FVII signal peptide mutant and application thereof

Abstract

The invention relates to an FVII signal peptide mutant and application thereof. According to the FVII signal peptide mutant, gene mutation is carried out on leucine at one or more sites of L12, L13 and L19 of the FVII signal peptide based on the FVII signal peptide, so that the activity output of the FVII signal peptide is increased. The invention also provides nucleic acid for coding the FVII signal peptide mutant and an expression vector containing the nucleic acid. According to the FVII signal peptide mutant disclosed by the invention, the activity output of protein is remarkably improved. In an enzyme-linked immunosorbent assay and a Western Blot experiment, a specific antibody is used for performing quantitative detection on carboxylated and expressed reporter protein, and the substrate can recognize a carboxylated FVII Gla structural domain and a functional structural domain, so that the expression and carboxylation level of the blood coagulation factor VII can be detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, in particular to a FVII signal peptide mutant and application thereof. BACKGROUND

[0002] Coagulation factor VII (encoded by F7) is located at the beginning of the extrinsic coagulation cascade, and is a key factor for the initiation of the extrinsic coagulation pathway, which is composed of a signal peptide, a propeptide, a Gla domain and a serine protease domain. Since FVII is located at a key position of the extrinsic coagulation pathway, severe FVII deficiency can cause central nervous system bleeding, gastrointestinal bleeding, etc., and complete deficiency can cause death of infants before and after birth. The treatment principle of FVII deficiency is similar to hemophilia B. However, due to the low content (0.5 μg / ml) and short half-life (only 3-4 h) of factor VII in plasma, replacement therapy should be performed every 4-6 h for severe bleeding. In addition to the treatment of FVII deficiency, FVII is more likely to be used for emergency treatment of hemophilia, clinical first aid for massive trauma bleeding and treatment of thrombocytopenia, etc.

[0003] Coagulation factor VII is secreted into the extracellular matrix after undergoing multiple specific post-translational modifications after synthesis. Gamma-glutamyl carboxylation modification is a necessary modification of coagulation factor VII, and its modification efficiency is the rate-limiting step for the production of factor VII activity. Meanwhile, the complex structure and post-translational modification of FVII are also an important factor hindering the development of recombinant coagulation factor VII. Currently, only two types of recombinant coagulation factor VII have been approved for clinical treatment on the market, namely NovoSeven approved by Novo Nordisk in 1999 and Coagulation factor VIIa (recombinant)-jncw / Sevenfact approved by LFB Biotechnology in 2020. The treatment drug prices of these two types of recombinant coagulation factor VII are high, among which the price of NovoSeven of Novo Nordisk reaches 6500 yuan / mg. However, patients need to be administered every 4-6 hours until hemostasis is achieved, and the injection dose and frequency should be determined according to the individual. Such injection frequency and treatment cost have brought great pressure to patients and the medical system.

[0004] Therefore, the establishment of a high-efficiency active coagulation factor VII expression system and a F7 gene therapy system will significantly reduce the production and treatment costs and effectively solve this problem. SUMMARY

[0005] The purpose of the present application is to provide a FVII signal peptide mutant and application thereof, and to efficiently produce active coagulation factor VII.

[0006] The present application provides a FVII signal peptide mutant, which is based on FVII signal peptide and has one or more of the following mutations in L12, L13 and L19 to increase the activity of the mutant.

[0007] The present application also provides a nucleic acid encoding the FVII signal peptide mutant.

[0008] The present application also provides an expression vector comprising the nucleic acid.

[0009] The present application also provides a cell line expressing the FVII signal peptide mutant.

[0010] The present application also provides a method for preparing the FVII signal peptide mutant, which comprises mutating the leucine at the target position of the wild-type FVII signal peptide to an amino acid that can significantly increase the activity of the FVII mutant, transiently transfecting the mutant plasmid into a cell line overexpressing VKOR (vitamin K epoxidase), constructing the cell line that can stably express the active FVII mutant, and expanding the cell line in a multi-layer flask, collecting the supernatant in large quantities, and purifying the protein.

[0011] Preferably, the sequence of the PCR cloning primer of the FVII mutant is shown in any one of SEQ ID NO: 2-17.

[0012] The present application also provides the use of the FVII signal peptide mutant, the nucleic acid, the expression vector or the cell line in the production of blood coagulation factor VII and / or FVII protein.

[0013] The present application also provides the use of the FVII signal peptide mutant, the nucleic acid, the expression vector or the cell line in the preparation of a medicament for treating blood coagulation-related diseases.

[0014] Preferably, the blood coagulation-related disease includes FVII deficiency or hemophilia.

[0015] Through the study of FVII signal peptide, the FVII signal peptide mutant of the present application significantly improves the activity of the protein. In the present application, the enzyme-linked immunosorbent assay (ELISA) and Western Blot experiment use specific antibodies to quantitatively detect the carboxylated and expressed reporter protein. The substrate can recognize the carboxylated FVII Gla domain and functional domain, thereby detecting the expression and carboxylation level of blood coagulation factor VII.

[0016] The FVII signal peptide mutant of the present application can be used for efficient production of blood coagulation factor VII and active FVII protein, has low production cost, and can be used for preparing a gene therapy drug for treating blood coagulation diseases, thereby providing a new treatment approach and idea for the diseases.

[0017] Hemophilia A and B can be treated prophylactically and on-demand by injection of the corresponding clotting factor. However, some hemophilia patients have inhibitors against the clotting factor VIII or IX, which makes the treatment difficult. In this case, the treatment of the clotting factor VIII or IX is no longer effective, and other methods such as injection of exogenous clotting factor of the extrinsic coagulation pathway are needed for hemostasis and bleeding prevention.

[0018] Furthermore, about 30% of severe hemophilia A patients have inhibitors, and thus have a high demand for prophylactic and on-demand treatment. In addition, there are a large number of other needs for hemophilia treatment, such as treatment of intracranial hemorrhage, varicose hemorrhage, etc., which further expands the market size of FVII. The preparation of clotting factor VII by genetic engineering technology is not only safe, but also easy to produce in large quantities. Therefore, the development of clotting factor VII with high output activity has very promising application prospects.

[0019] Due to the complexity of the post-translational modification of clotting factor VII, the two commonly used recombinant activated clotting factor VII products have the problems of low expression and imperfect post-translational modification processing of the protein. The preparation method of the present application modifies the signal peptide to obtain a high expression protein, which is simple, efficient and highly practical. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Schematic diagram for detecting the expression level of intracellular FVII L13 site mutant by immunoblotting.

[0021] Figure 2 Schematic diagram for detecting the expression level of extracellular FVII L13 mutant protein by immunoblotting.

[0022] Figure 3 Schematic diagram for detecting the carboxylation level of FVII L12, L13 and L19 site mutants by enzyme-linked immunosorbent assay, wherein A is the carboxylation level of FVII L12 site mutant, B is the carboxylation level of FVII L13 site mutant, and C is the carboxylation level of FVII L19 site mutant.

[0023] Figure 4 Schematic diagram for detecting the coagulation activity level of L12, L13 and L19 site mutants with increased FVII activity output by coagulation method. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described clearly and completely in combination with specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0025] The construction of the high-efficiency expression system includes the following steps: (1) Modification of FVII amino acid sequence Based on the wild-type coagulation factor VII gene sequence (as shown in SEQ ID NO: 1), site-directed codon mutation is carried out; on this basis, the L12, L13, L19 sites of the FVII signal peptide are saturatedly mutated by site-directed mutation technology.

[0026] (2) Detection of carboxylation and coagulation activity of FVII signal peptide L12, L13, L19 site after mutation Based on the common HEK293T cells, the wild-type FVII and its mutant expression plasmids are transiently transfected to compare the differences between the FVII mutant and the wild type. Enzyme-linked immunosorbent assay (ELISA) and Western Blot experiment use specific antibodies to quantitatively detect the carboxylation and expressed reporter protein. The substrate can recognize the carboxylated FVII Gla domain and functional domain, so as to detect the expression and carboxylation level of coagulation factor VII. For the coagulation activity of FVII, coagulation factor VII detection kit (coagulation method) is directly used for detection.

[0027] (3) Establishment of high-efficiency expression system of active coagulation factor VII The modified VKOR is transfected into HEK293 to construct a VKOR-flag HKE293 cell line which can significantly improve the expression of active coagulation factor. Therefore, we transfected the FVII mutant into the constructed VKOR-flag HKE293 cell line, and after verification, a high-efficiency expression system of active coagulation factor VII was established.

[0028] The reagents and instruments involved in the implementation are as follows: Multiskan F type enzyme label instrument was purchased from Thermo Fisher (Shanghai) Instrument Co., Ltd.; Restriction endonuclease Xho I, Kpn I and BamH I were purchased from NEB company; T4 DNA ligase was purchased from NEB company; phCMV1-WPRE plasmid was purchased from Invitrogen company; Mouse anti-human FVII monoclonal antibody (CaFVII-22) was purchased from Invitrogen company, and the item number was MA5-17631; Rabbit anti-human F7 polyclonal antibody was purchased from Proteintech company, and the item number was 23058-1-AP; Rabbit anti His-tag mAb was purchased from ABclonal company, and the item number was AE086; Goat anti-rabbit HRP was purchased from ABclonal company, and the item number was AS014; Goat anti-mouse HRP was purchased from Proteintech company, and the item number was SA00001-1; Mouse anti-GAPDH monoclonal antibody was purchased from Proteintech company, and the item number was 60004-1-Ig; ABTS color developing solution was purchased from Eracare company; Plasmid small extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; Escherichia coli F-DH5α was purchased from Shanghai Weidi Biotechnology Co., Ltd.; Factor VII activity assay kit (coagulation method) was purchased from SIEMENS Healthineers company.

[0029] Pancreatin, Opti-MEM® I medium, DMEM cell culture medium and 1×PBS buffer were purchased from Gibco company, and the DMEM medium contained 10% fetal bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin, and was stored in a 4 ℃ refrigerator for standby; PEI transfection reagent and 2×Prime Star Master Mix reagent were purchased from TAKARA company; HEK293T cells were purchased from Clontech company.

[0030] Solution preparation: TBST buffer: 20 mM Tris-HCl (pH 7.6), 100 mM sodium chloride and 0.1% Tween 20; 50 mM Carbonate buffer: 1.6 g sodium carbonate, 2.9 g sodium bicarbonate to 1 L; Cell lysis solution: 865 μL 1× PBS, 100 μL 10% Triton, 10 μL 100× PIC, 20 μL 100 mM PMSF and 5 μL 2-4 mg / mL DNase.

[0031] Other common reagents and instruments used in the examples were purchased in the market and are not specifically mentioned here.

[0032] Example 1 Step one, construction of FVII and mutant expression plasmid (1) Construction of FVII expression plasmid According to the information sequence of human FVII gene in GeneBank database (http: / / www.ncbi.nlm.nih.gov / genbank), the cDNA sequence is SEQ ID NO: 1. After double enzyme digestion, the FVII-8His (Xho I-BamH I) fragment and the FVII-MBP-6His (Xho I-Kpn I) fragment are connected to the phCMV-WPRE vector digested by the same enzyme through T4 connection to construct the recombinant plasmids FVII+8His phCMV1-WPRE and FVII+MBP 6His phCMV1-WPRE.

[0033] SEQ ID NO: 1:

[0034] (2) Construction of FVII mutant plasmid PCR cloning primers for constructing FVII mutant were designed, and the complete mutant plasmid was cloned by PCR using FVII MBP 6His-phCMV1-WPRE plasmid as a template according to the instructions of 2x Prime Star Master Mix (TAKARA) kit. The primers for FVII mutant were synthesized by GenScript Biotech Co., Ltd., and the sequence design is shown in Table 1.

[0035] Table 1

[0036] Step two, detection of L13 mutant FVII expression level (1) Transient transfection Cells HEK293T were seeded at 2.5x10 5 cells / well in 12 / 48 well plates, and cultured in DMEM medium containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin, and grown to about 70%~80% before starting the experiment.

[0037] Wild type and mutant plasmids were transiently transfected into cells using PEI, and HEK293T cells without plasmid transfection were set as controls. After 4~6 h of transfection, the culture medium containing 5 μg / ml Vitamin K was replaced and cultured for 48 h, and the supernatant was collected.

[0038] (2) Detection of FVII expression by immunoblotting The culture supernatant of transient transfection expression for 48 h was collected, and the cells were lysed using PIC cell lysis buffer, and the supernatant was collected after centrifugation. After protein denaturation, the culture supernatant and cell samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) at 60V, and then transferred to PVDF membrane. The primary antibody for detecting intracellular / extracellular protein expression level was MBP-Tag Polyclonal antibody (1:4000) / F7 Rabbit (1:2000), and the secondary antibody was Goat-rabbit-HRP (1:5000 / 1:4000). GAPDH (detection antibody was GAPDH Monoclonal antibody 1:6000, Goat-Mouse-HRP 1:5000) was used as an internal reference to compare the expression amount of intracellular mutant FVII, as shown in Figure 1 and Figure 2 .

[0039] The results show that the modification of L13P and L13R at the position L13 of the signal peptide enhances the secretion of the protein to some extent, and other mutations have no effect on the secretion and expression of the protein.

[0040] Step three, detecting the influence of FVII L12, L13, L19 site mutation on the carboxylation efficiency of active blood coagulation factor VII (1) Transient transfection The transient transfection method of step two is used.

[0041] (2) ELISA detection of carboxylation level of active blood coagulation factor VII in supernatant Primary antibody coating: dilute Rabbit anti His-tag mAb by 1:3000, take 100 μl into the enzyme-labeled plate, and stand at room temperature for 3 h; after washing with TBST, add 230 μl of 0.2% BSA solution, and stand at 4 ℃ overnight.

[0042] Tap the BSA solution and add 30 μl of CaCl2 and 100 μl of the culture supernatant collected after 48 h of transient transfection expression in step two, and stand at room temperature for 3 h. Discard the supernatant and wash with TBST-CaCl2, then add 100 μl of secondary antibody Factor VII Monoclonal Antibody (CaFVII-22) (1:2000), stand at room temperature for 1 h; discard the supernatant and wash with TBST-CaCl2, then add 100 μl of tertiary antibody Goat anti-mouse HRP (1:4000), stand at room temperature for 1 h; discard the supernatant, develop and use the enzyme-labeled instrument to detect the carboxylation level at 405 nm wavelength after washing with TBST-CaCl2. The detection results are shown in Figure 3 .

[0043] The results show that saturated mutation is performed on L12, L13 and L19, L12E at the position L12 significantly improves the FVII activity output; L13D, L13E, L13P and L13R at the position L13 significantly improve the FVII activity output, L19E, L19G and L19P at the position L19 significantly improve the FVII activity output, and other mutations have no significant effect on the FVII activity output.

[0044] Step four, detecting the influence of FVII high carboxylation mutation on the coagulation activity of active blood coagulation factor VII Collect the culture supernatant of transient transfection recombinant expression for 48 h, dilute the supernatant by 4 times, and then determine the coagulation activity of the mutant protein according to the instructions of the factor VII activity determination kit (coagulation method). The coagulation activity of the mutant protein is shown in Figure 4 .

[0045] The results show that the screened mutants (L12E, L13D, L13E, L13P, L13R, L19E, L19G, L19P) which can efficiently produce active FVII all improve the coagulation activity of FVII to different degrees, indicating that the modification of the signal peptide site can obtain the recombinant FVII protein with high activity.

[0046] Step five, construction of high-efficiency expression system of active coagulation factor VII The VKOR-flag HEK293 cells were inoculated into 12-well plates at 2.5×10 5 cells / well, and were cultured in DMEM containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin, and were grown to about 70%~80% for starting the experiment.

[0047] The FVII MBP6His-phCMV1-WPRE (and some mutants: such as L13P, L19P) plasmid was transfected into the cells using PEI, and after 4~6 h of transfection, the culture medium was replaced with DMEM containing 10% FBS and was cultured for 48 h. The infected cells were passaged into DMEM containing 0.1 mg / ml Hygromycin B (Hygro + ) and 0.8 mg / ml G418, and were cultured for 5 days, and were picked into 96-well plates containing DMEM containing 0.1 mg / ml Hygro + and 0.4 mg / ml G418; after 5 days of culture, they were passaged into 48-well plates, and were passaged into 24-well, 12-well, 6-well and T25 cell culture bottles in this order. The expression level of active FVII was detected, and the cell line with high expression of active coagulation factor VII was screened and was used for large-scale expression of the protein for protein purification.

[0048] Those skilled in the art should understand that the technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced with equivalents, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A FVII signal peptide mutant, characterized in that, FVII signal peptide is genetically mutated at one or more of the L12, L13, L19 sites to increase its activity output.

2. A nucleic acid encoding the FVII signal peptide mutant of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A cell line expressing the FVII signal peptide mutant of claim 1.

5. The method of claim 1, wherein the FVII signal peptide mutant is prepared by, After mutating the leucine at the target position of the wild-type FVII signal peptide to an amino acid that significantly increases the activity output of FVII, the mutant plasmid is transiently transfected into a cell line that overexpresses VKOR, and the cell line that stably and efficiently produces the active FVII mutant is constructed. The cell line is expanded in a multi-layer flask, and the supernatant is collected in large quantities for protein purification.

6. The production method according to claim 5, characterized by, The sequence of the PCR cloning primer of the FVII mutant is shown in any one of SEQ ID NOs: 2-17.

7. Use of the FVII signal peptide mutant of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, or the cell line of claim 4 in the production of coagulation factor VII and / or FVII protein.

8. Use of the FVII signal peptide mutant of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, or the cell line of claim 4 in the preparation of a medicament for treating a coagulation-related disease.

9. Use according to claim 8, characterized in that, The coagulation-related disease includes FVII deficiency or hemophilia.

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