Application of CHO cell-derived exosome miRNA in regulation of recombinant protein expression

Abstract

The application provides a kind of CHO cell-derived exosome miRNA in the application of regulating recombinant protein expression, and belongs to the technical field of biological medicine.The application provides a kind of CHO cell-derived exosome miRNA in the application of regulating recombinant protein expression, and the miRNA is cgr-miR-92a-3p, nucleotide sequence is shown as SEQ ID NO:1.The application finds that the level of exosome miR-92a-3p of CHO cell is down-regulated by transfecting miRNA inhibitor, and the expression level of adalimumab (recombinant protein) is significantly increased, which is expected to provide a new method for improving the expression of CHO cell recombinant protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of CHO cell-derived exosome miRNA in regulation of recombinant protein expression. BACKGROUND

[0002] Biological drugs are increasingly occupying a larger proportion in the pharmaceutical industry, and among them, recombinant protein drugs are used for diagnosis and treatment of various diseases due to their strong target specificity. Nearly 70% of recombinant protein drugs are produced by Chinese hamster ovary (CHO) cells. However, CHO cells often have problems such as poor cell viability and weak ability to secrete exogenous proteins in serum-free medium, resulting in high cost and long cycle of mammalian cell production of recombinant protein drugs.

[0003] In order to meet the growing demand for therapeutic recombinant proteins in the treatment of various diseases and clinical applications, significant progress has been made in cell line development, medium composition and culture condition optimization, but it still cannot fully meet market demand.

[0004] In recent years, exosomes have attracted much attention due to their important role in health and disease and their potential clinical applications in treatment and diagnosis. Almost all types of cells can secrete exosomes, which carry proteins, mRNAs, miRNAs and lipid substances involved in intracellular signal transduction, and participate in important regulatory processes of cell activities. Studies have found that exosomes can not only enhance the anti-apoptotic activity of CHO cells during culture, but also reduce the cost of serum-free suspension culture of cells. However, there are few reports on CHO cell exosome miRNA.

[0005] miRNA is involved in a series of cell processes such as cell division, death, metabolism and tissue differentiation, and CHO cell miRNA is closely related to cell characteristics related to recombinant protein production, such as cell growth, apoptosis, metabolism, glycosylation and epigenetics. Exosome miRNA can participate in the regulation of gene expression and can be used as a potential regulatory factor for cell communication, and is considered as a potential target for optimization of biological pharmaceutical production cell lines.

[0006] The functions of exosome miRNA can be roughly divided into two types: one is the conventional function, that is, miRNA regulates the expression level of target genes, causing specific changes in the expression level of target genes; the other is a new function found in some exosome miRNAs but not in intracellular miRNAs. There is no related research on the effect of CHO cell-derived exosome miR-92a-3p on recombinant protein expression. SUMMARY

[0007] The application aims to provide a CHO cell-derived exosome miRNA in the expression of recombinant proteins, and provide a new method for producing recombinant protein drugs.

[0008] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0009] The application provides a CHO cell-derived exosome miRNA in the expression of recombinant proteins, and the miRNA is cgr-miR-92a-3p.

[0010] Preferably, the nucleic acid sequence of the cgr-miR-92a-3p is shown in SEQ ID NO. 1 (5'-ACAGGCCGGGACAAGUGCAAUA-3').

[0011] Preferably, the primer probe group sequence for amplifying the cgr-miR-92a-3p is shown in SEQ ID NO. 2-4.

[0012] Forward primer: 5'-GCGTATTGCACTTGTCCCG-3' (SEQ ID NO. 2)

[0013] Reverse primer: 5'-AGTGCAGGGTCCGAGGTATT-3' (SEQ ID NO. 3)

[0014] RT primer: 5'

[0015] -GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGGC-3' (SEQ ID NO. 4)

[0016] Preferably, the CHO cell is CHO-S or CHO-K1.

[0017] Preferably, the recombinant protein is adalimumab.

[0018] Preferably, the expression level of the adalimumab is improved by down-regulating the expression level of the exosome miRNA.

[0019] The application finds that the expression level of adalimumab (recombinant protein) is significantly increased by down-regulating the level of CHO cell-derived exosome miR-92a-3p through transfection of miRNA inhibitor, and a new method for improving the expression of recombinant protein of CHO cell is expected to be provided. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1Transmission electron microscope image of exosomes of EGFP stable high / low expression CHO cells in Example 1.

[0021] Figure 2 NTA image of exosomes of EGFP stable high / low expression CHO cells in Example 1.

[0022] Figure 3 Western blot result of marker proteins of exosomes of EGFP stable high / low expression CHO cells in Example 1.

[0023] Figure 4 Expression level of miR-92a-3p in exosomes of EGFP stable high / low expression CHO cells in Example 1.

[0024] Figure 5 Effect of down-regulation of miR-92a-3p level on EGFP expression level detected by flow cytometry in Example 2.

[0025] Figure 6 Effect of down-regulation of miR-92a-3p level on viable cell density of CHO cells in Example 3.

[0026] Figure 7 Effect of down-regulation of miR-92a-3p level on expression amount of adalimumab detected by ELISA in Example 3. DETAILED DESCRIPTION

[0027] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0028] CHO cells used in the following examples were CHO-S purchased from Gibco Company; and exosome miR-92a-3p inhibitor and inhibitor NC used were purchased from GenePharma Company.

[0029] Example 1

[0030] Detection of expression level of miR-92a-3p in CHO cell-derived exosomes

[0031] (1) Construction of EGFP stable expression cells

[0032] CHO cells in logarithmic growth phase were selected, and 2×10 5 cells per well were inoculated into a 24-well culture plate. When the cell confluence reached 80-90%, Lipofectamine TMEGFP plasmid was transfected using 2000 transfection reagent. Forty-eight hours after transfection, cells were selected under pressure with blast fungicide (15 μg / mL) until all untransfected cells died. Cell pools were then obtained by pressure selection for two weeks using blast fungicide (10 μg / mL). The obtained cell pools were then used for single-clone cell line selection using a limiting dilution method to obtain cells with stable high and low EGFP expression.

[0033] (2) Cell Culture

[0034] EGFP-stable high-expressing and low-expressing CHO cells were cultured in 10cm cell culture dishes using DMEM / F12 medium containing 10% fetal bovine serum. The cell culture dishes were placed in a 37°C, 5% CO2 cell culture incubator. When the cell density reached 70%, the cell culture medium was aspirated, the cells were washed with PBS, and then cultured for another 48 hours in DMEM / F12 medium containing 10% exosome-free serum. The cell culture supernatant was then collected.

[0035] (3) Exosome extraction

[0036] Centrifuge the collected cell culture supernatant at 4°C and 2000g for 30 min; collect the supernatant and centrifuge again at 4°C and 10000g for 45 min to remove larger vesicles; collect the supernatant and filter it through a 0.22 μm filter membrane, and collect the filtrate; transfer the filtrate to a new centrifuge tube and centrifuge at 4°C and 100000g for 70 min; remove the supernatant, resuspend the precipitate in 10 mL of pre-chilled PBS, and centrifuge again at 4°C and 100000g for 70 min; remove the supernatant and resuspend the precipitate in 100 μl of pre-chilled PBS to obtain the exosomes.

[0037] (4) Exosome identification

[0038] The morphology of exosomes was observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown, the extracted exosomes from CHO cells with stable high / low EGFP expression have a double-membrane structure and exhibit a typical "cup-and-disc" morphology. NTA was used to identify the particle size and concentration of the exosomes, and the results are as follows: Figure 2 As shown, the extracted CHO cell exosomes have a particle size of approximately 130 nm. Figure 2 The calculated initial concentrations of exosomes were 4.3 × 10⁻⁶. 11 particles / mL and 3.9×10 10 particles / mL ( Figure 2 The concentrations shown are the diluted concentrations. Western blot analysis of the expression of exosomal marker proteins CD63, CD81, and TSG101 yielded the following results: Figure 3 As shown, the extracted CHO cell exosomes all showed expression of CD63, CD81 and TSG101 proteins.

[0039] (5) qPCR detection of the expression level of miR-92a-3p in CHO cell-derived exosomes

[0040] The total RNA of exosomes of EGFP stable high / low expression CHO cells was extracted using Trizol, and the RNA concentration was determined on Quantus Fluorometer. U6 was used as the miRNA internal standard. Reverse transcription reaction was performed using miRNA cDNA first strand synthesis kit (Aikewei Biological Engineering Co., Ltd.). qPCR reaction was performed according to the SYBR Green Pro Taq HS premix qPCR reagent kit instructions (the primers and probes used are shown in SEQ ID NO. 2-4). As shown in Figure 4 the expression level of miR-92a-3p in exosomes of EGFP stable low expression CHO cells was significantly higher than that in EGFP stable high expression CHO cells.

[0041] Example 2

[0042] Detection of the effect of CHO cell-derived exosome miR-92a-3p on EGFP expression level

[0043] (1) Cell transfection

[0044] EGFP stable low expression CHO cells were cultured in DMEM / F12 complete medium to the logarithmic growth phase, and inoculated into 6-well cell culture plates at a cell density of 8x10 3 cells / well, with 3 replicate wells for each group. When the cell density reached 70-80%, 30nM inhibitor NC and miR-92a-3p inhibitor were transfected using Lipofectamine 2000 transfection reagent, respectively, and the medium was changed 6h after transfection and cultured for 48h. TM

[0045] (2) Flow cytometry detection of EGFP expression level

[0046] After 48h of transfection, the cells were collected in a 1.5mL centrifuge tube by trypsin digestion, centrifuged at 1000rpm for 5min, and the cell pellet was collected. The supernatant was discarded, and the cell pellet was washed twice with PBS, then resuspended with 500μl PBS, and the mean fluorescence intensity of the cells was detected using Invitrogen Attune Nxt flow cytometer. The results are shown in Figure 5 ​As shown, the mean fluorescence intensities of cells transfected with inhibitor NC and miR-92a-3p inhibitor were 52489 and 74328, respectively. Downregulation of miR-92a-3p levels increased EGFP expression by 1.42-fold. This indicates that downregulation of exosomal miR-92a-3p levels can significantly increase EGFP expression.

[0047] Example 3

[0048] Detecting the effect of CHO cell-derived exosomes miR-92a-3p on adalimum antibody expression levels

[0049] (1) Constructing cells with stable low expression of adalimum antibody

[0050] Select CHO cells in the logarithmic growth phase, using 2 × 10⁶ cells per well. 5 Cells were seeded into 24-well culture plates, and when the cell confluence reached 80-90%, Lipofectamine was used. TM Adalimide antibody plasmid was transfected using 2000 transfection reagent. Forty-eight hours after transfection, cells were selected under pressure with blast fungicide (15 μg / mL) until all untransfected cells died. Cell pools were then obtained by pressure selection for two weeks using blast fungicide (10 μg / mL). The obtained cell pools were then used for monoclonal cell line selection using a limiting dilution method to obtain cells with stable low expression of adalimide antibody.

[0051] (2) Cell transfection

[0052] Adalimide antibody-stably low-expressing cells were cultured to the logarithmic growth phase using DMEM / F12 complete medium, followed by 8 × 10⁸ cells. 3 Cells were seeded at a density of 70-80% into 6-well cell culture plates, with 3 replicates per group. The plates were incubated at 37°C in a 5% CO2 incubator. When the cell density reached 70-80%, the cells were transfected with inhibitor NC and miR-92a-3-pinhibitor for 48 hours, respectively.

[0053] (3) Cell suspension culture

[0054] 48 hours after transfection, cells were digested with trypsin to form a single-cell suspension, and then cultured at a rate of 5 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 3 mL serum-free culture medium (purchased from Henan Punoyi Biological Products Research Institute Co., Ltd.) into 6-well cell culture plates. The plates were then incubated at 37°C on a shaker at 120 rpm in a 5% CO2 incubator. During the incubation period, 20 μL of the cell suspension was mixed daily with 0.2% trypan blue solution at a 1:1 ratio, and the viable cell density was measured using a Countstar Rigel S2 cell counter. Results are as follows:Figure 6 As shown, the down-regulation of miR-92a-3p level did not significantly change the viable cell density compared with the control group (inhibitor NC group). It is illustrated that the cells with down-regulated exosome miR-92a-3p level can grow normally.

[0055] (4) ELISA detection of adalimumab expression

[0056] After 7 days of cell suspension culture, the cell suspension was collected, centrifuged at 1000 rpm for 5 min, and the cell culture supernatant was collected. The expression amount of adalimumab in the supernatant was detected by ELISA. The results are shown in Figure 7 As shown, the expression amounts of adalimumab in the transfection inhibitor NC group and the miR-92a-3p inhibitor group were 8.51 μg / mL and 15.79 μg / mL, respectively. Compared with the control group (inhibitor NC group), the yield of adalimumab increased by 1.86 times after the down-regulation of miR-92a-3p level. It is illustrated that the inhibition of exosome miR-92a-3p expression can significantly improve the expression level of adalimumab.

[0057] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of an inhibitor of down-regulating CHO cell-derived exosomal miRNA in improving expression of a recombinant protein, characterized in that, The miRNA is cgr-miR-92a-3p; The nucleic acid sequence of the cgr-miR-92a-3p is shown as SEQ ID NO. 1; The recombinant protein is adalimumab; The expression level of the exosome miRNA is down-regulated by a cgr-miR-92a-3p inhibitor, and the expression level of the adalimumab is increased.

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