Figure 1 The specific method is: before the coagulation factor X gene sequence, add a start codon (ATG); before the start codon, add a Kozak sequence (GCCACC) (gene design as shown in Figure 1 (B) to enhance the translation efficiency of eukaryotic genes. The PCR method adds a Hind III restriction site upstream of the gene fragment and a BamH I restriction site downstream. Specifically, Primer 5.0 software was used to design PCR amplification primers; the upstream primer was named FX-Hind III-F (SEQ ID NO.2): 5'-CCCAAGCTT GCCACC ATGGGA AGACCCCTGCATCTGGT-3' (the bold part is the Hind III restriction site; the underlined part is the Kozak sequence); the downstream primer is named FX-BamH IR (SEQ ID NO.3): 5'-CGCGGATCCTCAATGATGGTGGTGGTGGTGCTTCAGGG-3' (the bold part is the BamH I restriction site). Amplification system: 1.5 μL of each upstream and downstream primers, 1 μL of synthetic human coagulation factor X gene sequence, 1 μL KOD high-fidelity enzyme, 10 μL dNTPs, 10 μL 2×PCR Buffer, 25 μL ddH2O. The PCR reaction program is: 95℃ pre-denaturation for 2 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 45 s, a total of 35 cycles; 72℃ final extension for 7 min. The PCR product obtained by amplification is a human coagulation factor X gene fragment containing a Kozak sequence, referred to as the FX fragment.

[0053] The FX fragment and the pcDNA3.1 empty vector were double-digested with HindⅢ restriction endonuclease and BamHI restriction endonuclease, respectively. The digestion system was: 3.5 μL each of HindⅢ restriction endonuclease and BamHI restriction endonuclease, 20 μL of FX fragment or pcDNA3.1 vector, and 3 μL of 10×GreenBuffer. After digestion at 37℃ for 10 minutes, the digestion products were electrophoresed on 1% agarose gel, cut and recovered using a rapid purification gel DNA extraction kit.

[0054] The recovered pcDNA3.1 vector and FX fragment were connected in a 16°C metal bath for 0.5 hours to obtain a connection product, which was a pcDNA3.1-FX recombinant expression vector. The connection system was: 2 μL of the recovered pcDNA3.1 vector, 3 μL of the recovered FX fragment, and 5 μL of solution I.

[0055] Transform the pcDNA3.1-FX recombinant expression vector into Trelief5α competent cells. ① Before transformation, add 1μL solutionⅢ to 10μL of pcDNA3.1-FX recombinant expression vector, add 40μL competent cells melted on ice (TSC-E01, Qingke Biotechnology), mix gently, and let stand on ice for 30min. ② Heat shock in a 42℃ metal bath for 60s, quickly transfer to an ice bath, and let stand for 2min; ③ Add 700μL of sterile LB liquid culture medium without antibiotics to the centrifuge tube, mix well, and resuscitate at 37℃, 200rpm for 60min. ④ Take 50μL of the resuscitation solution and evenly apply it to the LB solid culture medium containing 100μg / mL of ampicillin, and place the plate upside down in a 37℃ incubator for overnight culture.

[0056] Enzyme digestion verification of the recombinant expression vector pcDNA3.1-FX: The recombinant plasmid was extracted using an endotoxin-free plasmid extraction kit, and the plasmid was verified by enzyme digestion. The recombinant expression vector pcDNA3.1-FX was double-digested with HindⅢ restriction endonuclease and BamHI restriction endonuclease. The enzyme digestion system: 3.5μL each of HindⅢ restriction endonuclease and BamHI restriction endonuclease, 20μL of the recombinant expression vector pcDNA3.1-FX, and 3μL 10× Green Buffer. After enzyme digestion at 37℃ for 10min, the enzyme digestion product was electrophoresed by 1% agarose gel.

[0057] The results of enzyme digestion verification are as follows Figure 2 As shown, the pcDNA3.1-FX recombinant expression vector was analyzed by agarose gel electrophoresis and the target band between 6133bp and 8023bp was observed ( Figure 2 Middle (B); After the pcDNA3.1-FX recombinant expression vector was double-digested with HindIII and BamHI, the electrophoresis results showed that there was a FX target band between 1200bp and 2000bp, and there was a pcDNA3.1 vector plasmid fragment after enzyme digestion above 4500bp ( Figure 2 In (A), it was proved that the pcDNA3.1-FX recombinant expression vector was successfully constructed. On this basis, the above plasmid was verified by BGI sequencing.

[0058] Colony PCR verification of the recombinant expression vector pcDNA3.1-FX: Pick 5 single colonies for colony PCR verification. Colony PCR reaction system: 2.75μL of upstream and downstream primers, 55μL2×RapidTaqMaster Mix, 49.5μLddH2O. The PCR reaction program is: 95℃ pre-denaturation for 2min; 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 1min, a total of 35 cycles; 72℃ final extension for 7min. Colony PCR verification results are shown in Figure 3 ,Agarose gel electrophoresis analysis showed that the 5 pcDNA3.1-FX single colonies were between 1200bp-2000bp, and the band size was consistent with the target protein (1464bp), indicating that the 5 colonies contained successfully constructed recombinant expression vectors. Four positive colonies were selected and shaken on a constant temperature shaker at 37℃, 200r / min for 12h, and part of the bacterial solution was sent to BGI for further identification.

[0059] Example 2 HEK293 cell culture and transient transfection of pcDNA3.1-FX recombinant expression vector

[0060] Transient transfection process:

[0061] The MEM basal medium supplemented with 10% fetal bovine serum was used as the growth medium for HEK293 cells, and the cells were cultured in an adherent environment at 37°C and 5% CO2. When the cells grew to about 90%, they were digested with trypsin cell digestion solution (0.25% trypsin) for 1 min and centrifuged at 1200rpm for 3 min. HEK293 cells were used as the transfection cell line, and the pcDNA3.1-FX recombinant expression vector prepared in Example 1 was transfected into the cells, and the cells were plated one day before transfection. Taking a bottle of T75 culture flask as an example, the cell culture medium was replaced with serum-free MEM basal medium before transfection, 10 μg of pcDNA3.1-FX recombinant expression vector was taken into a 2mL EP tube, 1mL of serum-free MEM basal medium was added, and gentle mixing was performed to obtain a mixed recombinant expression vector. 25 μL of PEI transfection reagent was added to another 2mL EP tube, 1mL of serum-free MEM basal medium was added, and gentle mixing was performed to obtain a mixed PEI. Transfer the mixed recombinant expression vector to a PEI EP tube containing the mixture, mix gently (total volume is 2 mL), and incubate at room temperature for 30 minutes to form a transfection reagent-nucleic acid complex. Add the transfection reagent-nucleic acid complex evenly to the culture flask containing HEK293 cells. The negative control uses pcDNA3.1 empty plasmid.

[0062] Protein concentration and purification process:

[0063] 48 hours after transfection, the cell supernatant was collected, placed in a 30KDa ultrafiltration tube at 4°C for concentration, replaced with PBS three times, and mixed with HisTagNi-NTA resin (Roche) pre-equilibrated with Lysis Buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, 10% glycerol, pH 8.0), and then the mixture was placed on a shaker and incubated on ice at a speed of 65r / min for 2h. The mixture was transferred to an empty chromatography column. After filtration, it was washed with Washing Buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole, 10% glycerol, pH 8.0) to wash away unbound impurities. Finally, rhFX was eluted and collected with Elution Buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole, 10% glycerol, pH 8.0). Concentrated using a 30KDa ultrafiltration tube. After concentration, take 20 μL of sample and mix with 5 μL of protein loading buffer and boil for 5 minutes. The SDS-PAGE electrophoresis conditions are 130V, 50 minutes. Place the separation gel part in Coomassie Brilliant Blue fast staining solution for 40 minutes. Transfer the protein on the gel to the PVDF membrane using semi-dry electrotransfer method, the conditions are 350mA, 80 minutes. Rinse with TBST at room temperature for 30 seconds, and block with protein-free blocking solution at room temperature for 40 minutes. Add primary antibody and incubate slowly at 4°C overnight. Wash with TBST for 10 minutes, repeat 3 times, add secondary antibody and incubate slowly at room temperature for 1 hour, wash with TBST for 10 minutes, repeat 3 times; after completion, expose and develop under the imager.

[0064] Test results:

[0065] By Western blot ( Figure 4 (A)) and SDS-PAGE ( Figure 4 In (B), the collected Elution eluate was analyzed and identified. In the figure, plasmid 1, plasmid 2, plasmid 3 and plasmid 4 are proteins expressed by the recombinant expression vectors in the 4 positive colonies selected in Example 1, and the blank is the pcDNA3.1 empty vector. The results showed that specific bands were shown at 56 kDa; no specific bands were observed in the supernatant of cells transfected with the pcDNA3.1 empty plasmid, indicating that the rhFX enzyme was successfully expressed in HEK293.

[0066] Example 3 Optimization of conditions for transfecting HEK293 cells with the recombinant expression vector pcDNA3.1-FX

[0067] The transfection conditions were systematically optimized from the perspectives of HEK293 cell density, the ratio of plasmid to transfection reagent, and the number of days of transfection, and the expression intensity of rhFX was detected by Western Blot. ① Optimization of transfection days: The same number of HEK293 cells was added to the T75 cell culture flask at the same time for 6 consecutive days. The cells were transfected after 24 hours of attachment, and the 6 bottles of cells were numbered day6, day5, day4, day3, day2, and day1. When transfecting day1 on the 7th day, the five bottles of cells on day6, day5, day4, day3, and day2 were replaced with blank MEM basal medium. On the 8th day, the supernatants were taken for Western Blot detection, so that the six bottles of cells corresponded to the expression of rhFX on the sixth day, the fifth day, the fourth day, the third day, the second day, and the first day, respectively. The transfection method was transfected according to the method of Example 2. Except for the time of transfection, other conditions remained unchanged. The three optimized condition negative controls all used pcDNA3.1 empty plasmid transfection. ② Optimization of the ratio of recombinant expression vector and transfection reagent: The ratio of the amount of recombinant expression vector (μg) and PEI (μL) was set to 1:1, 1:2, 1:3, 1:4, 1:5, and transfection was performed. The transfection method was performed according to the method of Example 2. Except for the ratio of the recombinant expression vector and the transfection reagent, other conditions remained unchanged. After 48 hours of transfection, Western Blot was used to observe the protein expression and select the optimal transfection plasmid and PEI ratio. ③ Optimization of HEK293 cell density: HEK293 of different densities was spread in a 6-well plate so that on the third day, it could reach 60%, 70%, 80%, 90%, and 100% of the bottom area of ​​the well, respectively, and transfection was performed. The transfection method was performed according to the method of Example 2. Except for the HEK293 cell density, other conditions remained unchanged. After 48 hours of transfection, Western Blot was used to observe the protein expression.

[0068] The results of transfection condition optimization are as follows Figure 5 As shown in the figure, ① optimization of transfection days: the expression of recombinant human coagulation factor X gradually increased from day 1 to day 3, and the expression of recombinant human coagulation factor X began to gradually decrease from day 3 to day 6, indicating that the expression of rhFX enzyme was the highest on day 3 after transfection ( Figure 5 ② Optimization of the ratio of recombinant expression vector to transfection reagent: The results showed that the ratio of recombinant expression vector (μg) to PEI (μL) was 1:2, and the expression level of rhFX enzyme was the highest ( Figure 5 ③ Optimization of cell density: When the cell density was 80%, the expression level of rhFX enzyme was the highest ( Figure 5 Middle (C)). No specific band was observed in the supernatant of cells transfected with pcDNA3.1 empty plasmid.

[0069] Example 4 ELISA detection of rhFX in cell supernatant

[0070] The transfection conditions were optimized, that is, transfection was performed when the cell density reached 80% of the bottom area of ​​the culture flask, the ratio of the amount of the recombinant expression vector (μg) to PEI (μL) was 1:2, and the transfection steps and methods were the same as the transient transfection process in Example 2. Samples were taken on the 4th day after transfection, and the supernatant was concentrated and purified according to the protein concentration and purification process in Example 2. The purified sample was used as the rhFX concentration sample to be tested. The sample was prepared 3 times. According to the instructions of the human coagulation factor Ⅹ enzyme-linked immunosorbent assay kit, the standard curve was drawn with the standard product in the kit, 100 μL of the rhFX sample to be tested was added to the enzyme-labeled well, and incubated at 37°C for 80 min. Washed 3 times with washing buffer, patted dry, added 100 μL of biotinylated antibody working solution, and incubated at 37°C for 50 min. Washed 3 times with washing buffer, patted dry, added 100 μL of HRP enzyme working solution, and incubated at 37°C for 50 min. Wash 5 times with washing buffer, pat dry, add 90 μL TMB, and incubate at 37°C for 20 min. Add 50 μL stop solution and immediately measure the OD value at a wavelength of 450 nm. Use prism to draw a standard curve and calculate the concentration of rhFX in the sample.

[0071] The cell supernatant was collected and the rhFX concentration in the cell supernatant was detected using the rhFX ELISA kit. The standard curve drawn by the standard product was Y=0.1382×X+0.1228, R 2 =0.9896( Figure 6 ). Detect the supernatant of HEK293 cells transiently transfected three times, and bring the OD value of the sample to be tested into the standard curve ( Figure 6 ), 3 transient transfections, rhFX concentration was 5.00 ng·mL -1 , 4.80ng·mL -1 and 5.20 ng·mL -1 The concentration of rhFX enzyme obtained by the method of the present invention is more than 4 times higher than that of the prior art (Feng Yuanzheng. Study on coagulation factor X deficiency caused by compound mutations in a Chinese family and its function [D], 2022).

[0072] Example 5 Detection of coagulation activity of rhFX

[0073] The transfection conditions were optimized, i.e., transfection was performed when the cell density reached 80% of the bottom area of ​​the culture flask, the ratio of the amount of the recombinant expression vector (μg) to PEI (μL) was 1:2, and the transfection steps and methods were the same as those in Example 2. Samples were taken on the 4th day after transfection, and the supernatant was concentrated and purified according to the method of Example 2. The purified sample was used as the rhFX sample to be tested. The sample was prepared 3 times. The negative control selected the supernatant of cells transfected with the pcDNA3.1 empty plasmid, and the treatment method was the same as that of the experimental group.

[0074] The activity of rhFX was determined by referring to the "Human Coagulation Factor Titer Assay" numbered 3520 in the General Rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia. Take 50 μL of the 10-fold diluted rhFX sample or blank sample, add 50 μL of factor X-deficient plasma, place it on a semi-automatic coagulation analyzer and incubate at 37°C for 3 minutes, add 100 μL of pre-warmed PT prothrombin reagent, and record the coagulation time.

[0075] The results of the coagulation activity test of rhFX are shown in Tables 1 and Figure 7 As shown, the clotting time of the supernatant of the control group cells transfected with the empty plasmid of pcDNA3.1 was greater than 120s, and the clotting time of rhFX in the three transient transfection samples was 61.1s, 60.9s, and 60.2s, respectively; each sample was tested three times, and the error was within 1s. The results showed that the clotting time of the supernatant collected by the experimental group rhFX and the control group pcDNA3.1 empty vector transfection was significantly different (P<0.0001), proving that the rhFX enzyme has good procoagulant activity.

[0076] Table 1 Coagulation time and activity of different samples

[0077] Samples to be tested blank A B C Solidification time(s) >120 61.1±1 60.9±1 60.2±1

[0078] Example 6 Determination of rhFX enzyme activity by chromogenic substrate method

[0079] The transfection conditions were optimized, i.e., transfection was performed when the cell density reached 80% of the bottom area of ​​the culture flask, the ratio of the amount of the recombinant expression vector (μg) to PEI (μL) was 1:2, and the transfection steps and methods were the same as those in Example 2. Samples were taken on the 4th day after transfection, and the supernatant was concentrated and purified according to the method of Example 2. The purified sample was used as the rhFX sample to be tested.

[0080] In order to further detect the coagulation activity of rhFX, the chromogenic substrate method was used to verify the enzyme activity of rhFX based on the method of the 2020 edition of the Chinese Pharmacopoeia (Example 5). Specifically, (1) rhFX titration: After RVV activates rhFX of different concentrations, rhFX generates FXa; FXa reacts with S2765 to generate p-nitroaniline (pNA), and pNA has an absorption peak at 405nm. The OD value at 405nm is detected using an enzyme reader. When the trend of increasing (or decreasing) rhFX concentration is consistent with the trend of OD405 value, it indicates that rhFX has enzyme activity. (2) Positive drug verification: After adding the FXa inhibitor Edoxaban, the generation of pNA can be inhibited, which is manifested as a decrease in the absorption of light. The OD value at 405nm is detected by an enzyme reader, from which it is inferred that Edoxaban has inhibitory activity on FXa, that is, rhFX has enzyme activity. The stock concentrations of rhFX, RVV, and S2765 were 3.2 μM, 5 U / mL, and 6 mM, respectively. During the experiment, they were diluted with self-prepared buffer assay buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl).

[0081] (1) rhFX titration: The total reaction system is 80 μL, including 40 μL of rhFX and RVV mixture, and 40 μL of S2765. The specific method is: aspirate 2 μL of rhFX with a concentration of 3.2 μM and 10 μL of RVV with a concentration of 5 U / mL into the same EP tube, and add 68 μL of assay buffer into the EP tube. Mix gently, and incubate the rhFX and RVV mixture at 37°C for 10 min. Dilute the RVV and rhFX mixture by 2 times using the gradient dilution method. In a 384-well plate, add 40 μL of the diluted RVV and rhFX mixture to each well, and then add 40 μL of S2765 diluted with assay buffer (so that the final concentration of S2765 in the 80 μL system is 500 μM). Incubate at 37°C for 30 min, and immediately measure the OD value at a wavelength of 405 nm (see the test results). Figure 8 (A) in the figure.

[0082] (2) Verification of positive drugs: The reaction system is 80 μL, including 10 μL Edoxaban, 30 μL rhFX and RVV mixture, and 40 μL S2765. The final concentrations of rhFX, RVV, and S2765 in the 80 μL system are 2 nM, 0.31 U / mL, and 100 μM, respectively. The specific method is as follows: First, the experiment dilutes rhFX and RVV according to the final concentration requirements, and the diluted rhFX and RVV mixture is incubated at 37°C for 10 minutes. Within these 10 minutes, 200 μM S2765 is prepared, and Edoxaban is diluted 3 times from 1 μM to the specified concentration by gradient dilution method. In a 384-well plate, 10 μL of Edoxaban of different concentrations is added to each well, and 10 μL DMSO is added to the blank group. Then add 30 μL of the incubated rhFX and RVV mixture, and finally add 40 μL of 200 μM S2765. Incubate at 37°C for 30 min, and immediately measure the OD value at a wavelength of 405 nm. Use Prism to draw a curve and calculate the IC 50 (Test results see Figure 8 (B) in the figure.

[0083] rhFX enzyme titration results Figure 8 As shown in (A), rhFX has a large window at 2nM and shows a linear relationship below 5nM, indicating that the sample has high activity. The chromogenic substrate method was used to verify rhFX as a positive drug. The results are as follows Figure 8 As shown in (B), the IC of Edoxaban 50 It is 2.415nM, which is close to the 2.3nM reported in the prior art (HONDAY, KAMISATO C, MORISHIMAY. Edoxaban, a direct factor Xa inhibitor, suppresses tissue-factorinduced human platelet aggregation and clot-bound factor Xa in vitro: Comparison with an antithrombin-dependent factor Xa inhibitor, fondaparinux[J]. Thrombosis Research, 2016, 141: 17-21), proving that the purified rhFX has good activity.

[0084] It can be seen that the concentration of recombinant human coagulation factor X (rhFX) obtained by the method of the present invention can reach 5.2 ng / mL, which is more than 4 times higher than that of the prior art, and the rhFX obtained by the method of the present invention has good biological activity.

[0085] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for increasing the expression of recombinant human coagulation factor X in HEK293 cells, characterized in that: The following steps are involved: Inserting a human coagulation factor X gene fragment containing a Kozak sequence into a eukaryotic expression vector to obtain a recombinant expression vector; The recombinant expression vector is transformed into HEK293 cells to obtain recombinant HEK293 cells that highly express recombinant human coagulation factor X.

2. The method according to claim 1, characterized in that The nucleotide sequence of the human coagulation factor X gene fragment containing the Kozak sequence is shown in SEQ ID NO.

1.

3. The method according to claim 2, characterized in that The amplification primers for the human coagulation factor X gene fragment containing the Kozak sequence include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.2; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

3.

4. The method according to claim 3, characterized in that The eukaryotic expression vector includes pcDNA3.1 plasmid.

5. The method according to claim 4, characterized in that The transformation method comprises mixing the recombinant expression vector with PEI to obtain a transfection reagent-nucleic acid complex, and using the transfection reagent-nucleic acid complex to transfect HEK293 cells.

6. The method according to claim 5, characterized in that The mass volume ratio of the recombinant expression vector to PEI is 1 μg:1-5 μL.

7. The method according to claim 6, characterized in that After the recombinant expression vector is mixed with PEI, the method further comprises incubating the recombinant expression vector and PEI at room temperature, wherein the incubation time is 20 to 40 minutes.

8. The method according to claim 7, characterized in that The density of the HEK293 cells is 50% to 100%.

9. The method according to claim 8, characterized in that The transfection time is 1 to 7 days.

10. Use of the recombinant human coagulation factor X prepared by the method according to any one of claims 1 to 9 in the preparation of a drug for treating coagulation factor X deficiency.

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