Construction method of hepatocyte line with high albumin yield and plasmid

Abstract

The invention discloses a construction method of a hepatocyte line with high albumin yield, a plasmid and a hepatocyte line. According to the invention, a human serum albumin gene and a YAP gene are inserted into a genome of HepG2 human liver tumor cells so as to obtain a liver cell line with high albumin yield; the nucleotide sequence of the human serum albumin 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-H cell line constructed by the invention, under a specific culture condition, the concentration of albumin in a cell culture supernatant detected by ELISA (Enzyme-Linked Immunosorbent Assay) is obviously increased compared with that of an unmodified wild type HepG2 cell, the production efficiency is obviously improved, the large-scale extraction period of albumin is shortened, and the potential tumorigenic risk of a viral vector is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to genetic engineering and biopharmaceutical technology, in particular to a method for constructing an albumin high-yield hepatocyte cell line and a plasmid. BACKGROUND

[0002] Human serum albumin (HSA) is a key clinical emergency drug widely used in the medical field, involving the treatment of liver disease, kidney disease, shock, burns, and plasma expander and other aspects, with huge and growing market demand. The traditional production method mainly relies on plasma extraction, but this method has many drawbacks: on the one hand, the supply of plasma is limited by the amount of blood donation, region, season and other factors, and is prone to shortages; on the other hand, the quality and safety of plasma are difficult to completely control, and there is a risk of infectious diseases, such as hepatitis B and AIDS viruses that can be transmitted through plasma, which seriously threatens patient safety. In addition, the purity of albumin extracted from plasma is relatively low, which needs to be further refined, increasing the production cost and complexity.

[0003] YAP (Yes1 Associated Transcriptional Regulator) is a key effector factor in the Hippo signaling pathway, involved in regulating cell proliferation, differentiation and tissue regeneration. YAP5SA refers to the 5SA mutant of YAP protein, which may enhance the transcriptional activation ability of YAP, thereby affecting cell growth and proliferation, significantly shortening the cell cycle arrest time, and improving cell density, thereby expanding the production capacity of target proteins.

[0004] With the development of biotechnology, recombinant albumin expression systems have emerged, such as CHO (Chinese hamster ovary) cells. However, these systems have the limitations of low yield and high cost. Although CHO cells have certain advantages in protein expression, their albumin expression yield cannot meet the demand of large-scale production, and the culture conditions are complex and costly. In contrast, liver-derived HepG2 cells have the natural mechanism of synthesizing and secreting albumin due to their origin in the liver, and are theoretically more suitable for albumin production. However, the natural albumin expression of HepG2 cells is low and cannot be directly used for large-scale production. Therefore, it is necessary to modify them through genetic engineering technology to construct a high-efficiency albumin-secreting HepcellPro-H cell line, improve the expression of albumin, and realize efficient, low-cost, safe and stable albumin production, which has important economic value and clinical significance for alleviating market supply pressure, reducing medical costs, ensuring patient safety and promoting the development of the biopharmaceutical industry. SUMMARY

[0005] In order to fill the gap in the prior art, the present application provides a method for constructing an albumin high-yield hepatocyte cell line and a plasmid, and the cell line obtained by the construction is named HepcelIPro-H cell line.

[0006] The method for constructing the albumin high-yield hepatocyte cell line provided by the present application is to insert a human serum albumin gene and a YAP gene into the genome of HepG2 human hepatoma cells to obtain the albumin high-yield hepatocyte cell line.

[0007] Preferably, the human serum albumin gene and the YAP gene are inserted into the genome of the HepG2 human hepatoma cells by transfecting the HepG2 human hepatoma cells with eukaryotic expression vectors of both.

[0008] Specifically, a eukaryotic expression vector, pcDNA3.1(+)-HSA-YAP, is first constructed, which is based on a pcDNA3.1(+) plasmid containing an ampicillin selection tag, and loaded with a human serum albumin (HSA) gene and a YAP gene, the nucleotide sequence of the HSA gene is shown as SEQ ID No: 1, and the nucleotide sequence of the YAP gene is shown as SEQ ID No: 2. Then, the pcDNA3.1(+)-HSA-YAP (SEQ ID No: 3) is transfected into a liver-derived cell HepG2, thereby obtaining a HepcelIPro-H cell line that efficiently expresses albumin, which lays a foundation for further research on the expression capacity of the HSA gene on secretory recombinant proteins.

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

[0010] The present application also provides specific steps of the method for constructing the pcDNA3.1(+)-HSA-YAP eukaryotic expression vector: 1) The target gene is synthesized by Beijing Qianke Biotechnology Co., Ltd.; 2) The pcDNA3.1(+) plasmid is double-cut treated by using restriction endonucleases BamHI and EcoRI, and the cut fragments are extracted; 3) The target gene and the pcDNA3.1(+) plasmid after the double-cut treatment are recombined and cloned to obtain a pcDNA3.1(+)-HSA-YAP recombinant plasmid containing the HSA and YAP genes; 4) The pcDNA3.1(+)-HSA-YAP recombinant plasmid is transformed into competent cells, positive clones are screened by colony PCR, and the pcDNA3.1(+)-HSA-YAP recombinant plasmid is extracted. 5) DNA sequencing analysis of the extracted pcDNA3.1(+)-HSA-YAP recombinant plasmid, selection of bidirectional sequencing mode, detection of each base across the multiple cloning site region of the plasmid using the pcDNA3.1(+) plasmid universal primer, and comparison of the detection results with the HSA gene sequence.

[0011] The HepcellPro-H cell line constructed in the application has a significantly increased albumin concentration in the cell culture supernatant compared with the wild-type HepG2 cell that has not been modified, as detected by ELISA under specific culture conditions, thereby significantly improving the production efficiency, shortening the large-scale extraction period of albumin, and avoiding the potential tumorigenic risk of viral vectors. BRIEF DESCRIPTION OF DRAWINGS

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

[0013] Figure 2 Comparison chart of HepcellPro-H and HepG2 cell proliferation curves.

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

[0015] Figure 4 ELISA detection of albumin content in the cell culture supernatant of HepcellPro-H. DETAILED DESCRIPTION

[0016] The application will be described in detail below in conjunction with the embodiments and drawings, which are implemented on the premise of the technical solutions of the application, and give detailed implementation modes and specific operation processes, but the protection scope of the application is not limited to the following examples.

[0017] Example 1: Construction of vector (1) Plasmid double enzyme digestion treatment Take 10 μg of pcDNA3.1(+) plasmid, add restriction endonuclease BamHI (20 U) and EcoRI (20 U), 10×CutSmart Buffer 5 μL, and ddH2O to a total volume of 50 μL, and incubate at 37℃ for 4 hours. After the reaction is completed, separate the enzyme digestion product using 1% agarose gel electrophoresis (voltage 100 V, 30 minutes), and recover the linearized vector fragment by cutting the gel.

[0018] (2) HSA and YAP gene insertion and ligation The synthesized HSA and YAP genes (sequences shown in the sequence listing, synthesized by Beijing Qikexin Biotechnology Co., Ltd.) were mixed with the linearized vector described above at a molar ratio of 3: 1, 10 μL of T4 DNA ligase (5 U / μL) was added, 10 μL of 10×T4 buffer was added, ddH2O was added to a total volume of 100 μL, and 16°C ligation was performed 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 in an ice bath for 30 minutes, heated at 42°C for 45 seconds, incubated in an ice bath for 2 minutes, 500 μL of LB medium was added, and incubated at 37°C for 1 hour. Take 200 μL of bacterial solution and evenly coat it on LB solid medium containing ampicillin (100 μg / mL), and incubate at 37°C for 16 hours. Pick single colonies and use colony PCR to verify (the primers and amplification conditions used are well known to those skilled in the art and are not described here). After the positive clone was 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 Take the logarithmically growing HepG2 cells, digest with 0.25% trypsin-EDTA solution for 3 minutes, terminate digestion with DMEM medium containing 10% fetal bovine serum (FBS), centrifuge (1000 rpm, 5 minutes) to collect the cell pellet, and adjust the cell density to 1×10 6 cells / mL with Nucleofector buffer.

[0021] (2) Operation of electroporation Take 10 μg of pcDNA3.1(+)-HSA-YAP plasmid and 100 μL of Nucleofector electroporation buffer to resuspend the cell suspension, and transfer it to a Nucleofector 2b transfection system special electroporation cup. Set the electroporation program: Nucleofector program T-028. Immediately after electroporation, the cells were seeded in a 6-well plate (containing 10% FBS DMEM medium) and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0022] Example 3: Construction of stable cell lines (1) Puromycin screening After 48 hours of transfection, the screening medium containing 2 μg / mL puromycin was replaced, and the medium was replaced every 2 days for 7-10 days of continuous screening. After the untransfected cells died completely, the complete medium without puromycin was replaced to continue culturing for 24 hours to restore the cell 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 plasmid for constructing a high-albumin-yielding 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.

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

2.

4. The plasmid for constructing a high-albumin-yielding hepatocyte cell line according to claim 3, characterized in that: The plasmid is a eukaryotic expression vector.

5. The plasmid for constructing a high-albumin-yielding hepatocyte cell line 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 high-albumin-yielding hepatocyte cell line according to claim 5, characterized in that: The human serum albumin gene and the YAP gene are inserted between the restriction enzyme sites BamHI and EcoRI in the eukaryotic expression vector.

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

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