Hepatocyte line construction method with high blood coagulation factor yield and plasmid

Abstract

The invention discloses a construction method of a liver cell line with high blood coagulation factor yield, a plasmid and a cell line. According to the invention, the F10 gene and the YAP gene are inserted into the genome of HepG2 human liver tumor cells to obtain the liver cell line with high blood coagulation factor yield; the nucleotide sequence of the F10 gene is as shown in SEQ ID No: 1, and the nucleotide sequence of the YAP gene is as shown in SEQ ID No: 2. According to the HepcellPro-F cell line constructed by the invention, under specific culture conditions, the concentration of the blood coagulation factors in cell culture supernatant detected by ELISA is obviously increased compared with that of unmodified wild type HepG2 cells, so that the production efficiency is obviously improved, the large-scale extraction period of the blood coagulation factors is shortened, and the potential tumorigenic risk of a viral vector is avoided.

Description

Technical Field

[0001] This invention relates to genetic engineering and biopharmaceutical technology, specifically to a method for constructing a hepatocyte cell line with high coagulation factor production and a plasmid. Background Technology

[0002] Coagulation factors (such as VIII) are core biological agents for treating hereditary coagulation disorders such as hemophilia, and their clinical demand continues to grow. Currently, the production of coagulation factors mainly relies on human plasma extraction or recombinant expression in mammalian cells (such as CHO cells). However, the former is limited by plasma supply shortages and the risk of viral contamination, while the latter faces bottlenecks such as low expression levels (usually below 100 IU / mL), complex post-translational modifications, and high production costs. Taking CHO cells as an example, their long culture cycle, high culture medium costs, and reliance on fetal bovine serum result in insufficient economic viability and sustainability for large-scale production.

[0003] YAP (Yes1 Associated Transcriptional Regulator) is a key effector in the Hippo signaling pathway, involved in regulating cell proliferation, differentiation, and tissue regeneration. YAP5SA refers to the 5SA mutant of the YAP protein, which may enhance the transcriptional activation capacity of YAP, thereby affecting cell growth and proliferation.

[0004] The F10 gene encodes vitamin K-dependent coagulation factor X, a component of the blood coagulation cascade. This factor undergoes several processing steps before its precursor protein, the tripeptide RKR, is cleaved, transforming it into its mature two-chain form. The two chains of the factor are linked together by one or more disulfide bonds; the light chain contains two EGF-like domains, while the heavy chain contains a catalytic domain, its structure being homologous to other hemostatic serine proteases. The mature factor is activated by the cleavage of an activating peptide under the influence of factor IXa (intrinsic pathway) or factor VIIa (extrinsic pathway). Then, in the presence of factors Va, Ca2+, and phospholipids, the activated factor converts prothrombin to thrombin, participating in the blood coagulation process.

[0005] In recent years, research on constructing expression systems based on liver-derived cells has attracted much attention. HepG2 cells, as a human hepatocellular carcinoma cell line, not only retain the protein synthesis and secretion functions unique to hepatocytes but also possess the characteristics of easy culture and rapid proliferation, making them an ideal host for secretory recombinant proteins. However, their endogenous coagulation factor expression levels are extremely low, and traditional gene transfection methods (such as transient transfection with ordinary plasmids) suffer from low exogenous gene integration rates and short expression durations. While existing viral vector systems can improve transfection efficiency, they may introduce tumorigenic risks due to random genomic insertions, making it difficult to meet the compliance requirements for drug production. Therefore, developing a non-viral vector-based HepG2 cell engineering technology that combines high stability and high yield has become a key direction for overcoming the barriers to coagulation factor production. Summary of the Invention

[0006] To fill the gap in the prior art, this invention provides a method for constructing a hepatocyte cell line with high coagulation factor production and a plasmid. The resulting cell line is named HepcelIPro-F cell line.

[0007] The method for constructing a hepatocyte cell line with high coagulation factor yield provided by this invention involves inserting the F10 gene and the YAP gene into the genome of HepG2 human liver tumor cells to obtain a hepatocyte cell line with high coagulation factor yield.

[0008] Preferably, the F10 gene and the YAP gene are inserted into the genome of HepG2 human liver tumor cells by transfecting HepG2 human liver tumor cells with their eukaryotic expression vectors.

[0009] Specifically, a eukaryotic expression vector, pcDNA3.1(+)-F10-YAP, was first constructed. This vector is based on a pcDNA3.1(+) plasmid containing an ampicillin selection tag, and carries the coagulation factor X (F10) gene and the YAP gene. The nucleotide sequence of the F10 gene is shown in SEQ ID No:1, and the nucleotide sequence of the YAP gene is shown in SEQ ID No:2. Then, pcDNA3.1(+)-F10-YAP (SEQ ID No:3) was transfected into HepG2 liver-derived cells to obtain a HepcelIPro-F cell line that efficiently expresses coagulation factors, laying the foundation for further research on the expression ability of the F10 gene in secreting coagulation factors.

[0010] More specifically, the F10 gene and the YAP gene are inserted between the restriction enzyme sites BamHI and EcoRI in the pcDNA3.1(+) plasmid to obtain the eukaryotic expression vector.

[0011] This invention also provides specific steps for constructing the above-mentioned pcDNA3.1(+)-F10-YAP eukaryotic expression vector: 1) The target gene was synthesized by Beijing Qingke Biotechnology Co., Ltd.; 2) The pcDNA3.1(+) plasmid was double-digested with restriction endonucleases BamHI and EcoRI to extract the digested fragments; 3) The target gene was recombinantly cloned with the double-enzyme-digested pcDNA3.1(+) plasmid to obtain the pcDNA3.1(+)-F10-YAP recombinant plasmid containing the F10 and YAP genes; 4) Transform competent cells with the pcDNA3.1(+)-F10-YAP recombinant plasmid, screen positive clones by colony PCR, and extract the pcDNA3.1(+)-F10-YAP recombinant plasmid. 5) DNA sequencing analysis was performed on the extracted pcDNA3.1(+)-F10-YAP recombinant plasmid. Bidirectional sequencing mode was selected, and the universal primers for pcDNA3.1(+) plasmid were used to detect each base across the multiple cloning site region of the plasmid. The detection results were compared with the F10 and YAP gene sequences.

[0012] The beneficial effects of this invention are as follows: Under specific culture conditions, the HepcellPro-F cell line constructed by this invention shows a significant increase in the expression level of coagulation factors in the cell culture supernatant compared with the unmodified HepG2, as detected by ELISA. This significantly improves production efficiency, shortens the large-scale extraction cycle of coagulation factors, and avoids the potential tumorigenic risk of viral vectors. Attached Figure Description

[0013] Figure 1 The vector map of pcDNA3.1(+)-F10-YAP.

[0014] Figure 2 This is a comparison of the proliferation curves of HepcellPro-F and HepG2 cells.

[0015] Figure 3 The content of coagulation factors in the supernatant of HepG2-F10 cell culture was detected by ELISA.

[0016] Figure 4 The content of coagulation factors in the supernatant of HepcellPro-F cell culture was detected by ELISA. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0018] Example 1: Construction of the carrier (1) Construction of pcDNA3.1(+)-F10-YAP eukaryotic expression vector The F10 and YAP gene sequences were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0019] 2) Vector double enzyme digestion Take the pcDNA3.1(+) plasmid (containing an ampicillin resistance tag) and perform double digestion with restriction endonucleases BamHI and EcoRI. The digestion system (50 μL): 2 μg pcDNA3.1(+) plasmid, 5 μL 10× buffer, 1 μL BamHI, 1 μL EcoRI, and ddH2O to a final volume of 50 μL. Incubate at 37℃ for 2 h. Verify the digestion effect using 1% agarose gel electrophoresis and recover the target fragment from the gel.

[0020] 3) Recombinant clones The digested pcDNA3.1(+) vector and the F10-YAP gene fragment (molar ratio 1:3) were ligated using T4 DNA ligase. The ligation system (20 μL) consisted of 100 ng of vector, 300 ng of insert fragment, 1 μL of T4 ligase, and 2 μL of 10× buffer. Ligation was carried out overnight at 16°C.

[0021] 4) Transformation and positive clone screening The ligation product was transformed into DH5α competent cells, plated on LB plates containing ampicillin (100 μg / mL), and cultured at 37°C for 16 h.

[0022] 5) Select single colonies for colony PCR verification (the primers and amplification conditions used are well known to those skilled in the art and will not be described in detail). After confirming the correct insertion of the F10 gene by sequencing, extract the recombinant plasmid pcDNA3.1(+)-F10-YAP.

[0023] Example 2: Cell electroporation (1) Cell preparation HepG2 cells in logarithmic growth phase were harvested, trypsinized, and centrifuged (1000 rpm, 5 min), discarding the supernatant. The cells were resuspended in pre-chilled electroporation buffer (Nucleofector) and the density adjusted to 1 × 10⁶ cells / mL. 6 cells / mL.

[0024] (2) Electroporation Take 100 μL of cell suspension resuspended in Nucleofector electroporation buffer and mix it with 10 μg of pcDNA3.1(+)-F10-YAP plasmid. Transfer the mixture to a dedicated electroporation cuvette for the Nucleofector 2b transfection system. Electroporation program: Nucleofector program T-028. Immediately after electroporation, seed the cells into 6-well plates (DMEM medium containing 10% FBS) and incubate at 37°C and 5% CO2 for 48 h.

[0025] Example 3: Construction of stable cell lines (1) Screening for puromycin Forty-eight hours after transfection, the medium was replaced with selection medium containing 2 μg / mL puromycin, and the medium was changed every two days for 7-10 days. After the untransfected cells had completely died, the medium was replaced with complete medium without puromycin and cultured for another 24 hours to restore the cells to their original state.

[0026] (2) Monoclonal amplification Viable cells were diluted to 0.5 cells / well and seeded into 96-well plates. After 14 days of culture, single clones were picked. Clones with high coagulation factor expression levels (pre-screened by ELISA) were selected and expanded to T25 culture flasks to obtain stable HepcellPro-F cell lines.

[0027] Example 4: CCK-8 assay for cell proliferation (1) HepcellPro-F cells were seeded in 96-well plates (5×10³ cells / well), with untransfected HepG2 cells as the control.

[0028] CCK-8 reagent (10 μL) was added to each well at 0 h, 24 h, 48 h, 72 h, and 96 h, and the cells were incubated at 37 °C for 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader, and proliferation curves were plotted. The results showed that the proliferation rate of HepcellPro-F was significantly different from that of wild type (P<0.05).

[0029] (2) Detection of coagulation factor expression levels (ELISA) Collect the culture supernatant of HepcellPro-F and HepG2 cells after 72 h of culture in T25 flasks, centrifuge (1000×g, 20 min) to remove cell debris, calculate the volume, and dilute 1000-fold for immediate analysis. Trypsin digest and count the cells in the T25 flasks to obtain the number of HepcellPro-F and HepG2 cells after 72 h of culture.

[0030] Use the Human Coagulation Factor VIII ELISA Kit and follow the instructions.

[0031] The results showed that after culturing the HepcellPro-F cell line constructed using the above method in DMEM medium containing 10% FBS for 72 hours, ELISA analysis revealed that the secretion of coagulation factor VIII was significantly higher than that of wild-type HepG2 cells per unit time and per unit data of protein produced, indicating that F10 gene transfection affects the cell's coagulation factor VIII secretion capacity. CCK-8 assays showed that the proliferation rate of HepcellPro-F cells was significantly different from that of the parental cells (P<0.05), indicating that YAP gene transfection affects cell growth activity and enhances cell proliferation capacity.

[0032] Those skilled in the art can better understand and master this invention with the help of the embodiments. However, the protection and scope of the claims of this invention are not limited to the provided examples. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

Claims

1. A method for constructing a hepatocyte cell line with high coagulation factor production, characterized in that, The F10 gene and the YAP gene were inserted into the genome of HepG2 human liver tumor cells to obtain a hepatocyte line with high coagulation factor production; the nucleotide sequence of the F10 gene is shown in SEQ ID No:1, and the nucleotide sequence of the YAP gene is shown in SEQ ID No:

2.

2. The plasmid for constructing a hepatocyte cell line with high coagulation factor production according to claim 1, characterized in that: The F10 and YAP genes were inserted into the genome of HepG2 human liver tumor cells by transfecting HepG2 human liver tumor cells with their eukaryotic expression vectors.

3. A plasmid for constructing a hepatocyte cell line with high coagulation factor production, characterized in that, The plasmid contains the F10 gene and the YAP gene, the nucleotide sequence of the F10 gene is shown in SEQ ID No:1, and the nucleotide sequence of the YAP gene is shown in SEQ ID No:

2.

4. The plasmid for constructing a hepatocyte cell line with high coagulation factor production according to claim 3, characterized in that: The plasmid is a eukaryotic expression vector.

5. The plasmid for constructing a hepatocyte cell line with high coagulation factor production according to claim 4, characterized in that: The plasmid is a eukaryotic expression vector containing an ampicillin selection tag.

6. The plasmid for constructing a hepatocyte cell line with high coagulation factor production according to claim 5, characterized in that: The F10 gene is inserted between the restriction enzyme sites BamHI and EcoRI in the eukaryotic expression vector.

7. The plasmid for constructing a hepatocyte cell line with high coagulation factor production according to claim 3, characterized in that: Its complete nucleotide sequence is shown in SEQ ID No:3.

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