ShRNA (short hairpin ribonucleic acid) for knocking down MAT2A gene expression, vector, CHO (Chinese hamster ovary) cell line, recombinant protein expression system as well as construction method and application of recombinant protein expression system

Abstract

The invention provides shRNA for knocking down MAT2A gene expression, a vector, a CHO cell line, a recombinant protein expression system and a construction method and application of the recombinant protein expression system, and belongs to the technical field of gene engineering. The preparation method comprises the following steps: inserting an shRNA nucleic acid fragment for knocking down the MAT2A gene into the downstream of a starting vector U6 promoter to construct a vector for knocking down the MAT2A gene, and co-transfecting the vector for knocking down the MAT2A gene to a host cell of a CHO cell line under the action of recombinase to obtain the CHO cell line for knocking down the expression of the MAT2A gene; and transfecting an expression vector containing the recombinant protein coding gene to a CHO cell line with knock-down MAT2A gene expression to construct a recombinant protein expression system. By silencing / inhibiting the expression of the MAT2A gene in the CHO cell line, the gene expression of the cyclin-dependent kinase inhibition factor p21 is improved, and the expression quantity of recombinant protein can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a shRNA for knocking down MAT2A gene expression, a vector, a CHO cell line, a recombinant protein expression system, and a construction method and application thereof. Background Art

[0002] Chinese hamster ovary (CHO) cells have become the most reliable host cells for the production of recombinant therapeutic proteins (antibodies) in biomedicine. They have advantages such as human-like post-translational modifications, correct protein folding, higher productivity and biopharmaceutical development records, and are widely used as hosts for the production of recombinant therapeutic proteins. In recent years, CHO cell expression systems have gradually become the preferred production platform, and the demand for recombinant therapeutic proteins (antibodies) has increased significantly. Clonal heterogeneity of transgenic expression and productivity loss in long-term culture are the main bottleneck issues of recombinant CHO cells. Therefore, in order to improve the level and stability of transgenic expression, it is very important to construct an efficient CHO cell line homogeneous expression system using genetic and cell engineering methods, which helps to screen highly productive cell clones.

[0003] In the methylation process of nucleic acids, phospholipids, histones, biogenic amines and proteins, S-adenosylmethionine (SAM) is the main methyl donor for the synthesis of polyamines and glutathione. Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme that catalyzes the synthesis of SAM from ATP and methionine (MET). It is a rate-limiting enzyme in the methionine cycle and mainly catalyzes the synthesis of SAM from methionine and adenosine triphosphate (ATP). Studies have shown that restricting methionine in the diet of multiple sclerosis model mice can reduce the expansion of pathogenic Th17 cells in vivo, thereby reducing T cell-mediated neuroinflammation and the onset of the disease. Therefore, methionine, as a key nutritional factor, shapes Th cell proliferation and function through epigenetic modification. The cell cycle is a key determinant affecting cell growth: in mammalian cells, the regulation of methionine metabolism is mainly achieved through the expression regulation of related enzymes in the cell cycle, which is mainly affected by hormones, circulating metabolites and byproducts produced by related pathways. At present, there are no reports on the effects of expression regulation of MAT2A gene in CHO cells on the expression and production of recombinant proteins and the characteristics of cell growth itself. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a shRNA, vector, CHO cell line, recombinant protein expression system and construction method and application thereof for knocking down the expression of MAT2A gene, to construct a new cell line by knocking down the expression of MAT2A gene in CHO cell line, to overcome the heterogeneity of recombinant cell clones and the loss of productivity in subculture, and to provide a new solution for increasing the yield of recombinant protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a shRNA for knocking down the expression of MAT2A gene, and the nucleotide sequence of the shRNA is shown as SEQ ID NO.1.

[0007] The present invention also provides a vector for knocking down the expression of the MAT2A gene. The vector is constructed by inserting the shRNA into the downstream of the U6 promoter of the starting vector.

[0008] Preferably, the starting vector is a piggyBac vector; the piggyBac vector comprises an ITR element recognized by a transposase.

[0009] The present invention also provides a CHO cell line for knocking down the expression of the MAT2A gene. The CHO cell line is obtained by co-transfecting the vector into a CHO cell line host cell.

[0010] The present invention also provides a recombinant protein expression system, comprising a CHO cell line host cell, the vector, a transposase or an IVT mRNA expressing the transposase, and an expression vector comprising a recombinant protein encoding gene.

[0011] Preferably, the transposase is PBase transposase.

[0012] The present invention also provides a method for constructing the recombinant protein expression system, comprising the following steps:

[0013] Under the action of transposase, the vector is co-transfected into a CHO cell line host cell to obtain the CHO cell line;

[0014] The expression vector containing the recombinant protein encoding gene is transfected into the CHO cell line to construct a recombinant protein expression system.

[0015] The present invention also provides the use of the shRNA, the vector, the CHO cell line, the recombinant protein expression system or the recombinant protein expression system constructed by the construction method in improving the yield of recombinant protein.

[0016] Preferably, the recombinant protein comprises adalimumab.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention silences / inhibits the expression of MAT2A gene in a cell line (such as CHO cells) that can be used as a host cell for recombinant protein expression, thereby increasing the gene expression of the cyclin-dependent kinase inhibitor p21, arresting the cell cycle at the G1 / 0 phase, affecting cell growth and proliferation, and thus promoting the expression of recombinant proteins.

[0019] The present invention inserts a shRNA fragment targeting the MAT2A gene into the CHO cell genome to silence / inhibit the expression of the MAT2A gene, thereby constructing a cell line with low expression of the MAT2A gene. Specifically, a synthetic shRNA nucleic acid fragment is designed for the target gene MAT2A sequence of the CHO cell, and the encoding nucleic acid fragment is integrated into a piggyBac transposon vector to construct the shRNA transposon vector. PBase continuously and efficiently catalyzes the integration of the shRNA transposon vector into the genome of the CHO cell, thereby achieving a continuous interference effect of the gene in the CHO cell.

[0020] The CHO cell line constructed by the present invention can significantly reduce the mRNA and protein expression levels of MAT2A, and as a host cell for recombinant protein expression, can significantly increase the expression amount of recombinant protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the structure of the MAT2A gene knockdown transposon vector constructed in Example 1 of the present invention;

[0022] Figure 2 This is a graph showing the results of detecting the mRNA expression of the MAT2A gene in the CHO cells with low expression of the MAT2A gene constructed in Example 2 by qPCR in Example 3 of the present invention;

[0023] Figure 3 This is a graph showing the results of Western blot detection of MAT2A gene protein expression in CHO cells with low expression of MAT2A gene constructed in Example 2 in Example 3 of the present invention;

[0024] Figure 4 The CHO cells with low expression of the MAT2A gene constructed in Example 2 detected by the CCK-8 method in Example 4 of the present invention are morphology, growth state observation result graphs, cell proliferation graphs, and viable cell density result graphs (wherein A is a cell morphology, growth state observation result graph, and B is a cell proliferation graph (left graph) and a viable cell density result graph (right graph));

[0025] Figure 5The apoptosis detection result and cell cycle detection result of CHO cells with low expression of the MAT2A gene constructed by flow cytometry in Example 2 in Example 4 of the present invention (wherein A is the apoptosis detection result of CHO cells with low expression of the MAT2A gene, and B is the cell cycle detection result of CHO cells with low expression of the MAT2A gene);

[0026] Figure 6 This is a graph showing the result of detecting the mRNA expression of the cyclin-dependent kinase inhibitor p21 gene in CHO cells with low expression of the MAT2A gene constructed in Example 2 by qPCR in Example 4 of the present invention;

[0027] Figure 7 This is a graph showing the results of Western blot detection of the recombinant protein expression of recombinant adalimumab in CHO cells with low expression of the MAT2A gene constructed in Example 2 and CHO cells with normal expression of the MAT2A gene in Example 5 of the present invention. DETAILED DESCRIPTION

[0028] The present invention provides a shRNA for knocking down the expression of MAT2A gene. The nucleotide sequence of the shRNA is shown in SEQ ID NO.1, specifically: 5'-GTGGTTCGTGAAGCCATTAAA-3'.

[0029] The present invention also provides a vector for knocking down the expression of the MAT2A gene, wherein the vector is constructed by inserting the shRNA into the downstream of the U6 promoter of the starting vector. In the present invention, the starting vector is a piggyBac vector; the piggyBac vector comprises an ITR element recognized by a transposase.

[0030] The present invention also provides a CHO cell line for knocking down the expression of the MAT2A gene. The CHO cell line is obtained by co-transfecting the vector into a CHO cell line host cell.

[0031] The present invention also provides a recombinant protein expression system, comprising a CHO cell line host cell, the vector, a transposase or an IVT mRNA expressing a transposase, and an expression vector comprising a recombinant protein encoding gene. In the present invention, the transposase is a PBase transposase.

[0032] The present invention also provides a method for constructing the recombinant protein expression system, comprising the following steps:

[0033] Under the action of transposase, the vector is co-transfected into a CHO cell line host cell to obtain the CHO cell line;

[0034] The expression vector containing the recombinant protein encoding gene is transfected into the CHO cell line to construct a recombinant protein expression system.

[0035] The present invention also provides the use of the shRNA, the vector, the CHO cell line, the recombinant protein expression system or the recombinant protein expression system constructed by the construction method in increasing the yield of recombinant protein. In the present invention, the recombinant protein includes adalimumab.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] Construction of transposon vector for knocking down MAT2A gene

[0039] 1) According to the sequence of Chinese hamster MAT2A in NCBI GenBank (No.: NW003614172.1) and the design principles of shRNA vectors, the sequence of the nucleic acid fragment encoding shRNA was designed to be: 5'-GTGGTTCGTGAAGCCATTAAA-3' (SEQ ID NO. 1);

[0040] 2) Using piggyBac vector (purchased from Yunzhou Biotechnology Co., Ltd.) as the mother vector, the shRNA nucleic acid fragment synthesized in step 1) was inserted into the downstream of the U6 promoter of the starting vector by enzyme digestion and ligation, thus obtaining a transposon vector for knocking down the MAT2A gene. The schematic diagram of its structure is shown in the figure Figure 1 shown.

[0041] Example 2

[0042] Construction of CHO cell line with knockdown of MAT2A gene expression

[0043] 1) Cell culture preparation: CHO-S cells were grown in DMEM / F12 medium containing 10% inactivated fetal bovine serum at 37°C and 5% CO2. Before transfection, 2.0×10 5 CHO-S cells were seeded in 24-well culture plates and used for cell transfection when the cell confluence reached about 85% after 24 hours of culture.

[0044] 2) Cell transfection: Add 0.5 ml serum-free medium, 2.5 μg knockdown MAT2A gene transposon vector and 0.83 μg PBase transposase to each well (the mass ratio of knockdown MAT2A gene transposon vector to transposase is 3:1), add 50 μL DMEM / F12 medium, and add 2 μL lipo2000 to 50 μL DMEM / F12 medium and gently mix. Discard the medium inoculated with cultured cells in the above 24-well plate, mix the incubated plasmid DNA and liposome mixture, and place at room temperature for 5 minutes. After all cells were incubated in a 37°C and 5% CO2 incubator for 4 hours, the medium was discarded and replaced with fresh medium for continued culture; 48 hours after transfection, Geneticin (Geneticin G418) was used for screening. After the cell confluence reached 80%, the cells were passaged at 1:2 to 1:3, and the drug screening culture was continued for 7 to 14 days.

[0045] 3) Experimental groups: the knockdown MAT2A gene transposon vector + PBase transposase (shRNA group) prepared in Example 1 and a blank control group (Control group); wherein the blank control group is a blank transfection without adding a vector, but only adding PBase transposase, to obtain a blank control cell line (Control group).

[0046] Example 3

[0047] Detection of the efficiency of silencing / inhibiting expression of MAT2A gene

[0048] The CHO cell lines with low expression of the MAT2A gene screened after transfection in Example 2 (shRNA group) and the blank transfection control group cell line (Control group) were collected respectively, and the expression of the MAT2A gene in each group was detected at the mRNA and protein levels by qPCR and Western blot experiments respectively. The specific experimental methods are as follows.

[0049] 1) qPCR experiments

[0050] Primers for qPCR detection of the target gene MAT2A and the internal reference gene β-actin were designed and synthesized respectively. The cultured CHO cell lines of the Control group and the shRNA group were collected, and the total RNA of each group of cells was extracted with Trizol. Then, the total RNA was reverse transcribed into cDNA using the All-in-one 1st Strand cDNA Synthesis Super Mix Kit (purchased from Near Shore Protein Technology Co., Ltd.). cDNA was diluted at a ratio of 1:4 as a template; a 20 μL reaction system was used: 10 μL of SYBR Green Mix, 2 μL of the diluted template, 1 μL of primers (F+R) (target gene MAT2A primers: F: 5′-GGGGTCATTGTCAGGGATCT-3′ (SEQ ID NO.2), R: 5′-AGCCTACGCCATCAAGTCTG-3′ (SEQ ID NO.3)); (internal reference β-actin primers F: 5′-GTCGTACCACTGGCATTGTG-3′ (SEQ ID NO.4), R: 5′-AGGGCAACATAGCACAGCTT-3′ (SEQ ID NO.5)), and 6 μL of enzyme-free water. The reaction conditions were: 95°C pre-denaturation for 30 s, amplification stage (95°C for 10 s, 60°C for 30 s, 45 cycles). The melting curve stage was 95°C for 15 s, 60-95°C for 1 min. Finally, according to 2 -△△CT The algorithm calculated the relative expression of MAT2A gene mRNA respectively.

[0051] Experimental results: Figure 2 As shown, compared with the CHO cell line in the Control group, the mRNA expression level of the MAT2A gene in the CHO cell line in the shRNA group was significantly decreased.

[0052] 2) Western blot experiment

[0053] Western blot was used to analyze the expression of MAT2A protein in the CHO cell lines of the Control group and the shRNA group. Specifically, the cells were washed twice with ice-cold PBS and lysed with RIPA buffer. The supernatants of the two groups of CHO cells were added to 5×SDS sample buffer and boiled in a water bath for 10 min to fully denature. 10 μL of protein samples were taken respectively and separated by 10% SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membrane by wet transfer. The membrane was blocked in 5% skim milk TBST buffer for 2 h, and incubated with Anti-RASSF5 (1:1000) and Anti-β-actin (1:1000) overnight, and then incubated with Anti-Rabbit IgG (1:10000) for 2 h. After washing the membrane three times with TBST, the chemiluminescent colorimetric solution was added for development, and the image results were observed and collected on a gel imager to analyze the expression level of the protein to be tested.

[0054] Experimental results: Figure 3 As shown, compared with the Control group, the expression level of MAT2A protein in the shRNA group with MAT2A knockdown was significantly decreased.

[0055] The above detection results show that in the CHO cell line with MAT2A knockdown constructed in Example 2 of the present invention, the expression of MAT2A gene is significantly inhibited, and the CHO cell line with low expression of MAT2A gene is successfully constructed.

[0056] Example 4

[0057] Verification of biological characteristics of CHO cell lines with low expression of MAT2A gene

[0058] 1. Verify whether CHO cells with knockdown of MAT2A gene can grow and subculture normally by detecting cell proliferation and other cell biological characteristics;

[0059] Specifically, the cell growth characteristics of the Control group and shRNA group CHO cell lines constructed in Example 2 were compared and verified, including observation of cell morphology and growth status, detection of cell proliferation by CCK-8 method, and detection of viable cell density (VCD) by suspension counting.

[0060] The morphology and growth of adherent cells were observed using a fluorescence microscope (EVOS M5000 live cell fluorescence imaging system). Cell proliferation was detected using a CCK-8 kit (Cell Counting Kit-8 kit, Beyotime Biotechnology Co., Ltd.); CHO cells in the Control group and shRNA group were cultured at 5×10 5 / mL suspension for 7 days, and cells were collected every day. VCD was detected using a BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.).

[0061] Experimental results: Figure 4 As shown, there was no significant difference in the growth status, morphology and other biological characteristics of the shRNA group cells compared with the control group cells ( Figure 4 A), but the cell proliferation and viable cell density were affected to a certain extent compared with the cells in the Control group ( Figure 4 B).

[0062] 2. The apoptosis and cell cycle distribution of the CHO cell lines in the Control group and shRNA group constructed in Example 2 were analyzed using an Invitrogen flow cytometer (ThermoFisher).

[0063] Experimental results: Figure 5 As shown, knockdown of MAT2A had no significant effect on cell apoptosis. The results of cell cycle detection showed that compared with the untreated Control group cells, the number of MAT2A knockdown cells in the G1 / 0 phase was greater, while the number of cells in the S phase and G2 phase of the cell cycle was reduced ( Figure 5 B), which blocks the cell cycle at the G1 / 0 phase, thereby affecting cell proliferation and promoting recombinant protein expression; but has no negative impact on cell apoptosis ( Figure 5 A).

[0064] 3. The qPCR experimental method described in Example 3 was used to detect the effect of low expression of the MAT2A gene on the gene expression of the cyclin-dependent kinase inhibitor p21 in cells at the mRNA level:

[0065] Experimental results: Figure 6 As shown, compared with the CHO cell line in the Control group, the mRNA expression level of the p21 gene in the CHO cell line in the shRNA group was significantly increased.

[0066] Example 5

[0067] Expression of recombinant protein in CHO cell line with low expression of MAT2A gene

[0068] The CHO cell line with knockdown of the MAT2A gene screened and verified in Example 2 was applied to recombinant protein expression, including construction of a recombinant protein expression system. The specific construction process is as follows:

[0069] 1) Construction of recombinant adalimumab eukaryotic expression vector

[0070] The element sequences required for constructing the eukaryotic expression vector include U6 promoter, SV40, polyA, IRES, hPGK promoter, HC chain (GenBank No: LQ506328.1) and LC chain (GenBank No: LQ506329.1) of adalimumab, blasticidin (BSD) resistance gene, and puromycin (Puro) resistance gene. The above HC chain, LC chain, BSD, and Puro gene sequences were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0071] First, the HC sequence was cloned into The control eukaryotic expression vector pCHO-HL was obtained by adding promoters EF2 / CMV and CMV / EF1 of pCHO1.0 expression vector (Thermo Fisher Scientific). Subsequently, the pEF2 / CMV-LC-polyA-pCMV / EF1 and Puro sequences in the pCHO-HL expression vector were replaced with CMV promoter and BSD resistance gene sequences, respectively, to construct the vector pHC-BSD.

[0072] Then, the HC and BSD resistance gene sequences in the pHC-BSD vector were replaced with the LC and Puro resistance gene sequences, respectively, to construct the pLC-Puro vector. Finally, the LC-polyA-EF-1α expression cassette was cloned into the pHC-BSD vector to construct the pLC-pHC dual promoter eukaryotic expression vector expressing adalimumab.

[0073] 2) Transfection and recombinant protein expression detection

[0074] The recombinant adalimumab eukaryotic expression vector constructed in step 1) was transfected into the CHO cell line (shRNA group) with low expression of the MAT2A gene constructed in Example 2 and the Control group, and the transfected cells were cultured in a medium containing oryzacidin (1 mg / mL) for 15 days to screen the stably transfected recombinant cell pool. The recombinant CHO cells were then cultured in a six-well plate with 3 mL of protein-free, serum-free, chemically defined CD CHO medium (Henan Punoyi Biological Products Research Institute Co., Ltd.) for 6 days until the cell number reached 1×10 7 Cells / mL were collected every day and the cell density and viability were detected by BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.). The supernatant was collected by centrifugation on the 7th day and quantified with the same cell number for the detection and analysis of the expression of recombinant adalimumab.

[0075] Western blot was used to analyze the expression of adalimumab in the shRNA group and the Control group.

[0076] Experimental results: Figure 7 As shown, the shRNA group increased the expression level of recombinant adalimumab compared with the Control group, and the expression amount increased by 1.58±0.12 times. This shows that the CHO cell line with knockdown of MAT2A gene expression as a host cell for recombinant protein and the construction of a recombinant protein expression system can increase the yield of recombinant protein.

[0077] It can be seen from the above embodiments that the present invention can increase the gene expression of the cyclin-dependent kinase inhibitor p21 by silencing / inhibiting the expression of the MAT2A gene in the CHO cell line that can be used as a host cell for recombinant protein expression, thereby arresting the cell cycle at the G1 / 0 phase, affecting cell growth and proliferation, and thus significantly increasing the expression amount of the recombinant protein.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A shRNA for knocking down the expression of MAT2A gene, characterized in that: The nucleotide sequence of the shRNA is shown in SEQ ID NO.

1.

2. A vector for knocking down the expression of the MAT2A gene, characterized in that: The vector is constructed by inserting the shRNA described in claim 1 into the downstream of the U6 promoter of the starting vector.

3. The carrier according to claim 2, characterized in that The starting vector is a piggyBac vector; the piggyBac vector comprises an ITR element recognized by a transposase.

4. A CHO cell line with knockdown of MAT2A gene expression, characterized in that: The CHO cell line is obtained by co-transfecting the vector according to claim 2 or 3 into a CHO cell line host cell.

5. A recombinant protein expression system, characterized in that: It comprises a CHO cell line host cell, the vector according to claim 2 or 3, a transposase or an IVT mRNA expressing a transposase, and an expression vector comprising a recombinant protein encoding gene.

6. The recombinant protein expression system according to claim 5, characterized in that: The transposase is PBase transposase.

7. The method for constructing a recombinant protein expression system according to claim 5 or 6, characterized in that: The following steps are involved: Under the action of transposase, co-transfecting the vector according to claim 2 or 3 into a CHO cell line host cell to obtain the CHO cell line according to claim 4; The expression vector containing the recombinant protein encoding gene is transfected into the CHO cell line according to claim 4 to construct a recombinant protein expression system.

8. Use of the shRNA according to claim 1, the vector according to any one of claims 2 to 3, the CHO cell line according to claim 4, the recombinant protein expression system according to any one of claims 5 to 6, or the recombinant protein expression system constructed by the construction method according to claim 7 in increasing the yield of recombinant proteins.

9. The use according to claim 8, characterized in that The recombinant protein includes adalimumab.

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