Methods for constructing hepatocyte cell lines with high albumin production and plasmids

Abstract

This invention discloses a method for constructing a hepatocyte cell line with high albumin production, as well as plasmids and cell lines. The invention inserts the human serum albumin gene and the YAP gene into the genome of HepG2 human liver tumor cells to obtain a hepatocyte cell line with high albumin production. The nucleotide sequence of the human serum albumin gene is shown in SEQ ID No:1, and the nucleotide sequence of the YAP gene is shown in SEQ ID No:2. Under specific culture conditions, the HepcellPro-H cell line constructed by this invention shows a significantly higher albumin concentration in the cell culture supernatant than that of unmodified wild-type HepG2 cells, as detected by ELISA. This significantly improves production efficiency, shortens the large-scale albumin extraction cycle, and avoids the potential tumorigenic risks of viral vectors.

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 albumin production and a plasmid. Background Technology

[0002] Human serum albumin (HSA), a crucial clinical emergency drug, has wide applications in the medical field, including the treatment of liver disease, kidney disease, shock, burns, and as a plasma volume expander. Market demand is substantial and continues to grow. Traditional production methods primarily rely on plasma extraction; however, this method has several drawbacks: firstly, plasma supply is limited by factors such as blood donation volume, geographical location, and season, making shortages likely; secondly, plasma quality and safety are difficult to fully control, posing a risk of infectious diseases, such as hepatitis B and HIV, which can be transmitted through plasma, seriously threatening patient safety. Furthermore, albumin extracted from plasma has relatively low purity, requiring further purification, increasing production costs and complexity.

[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. This mutation may enhance the transcriptional activation capacity of YAP, thereby affecting cell growth and proliferation, significantly shortening cell cycle arrest time, increasing cell density, and thus expanding the energy production base of the target protein.

[0004] With the development of biotechnology, recombinant albumin expression systems have emerged, such as CHO (Chinese hamster ovary) cells. However, these systems have limitations such as low yield and high cost. Although CHO cells have certain advantages in protein expression, their albumin production is insufficient to meet the needs of large-scale production, and the culture conditions are complex and costly. In contrast, HepG2 liver-derived cells, originating from the liver, possess a natural mechanism for synthesizing and secreting albumin, theoretically making them more suitable for albumin production. However, HepG2 cells have low natural albumin expression levels, making them unsuitable for direct large-scale production. Therefore, it is urgent to modify them using genetic engineering technology to construct a highly efficient albumin-secreting HepcellPro-H cell line, thereby increasing albumin expression levels and achieving efficient, low-cost, safe, and stable albumin production. This has significant economic and clinical value in alleviating market supply pressure, reducing medical costs, ensuring patient safety, and promoting the development of the biopharmaceutical industry. Summary of the Invention

[0005] To fill the gap in the prior art, this invention provides a method for constructing a hepatocyte cell line with high albumin production and a plasmid. The resulting cell line is named HepcelIPro-H cell line.

[0006] The method for constructing a high-albumin-producing hepatocyte cell line provided by this invention involves inserting the human serum albumin gene and the YAP gene into the genome of HepG2 human liver tumor cells to obtain a high-albumin-producing hepatocyte cell line.

[0007] Preferably, the human serum albumin 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.

[0008] Specifically, a eukaryotic expression vector, pcDNA3.1(+)-HSA-YAP, was first constructed. This vector is based on a pcDNA3.1(+) plasmid containing an ampicillin selection tag, and carries the human serum albumin (HSA) gene and the YAP gene. The nucleotide sequence of the HSA 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(+)-HSA-YAP (SEQ ID No:3) was transfected into HepG2 liver-derived cells to obtain the HepcelIPro-H cell line, which efficiently expresses albumin. This lays the foundation for further research on the expression ability of the HSA gene for secretory recombinant proteins.

[0009] More specifically, the HSA and YAP genes are inserted between the restriction enzyme sites BamHI and EcoRI in the pcDNA3.1(+) plasmid to obtain the eukaryotic expression vector.

[0010] This invention also provides specific steps for constructing the above-mentioned pcDNA3.1(+)-HSA-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, and the digested fragments were extracted. 3) The target gene was recombinantly cloned with the double-enzyme-digested pcDNA3.1(+) plasmid to obtain the pcDNA3.1(+)-HSA-YAP recombinant plasmid containing the HSA and YAP genes; 4) Transform competent cells with the pcDNA3.1(+)-HSA-YAP recombinant plasmid, screen positive clones by colony PCR, and extract the pcDNA3.1(+)-HSA-YAP recombinant plasmid; 5) DNA sequencing analysis was performed on the extracted pcDNA3.1(+)-HSA-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 HSA gene sequence.

[0011] The beneficial effects of this invention are as follows: Under specific culture conditions, the HepcellPro-H cell line constructed by this invention shows a significantly higher albumin concentration in the cell culture supernatant than that of unmodified wild-type HepG2 cells as detected by ELISA, which significantly improves production efficiency, shortens the large-scale extraction cycle of albumin, and avoids the potential tumorigenic risk of viral vectors. Attached Figure Description

[0012] Figure 1 The vector map of pcDNA3.1(+)HSA-YAP.

[0013] Figure 2 This is a comparison of the proliferation curves of HepcellPro-H and HepG2 cells.

[0014] Figure 3 To detect the albumin content in the supernatant of HepG2-HSA cell culture using ELISA.

[0015] Figure 4 To detect the albumin content in the supernatant of HepcellPro-H cell culture using ELISA. Detailed Implementation

[0016] 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.

[0017] Example 1: Construction of the carrier (1) Plasmid double enzyme digestion treatment Take 10 μg of pcDNA3.1(+) plasmid, add restriction endonucleases BamHI (20 U) and EcoRI (20 U), 5 μL of 10×CutSmart Buffer, and add ddH2O to a total volume of 50 μL. Incubate at 37°C for 4 hours. After the reaction, separate the digestion products by 1% agarose gel electrophoresis (100V, 30 minutes), and recover the linearized vector fragment by gel excision.

[0018] (2) Insertion and ligation of HSA and YAP genes The synthesized HSA and YAP genes (sequences shown in the sequence listing, synthesized by Beijing Qingke Biotechnology Co., Ltd.) were mixed with the above linearized vector at a molar ratio of 3:1, 10 μL of T4 DNA ligase (5 U / μL) and 10 μL of 10×T4 Buffer were added, and ddH2O was added to a total volume of 100 μL. The mixture was ligated at 16°C for 12 hours.

[0019] (3) Transformation and positive clone screening The ligation product was transformed into DH5α competent cells: 50 μL of competent cells were mixed with 10 μL of ligation product, incubated on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, incubated on ice for 2 minutes, and then 500 μL of LB medium was added. The cells were incubated at 37°C with shaking for 1 hour. 200 μL of the bacterial culture was evenly spread on LB solid medium containing ampicillin (100 μg / mL) and incubated upside down at 37°C for 16 hours. Single colonies were picked and verified by colony PCR (the primers and amplification conditions used are well known to those skilled in the art and will not be described in detail). After positive clones were confirmed by sequencing to have the HSA gene correctly inserted, the recombinant plasmid pcDNA3.1(+)-HSA-YAP was extracted.

[0020] Example 2: Cell electroporation (1) Preparation of HepG2 cells HepG2 cells in logarithmic growth phase were digested for 3 minutes with EDTA solution containing 0.25% trypsin. Digestion was terminated by adding DMEM medium containing 10% fetal bovine serum (FBS). The cell pellet was collected by centrifugation (1000 rpm, 5 minutes), and the cell density was adjusted to 1 × 10⁶ cells / year with Nucleofector buffer. 6 cells / mL.

[0021] (2) Electroporation operation Mix 10 μg of pcDNA3.1(+)-HSA-YAP plasmid with 100 μL of cell suspension resuspended in Nucleofector electroporation buffer, and transfer to the dedicated electroporation cuvette of the Nucleofector 2b transfection system. Set the electroporation program to Nucleofector program T-028. Immediately after electroporation, seed cells into 6-well plates (DMEM medium containing 10% FBS) and incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0022] 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.

[0023] (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 albumin expression levels (pre-screened by ELISA) were selected and expanded to T25 culture flasks to obtain stable HepcellPro-H cell lines.

[0024] Example 4: CCK-8 assay for cell proliferation (1) Cell inoculation and culture HepcellPro-H cells and wild-type HepG2 cells were seeded at 5×10³ cells / well in 96-well plates with 3 replicates per group. DMEM medium containing 10% FBS was added and the cells were cultured at 37°C for 0, 24, 48, 72 and 96 hours.

[0025] (2) CCK-8 detection Add 10 μL of CCK-8 reagent to each well at 0 h, 24 h, 48 h, 72 h, and 96 h respectively, and incubate at 37 °C for 2 h.

[0026] 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-H was significantly different from that of wild-type (P<0.05).

[0027] (3) ELISA detection of albumin expression level Collect the culture supernatant of HepG2-HSA, HepcellPro-H, and HepG2 cells from T25 culture flasks after 72 h of culture. Centrifuge (1000×g, 10 min) to remove cell debris, calculate the volume, and dilute 1000-fold for immediate analysis. Trypsin digest and count the cells in the T25 culture flasks to obtain the number of HepG2-HSA, HepcellPro-H, and HepG2 cells after 72 h of culture.

[0028] Use the Human Serum Albumin (HSA) ELISA Kit and follow the instructions.

[0029] The results showed that the HepcellPro-H cell line constructed using the above method, after being cultured in DMEM medium containing 10% FBS for 72 hours, showed significantly higher albumin secretion per unit time and per unit data of cells after simultaneous transfection of HSA and YAP genes compared to wild-type HepG2 cells. Compared with the HepG2-HSA cell line transfected only with HSA gene, this indicates that the synergistic effect of HSA and YAP genes on cellular albumin secretion capacity is significant. The CCK-8 assay showed that the proliferation rate of HepcellPro-H cells was significantly different from that of parental cells (P<0.05), indicating that YAP gene transfection affects cell growth activity and enhances cell proliferation capacity.

[0030] 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 albumin production, characterized in that, The human serum albumin gene and the YAP gene were inserted into the genome of HepG2 human liver tumor cells to obtain a hepatocyte line with high albumin production; the nucleotide sequence of the human serum albumin 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 method for constructing a high-albumin-producing hepatocyte cell line according to claim 1, characterized in that, The human serum albumin gene and YAP gene were inserted into the genome of HepG2 human liver tumor cells by transfecting HepG2 human liver tumor cells with their eukaryotic expression vectors.

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