Use of cho cell lines knocked down for suv39h1 expression, recombinant protein expression systems

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to application of a CHO cell line with knocked-down SUV39H1 gene expression and a recombinant protein expression system. The present application constructs an shRNA expression vector targeting SUV39H1 gene, realizes efficient and stable transfection of the interference sequence DNA vector by using an SSR recombinase, catalyzes the integration of the shRNA vector into the cell genome, and can obviously improve the interference efficiency; the SUV39H1 gene expression is knocked down, and the recombinant protein expression system is applied, so that the expression level of the target protein can be obviously improved, and the problem of low expression level in the CHO cell expression system in the biological pharmaceutical production process is effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of CHO cell lines with knockdown of SUV39H1 expression and recombinant protein expression systems. Background Technology

[0002] Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for recombinant protein drug production. However, a significant decline in recombinant protein expression persists during long-term culture, making the selection of stable and efficient CHO cell lines time-consuming and laborious. Therefore, constructing efficient CHO cell expression systems through genetic engineering techniques involving cell line modification is of great significance. Many studies have reported improving recombinant protein expression in CHO cells as host cells by knocking out genes affecting cell differentiation and growth capacity or introducing exogenous genes that promote cell proliferation and differentiation.

[0003] Epigenetic regulation, such as DNA methylation and histone methylation, plays a crucial role in gene transcription activity and gene expression silencing. Current research on the effects of DNA methylation on transgenic expression in CHO cells mainly focuses on promoter modification, such as mutating cytosine in the promoter region, using CpG-free or synthetic promoters, or inserting core CpG island elements into the promoter. Additionally, cell engineering techniques such as gene knockout, gene overexpression, or gene silencing can also alter gene expression levels related to recombinant protein expression or activity. In recent years, CHO cells have also been modified by silencing genes related to DNA methylation. For example, patent CN110257340A discloses a Dnmt3b gene-deficient CHO cell line. In this cell line, the epigenetic modification mediated by the missing DNA methyltransferase Dnmt3b is more closely related to gene expression regulation, and exogenous protein expression is more stable and significantly increased in the absence of selection pressure.

[0004] Trimethylation of histone 3 at position 9 (H3K9me3) is associated with gene repression and heterochromatin formation. Histone methyltransferase SUV39H1 mainly modifies H3K9 through trimethylation, thereby leading to transcriptional silencing and playing a significant role in the regulation of heterochromatin formation and gene expression. However, techniques for constructing CHO cell lines by knocking down or silencing SUV39H1 expression to enhance recombinant protein expression have not yet been reported. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide the application of shRNA expression vectors targeting the SUV39H1 gene in the construction of recombinant protein expression systems, which can improve the expression level of recombinant proteins.

[0006] The second objective of this invention is to use the CHO cell line with knockdown of SUV39H1 expression as a host cell for recombinant protein expression, thereby increasing the expression level of recombinant proteins.

[0007] Meanwhile, a third objective of this invention is to provide a recombinant protein expression system that uses the CHO cell line with knocked-down SUV39H1 expression provided by this invention as the host cell to enhance the expression level of recombinant proteins.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0009] Application of shRNA expression vectors targeting the SUV39H1 gene in the construction of recombinant protein expression systems.

[0010] Application of CHO cell lines with knockdown of SUV39H1 gene expression as host cells for recombinant protein expression.

[0011] Optionally, the recombinant protein is selected from adalimumab and human serum albumin.

[0012] A recombinant protein expression system is constructed from CHO cell line host cells, an shRNA expression vector targeting the SUV39H1 gene, and an expression vector containing a recombinant protein encoding gene.

[0013] Optionally, it also includes an SSR recombinase (Site-specific Recombinase); the shRNA expression vector targeting the SUV39H1 gene contains an SSR element.

[0014] Optionally, the construction method includes first constructing a CHO cell line with knockdown of SUV39H1 expression: under the action of recombinase, the shRNA expression vector is co-transfected into the host cells of the CHO cell line to obtain host cells with knockdown of SUV39H1 expression;

[0015] The expression vector containing the recombinant protein-coding gene was then transfected into host cells with knockdown of SUV39H1 expression.

[0016] Alternatively, other construction methods can be used, including first transfecting an expression vector containing the recombinant protein encoding gene into a CHO cell line to obtain a recombinant protein low-expression cell line; then, under the action of recombinase, co-transfecting a shRNA expression vector targeting the SUV39H1 gene into the recombinant protein low-expression cell line.

[0017] Optionally, it also includes first constructing an shRNA expression vector: designing shRNA based on the sequence of SUV39H1, and ligating shRNA nucleic acid fragments into a starting vector with SSR elements.

[0018] Optionally, the shRNA sequence targeting the SUV39H1 gene is: 5'-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3'.

[0019] Optionally, the starting carrier is a piggy BAC carrier.

[0020] Specifically, the nucleic acid fragment encoding shRNA is inserted downstream of the U6 promoter.

[0021] Beneficial effects of this invention:

[0022] RNAi technology can highly specifically and selectively knock out the expression of target genes. By introducing shRNA into mammalian cells through vector transfection, shRNA can be stably integrated and target genes can be knocked out for a long time, thereby affecting the transcription and protein expression levels of recombinant proteins. This invention uses RNAi technology to achieve SUV39H1 gene knockdown and silence SUV39H1 gene expression, which has the following beneficial effects:

[0023] 1) This invention designs and synthesizes shRNA (5'-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3') targeting the SUV39H1 gene sequence of Chinese hamsters, and uses SSR recombinase to achieve efficient and stable transfer of the interference sequence DNA vector, catalyzing the integration of the shRNA vector into the cell genome, which can significantly improve the interference efficiency and realize the construction of a stable cell line for interference of the target gene SUV39H1;

[0024] 2) This invention can significantly reduce the expression levels of SUV39H1 mRNA and protein in CHO cells through SSR gene recombination technology, and has a significant knockdown effect on the target gene;

[0025] 3) The SUV39H1 stably knocked-down cell line constructed in this invention has the same growth ability as normal cells. As the host cell of the recombinant protein expression system, it can significantly improve the expression level of the target protein, effectively solving the problem of low expression level in the CHO cell expression system during biopharmaceutical production.

[0026] 4) Meanwhile, the embodiments of the present invention demonstrate that transfecting the shRNA expression vector targeting the SUV39H1 gene into the recombinant protein expression system can increase the recombinant protein expression level. Attached Figure Description

[0027] Figure 1Figure 1 shows the results of qPCR detection of SUV39H1 gene mRNA expression in stable knockdown cell lines and control cells.

[0028] Figure 2 The figure shows the results of detecting SUV39H1 gene protein expression in stable knockdown cell lines and control cells using the Digital Western (Jess) method.

[0029] Figure 3 Figure showing the results of ELISA detection of recombinant adalimumab protein expression in SUV39H1 stably knocked-down cell lines and control cells expressing adalimumab;

[0030] Figure 4 The figure shows the results of SUV39H1 gene protein expression detection in stable knockdown cell lines expressing human serum albumin and control cells using the Digital Western (Jess) method.

[0031] Figure 5 The figure shows the protein expression results of human serum albumin in the SUV39H1 stably knocked-down cell line and the control group cells as detected by ELISA. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto;

[0033] Example 1: Construction of an shRNA expression vector targeting the SUV39H1 gene

[0034] The shRNA expression vector targeting the SUV39H1 gene was constructed using the following method:

[0035] 1) Based on the NCBI GenBank Chinese hamster SUV39H1 gene sequence (GeneID:100764568), and in accordance with the design principles of shRNA, the shRNA encoding nucleic acid fragment sequence was designed as 5'-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3';

[0036] 2) Using the piggy BAC vector containing SSR elements (purchased from Haixing Biotechnology Co., Ltd.) as the starting vector, the nucleic acid fragment synthesized in step 1) is inserted downstream of the U6 promoter of the starting vector by enzyme digestion and ligation to construct an shRNA transposon vector containing SSR elements, which is the shRNA expression vector targeting the SUV39H1 gene.

[0037] Example 2: Construction of a CHO cell line with knockdown of SUV39H1 expression

[0038] This embodiment provides a CHO cell line with knocked-down SUV39H1 expression, and the specific steps for its construction are as follows:

[0039] 1) Cell Culture Preparation: CHO-S cells were cultured in a 37°C, 5% CO2 incubator using DMEM / F12 medium (containing 10% fetal bovine serum). Resuscitated cells were passaged at least three times, and the cell culture medium was changed the day before transfection. The day before transfection, cells were trypsin-digested and seeded in two wells of a 24-well plate. Cells with approximately 60% confluence and in optimal proliferative condition were used for transfection. The medium was changed to antibiotic-free, low-serum medium before transfection.

[0040] 2) Cell transfection: Add 50 μL of antibiotic-free, low-serum medium, 2.5 μg of vector and SSR recombinase to each well, along with 50 μL of Opti-MEM medium. Add 2 μL of Lipo2000 to the Opti-MEM medium and mix gently. Discard the culture medium from the 24-well plate containing the cells. Mix the incubated plasmid DNA and liposome mixture and incubate at room temperature for 5 min. Gently shake the culture dish to distribute the mixture evenly, and label and record the time. Finally, incubate the culture plate containing the transfection complex and cells in a constant temperature incubator (37℃, 5% CO2) overnight, then replace with DMEM / F12 complete medium. 48 h after transfection, select using Puromycin; once cell confluence reaches 80%, passage at a 1:2 ratio, and perform drug selection for 7-14 days.

[0041] 3) The experiment was divided into two groups: shRNA1+SSR recombinase interference group (R1 group) and blank control group (control). The shRNA1+SSR recombinase interference group used the vector provided in Example 1 to transfect cells according to the above steps to obtain a stable knockdown cell line of SUV39H1 (R1 group); the blank control group was a blank transfection without the addition of the vector to obtain a blank control cell line (control).

[0042] Example 3: Detection of Effective shRNA Interference Efficiency

[0043] 1) qPCR experiment

[0044] Primers for qPCR detection of the target gene SUV39H1 and the internal reference gene GAPDH were designed and synthesized, as shown in Table 1:

[0045] Table 1. DNA Primer Sequence List

[0046]

[0047] Stable knockout cell line SUV39H1 (R1) and control group were collected. mRNA was extracted by Trizol lysis and reverse transcribed into cDNA. The mRNA expression of the SUV39H1 gene in R1 and control cells was detected using SUV39H1 primers. The reaction volume was 20 μL, and the reaction conditions were: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles. The standardized internal control was GAPDH. -ΔΔCt The method calculates the relative expression level of the target gene. For example... Figure 1 As shown, the results indicate that the mRNA expression level of the SUV39H1 gene in the shRNA1+SSR recombinase interference group R1 was reduced to 26% of the control level.

[0048] 2) Digital Western blot experiment

[0049] The Jess fully automated protein expression analysis system (ProteinSimple) was used to detect SUV39H1 protein expression.

[0050] Sample and antibody preparation: Proteins extracted from lysed cells in each group were stored in lysis buffer and their concentrations were determined using a BCA kit.

[0051] Prepare the Jess instrument: First, turn on Jess and connect it to the computer. Then, use Self-Test to perform a hardware self-test on Jess.

[0052] Prepare reagents: Take out kits PS-ST01-EZ-8 and DM-001, take out DTT powder and add 40 μL of deionized water to prepare DTT solution; take out Master Mix powder and add 20 μL of DTT solution and 20 μL of 10×Sample Buffer to prepare 5×Master Mix (i.e., Loading Buffer); take out Ladder powder and add 20 μL of deionized water to prepare Ladder (i.e., marker, molecular weight standard).

[0053] Add sample: Add 3 μL of sample (prepared with Master Mix) to each well. Calculate the required total sample volume based on the final sample concentration and the number of wells. Denature the prepared sample in a 95°C water bath for 5 min. Place the primary antibody (10 μL / well) and secondary antibody (10 μL / well) on ice for later use. Prepare 200 μL each of Lumino-S and Peroxide to form a chemiluminescent solution, and place it on ice to protect from light.

[0054] Finally, following the instructions, use the reverse-phase aspiration method to add the prepared reagents sequentially into the plate. After balancing, centrifuge at 2500 rpm for 5 minutes at room temperature; finally, perform the operation by removing the capillary tube and plate, placing them in Jess, and clicking Start to begin the process.

[0055] like Figure 2 As shown in Jess's results, transfection with the shRNA interference vector significantly reduced the expression of SUV39H1 protein in stably knocked-down cell lines.

[0056] Example 4: Construction of CHO cell proliferation assay with knockdown of SUV39H1 gene expression

[0057] The ability of the SUV39H1 knockdown CHO cell line to grow and be passaged normally was verified by detecting its cell biological characteristics.

[0058] First, the cell morphology was observed under a microscope to determine if it was normal. Cell proliferation was then detected using the CCK-8 assay (Cell Counting Kit-8, Beyotime Biotechnology Co., Ltd.): R1 group and control group cells were seeded in 96-well plates, and the absorbance was measured at 450 nm wavelength using an ELISA reader at 24, 48 and 72 h, respectively, and growth curves were plotted.

[0059] Experimental results show that CHO cells with knockdown of the SUV39H1 gene expression can grow and proliferate normally.

[0060] Example 5 Recombinant Protein Expression

[0061] 1. Recombinant adalimumab (ADM)

[0062] 1) Construction of a stable knockdown cell line expressing adalimum SUV39H1 and detection of its cell biological characteristics

[0063] To investigate the effect of SUV39H1 stably knocked-down CHO cell lines on the expression of recombinant proteins (antibodies), SUV39H1 stably knocked-down cells (R1 group in Example 2) and CHO-S cells (control group in Example 2) were transfected using a eukaryotic expression vector for recombinant adalimumab. The transfected cell pools were stabilized for 14 days using blastcin, and SUV39H1 stably knocked-down cells expressing ADM (ADM-R1) and control cells were constructed.

[0064] For adherent ADM-R1 cells and control cells, cell proliferation was detected using the CCK-8 assay; for suspension culture cells, recombinant CHO cells (ADM-R1 and control) were cultured in six-well plates at a rate of 3 × 10⁻⁶ cells / well. 5Cells were seeded at a concentration of [cell / mL] in CD CHO medium (Henan Punoyi Biological Products Research Institute Co., Ltd.) with a defined chemical composition and cultured in suspension for 7 days. Cells were collected daily and stained using the trypan blue method. The BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.) was used to detect cell viability and viable cell density. Experimental results showed that the biological characteristics of ADM-R1 cells, such as growth status, morphology, cell proliferation, and viable cell density, were not significantly different from those of normal CHO cells.

[0065] 2) ELISA detection of recombinant ADM expression level

[0066] ADM-R1 cells and control cells were cultured in suspension in CD CHO with a defined chemical composition for 7 days. The supernatant was collected by centrifugation on day 7, and the expression of recombinant ADM was detected by ELISA. Figure 3 As shown, the results indicate that the expression level of recombinant ADM in the SUV39H1 stably knocked-down cell line was increased by 1.45 times compared with the control cell group.

[0067] 2. Human Serum Albumin (HSA)

[0068] 1) Construction of SUV39H1 stably knocked-down cells expressing human serum albumin and detection of cell biological characteristics.

[0069] This invention utilizes a eukaryotic vector expressing recombinant human serum albumin to transfect CHO-S cells. The transfected cell pool is stabilized and screened with blastomycin for 14 days to obtain a CHO cell pool expressing HSA. The limiting dilution method is used to obtain a single clonal cell line (control) with low HSA expression.

[0070] The shRNA expression vector targeting the SUV39H1 gene constructed in Example 1 was transfected into HSA-low-expressing monoclonal antibodies using SSR recombinase. Stable knockdown cell lines of SUV39H1 expressing HSA (HSA-R1) were obtained using Puromycin selection. Figure 4 As shown, the control group consists of single-clone cell lines with low HSA expression;

[0071] CCK-8 assay was used to detect the proliferation of adherent HSA-R1 cells; for suspension culture cells, HSA-R1 cells and HSA-low expression monoclonal cells (control) were cultured in six-well plates at a density of 3 × 10⁻⁶ cells / well. 5 HSA-R1 cells were seeded at 1 / mL in CD CHO medium, and the viable cell density and viability were detected by trypan blue staining. The results showed that the proliferation of HSA-R1 cells was not significantly different from that of the control group.

[0072] 2) ELISA detection of HSA expression changes

[0073] On day 7 of suspension culture, the supernatant was collected by low-temperature centrifugation. ELISA was used to detect changes in recombinant protein expression in the stable knockdown SUV39H1 cell line HSA-R1 and the control group HSA-low-expressing monoclonal cell line. Figure 5 As shown, the results indicate that the expression level of HSA in the SUV39H1 stably knocked-down cell line was increased by 2.63 times compared with the control cell group.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of shRNA expression vectors targeting the SUV39H1 gene in constructing recombinant protein expression systems, characterized in that, The host cell of the recombinant protein expression system is the CHO cell line; the recombinant protein is selected from adalimumab and human serum albumin.

2. A recombinant protein expression system, characterized in that, It was constructed from CHO cell line host cells, an shRNA expression vector targeting the SUV39H1 gene, and an expression vector containing a recombinant protein encoding gene; the recombinant protein was selected from adalimumab and human serum albumin.

3. The recombinant protein expression system as described in claim 2, characterized in that, The shRNA expression vector targeting the SUV39H1 gene contains an SSR element.

4. The recombinant protein expression system as described in claim 3, characterized in that, The construction method includes first constructing a CHO cell line with knockdown of SUV39H1 expression: co-transfecting the shRNA expression vector targeting the SUV39H1 gene into the host cells of the CHO cell line to obtain host cells with knockdown of SUV39H1 expression; The expression vector containing the recombinant protein-coding gene was then transfected into host cells with knockdown of SUV39H1 expression.

5. The recombinant protein expression system as described in claim 4, characterized in that, It also includes first constructing an shRNA expression vector: based on the sequence of SUV39H1, shRNA is designed, and the shRNA nucleic acid fragment is ligated and inserted into a starting vector with SSR elements.

6. The recombinant protein expression system as described in claim 5, characterized in that, The shRNA sequence targeting the SUV39H1 gene is: 5'-GGTTAAGTGGCGTGGATATCCCTCGAGGGATATCCACGCCACTTAACC-3'.

7. The recombinant protein expression system as described in claim 6, characterized in that, The starting vector is the piggyBAC vector; the nucleic acid fragment encoding shRNA is inserted downstream of the U6 promoter.

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