Use of let-7a in CHO cell recombinant protein expression and expression system

Abstract

The present invention relates to the technical field of genetic engineering, and in particular to a use of let-7a in CHO cell recombinant protein expression and an expression system. According to the present invention, the amount of miRNA let-7a in a CHO cell recombinant protein expression system is increased to increase the expression level of a recombinant protein; and an experiment is designed to verify that the direct transfection of let-7a mimics into the CHO cell recombinant protein expression system can increase the expression level of a target protein, for example, adalimumab. In addition, experiments verify that the insertion of a let-7a mimics encoding nucleic acid sequence into a starting vector to construct a let-7a overexpression vector, the transfection of the let-7a overexpression vector into the CHO cell recombinant protein expression system, and the increase of the amount of miRNA let-7a more efficiently increase the expression level of the target protein.

Description

Application and expression system of let-7a in recombinant protein expression in CHO cells Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to the application of let-7a in CHO cell recombinant protein expression and an expression system. Background Art

[0002] Recombinant antibody drugs have become a hot topic in drug development due to their strong targeting and minimal side effects. Using Chinese hamster ovary (CHO) cells to express therapeutic recombinant protein drugs (antibodies) is currently a key platform for biopharmaceutical research and development and production. However, low expression levels have been a major factor hindering the large-scale production of recombinant protein drugs. During cell culture, nutrient depletion, accumulation of harmful metabolites, and increased osmotic pressure can lead to cell apoptosis, severely affecting viable cell density and the yield and quality of the target product. By constructing efficient expression vectors, modifying host cell lines, and optimizing production processes, recombinant protein expression levels can be effectively increased, cell productivity can be enhanced, and high viable cell density can be maintained for an extended period of time.

[0003] MicroRNAs (miRNAs) are a class of evolutionarily conserved single-stranded non-coding RNAs that participate in post-transcriptional gene expression regulation by binding to target gene mRNAs, causing degradation or translational inhibition. Currently, the expression and biological functions of miRNAs in CHO cells have attracted widespread attention, and they can serve as potential cell therapy or engineering targets to improve cell productivity and stress resistance. Compared to most methods that rely on gene overexpression or gene knockout, miRNAs can simultaneously regulate multiple different cellular pathways to maintain cellular homeostasis. By overexpressing or inhibiting specific miRNAs, multiple pathways such as protein synthesis and secretion, and cellular metabolism can be simultaneously controlled, thereby achieving the goal of increasing production.

[0004] The let-7 family of miRNAs was among the first to be discovered in Caenorhabditis elegans. They are involved in regulating various physiological and pathological processes, including cell proliferation, differentiation, apoptosis, immune response, tumorigenesis, and metastasis. Let-7 regulates target genes in two ways: when let-7 binds to the target gene mRNA 3'UTR with perfect complementarity, it can directly cleave and degrade the target mRNA; when let-7 binds to the target gene mRNA 3'UTR with imperfect complementarity, it can block post-transcriptional translation. Studies have shown that the let-7 family, which is associated with apoptosis pathways, may serve as potential targets for cell engineering. However, studies on the effect of let-7a on recombinant protein expression in CHO cells have not yet been reported.

[0005] Although the current production of recombinant proteins has reached a high level, with the continuous development of the biopharmaceutical industry, the demand for recombinant proteins produced by mammalian cells is increasing, and further increasing the production of mammalian cells has become an urgent problem to be solved.

[0006] Summary of the Invention

[0007] In order to overcome the defects of the prior art, one of the objectives of the present invention is to provide an application of let-7a in the expression of recombinant proteins in CHO cells, thereby increasing the amount of recombinant protein expression by increasing the amount of miRNA let-7a.

[0008] A second object of the present invention is to provide a recombinant protein expression system, by constructing CHO cells that highly express let-7a, thereby increasing the expression level of the recombinant protein.

[0009] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0010] The application of let-7a in the expression of recombinant proteins in CHO cells increases the amount of miRNA let-7a in the CHO cell recombinant protein expression system, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0011] Alternatively, the amount of the miRNA in the CHO cell recombinant protein expression system is increased by transfecting let-7a mimics in the CHO cell recombinant protein expression system.

[0012] Specifically, the transfection amount of the let-7a mimics is 30 nM.

[0013] Optionally, the amount of the miRNA in the CHO cell recombinant protein expression system is increased by transfecting a let-7a overexpression vector into the CHO cell recombinant protein expression system.

[0014] Optionally, the let-7a overexpression vector is an expression vector comprising a let-7a mimics coding sequence, and the let-7a mimics coding sequence is shown as SEQ ID NO.2.

[0015] A recombinant protein expression system is constructed by transfecting let-7a mimics into a CHO cell recombinant protein expression system;

[0016] Alternatively, it is constructed by transfecting the let-7a overexpression vector into the CHO cell recombinant protein expression system;

[0017] let-7a mimics and let-7a overexpression vectors increase the amount of miRNA in the recombinant protein expression system. The miRNA is cgr-let-7a, and its nucleotide sequence is shown in SEQ ID NO.1.

[0018] Specifically, the transfection amount of the let-7a mimics was 30 nM;

[0019] Alternatively, the let-7a overexpression vector is an expression vector comprising a let-7a mimics coding sequence, and the let-7amimics coding sequence is shown as SEQ ID NO.2.

[0020] Optionally, the above-mentioned recombinant protein expression system is used to prepare a preparation containing the target protein;

[0021] The preparation is selected from protein detection reagents, target protein drugs for treating or preventing diseases, and gene drugs carrying target proteins.

[0022] As an example, in a specific embodiment of the present invention, the recombinant protein is adalimumab.

[0023] It should be noted that the let-7a described in the present invention is cgr-let-7a.

[0024] Beneficial effects of the present invention:

[0025] The present invention increases the amount of recombinant protein expression by increasing the amount of miRNA let-7a in a CHO cell recombinant protein expression system, and designs experiments to verify that directly transfecting let-7a mimics in the CHO cell recombinant protein expression system can increase the expression amount of a target protein, such as adalimumab. At the same time, through experiments, it is verified that the nucleic acid sequence encoding the let-7a mimics is inserted into a starting vector to construct a let-7a overexpression vector, and the let-7a overexpression vector is transfected into the CHO cell recombinant protein expression system, thereby increasing the amount of miRNA let-7a and more efficiently improving the expression amount of the target protein.

[0026] The present invention provides a new strategy for improving the expression of recombinant proteins in CHO cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a Western blot analysis of the effect of transfected let-7a mimics on adalimumab expression in Example 1;

[0028] FIG2 is an ELISA test showing the effect of transfection of let-7a mimics on the expression of adalimumab in Example 1;

[0029] Figure 3 is a diagram of the let-7a overexpression vector in Example 2;

[0030] FIG4 is a graph showing the expression level of let-7a in a pool of CHO cells stably expressing let-7a detected by qPCR in Example 2;

[0031] FIG5 shows the effect of stable overexpression of let-7a on the expression level of adalimumab detected by ELISA in Example 3. DETAILED DESCRIPTION

[0032] Unless otherwise specified, the relevant operations in the embodiments and test examples are all conventional technical means in the field; the various culture media, reagents, starting vectors, cell lines, tool enzymes, etc. used in the embodiments and test examples are all commercially available products.

[0033] The CHO cells used in the following examples were CHO-S cells purchased from Gibco;

[0034] The let-7a mimics and mimics NC used were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.;

[0035] Adalimumab plasmid was independently constructed, and the specific method was as follows: The pCHO1.0 vector (Invitrogen, Thermo Fisher Scientific) was used as the starting vector, and the light chain (LC) and heavy chain (HC) sequences of adalimumab were cloned downstream of the promoters EF2 / CMV and CMV / EF1, respectively. After a series of vector optimization, the pLC-pHC dual-promoter eukaryotic expression vector was independently constructed.

[0036] Adalimumab stably expressing CHO cells were obtained by self-construction. The specific method is as follows: CHO cells were transfected with the self-constructed adalimumab plasmid, and cell pools were obtained by blasticidin screening for 2-3 weeks. Monoclonal cell lines were screened by limiting dilution method to obtain cells stably expressing adalimumab.

[0037] The following examples are only used to further illustrate the present invention, but are not intended to limit the present invention in any way.

[0038] Example 1 Detection of the effect of transfection of let-7a mimics on adalimumab expression

[0039] (1) Cell transfection

[0040] Adalimumab stably expressed CHO cells were cultured in DMEM / F12 complete medium until the logarithmic growth phase, and 5 × 10 cells were added per well one day before transfection. 5Cells were seeded into 12-well cell culture plates, 1 mL of DMEM / F12 complete medium was added to each well, and the plates were cultured in a 37°C, 5% CO2 incubator for 24 h. The next day, when the cells reached 70%-80% confluency, they were divided into two groups and transfected with let-7a mimics and mimics NC, respectively.

[0041] The transfection steps are as follows:

[0042] ① Add 50 μl of DMEM / F12 basal medium and 30 nM let-7a mimics to a 1.5 mL centrifuge tube and mix well to prepare mimics dilution solution. Mimics NC served as the control group.

[0043] ② Prepare another 1.5 mL centrifuge tube, add 50 μl DMEM / F12 basal medium and 2.0 μl Lipofectamine 2000, mix well to make transfection reagent diluent, and let it stand at room temperature for 5 minutes;

[0044] ③ Add the transfection reagent diluted in step ② to the MIMCs diluted solution in step ①, mix gently, and incubate at room temperature for 20 minutes to prepare the transfection complex;

[0045] ④ Add the transfection complex dropwise to the cell culture plate and gently shake the plate to mix;

[0046] ⑤ Place the culture plate in a 37°C, 5% CO2 incubator for 4-6 hours, then replace the culture medium and continue culturing for 48 hours.

[0047] (2) Cell suspension culture

[0048] 48 h after transfection, cells were digested with trypsin to form a single-cell suspension, and 1 mL of CHO cell serum-free medium (purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd.) was added to resuspend the cells. The cell suspension was mixed with 0.2% trypan blue solution at a ratio of 1:1 and added to a cell counting plate. The viable cell density was detected using a Countstar Rigel S2 cell counter; the number of cells was 5 × 10 5 The viable cell density of 10 cells / mL was inoculated into a 12-well cell culture plate, 2 mL of serum-free culture medium (purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd.) was added to each well, and the culture plate was placed in a 37°C, 5% CO2 incubator shaker at 120 rpm for suspension culture.

[0049] (3) Detection of adalimumab expression level

[0050] After 7 days of cell suspension culture, the cell suspension was harvested and centrifuged at 1000 rpm for 5 minutes. The supernatant was collected and boiled in 5× protein loading buffer for 10 minutes. The expression of adalimumab was analyzed by Western blot. As shown in Figure 1, the expression level of adalimumab was higher in the group transfected with let-7a mimics than in the group transfected with NC mimics.

[0051] Cell culture supernatants were collected and assayed for adalimumab expression according to the instructions for the Human Immunoglobulin G1 Enzyme-Linked Immunosorbent Assay Kit (Kelu Biotechnology Co., Ltd.). As shown in Figure 2, adalimumab expression levels were 8.20 μg / mL and 13.51 μg / mL in the let-7a mimics and mimics NC groups, respectively. Compared with the mimics NC control group, adalimumab expression in the let-7a transfection group increased by 1.65-fold. This suggests that upregulating let-7a expression can increase adalimumab expression.

[0052] Example 2 Construction of let-7a overexpressing CHO cells

[0053] (1) Construction of let-7a overexpression vector

[0054] The pGCMV / EGFP / miR / Blasticidin vector (Shanghai Jima Pharmaceutical Technology Co., Ltd.) was used as the starting vector, and the let-7a mimics coding sequence was inserted downstream of the EGFP reporter gene in the starting vector to construct the let-7a overexpression vector pGCMV-let-7a mimics. pGCMV-NC was used as the control vector. The let-7a mimics coding sequence is shown in SEQ ID NO. 2. The vector map is shown in Figure 3.

[0055] (2) Screening of stable cell pools

[0056] One day before transfection, 5 × 10 5The amount of cells was inoculated into a 12-well cell culture plate in the logarithmic growth phase of CHO cells, 1 mL of DMEM / F12 complete medium was added to each well, and the cells were cultured overnight at 37°C in a 5% CO2 incubator. When the cell density reached 70%-80%, the overexpression vector pGCMV-let-7a mimics and the control vector pGCMV-NC were transfected using Lipofectamine2000 transfection reagent. 48 hours after transfection, 15 μg / mL blasticidin was added for screening. When all the untransfected cells died, 10 μg / mL blasticidin was used to continue screening for 2-3 weeks to obtain a CHO cell pool with stable high expression of let-7a (transfected with the overexpression vector pGCMV-let-7a mimics) and a control group of CHO cells (transfected with the control vector pGCMV-NC);

[0057] (3) qPCR detection of let-7a expression level

[0058] Design and synthesis of let-7a detection primers:

[0059] Forward primer:5'-GGGCGTGAGGTAGTAGGTTGT-3'(SEQ ID NO.3)

[0060] Reverse primer:5'-AGTGCAGGGTCCGAGGTATT-3'(SEQ ID NO.4)

[0061] RT Primer: 5'

[0062] -GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT-3'(SEQ ID NO.5)

[0063] Total RNA from CHO cells with stable high expression of let-7a and control cells was extracted using Trizol, and the total RNA was reverse transcribed into cDNA using the miRNA cDNA First Chain Synthesis Kit (Aikerui Biotechnology Co., Ltd.). qPCR reaction was performed according to the instructions of the SYBR Green Pro Taq HS Premix qPCR Kit. The qPCR reaction system was: 2×SYBR Green Pro Taq HS Premix (ROX plus) 10μl, cDNA 2μl, primer F 0.4μl, primer R 0.4μl, RNase-free water 7.2μl. Reaction conditions were: 95℃30s; 95℃5s, 60℃30s, 40 cycles; 95℃15s, 60℃1min. According to 2 -△△Ct The expression level of let-7a was calculated.

[0064] The results are shown in Figure 4. The expression level of let-7a in CHO cells with stable high expression of let-7a (transfected with the overexpression vector pGCMV-let-7amimics) was significantly higher than that in the control group cells (transfected with the control vector pGCMV-NC). Compared with the control group, the expression level of let-7a increased by 8.22 times.

[0065] Example 3 Detection of the effect of stable overexpression of let-7a on the expression level of adalimumab

[0066] (1) Cell transfection

[0067] 5×10 per well 5 Cell number: CHO cells with stable high expression of let-7a constructed in Example 2 and control group cells were inoculated into 12-well cell culture plates and cultured overnight in a 37°C, 5% CO2 incubator. When the cell density reached 70%-80%, adalimumab plasmid was transfected using Lipofectamine 2000 transfection reagent. 48 hours after transfection, cells were washed with PBS, digested with trypsin, and counted. The number of cells was calculated as 5×10 5 The viable cell density of 10 cells / mL was inoculated into a 12-well cell culture plate, 2 mL of CHO cell serum-free culture medium was added to each well, and the culture plate was placed in a 37°C, 5% CO2 incubator shaker at 120 rpm for suspension culture.

[0068] (2) ELISA detection of adalimumab antibody expression

[0069] After 7 days of cell suspension culture, the cell suspension was collected in a 1.5 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. The cell supernatant was collected and the expression level of adalimumab in the supernatant was detected by ELISA.

[0070] The results are shown in Figure 5. The expression levels of adalimumab in CHO cells stably overexpressing let-7a and in the control group were 21.69 μg / mL and 9.50 μg / mL, respectively. Compared with the control group, the expression level of adalimumab in cells stably overexpressing let-7a increased by 2.28 times, indicating that stable overexpression of let-7a can significantly increase the expression level of adalimumab.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of let-7a in the expression of recombinant proteins in CHO cells, characterized in that: The amount of miRNA let-7a in the CHO cell recombinant protein expression system is increased, and the nucleotide sequence thereof is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The amount of the miRNA in the CHO cell recombinant protein expression system was increased by transfecting let-7a mimics in the CHO cell recombinant protein expression system.

3. The use according to claim 2, characterized in that: The transfection amount of the let-7a mimics was 30 nM.

4. The use according to claim 1, characterized in that: The amount of the miRNA in the CHO cell recombinant protein expression system was increased by transfecting the let-7a overexpression vector in the CHO cell recombinant protein expression system.

5. The use according to claim 4, characterized in that: The let-7a overexpression vector is an expression vector comprising a let-7a mimics coding sequence, and the let-7a mimics coding sequence is shown in SEQ ID NO.

2.

6. A recombinant protein expression system, characterized in that: It was constructed by transfecting let-7a mimics into a CHO cell recombinant protein expression system; Or it was constructed by transfecting the let-7a overexpression vector into the CHO cell recombinant protein expression system; let-7a mimics and let-7a overexpression vectors increase the amount of miRNA in the recombinant protein expression system. The miRNA is cgr-let-7a, and its nucleotide sequence is shown in SEQ ID NO.

1.

7. The recombinant protein expression system according to claim 6, characterized in that: The transfection amount of the let-7a mimics was 30 nM; Alternatively, the let-7a overexpression vector is an expression vector comprising a let-7a mimics coding sequence, and the let-7amimics coding sequence is shown in SEQ ID NO.

2.

8. Use of the expression system according to claims 6 to 7, characterized in that: Used for preparing a preparation containing a target protein; The preparation is selected from protein detection reagents, target protein drugs for treating or preventing diseases, and gene drugs carrying target proteins.

9. The use according to claim 8, characterized in that The target protein is adalimumab.

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